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WO2025221789A1 - Compositions et méthodes destinées à être utilisées dans des thérapies cellulaires car/tcr - Google Patents

Compositions et méthodes destinées à être utilisées dans des thérapies cellulaires car/tcr

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Publication number
WO2025221789A1
WO2025221789A1 PCT/US2025/024771 US2025024771W WO2025221789A1 WO 2025221789 A1 WO2025221789 A1 WO 2025221789A1 US 2025024771 W US2025024771 W US 2025024771W WO 2025221789 A1 WO2025221789 A1 WO 2025221789A1
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WO
WIPO (PCT)
Prior art keywords
cells
cell
car
acid sequence
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/024771
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English (en)
Inventor
Lu YAO
Sergio QUINONES-PARRA
Devon SHEDLOCK
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Poseida Therapeutics Inc
Original Assignee
Poseida Therapeutics Inc
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Publication date
Application filed by Poseida Therapeutics Inc filed Critical Poseida Therapeutics Inc
Publication of WO2025221789A1 publication Critical patent/WO2025221789A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4256—Tumor associated carbohydrates
    • A61K40/4257—Mucins, e.g. MUC-1
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/23—On/off switch
    • A61K2239/25—Suicide switch

Definitions

  • polynucleotides comprising in the 5’ to 3’ direction: a promoter; a nucleic acid sequence encoding iCas9 safety switch; a nucleic acid sequence encoding a T-cell receptor gamma chain (gTCR) and a nucleic acid sequence encoding a T-cell receptor delta chain (dTCR); a nucleic acid sequence encoding a chimeric antigen receptor (CAR); and a nucleic acid encoding a selectable marker.
  • a promoter a nucleic acid sequence encoding iCas9 safety switch
  • gTCR T-cell receptor gamma chain
  • dTCR T-cell receptor delta chain
  • CAR chimeric antigen receptor
  • polynucleotides comprising in the 5’ to 3’ direction: a promoter; a nucleic acid sequence encoding iCas9 safety switch; a nucleic acid sequence encoding a dTCR; a nucleic acid sequence encoding a gTCR; a nucleic acid sequence encoding a chimeric antigen receptor (CAR); and a nucleic acid encoding a selectable marker.
  • the nucleic acid sequence encoding iCas9 safety switch further comprises a 3’ 2A sequence.
  • the nucleic acid sequence encoding gTCR further comprises a 3’ 2A sequence.
  • the nucleic acid sequence encoding dTCR further comprises a 3’ 2A sequence.
  • the nucleic acid sequence encoding a chimeric antigen receptor (CAR) further comprises a 3’ 2A sequence.
  • polynucleotides comprising in the 5’ to 3’ direction: a promoter; a nucleic acid sequence encoding iCas9 safety switch; a nucleic acid sequence encoding a bTCR; a nucleic acid sequence encoding an aTCR; a nucleic acid sequence encoding a chimeric antigen receptor (CAR); and a nucleic acid encoding a selectable marker.
  • the nucleic acid sequence encoding iCas9 safety switch further comprises a 3’ 2A sequence.
  • the nucleic acid sequence encoding aTCR further comprises a 3’ 2A sequence.
  • the nucleic acid sequence encoding bTCR further comprises a 3’ 2A sequence. In certain aspects, the nucleic acid sequence encoding a chimeric antigen receptor (CAR) further comprises a 3’ 2A sequence.
  • the promoter is the EF1a promoter. In certain embodiments, the EF1a promoter comprises the nucleic acid sequence set forth in SEQ ID No: 83. In one embodiment, the iCas9 safety switch comprises the nucleic acid sequence set forth in SEQ ID No: 84. In one embodiment, the iCas9 safety switch comprises the amino acid sequence set forth in SEQ ID No: 93.
  • the T-cell receptor gamma chain is a G115 variant.
  • the G115 variant is gene optimized and comprises the nucleic acid sequence set forth in SEQ ID No: 49.
  • the T-cell receptor delta chain is a G115 variant.
  • the G115 variant is gene optimized and comprises the nucleic acid sequence set forth in SEQ ID No: 51.
  • the nucleic acid sequence encoding aTCR is gene optimized and comprises the nucleic acid sequence set forth in SEQ ID No: 53.
  • the nucleic acid sequence encoding bTCR is gene optimized and comprises the nucleic acid sequence set forth in SEQ ID No: 55.
  • the selectable marker is a recombinant dihydrofolate reductase mutein gene (mDHFR).
  • the mDHFR comprises the nucleic acid sequence set forth in SEQ ID NO: 86.
  • the mDHFR comprises the amino acid sequence set forth in SEQ ID NO: 94.
  • the CAR targets an oncogenic gene product comprising BCMA, CD7, CD19, CD20, MUC1C, PSMA, CD70, mesothelin or c-kit.
  • transposons comprising any of the polynucleotides of the present disclosure.
  • the transposon is a piggyBac transposon.
  • the piggyBac left end ITR of the piggyBac transposon comprises the nucleic acid sequence set forth in SEQ ID NO: 82.
  • the piggyBac right end ITR of the piggyBac transposon comprises the nucleic acid sequence set forth in SEQ ID NO: 88.
  • vectors comprising any of the polynucleotides of the present disclosure.
  • the vector comprises any of the transposons of the present disclosure.
  • cells comprising the polynucleotides, transposons or vectors of the present disclosure.
  • the cell expresses an iCasp9 safety switch, a functional CAR, a functional, recombinant gdTCR or abTCR and a selectable marker.
  • compositions comprising a T-cell population isolated from at least one healthy human donor; wherein the T-cell population a) does not express a functional endogenous T-cell receptor; b) expresses a chimeric antigen receptor, a recombinant T-cell receptor (abTCR or gdTCR); and a selectable marker.
  • compositions comprising a T-cell population isolated from a patient having a disease or disorder; wherein the T-cell population a) does not express a functional endogenous T-cell receptor and b) expresses a chimeric antigen receptor and a recombinant T-cell receptor (abTCR or gdTCR); and mDHFR.
  • a composition comprising cells expressing a chimeric antigen receptor and a recombinant T cell receptor (CAR/TCR cells), wherein the CAR cells comprise a chimeric antigen receptor (CAR), a recombinant T-cell receptor (abTCR or gdTCR) and a recombinant mutein dihydrofolate reductase (mDHFR), and wherein the CAR/TCR cells exhibit enhanced cytolytic activity compared to CAR-T cells.
  • CAR/TCR cells comprise a chimeric antigen receptor (CAR), a recombinant T-cell receptor (abTCR or gdTCR) and a recombinant mutein dihydrofolate reductase (mDHFR)
  • CAR/TCR cells a chimeric antigen receptor
  • abTCR or gdTCR a recombinant T-cell receptor
  • mDHFR recombinant mutein dihydrofolate reductase
  • step (b) can occurs within 15 minutes, within 30 minutes, within an hour, within two hours, within four hours, within six hours, within eight hours, within 12 hours, within 18 hours, within 24 hours, within 36 hours, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within a week, within 2 weeks, within 3 weeks, within 4 weeks, with 6 weeks, or within 8 weeks of step (a).
  • the method further comprises, following step (b), a step (c) administering to the subject an effective amount of a bispecific antibody (also referred to herein as a Bispecific T-Cell Engager (TCE)).
  • a bispecific antibody also referred to herein as a Bispecific T-Cell Engager (TCE)
  • the bispecific antibody binds to the same target cells as the CAR and a CD3 domain of the recombinant TCR of the CAR/TCR cell. In some embodiments, the bispecific antibody binds to a different target cell than the CAR and a CD3 domain of the recombinant TCR of the CAR/TCR cell. In one embodiment, the bispecific antibody binds to the target cell and the CD3 domain of the recombinant TCR (abTCR or gdTCR) of the CAR-T/TCR-T cell resulting in stimulation of the CAR-T/TCR-T cell. [0019] In some embodiments, step (c) occurs at a pre-determined time after following step (b).
  • step (c) occurs after disease progression in the subject.
  • disease progression is characterized by increase in the number of disease episodes and/or symptoms; increase in lesion size; increase in disease cell infiltration into adjacent peripheral organs and/or tissues; increase in disease spread; increase in auto-immune response; increase in one or more of the following symptoms: congestion: fluid/edema, wheezing, coughing, hypoxemia, low oxygen saturation, lung stiffness, shortness of breath, shortness of breath during exercise, dry hacking cough, fast shallow breathing, weight loss, tiredness, aching joints, aching muscles, and clubbing.
  • a bispecific antibody binds to the same target cells as the CAR and a CD3 domain of the recombinant TCR of the CAR/TCR cell.
  • the bispecific antibody binds to the target cell of the CAR and the CD3 domain of the recombinant TCR (abTCR or gdTCR) of the CAR-T/TCR-T cell resulting in stimulation of the CAR-T/TCR-T cell.
  • a bispecific antibody binds to different target cells than the CAR and a CD3 domain of the recombinant TCR of the CAR/TCR cell.
  • the bispecific antibody binds to the target cell and the CD3 domain of the recombinant TCR (abTCR or gdTCR) of the CAR-T/TCR-T cell resulting in stimulation of the CAR-T/TCR-T cell.
  • the cells expressing a chimeric antigen receptor are allogenic cells or autologous cells.
  • the disease or disorder is cancer.
  • the CAR targets an oncogenic gene product comprising BCMA, CD7, CD19, CD20, MUC1C, PSMA, CD70, mesothelin or c-kit.
  • the disease or disorder is an autoimmune disorder.
  • the autoimmune disease is selected from the group consisting of: autoimmune neutropenia, Guillain-Barré syndrome, epilepsy, autoimmune encephalitis, Isaacs' syndrome, nevus syndrome, pemphigus vulgaris, deciduous pemphigus, bullous pemphigoid, acquired epidermolysis bullosa, gestational pemphigoid, mucous membrane pemphigoid, antiphospholipid syndrome, autoimmune anemia, myasthenia gravis, autoimmune Graves' disease, thyroid eye disease (TED), Goodpasture syndrome, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura (ITP), warm autoimmune hemolytic anemia (WAIHA), chronic inflammatory demyelinating polyneuropathy (CIDP), lupus nephritis, and membranous nephropathy.
  • autoimmune neutropenia Guillain-Barré syndrome, epilepsy
  • FIG.1 is an illustration of a transposon of the present disclosure comprising: a left end piggyBac inverse terminal repeat (ITR); a EF1a promoter; a gene encoding a safety switch; genes encoding TCR chains (gamma delta or alpha beta); a gene encoding a chimeric antigen receptor (CAR); a gene encoding a selectable marker; a polyA tail; and a right end piggyBac ITR.
  • ITR left end piggyBac inverse terminal repeat
  • EF1a promoter a gene encoding a safety switch
  • genes encoding TCR chains gamma delta or alpha beta
  • CAR chimeric antigen receptor
  • FIG. 2 is a representative graph of the results of a restimulation assay demonstrating that cells expressing a CAR and a gdTCR receptor demonstrated enhanced cell proliferation compared to cells expressing only a CAR or cells expressing only a gdTCR.
  • FIG. 3 is a representative graph of the results of a restimulation assay demonstrating that cells expressing a CAR and a gdTCR receptor demonstrated enhanced cytotoxicity compared to cells expressing only a CAR or cells expressing only a gdTCR.
  • FIG. 3 is a representative graph of the results of a restimulation assay demonstrating that cells expressing a CAR and a gdTCR receptor demonstrated enhanced cytotoxicity compared to cells expressing only a CAR or cells expressing only a gdTCR.
  • FIGs. 4 are representative graphs of the results of a restimulation assay demonstrating that cells expressing a CAR targeting MUC1C or a CAR targeting MUC1C and abTCR each demonstrated cytolytic activity upon restimulation; however, only CAR+abTCR positive T- cells exhibited the capacity to kill double TCR-Ag+CAR-Ag+ as well as single TCR- Ag+CAR-Ag–, TCR-Ag–CAR-Ag+ A375 tumor cells. [0029] FIGs.
  • FIG. 5A and 5B are representative graphs of the results of a reactivation assay demonstrating that pre-existing MUC1C expressing CAR-T+abTCR/gdTCR cells may be reactivated in vivo to eliminate tumors expressing a different tumor antigen targeted by the MUC1C CAR by administration of a bispecific CD3-comprising T-cell engager (TCE).
  • FIG. 5A illustrates a primary challenge, measuring GFP+A375.MUC1C tumor volume over time.
  • FIG. 5B illustrates a secondary challenge, measuring b2M –/– GFP+A375 tumor volume over time.
  • FIG.6A shows the level of CD25 expression and cell size in CAR T cells, TCR T cell and CAR+TCR- T cells.
  • FIG. 6B shows the enrichment of genes associated with TSCM and Teff phenotypes in CAR+TCR-T cells compared to CAR-T cells.
  • the chimeric antigen receptor comprises (a) an ectodomain comprising a ligand recognition region, wherein the ligand recognition region comprises at least scaffold protein; (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain.
  • the ectodomain of (a) further comprises a signal peptide. In certain embodiments, the ectodomain of (a) further comprises a hinge between the ligand recognition region and the transmembrane domain.
  • the signal peptide comprises a sequence encoding a human CD2, CD3 , CD3 , CD3 , CD3 , CD4, CD8 , CD19, CD28, 4-1BB or GM-CSFR signal peptide. In certain embodiments, the signal peptide comprises a sequence encoding a human CD8 signal peptide. In certain embodiments, the signal peptide comprises an amino acid sequence comprising: MALPVTALLLPLALLLHAARP (SEQ ID NO: 1).
  • the signal peptide is encoded by a nucleic acid sequence comprising: atggcactgccagtcaccgccctgctgctgcctctggctctgctgctgcacgcagctagacca (SEQ ID NO: 2).
  • the transmembrane domain comprises a sequence encoding a human CD2, CD3 , CD3 , CD3 , CD4, CD8 , CD19, CD28, 4-1BB or GM-CSFR transmembrane domain.
  • the transmembrane domain comprises a sequence encoding a human CD8 transmembrane domain.
  • the transmembrane domain comprises an amino acid sequence comprising: IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 3). In certain embodiments, the transmembrane domain is encoded by a nucleic acid sequence comprising: atctacatttgggcaccactggccgggacctgtggagtgctgctgagcctggtcatcacactgtactgc (SEQ ID NO: 4). [0035] In certain embodiments, the endodomain comprises a human CD3 endodomain.
  • the at least one costimulatory domain comprises a human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof.
  • the at least one costimulatory domain comprises a human CD28 and/or a 4-1BB costimulatory domain.
  • the CD28 costimulatory domain comprises an amino acid sequence comprising: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 5).
  • the CD28 costimulatory domain is encoded by a nucleic acid sequence comprising: agaagcaagcggagccggctgctgcacagcgactacatgaacatgacccctagacggcccggacctaccagaaagcactaccag ccttacgctcctcctagagacttcgccgcctaccggtcc (SEQ ID NO: 6).
  • the 4-1BB costimulatory domain comprises an amino acid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 7).
  • the 4-1BB costimulatory domain is encoded by a nucleic acid sequence comprising: aagagaggcaggaagaaactgctgtatattttcaaacagcccttcatgcgccccgtgcagactacccaggaggaagacgggtgctcc tgtcgattccctgaggaagaggaaggcgggtgtgagctg (SEQ ID NO: 8).
  • the 4-1BB costimulatory domain is located between the transmembrane domain and the CD28 costimulatory domain.
  • the hinge comprises a sequence derived from a human CD8 , IgG4, and/or CD4 sequence. In certain embodiments, the hinge comprises a sequence derived from a human CD8 sequence. In certain embodiments, the hinge comprises an amino acid sequence comprising: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 9).
  • the hinge is encoded by a nucleic acid sequence comprising: actaccacaccagcacctagaccaccaactccagctccaaccatcgcgagtcagcccctgagtctgagacctgaggcctgcaggcc agctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 10).
  • the at least one protein scaffold specifically binds the ligand.
  • the chimeric ligand receptor comprises (a) an ectodomain comprising a ligand recognition region, wherein the ligand recognition region comprises at least scaffold protein; (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain.
  • the at least one protein scaffold comprises an antibody, an antibody fragment, a single domain antibody, a single chain antibody, an antibody mimetic, or a Centyrin.
  • the ligand recognition region comprises one or more of an antibody, an antibody fragment, a single domain antibody, a single chain antibody, an antibody mimetic, and a Centyrin.
  • the single domain antibody comprises or consists of a VHH.
  • the antibody mimetic comprises or consists of an affibody, an afflilin, an affimer, an affitin, an alphabody, an anticalin, an avimer, a DARPin, a Fynomer, a Kunitz domain peptide or a monobody.
  • the Centyrin comprises or consists of a consensus sequence of at least one fibronectin type III (FN3) domain.
  • scFv CARs [0042]
  • the chimeric antigen receptor (CAR) is an scFv CAR.
  • the scFv CAR comprises an ectodomain comprising antigen recognition region, wherein the antigen recognition region comprises: (a) at least one single chain variable fragment (scFv), (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain.
  • the CAR can further comprise a hinge region between the antigen recognition domain and the transmembrane domain.
  • An scFv generally consists of a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody connected by a linker.
  • an scFv CAR disclosed herein comprises an antigen-binding domain that is an anti-MUC1 scFv.
  • the antigen recognition region comprises at least two anti-MUC1 scFv.
  • the antigen recognition region comprises at least three anti-MUC1 scFv.
  • a CAR of the disclosure is a bi-specific CAR comprising at least two scFvs that specifically bind two distinct antigens.
  • the scFv CAR comprises an scFv comprising a heavy chain variable region comprising the amino acid sequence of: QVQLVQSGAEVKKPGSSVKX 1 SCKTSGYAFSNFWMNWVX 2 QX 3 PGQGLEWIGQIYPG DGDTNYNX 4 KFKGRX 5 TLTADKSX 6 STAYMELSSLRSEX 7 TAVYFCARSYYRSAWFA YWGQGTLVTVSS (SEQ ID NO:11), wherein X1 of SEQ ID NO: 11 is V or I, wherein X2 of SEQ ID NO: 11 is R or K, wherein X3 of SEQ ID NO: 11 is A or R, wherein X4 of SEQ ID NO: 11 is G or A, wherein X5 of SEQ ID NO: 11 is V or A, wherein X6 of SEQ ID NO: 11 is T or S, and wherein X7 of SEQ ID NO: 11 is D or A; and
  • the ectodomain of a CAR provided herein can further comprise a signal peptide.
  • the signal peptide can be derived from a human CD2, CD3 , CD3 , CD3 , CD3 , CD4, CD8 , CD19, CD28, 4-1BB or GM-CSFR signal peptide.
  • the signal peptide comprises, consists essentially of, or consists of a human CD8 alpha (CD8 or CD8a) signal peptide (SP) or a portion thereof.
  • the human CD8 SP comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 1. In some embodiments, the human CD8 SP comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 1.
  • the human CD8 SP is encoded by a polynucleotide comprising, consisting essentially of or consisting of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 13.
  • the human CD8 SP is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 13.
  • the hinge domain or hinge region can comprise a human CD8 , IgG4, CD4 sequence, or a combination thereof.
  • the hinge can comprise, consist essentially of, or consist of a human CD8 alpha (CD8 ) hinge or a portion thereof.
  • the human CD8a hinge comprises, consists essentially, of or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 9.
  • the human CD8 hinge domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 9.
  • the human CD8 hinge is encoded by a polynucleotide comprising, consisting essentially of or consisting of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 13 or SEQ ID NO: 14.
  • the human CD8 hinge domain is encoded by a polynucleotide comprising, consisting essentially of or consisting of the nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14.
  • the transmembrane domain can comprise, consist essentially of, or consist of a human CD2, CD3 , CD3 , CD3 , CD4, CD8 , CD19, CD28, 4-1BB or GM-CSFR transmembrane domain.
  • the transmembrane domain can comprise, consist essentially of, or consist of a human CD8 alpha (CD8 ) transmembrane domain, or a portion thereof.
  • the CD8 transmembrane domain comprises, consists essentially of or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 3.
  • the human CD8 transmembrane domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3.
  • the CD8 transmembrane domain is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 4 or SEQ ID NO: 15.
  • the CD8 transmembrane domain is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 4 or SEQ ID NO: 15.
  • the at least one costimulatory domain can comprise, consist essentially of, or consist of a human 4-1BB, CD28, CD3 zeta (CD3 or CD3z), CD40, ICOS, MyD88, OX-40 intracellular domain, or any combination thereof.
  • the at least one costimulatory domain comprises a CD3 , a 4-1BB costimulatory domain, or a combination thereof.
  • the 4-1BB intracellular domain comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 7.
  • the 4-1BB intracellular domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 7.
  • the 4-1BB intracellular domain is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 8 or SEQ ID NO: 16.
  • the 4-1BB intracellular domain is encoded by a polynucleotide comprising, consisting essentially of or consisting of the nucleic acid sequence of SEQ ID NO: 8 or SEQ ID NO: 16.
  • the CD3 intracellular domain comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR (SEQ ID NO: 95).
  • the CD3 intracellular domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 95.
  • the CD3 intracellular domain is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 17 or SEQ ID NO: 18.
  • the CD3 intracellular domain is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18.
  • a composition of the present disclosure may bind human MUC1 with at least one affinity selected from a KD of less than or equal to 10 9M, less than or equal to 10 10M, less than or equal to 10 11M, less than or equal to 10 12M, less than or equal to less than or equal to 10 14M, and less than or equal to 10 15M.
  • the KD may be determined by any means, including, but not limited to, surface plasmon resonance.
  • the disclosure further provides a chimeric antigen receptor (CAR) comprising: (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one VH, (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain.
  • a CAR comprising a VH is referred to as a VCAR.
  • the VCAR further comprises a hinge domain between the transmembrane domain and the ectodomain.
  • a VCAR may further comprise a signal peptide.
  • the transmembrane domain may comprise a sequence encoding a human CD2, CD3d, CD3e, CD3g, CD3z, CD4, CD8a, CD19, CD28, 4-1BB or GM-CSFR transmembrane domain.
  • the transmembrane domain may comprise a sequence encoding a human CD8a transmembrane domain.
  • the CD8a transmembrane domain may comprise an amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 3) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 3).
  • the CD8a transmembrane domain may be encoded by the nucleic acid sequence comprising: atctacatttgggcaccactggccgggacctgtggagtgctgctgctgagcctggtcatcacactgtactgc (SEQ ID NO: 4).
  • the endodomain may comprise a human CD3z endodomain.
  • the at least one costimulatory domain may comprise a human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof.
  • the at least one costimulatory domain may comprise a CD28 and/or a 4-1BB costimulatory domain.
  • the CD3z costimulatory domain may comprise an amino acid sequence comprising: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR (SEQ ID NO: 5) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR (SEQ ID NO: 5).
  • the CD3z costimulatory domain may be encoded by the nucleic acid sequence comprising: cgcgtgaagtttagtcgatcagcagatgccccagcttacaaacagggacagaaccagctgtataacgagctgaatctgggccgccga gaggaatatgacgtgctggataagcggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaccctcaggaagg cctgtataacgagctgcagaaggacaaatggcagaagcctattctgagatcggcatgaagggggagcgacggagaggcaaagg gcacgatgggctaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgcagg
  • the 4-1BB costimulatory domain may comprise an amino acid sequence comprising: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 7) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 7).
  • the 4-1BB costimulatory domain may be encoded by the nucleic acid sequence comprising: aagagaggcaggaagaaactgctgtatattttcaaacagcccttcatgcgccccgtgcagactacccaggaggaagacgggtgctcc tgtcgattccctgaggaagaggaaggcgggtgtgagctg (SEQ ID NO: 8).
  • the 4-1BB costimulatory domain may be located between the transmembrane domain and the CD28 costimulatory domain.
  • the hinge may comprise a sequence derived from a human CD8a, IgG4, and/or CD4 sequence. In certain embodiments of the VCARs of the disclosure, the hinge may comprise a sequence derived from a human CD8a sequence.
  • the hinge may comprise a human CD8a amino acid sequence comprising: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 9) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence comprising: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 9).
  • the human CD8a hinge amino acid sequence may be encoded by the nucleic acid sequence comprising: actaccacaccagcacctagaccaccaactccagctccaaccatcgcgagtcagcccctgagtctgagacctgaggcctgcaggcc agctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 10).
  • the signal peptide may comprise a sequence encoding a human CD2, CD3d, CD3e, CD3g, CD3z, CD4, CD8a, CD19, CD28, 4-1BB or GM-CSFR signal peptide.
  • the signal peptide may comprise a sequence encoding a human CD8a signal peptide.
  • the human CD8a signal peptide may comprise an amino acid sequence comprising: MALPVTALLLPLALLLHAARP (SEQ ID NO: 1).
  • the human CD8a signal peptide may comprise an amino acid sequence comprising: MALPVTALLLPLALLLHAARP (SEQ ID NO: 1) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the an amino acid sequence comprising: MALPVTALLLPLALLLHAARP (SEQ ID NO: 1).
  • the human CD8a signal peptide may be encoded by a nucleic acid sequence comprising: atggcactgccagtcaccgccctgctgctgcctctggctctgctgctgcacgcagctagacca (SEQ ID NO: 2).
  • VHHs and/or VCARs of the disclosure may bind an antigen with at least one affinity selected from a KD of less than or equal to 10 -9 M, less than or equal to 10 -10 M, less than or equal to 10 -11 M, less than or equal to 10 -12 M, less than or equal to 10 -13 M, less than or equal to 10 -14 M, and less than or equal to 10 -15 M.
  • the KD may be determined by surface plasmon resonance.
  • the disclosure provides an anti-BCMA VCAR.
  • the disclosure provides an anti-MUC1C VCAR.
  • the disclosure provides an anti-CD19 VCAR.
  • the disclosure provides an anti-CD20 VCAR. In certain embodiments, the disclosure provides an anti-PSMA VCAR. In certain embodiments, the disclosure provides an anti-CD70 VCAR. In certain embodiments, the disclosure provides an anti-c-kit VCAR. [0069] In certain embodiments, the antigen recognition region may comprise two VHs to produce a bi-specific or tandem VCAR. In certain embodiments, the antigen recognition region may comprise three VHs to produce a tri-specific VCAR. In certain embodiments, the ectodomain may further comprise a signal peptide. Alternatively, or in addition, in certain embodiments, the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain.
  • the ectodomain may further comprise a signal peptide. Alternatively, or in addition, in certain embodiments, the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain.
  • the VCAR comprises a single domain antibody, a VHH, a VH, or a combination thereof.
  • the single domain antibody, VHH or VH comprises or consists of a recombinant sequence.
  • the single domain antibody, VHH or VH comprises or consists of a chimeric sequence.
  • the single domain antibody, VHH or VH comprises or consists of a human sequence.
  • the single domain antibody, VHH or VH comprises or consists of a humanized sequence.
  • the VCAR comprises a single domain antibody.
  • the single domain antibody is a VHH or a VH antibody.
  • the VH antibody is a UniDab antibody.
  • VH antibody is not a fragment of a naturally occurring monoclonal antibody.
  • the VH comprises a human or a humanized sequence.
  • the VH comprises a non-naturally occurring sequence.
  • the VH is not naturally occurring.
  • the VH comprises a recombinant or chimeric sequence.
  • the VH is produced by an in vitro procedure of affinity selection and recombination.
  • Examples of sequences of antigen-binding regions targeting BCMA, CD19, CD20, PSMA, MUC1C and c-kit are set forth below. However, a person of skill in the art will recognize that the cells disclosed herein may comprise any CAR construct known in the art.
  • BCMA Binder Sequences [0078] In some aspects, a VCAR disclosed herein binds to BCMA. In certain embodiments, the VCAR comprises an anti-BCMA VHH sequence.
  • the VHH comprises or consists of the amino acid sequence: MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMN WVRQAPGKGLEWVAGIIGSGGSTYYADSVKGRFSISRDNSKNTLDLQMNSLRAEDT AVYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR (VH-A; SEQ ID NO: 19).
  • the VHH is encoded by a polynucleotide comprising or consisting of the nucleic acid sequence: atggctctgcctgtgacagctctgctgctgcctctggctctgcttctcatgcggcgcgccctgaagttcagctgcttgaatctggcggag gcctggttcaacctggcggatctctgagactgagctgtgccgccagcggcttcacctttagcagctacgccatgaactgggtccgaca ggccctggcaaaggactggaatgggtggccggaatcatcggcagcggcggcagcacatattacgccgattctgtgaagggccgct tcagcatcagcatcagcccggga
  • the anti-BCMA VHH comprises or consists of the amino acid sequence: MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLTLSCAASGFTFSNYAMN WVRQAPGKGLEWVSGIIGSGATTYYADSVKGRFTISRDNSKNTLNLQMNSLRAEDT AIYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR (VH-B; SEQ ID NO: 21).
  • the VHH is encoded by a polynucleotide comprising or consisting of the nucleic acid sequence: atggctctgcctgtgacagctctgctgctgcctctggctctgcttctcatgcggcgcgccctgaagttcagctgcttgaatctggcggag gcctggttcaacctggcggatctctgacactgagctgtgccgccagcggcttcaccttcagcaactacgccatgaactgggtccgaca ggcccctggcaaaggccttgaatgggtgtccggcatcattggctctggcgcaccacctactacgccgattctgtgaagggcagattc accatcagccggcgaagggcagatt
  • the anti-BCMA VHH comprises or consists of the amino acid sequence: MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGESLRLSCAASGFTFSNYAMN WVRQAPGKGLEWVSGIVGGGGTSYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDT AVYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR (VH-C; SEQ ID NO: 23).
  • the VHH is encoded by a polynucleotide comprising or consisting of the nucleic acid sequence: atggctctgcctgtgacagctctgctgctgcctctggctctgcttctcatgcggcgcgccctgaagttcagctgcttgaatctggcggag gcctggttcagcctggcgaatctctgagactgagctgtgccgccagcggcttcaccttcagcaactacgccatgaactgggtccgaca ggcccctggcaaaggccttgaatgggtgtccggaatcgtggcggcggaggcacaagctactacgccgattctgtgcggggcagatt caccatca
  • the anti-BCMA VHH comprises or consists of the amino acid sequence: MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMT WIRQAPGKGLEWVSGITGDGGSTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT AVYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR (VH-D; SEQ ID NO: 25).
  • the VHH is encoded by a polynucleotide comprising or consisting of the nucleic acid sequence: atggctctgcctgtgacagctctgctgctgcctctggctctgcttctcatgcggcgcgccctgaagttcagctgcttgaatctggcggag gcctggttcaacctggcggatctctgagactgagctgtgccgccagcggcttcaccttcagcaattacgccatgacctggatcagaca ggccctggcaaaggcctggaatgggtgtccggaattacaggcgacggcggcagcaccttttacgccgattctgtgaagggcagatt caccatcagccgggacaa
  • the anti-BCMA VHH comprises or consists of the amino acid sequence: MALPVTALLLPLALLLHAARPEVQLLESGGGLAQPGGSLRLSCAASGFTFSSYAMN WIRQAPGKGLEWVSGISGSGGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDT AVYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR (VH-E; SEQ ID NO: 27).
  • the VHH is encoded by a polynucleotide comprising or consisting of the nucleic acid sequence: atggcactgcctgtgacagccctgctgctgcctctggccctgctgcacgcagcacggcccgaggtgcagctgctggagtccgga ggaggcctggcccagcctggcggcagcctgaggctgtcctgcgcctctggcttggcacctttagctcctacgccatgaactggatca gacaggcccctggcaagggcctggagtgggtgtccggcatctccggctctggaggctctacatactatgccgacagcgtgaagggccaccatcagccaccatcagccctgacaagggcct
  • the anti-BCMA VHH comprises or consists of the amino acid sequence: MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGRSLRLSCAASGFTFTNYAMN WVRQAPGKGLEWVSGISGGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT AVYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR (VH-F; SEQ ID NO: 29).
  • the VHH is encoded by a polynucleotide comprising or consisting of the nucleic acid sequence: atggcactgcctgtgacagccctgctgctgcctctggccctgctgcacgcagcacggcccgaggtgcagctgctggagtctgga ggaggcctggtgcagcccggccctgagactgtcttgcgcgccagcggcttcacctttacaaactacgccatgaattgggtgc ggcaggcccctggcaagggcctggagtgggtgtgtggcatcagcggaggaggaggaggcagcacctactatgcagactcccgtgaagg gcagcacctactatgcagactcccgtgaagg gcagcacct
  • the VCAR comprises an anti-BCMA VH sequence.
  • the VH comprises or consists of the amino acid sequence: MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMN WVRQAPGKGLEWVAGIIGSGGSTYYADSVKGRFSISRDNSKNTLDLQMNSLRAEDT AVYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTY
  • the VH is encoded by a polynucleotide comprising or consisting of the nucleic acid sequence: atggctctgcctgtgacagctctgctgctgcctctggctctgcttctcatgcggcgcgccctgaagttcagctgcttgaatctggcggag gcctggttcaacctggcggatctctgagactgagctgtgccgccagcggcttcacctttagcagctacgccatgaactgggtccgaca ggccctggcaaaggactggaatgggtggccggaatcatcggcagcggcggcagcacatattacgccgattctgtgaagggccgct tcagcatcagcccggga
  • the VH comprises or consists of the amino acid sequence: MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLTLSCAASGFTFSNYAMN WVRQAPGKGLEWVSGIIGSGATTYYADSVKGRFTISRDNSKNTLNLQMNSLRAEDT AIYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR (VH-B; SEQ ID NO: 21).
  • the VH is encoded by a polynucleotide comprising or consisting of the nucleic acid sequence: atggctctgcctgtgacagctctgctgctgcctctggctctgcttctcatgcggcgcgccctgaagttcagctgcttgaatctggcggag gcctggttcaacctggcggatctctgacactgagctgtgccgccagcggcttcaccttcagcaactacgccatgaactgggtccgaca ggcccctggcaaaggccttgaatgggtgtccggcatcattggctctggcgcaccacctactacgccgattctgtgaagggcagattc accatcagccggcgcgaagggca
  • the VH comprises or consists of the amino acid sequence: MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGESLRLSCAASGFTFSNYAMN WVRQAPGKGLEWVSGIVGGGGTSYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDT AVYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR (VH-C; SEQ ID NO:
  • the VH is encoded by a polynucleotide comprising or consisting of the nucleic acid sequence: atggctctgcctgtgacagctctgctgctgcctctggctctgcttctcatgcggcgcgccctgaagttcagctgcttgaatctggcggag gcctggttcagcctggcgaatctctgagactgagctgtgccgccagcggcttcaccttcagcaactacgccatgaactgggtccgaca ggcccctggcaaaggccttgaatgggtgtccggaatcgtggcggcggaggcacaagctactacgccgattctgtgcggggcagatt caccatca
  • the VH comprises or consists of the amino acid sequence: MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMT WIRQAPGKGLEWVSGITGDGGSTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT AVYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR(VH-D; SEQ ID NO: 25)
  • the VH comprises or consists of the amino acid sequence: MALPVTALLLPLALLLHAARPEVQLLESGGGLAQPGGSLRLSCAASGFTFSSYAMN WIRQAPGKGLEWVSGISGSGGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDT AVYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR (VH-E; SEQ ID NO: 27
  • the VH comprises or consists of the amino acid sequence: MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGRSLRLSCAASGFTFTNYAMN WVRQAPGKGLEWVSGISGGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT AVYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR (VH-F; SEQ ID NO: 29
  • the VH is encoded by a polynucleotide comprising or consisting of the nucleic acid sequence: atggcactgcctgtgacagccctgctgctgcctctggccctgctgcacgcagcacggcccgaggtgcagctgctggagtctgga ggaggcctggtgcagcccggtccctgagactgtcttgcgcgccagcggcttcacctttacaaactacgccatgaattgggtgc ggcaggcccctggcaagggcctggagtgggtgtgtggcatcagcggaggaggaggaggcagcacctactatgcagactcccgtgaagg gcaggttcaccatctccccgcgcgcgcgcgcg
  • a VCAR disclosed herein binds to CD19.
  • the VCAR comprises an anti-BCMA VH sequence.
  • the anti-CD19 VH comprises the amino acid sequence: QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYMSSSGS TIYYADSVKGRFTISRDNAKNSLYLQMTSLRAEDTAVYYCARGGIAATGTWGQGTL VTVSS (SEQ ID NO: 31).
  • the anti-CD19 VH heavy chain variable region comprises the amino acid sequence: QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYMSSSGS TIYYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCARGGIAAAGTWGQGTL VTVSS (SEQ ID NO: 32).
  • the anti-CD19 VH heavy chain variable region comprises the amino acid sequence further comprising a T29D mutation relative to the sequence set forth in SEQ ID NO: 32.
  • the anti-CD19 VH heavy chain variable region comprises the amino acid sequence: QVQLVESGGGLVKPGGSLRLSCAASGFDFSDYYMSWIRQAPGKGLEWVSYMSSSGS TIYYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCARGGIAAAGTWGQGTL VTVSS (SEQ ID NO: 33).
  • the anti-CD19 VH heavy chain variable region comprises the amino acid sequence further comprising a S55D mutation relative to SEQ ID NO: 32.
  • the anti-CD19 VH heavy chain variable region comprises the amino acid sequence: QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYMSSDGS TIYYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCARGGIAAAGTWGQGTL VTVSS (SEQ ID NO: 34).
  • CD20 Binder Sequences [0106] In some aspects, a VCAR disclosed herein binds to CD20. In certain embodiments, the VCAR comprises an anti-CD20 VH sequence.
  • the anti-CD20 VH heavy chain variable region comprises the amino acid sequence: EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSSINWNG GSKGYADSVKGRFTISRDNAKNSLYLQMNSLRVEDTALYQCARERGYRIGHDSFDI WGQGTLVTVSS (SEQ ID NO: 35).
  • the anti-CD20 VH heavy chain variable region comprises the amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGNYYWNWIRQPPGKGLEWIGYIYSSG STKYNPSLKSRVTILVDTSKNQFSLKLSSVTAADTAVYYCARSRLNGDGLFDDRGQG TLVTVSS (SEQ ID NO: 36).
  • PSMA Binder Sequences [0108] In some aspects, a VCAR disclosed herein binds to PSMA. In certain embodiments, the VCAR comprises an anti-PSMA VH sequence.
  • the anti-PSMA VH heavy chain variable region comprises the amino acid sequence: EVQLLESGGGVVQPGRSLRLSCAASGFSFSGYGMHWVRQAPGKEREWVAVISYDGS NKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDANWGQHPDHTSF DYRGQGTLVTVSS (SEQ ID NO: 37).
  • the anti-PSMA VH heavy chain variable region comprises the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFKFYAMSWVRQAPGKGPEWVSVISGSGG STYYADSVKGRFTISRDNSKNTLHLQMNSLRAEDTAVYFCAKEIAEASRGFDYRGQG TLVTVSS (SEQ ID NO: 38).
  • MUC1C Binder Sequences [0110] In some aspects, a VCAR disclosed herein binds to MUC1C. In certain embodiments, the VCAR comprises an anti-MUC1C VH sequence.
  • the anti-MUC1C VH heavy chain variable region comprises the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCTASGFAFSGNSMNWVRQAPGKGLEWVAFITSSGR SIKYADSVKGRFTISRDNAKNSLYLQMNTLRDEDTALYYCATGGTGTSLFDYRGQG TLVTVSS (SEQ ID NO: 39).
  • the anti-MUC1C VH heavy chain variable region comprises the amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHSMNWVRQAPGKGLEWVSFISSSSNI KKYADSVKGRFTISRDNAKNSLFLQMNSLRDEDTAVYYCATGGTGITVLDYRGQGT LVTVSS (SEQ ID NO: 40).
  • C-kit Binder Sequences [0112] In some aspects, a VCAR disclosed herein binds to c-kit. In certain embodiments, the VCAR comprises an anti-c-kit VH sequence.
  • the anti-c-kit VH heavy chain variable region comprises the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGLTISTYAMSWVRQAPGKGLEWVSAISTGGSS TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATGYDSSGHYYGGFDYR GQGTLVTVSS (SEQ ID NO: 41).
  • the anti-c-kit VH heavy chain variable region comprises the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFDSYAMSWVRQAPGKGLEWVSAISVRGG STYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYSCATGYDPSGHYYGGFDY RGQGTLVTVSS (SEQ ID NO: 42).
  • the anti-c-kit VH heavy chain variable region comprises the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGLTISSYAMSWVRQAPGKGLEWVSAISTGGSR TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCATGYDSSGHYYGGFDYR GQGTLVTVSS (SEQ ID NO: 43).
  • the anti-c-kit VH heavy chain variable region comprises the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFDSYAMSWVRQAPGEGLEWVSAISTGGG STYYADSVKGRFTISRDNSKNMLFLQMNSLRAEDTAVYSCATGYDSSGYYYGGFDY RGQGTLVTVSS (SEQ ID NO: 44).
  • the anti-c-kit VH heavy chain variable region comprises the amino acid sequence: QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIDYNGG TYNNPSLKSRVTISVDTSKNQFSLKLSYVTAADTAVYYCARQNPRRYASGASDYRG QGTLVTVSS (SEQ ID NO: 45).
  • Recombinant T-cell Receptors the disclosure provides recombinant T-cell receptor chains for expressing cell surface recombinant T-cell receptors (gdTCR or abTCR) in CAR expressing cells lacking a functional endogenous TCR.
  • the recombinant TCR expressed in the cell comprises the T-cell receptor gamma and delta chains (gdTCR). [0119] In certain aspects, the recombinant TCR expressed in the cell comprises a G115 gamma chain.
  • the G115 gamma chain comprises the amino acid sequence: MLSLLHTSTLAVLGALCVYGAGHLEQPQISSTKTLSKTARLECVVSGITISATSVYWY RERPGEVIQFLVSISYDGTVRKESGIPSGKFEVDRIPETSTSTLTIHNVEKQDIATYYCA LWEAQQELGKKIKVFGPGTKLIITDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLE KFFPDVIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEKSLDKEHRCIVRH ENNKNGVDQEIIFPPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKS VVYFAIITCCLLRRTAFCCNGEKS (SEQ ID NO: 46).
  • the G115 gamma chain comprises an amino acid sequence that is at least about 80%, at least about 85%, at least 90%, or at least about 95% identical to the sequence set forth in SEQ ID NO: 46. In some embodiments, the G115 gamma chain comprises an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 46. In some embodiments, the G115 gamma chain comprises an amino acid sequence that comprises one, two or three conservative amino acid substitutions relative to the sequence set forth in SEQ ID NO: 46.
  • the G115 gamma chain of the gdTCR is encoded by the nucleic acid sequence: atgctgagcctgctgcacacatctacactggctgtgctgggagccctgtgtgtgtatggcgctggacatctggaacagccccagatca gcagcaccaagacactgagcaagaccgccagactggaatgcgtggtgtccggcatcacacaatcagcgccacaagcgtgtactggtat cgcgagaggcctggcgaagtgatccagttcctggtgtctatcagctacgacggcaccgtgcggaaagagagcggaatccccttccgg caagttcgaggtggacagaatcccccccgg caagttcgaggtggagga
  • the gamma chain of the gdTCR is a G115 variant.
  • the G115 gamma chain variant of the gdTCR comprises the amino acid sequence: MLSLLHTSTLAVLGALCVYGAGHLEQPQISSTKTLSKTARLECVVSGITISATSVYWY RERPGEVIQFLVSISYDGTVRKESGIPSGKFEVDRIPETSTSTLTIHNVEKQDIATYYCA LWEAQQELGKKIKVFVHELGKKIKVFGPGTKLIITDKQLDADVSPKPTIFLPSIAETKL QKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEKS LDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAY YMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS (SEQ ID NO: 48).
  • the G115 gamma chain variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least 90%, or at least about 95% identical to the sequence set forth in SEQ ID NO: 48. In some embodiments, the G115 gamma chain variant comprises an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 48. In some embodiments, the G115 gamma chain variant comprises an amino acid sequence that comprises one, two or three conservative amino acid substitutions relative to the sequence set forth in SEQ ID NO: 48.
  • the gamma chain of the gdTCR is encoded by the nucleic acid sequence: atgctgagcctgctgcacacatctacactggctgtgctgggagccctgtgtgtgtatggcgctggacatctggaacagccccagatca gcagcaccaagacactgagcaagaccgccagactggaatgcgtggtgtccggcatcacacaatcagcgccacaagcgtgtactggtat cgcgagaggcctggcgaagtgatccagttcctggtgtctatcagctacgacggcaccgtgcggaaagagagcggaatcccttccgg caagttcgaggtggacagaatccccccgagacaagcaccagcacactgaccatccacgct
  • the delta chain of the gdTCR is a G115 delta chain.
  • the G115 delta chain of the gdTCR comprises the amino acid sequence: MQRISSLIHLSLFWAGVMSAIELVPEHQTVPVSIGVPATLRCSMKGEAIGNYYINWYR KTQGNTMTFIYREKDIYGPGFKDNFQGDIDIAKNLAVLKILAPSERDEGSYYCACDTL GMGGEYTDKLIFGKGTRVTVEPRSQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINL VSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDST DHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTA KLFFL (SEQ ID NO: 50).
  • the G115 delta chain comprises an amino acid sequence that is at least about 80%, at least about 85%, at least 90%, or at least about 95% identical to the sequence set forth in SEQ ID NO: 50. In some embodiments, the G115 delta chain comprises an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 50. In some embodiments, the G115 delta chain comprises an amino acid sequence that comprises one, two or three conservative amino acid substitutions relative to the sequence set forth in SEQ ID NO: 50.
  • the delta chain of the gdTCR is encoded by the nucleic acid sequence: atgcagcggatcagctctctgatccacctgagcctgttttgggctggcgtgatgtctgccatcgagctggtgcctgaacaccagaccgt gcctgtgtctattggcgtgccagccacactgcggtgtagcatgaagggcgaagccatcggcaactactacatcaactggtacagaaa gacccagggcaacaccatgaccttcatctacagagagaaggacatctacggccttcaaggacaacttccagggcgacatcg acattgccaagaacctggccgtgtgaagattctggccctagcgagagagatgagggcagct
  • the recombinant TCR expressed in the cell comprises the T-cell receptor alpha and beta chains (abTCR).
  • the alpha chain of the abTCR comprises the amino acid sequence: METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQ DPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLY GGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDS DVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSSDVPCDV KLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 52).
  • the alpha chain of the abTCR comprises an amino acid sequence that is at least about 80%, at least about 85%, at least 90%, or at least about 95% identical to the sequence set forth in SEQ ID NO: 52. In some embodiments, the alpha chain of the abTCR comprises an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 52. In some embodiments, the alpha chain of the abTCR comprises an amino acid sequence that comprises one, two or three conservative amino acid substitutions relative to the sequence set forth in SEQ ID NO: 52.
  • the alpha chain of the abTCR is encoded by the nucleic acid sequence: atggaaacactgctgggcctgctgatcctgtggctgcaactgcaatgggtgtccagcaagcaagaagtgacacagatccctgccgct ctgtctgagggcgaaacctggtgctgaactgctccttcaccgacagcgccatctacaacctccagtggttcagacaggacc ccggcaagggactgacaagcctgctgattcagagcagccagagagagcagaccagcggcagactgaatgccagcctggataa gtcctcggcagaagcaccctgtatatcgccgcttctcagctggcgatag
  • the beta chain of the abTCR comprises the amino acid sequence: MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWY RQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCAS SYVGNTGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSKAEIAHTQKATLVCLATGFFP DHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHF RCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEIL LGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 54).
  • the beta chain of the abTCR comprises an amino acid sequence that is at least about 80%, at least about 85%, at least 90%, or at least about 95% identical to the sequence set forth in SEQ ID NO: 54. In some embodiments, the beta chain of the abTCR comprises an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to the sequence set forth in SEQ ID NO: 54. In some embodiments, the beta chain of the abTCR comprises an amino acid sequence that comprises one, two or three conservative amino acid substitutions relative to the sequence set forth in SEQ ID NO: 54.
  • the beta chain of the abTCR is encoded by the nucleic acid sequence: atgtctatcggcctgctgtgttgtgccgctctgtctctgggccggacctgttaatgccggcgtgacccagacacctaagttccagg tgctgaaaaccggccagagcatgaccctgcaatgcgcccaggacatgaaccacgagtacatgagctggtacagacaggaccctgg catgggcctgagactgatccactattctgtcggagccggcatcaccgaccagggcgaagttcctaatggctacaacgtgtccagaagc accaccgaggacttcccactgagactgctgtgtgctctgctctagcca
  • Cells and Modified Cells are cells modified to comprise the CARs and modified TCRs provided herein.
  • Cells and modified cells of the disclosure can be mammalian cells. In some embodiments, the cells and modified cells are human cells.
  • Cells and modified cells of the disclosure can be immune cells.
  • the immune cells of the disclosure can be iPSCs, lymphoid progenitor cells, natural killer (NK) cells, T lymphocytes (T-cell), stem memory T cells (TSCM cells), central memory T cells (TCM), stem cell-like T cells, B lymphocytes (B-cells), antigen presenting cells (APCs), cytokine induced killer (CIK) cells, myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, macrophages, platelets, erythrocytes, red blood cells (RBCs), megakaryocytes or osteoclasts.
  • the cells of the disclosure are immune precursor cells.
  • the immune precursor cells can be any cells which can differentiate into one or more types of immune cells.
  • the immune precursor cells can be multipotent stem cells that can self-renew and develop into immune cells.
  • the immune precursor cells can be hematopoietic stem cells (HSCs) or descendants thereof.
  • the immune precursor cells can be precursor cells that can develop into immune cells. [0138]
  • the immune precursor cells are hematopoietic progenitor cells (HPCs).
  • HSCs hematopoietic progenitor cells
  • HSCs Hematopoietic stem cells
  • HSCs can be found in adult bone marrow, peripheral blood, mobilized peripheral blood, peritoneal dialysis effluent and umbilical cord blood.
  • HSCs can be isolated or derived from a primary or cultured stem cell.
  • HSCs can be isolated or derived from, for example, an embryonic stem cell, a multipotent stem cell, a pluripotent stem cell, an adult stem cell, or an induced pluripotent stem cell (iPSC).
  • Immune precursor cells can be HSCs or HSC descendent cells.
  • HSC descendent cells include multipotent stem cells, lymphoid progenitor cells, natural killer (NK) cells, T lymphocyte cells (T-cells), B lymphocyte cells (B-cells), myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes and macrophages.
  • NK natural killer
  • T-cells T lymphocyte cells
  • B-cells B lymphocyte cells
  • myeloid progenitor cells neutrophils, basophils, eosinophils, monocytes and macrophages.
  • HSCs produced by the disclosed methods can retain features of “primitive” stem cells that, while isolated or derived from an adult stem cell and while committed to a single lineage, share characteristics of embryonic stem cells.
  • the “primitive” HSCs produced by the disclosed methods may retain their “stemness” following division and may not differentiate.
  • Primitive HSCs produced by the disclosed methods are believed to not only replenish their numbers, but also expand in vivo. “Primitive” HSCs produced by disclosed the methods can be therapeutically-effective when administered as a single dose.
  • Primitive HSCs can be CD34+.
  • Primitive HSCs can be CD34+ and CD38-.
  • Primitive HSCs can be CD34+, CD38- and CD90+.
  • Primitive HSCs can be CD34+, CD38-, CD90+ and CD45RA-.
  • Primitive HSCs can be CD34+, CD38-, CD90+, CD45RA-, and CD49f+.
  • Primitive HSCs can be CD34+, CD38-, CD90+, CD45RA-, and CD49f+.
  • Primitive HSCs, HSCs, and/or HSC descendent cells can be modified according to the disclosed methods to express an exogenous sequence (e.g., a chimeric antigen receptor or therapeutic protein).
  • Modified primitive HSCs, modified HSCs, and/or modified HSC descendent cells can be forward differentiated to produce a modified immune cell including, but not limited to, a modified T cell, a modified natural killer cell and/or a modified B-cell.
  • the modified immune or immune precursor cells can be NK cells.
  • the NK cells can be cytotoxic lymphocytes that differentiate from lymphoid progenitor cells.
  • Modified NK cells can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.
  • non-activated NK cells are derived from CD3-depleted leukapheresis (containing CD14/CD19/CD56+ cells).
  • the modified immune or immune precursor cells can be B cells.
  • B cells are a type of lymphocyte that express B cell receptors on the cell surface. B cell receptors bind to specific antigens.
  • Modified B cells can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.
  • Modified T cells of the disclosure may be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs. Unlike traditional biologics and chemotherapeutics, the disclosed modified-T cells may retain the capacity to rapidly reproduce upon antigen recognition, thereby potentially obviating the need for repeat treatments. To achieve this, in some embodiments, modified-T cells not only drive an initial response, but also persist in the patient as a stable population of viable memory T cells to prevent potential relapses. Alternatively, in some aspects, when it is not desired, the modified T cells do not persist in the patient.
  • HSPCs modified hematopoietic stem and progenitor cells
  • modified-T cells may retain the capacity to rapidly reproduce upon antigen recognition, thereby potentially obviating the need for repeat treatments.
  • modified-T cells not only drive an initial response, but also persist in the patient as a stable population of viable memory T cells to prevent potential relapses.
  • the modified T cells do not persist in the patient.
  • the modified T cell further comprise an inducible safety switch that may be activated by administering to the subject a ligand which results in the death of the modified T cell.
  • an inducible proapoptotic polypeptide operably linked to a ligand binding region that may be optimized to bind a chemical inducer of dimerization.
  • pro-apoptotic target molecules can become cross-linked, and, consequently, activated to selectively induce apoptosis in a cell containing an inducible proapoptotic polypeptide including, but not limited to, inducible caspase polypeptides including inducible caspase 9 polypeptides.
  • the inducible caspase 9 polypeptides may comprise a truncated caspase 9 polypeptide encoded by a truncated or modified amino acid and/or nucleic acid sequence encoding the truncated caspase 9 polypeptide.
  • the inducible proapoptotic polypeptide can comprise (a) a ligand binding region, (b) a linker, and (c) a proapoptotic polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence.
  • the non-human sequence comprises a restriction site.
  • the ligand binding region may be a multimeric ligand binding region.
  • T SCM stem cell memory
  • T SCM stem cell memory
  • Stem cell-like modified-T cells of the disclosure exhibit the greatest capacity for self-renewal and multipotent capacity to derive central memory (T CM ) T cells or T CM like cells, effector memory (T EM ) and effector T cells (T E ), thereby producing better tumor eradication and long-term modified-T cell engraftment.
  • T N Na ⁇ ve T cells (T N ) > T SCM > T CM > T EM > T E > T TE , whereby T N is the parent precursor cell that directly gives rise to T SCM , which then, in turn, directly gives rise to TCM, etc.
  • Compositions of T cells of the disclosure can comprise one or more of each parental T cell subset with TSCM cells being the most abundant (e.g., TSCM > TCM > TEM > TE > TTE).
  • the immune cell precursor can be differentiated into or is capable of differentiating into an early memory T cell, a stem cell like T-cell, a Na ⁇ ve T cells (TN), a TSCM, a TCM, a TEM, a TE, or a TTE.
  • the immune cell precursor can be a primitive HSC, an HSC, or a HSC descendent cell of the disclosure.
  • the immune cell can be an early memory T cell, a stem cell like T-cell, a Na ⁇ ve T cells (TN), a TSCM, a TCM, a TEM, a TE, or a TTE.
  • the methods of the disclosure can be used to modify and/or produce a population of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of a plurality of modified T cells in the population expresses one or more cell-surface marker(s) of an early memory T cell.
  • at least 20% of cells in the population express one or more cell-surface marker(s) of an early memory T cell.
  • at least 25% of cells in the population express one or more cell-surface marker(s) of an early memory T cell.
  • the population of modified early memory T cells comprises a plurality of modified stem cell-like T cells. In some embodiments, the population of modified early memory T cells comprises a plurality of modified stem cell memory T cells (TSCM cells).
  • the population of modified early memory T cells comprises a plurality of modified TCM cells.
  • the methods of the disclosure can be used to modify and/or produce a population of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells in the population expresses one or more cell-surface marker(s) of a stem cell-like T cell.
  • the population of modified stem cell-like T cells comprises a plurality of modified T SCM cells.
  • the population of modified stem cell-like T cells comprises a plurality of modified T CM cells.
  • TSCM stem memory T cell
  • the cell-surface markers of a TSCM or a TSCM-like cell can also comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2R .
  • the cell-surface markers of a TSCM or a TSCM-like cell can also comprise one or more of CD45RA, CD95, IL-2R , CCR7, and CD62L.
  • T CM central memory T cell
  • T CM central memory T cell
  • the cell-surface markers of a T CM or a T CM -like cell can also comprise one or more of CD45RO, CD95, IL-2R , CCR7, and CD62L.
  • the methods of the disclosure can be used to modify and/or produce a population of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells in the population expresses one or more cell-surface marker(s) of a na ⁇ ve T cell (TN).
  • TN na ⁇ ve T cell
  • the cell-surface markers of a TN cell can comprise one or more of CD45RA, CCR7 and CD62L.
  • the methods of the disclosure can be used to modify and/or produce a population of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells in the population expresses one or more cell-surface marker(s) of an effector T-cell (modified T EFF ).
  • the cell-surface markers of a modified T EFF can comprise one or more of CD45RA, CD95, and IL-2R .
  • the methods of the disclosure can modify and/or produce a population of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells of the population expresses one or more cell-surface marker(s) of a stem cell- like T cell, a stem memory T cell (T SCM ) or a central memory T cell (T CM ).
  • the modified cells of the population may comprise a transgene.
  • the transgene may be, for example, a gene encoding a CAR, a gene encoding an abTCR or a gene encoding a gdTCR.
  • the populations of cell sdisclosed herein may be characterized by their expression of certain cell surface markers, for example, CD34, CD90, CD38, CD45RA and CD49f.
  • At least 75%, 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%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise a transgene, and 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%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express CD34 (i.e., the cells have the cell-surface marker phenotype CD34+).
  • At least 75%, 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%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise the transgene and at least about 70% to about 99%, about 75% to about 95% or about 85% to about 95% of the population of modified cells express CD34 (i.e., the cells have the cell-surface marker phenotype CD34+).
  • 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%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express both the protein encoded by the transgene and CD34.
  • the transgene encodes a CAR. In some embodiments, the transgene encodes an abTCR. In some embodiments, the transgene encodes a gdTCR. [0161] In some embodiments, at least 75%, 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%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise a transgene, and wherein 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 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%, at least 99.5%, at least 99
  • At least 75%, 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%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise the transgene and at least about 45% to about 90%, about 50% to about 80% or about 65% to about 75% of the population of modified cells express CD34 and do not express CD38 (i.e., the cells have the cell-surface marker phenotype CD34+ and CD38-).
  • 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%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express both the protein encoded by the transgene and CD34, but do not express CD38.
  • the transgene encodes a CAR.
  • the transgene encodes an abTCR.
  • the transgene encodes a gdTCR.
  • At least 75%, 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%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise a transgene, and at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, 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 91%, at least
  • 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%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express the protein encoded by the transgene, CD34 and CD90, but do not express CD38.
  • the transgene encodes a CAR.
  • the transgene encodes an abTCR.
  • the transgene encodes a gdTCR.
  • At least 75%, 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%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise a transgene, and at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, 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 91%, at least
  • At least 75%, 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%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise the transgene and at least about 0.2% to about 40%, about 0.2% to about 30%, about 0.2% to about 2% or 0.5% to about 1.5% of the population of modified cells express CD34 and CD90 and do not express CD38 or CD45RA (i.e., the cells have the cell-surface marker phenotype CD34+, CD38-, CD90+, and CD45RA- ).
  • 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%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express the protein encoded by the transgene, CD34 and CD90 but do not express CD38 or CD45RA.
  • the transgene encodes a CAR.
  • the transgene encodes an abTCR.
  • the transgene encodes a gdTCR.
  • At least 75%, 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%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise a transgene, and at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%
  • At least 75%, 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%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise the transgene and at least about 0.02% to about 30%, about 0.02% to about 2%, about 0.04% to about 2% or about 0.04% to about 1% of the population of modified cells express CD34, CD90 and CD49f and do not express CD38 or CD45RA (i.e.., the cells have the cell-surface marker phenotype CD34+, CD38-, CD90+, CD45RA- and CD49f+).
  • 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%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express the protein encoded by the transgene, CD34, CD90 and CD49f, but does not express CD38 or CD45RA.
  • the transgene encodes a CAR.
  • the transgene encodes an abTCR.
  • the transgene encodes a gdTCR.
  • At least 75%, 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%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise a transgene, and at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%
  • At least 75%, 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%, at least 99.5%, at least 99.9% or 100% of the plurality of cells of the population comprise the transgene and at least about 0.2% to about 5%, about 0.2% to about 3% or about 0.4% to about 3% of the population of modified cells express CD34 and CD90 and do not express CD45RA (i.e., the cells have the cell-surface marker phenotype CD34+, CD90+ and CD45RA-).
  • 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%, at least 99.5%, at least 99.9% or 100% of the population of modified cells express both the protein encoded by the transgene, CD34 and CD90, but do not express CD45RA.
  • the transgene encodes a CAR.
  • the transgene encodes an abTCR.
  • the transgene encodes a gdTCR.
  • compositions and methods of producing and/or expanding the immune cells or immune precursor cells e.g., the disclosed CAR-T-cells
  • buffers for maintaining or enhancing a level of cell viability and/or a stem-like phenotype of the immune cells or immune precursor cells are disclosed elsewhere herein and are disclosed in more detail in U.S. Patent No.10,329,543 and PCT Publication No. WO 2019/173636, each of which is incorporate herein by reference in its entirety.
  • Cells and modified cells of the disclosure can be somatic cells. Cells and modified cells of the disclosure can be differentiated cells.
  • Cells and modified cells of the disclosure can autologous cells or allogenic cells. Allogeneic cells are engineered to prevent adverse reactions to engraftment following administration to a subject. Allogeneic cells may be any type of cell. Allogenic cells can be stem cells or can be derived from stem cells. Allogeneic cells can be differentiated somatic cells. Methods of Introducing Nucleic Acids into Cells [0168] A CAR cell can be produced by introducing a nucleic acid encoding a CAR into the cell. In some embodiments, a transgene and/or genomic editing construct are also introduced into the cell.
  • Introducing a nucleic acid sequence, transgene and/or genomic editing construct into a cell ex vivo, in vivo, in vitro or in situ can comprise one or more of topical delivery, adsorption, absorption, electroporation, spin-fection, co-culture, transfection, mechanical delivery, sonic delivery, vibrational delivery, magnetofection or by nanoparticle-mediated delivery.
  • Introducing a nucleic acid sequence, transgene and/or genomic editing construct into a cell ex vivo, in vivo, in vitro or in situ can comprise liposomal transfection, calcium phosphate transfection, fugene transfection, and dendrimer-mediated transfection.
  • Introducing a nucleic acid sequence, transgene and/or genomic editing construct into a cell ex vivo, in vivo, in vitro or in situ by mechanical transfection can comprise cell squeezing, cell bombardment, or gene gun techniques.
  • Introducing a nucleic acid sequence, transgene and/or genomic editing construct into a cell ex vivo, in vivo, in vitro or in situ by nanoparticle-mediated transfection can comprise liposomal delivery, delivery by micelles, and delivery by polymerosomes.
  • Gene editing tools can also be delivered to cells using one or more poly(histidine)-based micelles.
  • Poly(histidine) (e.g., poly(L-histidine)), is a pH-sensitive polymer due to the imidazole ring providing an electron lone pair on the unsaturated nitrogen. That is, poly(histidine) has amphoteric properties through protonation-deprotonation.
  • poly(histidine)-containing triblock copolymers may assemble into a micelle with positively charged poly(histidine) units on the surface, thereby enabling complexing with the negatively-charged gene editing molecule(s).
  • Using these nanoparticles to bind and release proteins and/or nucleic acids in a pH-dependent manner may provide an efficient and selective mechanism to perform a desired gene modification.
  • this micelle-based delivery system provides substantial flexibility with respect to the charged materials, as well as a large payload capacity, and targeted release of the nanoparticle payload.
  • site-specific cleavage of the double stranded DNA is enabled by delivery of a nuclease using the poly(histidine)-based micelles.
  • the hydrophobic blocks aggregate to form a core, leaving the hydrophilic blocks and poly(histidine) blocks on the ends to form one or more surrounding layer.
  • the disclosure provides triblock copolymers made of a hydrophilic block, a hydrophobic block, and a charged block.
  • the hydrophilic block may be poly(ethylene oxide) (PEO)
  • the charged block may be poly(L-histidine).
  • An example tri- block copolymer that can be used is a PEO-b-PLA-b-PHIS, with variable numbers of repeating units in each block varying by design.
  • Diblock copolymers that can be used as intermediates for making triblock copolymers can have hydrophilic biocompatible poly(ethylene oxide) (PEO), which is chemically synonymous with PEG, coupled to various hydrophobic aliphatic poly(anhydrides), poly(nucleic acids), poly(esters), poly(ortho esters), poly(peptides), poly(phosphazenes) and poly(saccharides), including but not limited by poly(lactide) (PLA), poly(glycolide) (PLGA), poly(lactic-co-glycolic acid) (PLGA), poly( -caprolactone) (PCL), and poly (trimethylene carbonate) (PTMC).
  • PEO poly(ethylene oxide)
  • PEG poly(ethylene oxide)
  • PEG poly(ethylene oxide)
  • PTMC poly(trimethylene carbonate)
  • Polymeric micelles comprised of 100% PEGylated surfaces possess improved in vitro chemical stability, augmented in vivo bioavailablity, and prolonged blood circulatory half-lives.
  • Polymeric vesicles, polymersomes and poly(Histidine)-based micelles, including those that comprise triblock copolymers, and methods of making the same, are described in further detail in U.S. Patent Nos.7,217,427; 7,868,512; 6,835,394; 8,808,748; 10,456,452; U.S. Publication Nos.2014/0363496; 2017/0000743; and 2019/0255191; and PCT Publication No. WO 2019/126589, each of which are incorporated herein by reference in its entirety.
  • introducing a nucleic acid sequence, transgene and/or genomic editing construct into a cell ex vivo, in vivo, in vitro or in situ can comprise a non-viral vector.
  • the non-viral vector can comprise a nucleic acid encoding a CAR.
  • the non-viral vector can comprise plasmid DNA, linear double-stranded DNA (dsDNA), linear single-stranded DNA (ssDNA), DoggyBoneTM DNA, nanoplasmids, minicircle DNA, single-stranded oligodeoxynucleotides (ssODN), DDNA oligonucleotides, single-stranded mRNA (ssRNA), and double-stranded mRNA (dsRNA).
  • the non-viral vector can comprise a transposon as described herein.
  • Introducing a nucleic acid sequence, transgene and/or genomic editing construct into a cell ex vivo, in vivo, in vitro or in situ can comprise a viral vector.
  • the viral vector can be a non-integrating non-chromosomal vector.
  • Non-limiting examples of non-integrating non- chromosomal vectors include adeno-associated virus (AAV), adenovirus, and herpes viruses.
  • the viral vector can be an integrating chromosomal vector.
  • Non-limiting examples of integrating chromosomal vectors include adeno-associated vectors (AAV), Lentiviruses, and gamma-retroviruses.
  • Introducing a nucleic acid sequence, transgene and/or genomic editing construct into a cell ex vivo, in vivo, in vitro or in situ can comprise a combination of vectors.
  • vector combinations include viral and non-viral vectors, a plurality of non-viral vectors, or a plurality of viral vectors.
  • vector combinations include a combination of a DNA-derived and an RNA-derived vector, a combination of an RNA and a reverse transcriptase, a combination of a transposon and a transposase, a combination of a non- viral vector and an endonuclease, and a combination of a viral vector and an endonuclease.
  • Genome modification can comprise introducing a nucleic acid sequence, transgene and/or genomic editing construct into a cell ex vivo, in vivo, in vitro or in situ to stably integrate a nucleic acid sequence, transiently integrate a nucleic acid sequence, produce site-specific integration of a nucleic acid sequence, or produce a biased integration of a nucleic acid sequence.
  • the nucleic acid sequence can encode a CAR.
  • the nucleic acid sequence or transgene can be about 1kb to about 15kb in size.
  • the nucleic acid sequence or transgene can be at least 1 kb, at least 2 kb, at least 3kb, at least 4kb, at least 5kb, at least 6kb, at least 7kb, at least 8kb, at least 9kb, at least 10 kb, at least 11 kb, at least 12 kb, at least 13 kb, at least 14 kb, at least 15 kb in size.
  • the nucleic acid sequence or transgene can be about 1 kb, about 2 kb, about 3 kb, about 4 kb, about 5 kb, about 6 kb, about 7 kb, about 8 kb, about 9 kb, about 10 kb, about 11 kb, about 12 kb, about 13 kb, about 14 kb or about 15 kb in size.
  • Another means for introducing a nucleic acid encoding a CAR includes using a transposon system.
  • the present disclosure provides a transposon comprising a nucleic acid encoding a CAR.
  • the transposon is a plasmid DNA transposon comprising a nucleotide sequence encoding the CAR (e.g., s scFv CAR or VCAR) as disclosed herein flanked by two cis-regulatory insulator elements.
  • the present disclosure also provides a composition comprising a transposon.
  • the composition comprising the transposon further comprises a plasmid comprising a nucleotide sequence encoding a transposase.
  • the nucleotide sequence encoding the transposase may be a DNA sequence or an RNA sequence.
  • the sequence encoding the transposase is an mRNA sequence.
  • a transposon of the present disclosure can be a piggyBacTM (PB) transposon.
  • the transposase is a piggyBacTM (PB) transposase a piggyBac-like (PBL) transposase or a Super piggyBacTM (SPB) transposase.
  • the sequence encoding the SPB transposase is an mRNA sequence.
  • PB transposons and PB, PBL and SPB transposases are described in detail in U.S. Patent No. 6,218,182; U.S. Patent No. 6,962,810; U.S. Patent No.
  • the PB, PBL and SPB transposases recognize transposon-specific inverted terminal repeat sequences (ITRs) on the ends of the transposon, and inserts the contents between the ITRs at the sequence 5’-TTAT-3’ within a chromosomal site (a TTAT target sequence) or at the sequence 5’-TTAA-3’ within a chromosomal site (a TTAA target sequence).
  • ITRs inverted terminal repeat sequences
  • the target sequence of the PB or PBL transposon can comprise or consist of 5’-CTAA-3’, 5’-TTAG-3’, 5’-ATAA-3’, 5’-TCAA-3’, 5’AGTT-3’, 5’-ATTA-3’, 5’-GTTA-3’, 5’-TTGA-3’, 5’-TTTA-3’, 5’-TTAC-3’, 5’-ACTA-3’, 5’-AGGG-3’, 5’-CTAG-3’, 5’-TGAA-3’, 5’-AGGT-3’, 5’-ATCA- 3’, 5’-CTCC-3’, 5’-TAAA-3’, 5’-TCTC-3’, 5’TGAA-3’, 5’-AAAT-3’, 5’-AATC-3’, 5’- ACAA-3’, 5’-ACAT-3’, 5’-ACTC-3’, 5’-AGTG-3’, 5’-ATAG-3’, 5’-CAAA-3’, 5’-CACA-3’, 5’-CATA
  • PB, PBL transposon system has no payload limit for the genes of interest that can be included between the ITRs.
  • Exemplary amino acid sequence for one or more PB, PBL and SPB transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810 and U.S. Patent No. 8,399,643, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in the compositions and methods described herein.
  • the PB transposase comprises or consists of an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 56: MGSSLDDEHILSALLQSDDELVGEDSDSEISDHVSEDDVQSDTEEAFIDEVHEVQPTS SGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRS QRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTGATFRDTNEDEI YAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRP TLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRMYIPNKPSKY GIKILMMCDSGYKYMINGMPYL
  • the PB transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56 with one, two, three, four or five conservative amino acid substitutions. In some embodiments, the PB transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56. [0184]
  • the PB or PBL transposase can also comprise or consist of the amino acid sequence of SEQ ID NO: 56 with amino acid substitution at positions 30, 165, 282, and/or 538 of the sequence of SEQ ID NO: 56.
  • the transposase can comprises or consists of the amino acid sequence of the sequence of SEQ ID NO: 56 comprising one, two, three or all of the following amino acid substitutions: I30V, G165S, M282V, N538K.
  • the SPB transposase comprises or consists of an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 57: MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTS SGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRS QRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEI YAFFGILVMTAVRKDNHMST
  • the SPB transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57 with one, two, three, four or five conservative amino acid substitutions. In some embodiments, the SPB transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57.
  • the transposases can further comprise an amino acid substitution at one or more of positions 3, 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570 and 591 of the sequence of SEQ ID NO: 56 or SEQ ID NO: 57.
  • Such mutations are described in more detail in PCT Publications No. WO 2019/173636 and No.
  • the PB, PBL or SPB transposases can be isolated or derived from an insect, vertebrate, crustacean or urochordate as described in more detail in PCT Publications No. WO 2019/173636 and No. WO 2020/051374, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in the methods and compositions disclosed herein.
  • the PB, PBL or SPB transposases is be isolated or derived from the insect Trichoplusia ni (GenBank Accession No.
  • a hyperactive PB or PBL transposase is a transposase that is more active than the naturally occurring variant from which it is derived.
  • a hyperactive PB or PBL transposase is isolated or derived from Bombyx mori or Xenopus tropicalis. Examples of hyperactive PB or PBL transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No.6,962,810, U.S.
  • Patent No.8,399,643 and WO 2019/17363 each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in the methods and compositions disclosed herein.6.
  • a list of hyperactive amino acid substitutions is disclosed in U.S. Patent No. 10,041,077, which is incorporated herein by reference in its entirety for examples of mutations that may be introduced into the transposases disclosed herein..
  • the PB or PBL transposase is integration deficient.
  • An integration deficient PB or PBL transposase is a transposase that can excise its corresponding transposon, but that integrates the excised transposon at a lower frequency than a corresponding wild type transposase.
  • Examples of integration deficient PB or PBL transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643 and WO 2019/173636, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in the methods and compositions disclosed herein.
  • the PB or PBL transposase is fused to a nuclear localization signal.
  • Examples of PB or PBL transposases fused to a nuclear localization signal are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643 and WO 2019/173636, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in the methods and compositions disclosed herein.
  • a transposon of the present disclosure can be a Sleeping Beauty transposon.
  • the transposase when the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase (for example as disclosed in U.S. Patent No. 9,228,180) or a hyperactive Sleeping Beauty (SB100X) transposase.
  • the Sleeping Beauty transposase comprises or consists of an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 58.
  • the Sleeping Beauty transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58 with one, two, three, four or five conservative amino acid substitutions. In some embodiments, the Sleeping Beauty transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58. In some embodiments, a hyperactive Sleeping Beauty (SB100X) transposase comprises or consists of an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the sequence set forth in SEQ ID NO: 59.
  • SB100X hyperactive Sleeping Beauty
  • the SB100X transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59 with one, two, three, four or five conservative amino acid substitutions. In some embodiments, the SB100X transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59.
  • a transposon of the present disclosure can be a Helraiser transposon.
  • An exemplary Helraiser transposon includes Helibat1.
  • the Helibat1 transposon comprises or consists of a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 60.
  • the Helibat1 transposon comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 60.
  • the transposase is a Helitron transposase (for example, as disclosed in WO 2019/173636).
  • the Helitron transposase comprises or consists of an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 61.
  • the Helitron transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61 with one, two, three, four or five conservative amino acid substitutions.
  • the Helitron transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61.
  • An Tol2 transposon may include inverted repeats, subterminal sequences.
  • the Tol2 transposon comprises or consists of a nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 62.
  • the Tol2 transposon comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 62.
  • the transposase is a Tol2 transposase (for example, as disclosed in WO 2019/173636).
  • the Tol2 transposase comprises or consists of an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63.
  • the Tol2 transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63 with one, two, three, four or five conservative amino acid substitutions.
  • the Tol2 transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63.
  • a transposon of the present disclosure can be a TcBuster transposon.
  • the transposase when the transposon is a TcBuster transposon, the transposase is a TcBuster transposase or a hyperactive TcBuster transposase (for example, as disclosed in WO 2019/173636).
  • the TcBuster transposase can comprise or consist of a naturally occurring amino acid sequence or a non-naturally occurring amino acid sequence.
  • a TcBuster transposase comprises or consists of an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%,or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 64.
  • the TcBuster transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64 with one, two, three, four or five conservative amino acid substitutions.
  • the TcBuster transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64.
  • the polynucleotide encoding a TcBuster transposase can comprise or consist of a naturally occurring nucleic acid sequence or a non-naturally occurring nucleic acid sequence.
  • a TcBuster transposase is encoded by a polynucleotide comprising or consisting of an nucleic acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 65.
  • the TcBuster transposase is encoded by the nucleic acid sequence set forth in SEQ ID NO: 65.
  • a mutant TcBuster transposase comprises one or more sequence variations when compared to a wild type TcBuster transposase as described in more detail in PCT Publications No. WO 2019/173636 and No. WO 2020/051374 , each of which is incorporated herein by reference in its entirety for examples of mutations that may be introduced into the transposases disclosed herein.
  • the transposon can be a nanotransposon.
  • a nanotransposon can comprise, consist essential of, or consist of (a) a sequence encoding a transposon insert, comprising a sequence encoding a first inverted terminal repeat (ITR), a sequence encoding a second inverted terminal repeat (ITR), and an intra-ITR sequence; (b) a sequence encoding a backbone, wherein the sequence encoding the backbone comprises a sequence encoding an origin of replication having between 1 and 450 nucleotides, inclusive of the endpoints, and a sequence encoding a selectable marker having between 1 and 200 nucleotides, inclusive of the endpoints, and (c) an inter-ITR sequence.
  • the inter-ITR sequence of (c) comprises the sequence of (b).
  • the intra-ITR sequence of (a) comprises the sequence of (b).
  • the sequence encoding the backbone can comprise between 1 and 600 nucleotides, inclusive of the endpoints.
  • the sequence encoding the backbone consists of between 1 and 50 nucleotides, between 50 and 100 nucleotides, between 100 and 150 nucleotides, between 150 and 200 nucleotides, between 200 and 250 nucleotides, between 250 and 300 nucleotides, between 300 and 350 nucleotides, between 350 and 400 nucleotides, between 400 and 450 nucleotides, between 450 and 500 nucleotides, between 500 and 550 nucleotides, between 550 and 600 nucleotides, each range inclusive of the endpoints.
  • the inter-ITR sequence can comprise between 1 and 1000 nucleotides, inclusive of the endpoints.
  • the inter-ITR sequence consists of between 1 and 50 nucleotides, between 50 and 100 nucleotides, between 100 and 150 nucleotides, between 150 and 200 nucleotides, between 200 and 250 nucleotides, between 250 and 300 nucleotides, between 300 and 350 nucleotides, between 350 and 400 nucleotides, between 400 and 450 nucleotides, between 450 and 500 nucleotides, between 500 and 550 nucleotides, between 550 and 600 nucleotides, between 600 and 650 nucleotides, between 650 and 700 nucleotides, between 700 and 750 nucleotides, between 750 and 800 nucleotides, between 800 and 850 nucleotides, between 850 and 900 nucleotides, between 900 and 950 nucleotides, or between 950 and 1000 nucleot
  • the nanotransposon can be a short nanotransposon (SNT), wherein the inter-ITR sequence comprises between 1 and 200 nucleotides, inclusive of the endpoints.
  • the inter-ITR sequence can consist of between 1 and 10 nucleotides, between 10 and 20 nucleotides, between 20 and 30 nucleotides, between 30 and 40 nucleotides, between 40 and 50 nucleotides, between 50 and 60 nucleotides, between 60 and 70 nucleotides, between 70 and 80 nucleotides, between 80 and 90 nucleotides, or between 90 and 100 nucleotides, each range inclusive of the endpoints.
  • the selectable marker having between 1 and 200 nucleotides, inclusive of the endpoints can comprise a sequence encoding a sucrose-selectable marker.
  • the sequence encoding a sucrose-selectable marker can comprise a sequence encoding an RNA-OUT sequence.
  • the sequence encoding an RNA-OUT sequence can comprise or consist of 137 base pairs (bp).
  • the selectable marker having between 1 and 200 nucleotides, inclusive of the endpoints can comprise a sequence encoding a fluorescent marker.
  • the selectable marker having between 1 and 200 nucleotides, inclusive of the endpoints can comprise a sequence encoding a cell surface marker.
  • the sequence encoding an origin of replication having between 1 and 450 nucleotides, inclusive of the endpoints can comprise a sequence encoding a mini origin of replication.
  • the sequence encoding an origin of replication having between 1 and 450 nucleotides, inclusive of the endpoints comprises a sequence encoding an R6K origin of replication.
  • the R6K origin of replication can comprise an R6K gamma origin of replication.
  • the R6K origin of replication can comprise an R6K mini origin of replication.
  • the R6K origin of replication can comprise an R6K gamma mini origin of replication.
  • the R6K gamma mini origin of replication can comprise or consist of 281 base pairs (bp).
  • the sequence encoding the backbone does not comprise a recombination site, an excision site, and/or a ligation site. In some aspects, neither the nanotransposon nor the sequence encoding the backbone comprises a product of a recombination site, an excision site, and/or a ligation site. In some aspects, neither the nanotransposon nor the sequence encoding the backbone is derived from a recombination site, an excision site, and/or a ligation site. [0204] In some aspects of the nanotransposon, a recombination site comprises a sequence resulting from a recombination event.
  • a recombination site comprises a sequence that is a product of a recombination event.
  • the recombination event comprises an activity of a recombinase (e.g., a recombinase site).
  • the sequence encoding the backbone does not further comprise a sequence encoding foreign DNA.
  • the inter-ITR sequence does not comprise a recombination site, an excision site, a ligation site or a combination thereof.
  • the inter-ITR sequence does not comprise a product of a recombination event, an excision event, a ligation event or a combination thereof. In some aspects, the inter-ITR sequence is not derived from a recombination event, an excision event, a ligation event or a combination thereof. In some aspects, the inter-ITR sequence comprises a sequence encoding foreign DNA. In some aspects, the intra-ITR sequence comprises at least one sequence encoding an insulator and a sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell. The mammalian cell can be a human cell.
  • the intra-ITR sequence comprises a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell and a second sequence encoding an insulator. In some aspects, the intra-ITR sequence comprises a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell, a polyadenosine (polyA) sequence and a second sequence encoding an insulator.
  • polyA polyadenosine
  • the intra-ITR sequence comprises a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell, at least one exogenous sequence, a polyadenosine (polyA) sequence and a second sequence encoding an insulator.
  • polyA polyadenosine
  • Nanotransposons are described in more detail in International Patent Application Publication No. WO 2020/132396, which is incorporated herein by reference in its entirety for examples for nanotransposons that may be used in the methods and compositions described herein.
  • vectors may be used to introduce nucleic acids into cells.
  • a vector of the present disclose can be a viral vector or a recombinant vector.
  • Viral vectors can comprise a sequence isolated or derived from a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus or any combination thereof.
  • the viral vector may comprise a sequence isolated or derived from an adeno-associated virus (AAV).
  • the viral vector may comprise a recombinant AAV (rAAV).
  • Exemplary adeno-associated viruses and recombinant adeno-associated viruses comprise two or more inverted terminal repeat (ITR) sequences located in cis next to a sequence encoding an scFv or a CAR of the disclosure.
  • Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to all serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9).
  • Exemplary adeno- associated viruses and recombinant adeno-associated viruses include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids containing the genome of one serotype and the capsid of another serotype (e.g., AAV2/5, AAV-DJ and AAV-DJ8).
  • a vector of the present disclose can be a nanoparticle.
  • nanoparticle vectors include nucleic acids (e.g., RNA, DNA, synthetic nucleotides, modified nucleotides or any combination thereof ), amino acids (L-amino acids, D-amino acids, synthetic amino acids, modified amino acids, or any combination thereof), polymers (e.g., polymersomes), micelles, lipids (e.g., liposomes), organic molecules (e.g., carbon atoms, sheets, fibers, tubes), inorganic molecules (e.g., calcium phosphate or gold) or any combination thereof.
  • nucleic acids e.g., RNA, DNA, synthetic nucleotides, modified nucleotides or any combination thereof
  • amino acids L-amino acids, D-amino acids, synthetic amino acids, modified amino acids, or any combination thereof
  • polymers e.g., polymersomes
  • micelles lipids (e.g., liposomes)
  • Genome modification can comprise introducing a nucleic acid sequence, transgene and/or a genomic editing construct into a cell ex vivo, in vivo, in vitro or in situ to stably integrate a nucleic acid sequence.
  • the stable chromosomal integration can be a random integration, a site-specific integration, or a biased integration.
  • the site-specific integration can be non-assisted or assisted.
  • the assisted site-specific integration is co-delivered with a site- directed nuclease.
  • the site-directed nuclease comprises a transgene with 5’ and 3’ nucleotide sequence extensions that contain a percentage homology to upstream and downstream regions of the site of genomic integration.
  • the transgene with homologous nucleotide extensions enable genomic integration by homologous recombination, microhomology-mediated end joining, or nonhomologous end-joining.
  • the site-specific integration can occur at a safe harbor site. Genomic safe harbor sites are able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements function reliably (for example, are expressed at a therapeutically effective level of expression) and do not cause deleterious alterations to the host genome that cause a risk to the host organism.
  • Non-limiting examples of potential genomic safe harbors include intronic sequences of the human albumin gene, the adeno-associated virus site 1 (AAVS1), a naturally occurring site of integration of AAV virus on chromosome 19, the site of the chemokine (C-C motif) receptor 5 (CCR5) gene and the site of the human ortholog of the mouse Rosa26 locus.
  • the site-specific transgene integration can occur at a site that disrupts expression of a target gene. Disruption of target gene expression can occur by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.
  • Non-limiting examples of target genes targeted by site-specific integration include TRAC, TRAB, PDI, any gene encoding an immunosuppressive protein, and genes encoding proteins involved in allo-rejection.
  • the site-specific transgene integration can occur at a site that results in enhanced expression of a target gene. Enhancement of target gene expression can occur by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.
  • Enzymes can be used to create strand breaks in the host genome to facilitate delivery or integration of the transgene. Enzymes can create single-strand breaks or double-strand breaks.
  • Non-limiting examples of break-inducing enzymes include transposases, integrases, endonucleases, CRISPR/Cas9, transcription activator-like effector nucleases (TALEN), zinc finger nucleases (ZFN), Cas-CLOVERTM, and CPF1.
  • Break-inducing enzymes can be delivered to the cell encoded in DNA, encoded in mRNA, as a protein, or as a nucleoprotein complex with a guide RNA (gRNA).
  • gRNA guide RNA
  • Non-limiting examples of break-inducing enzymes are described in International Patent Application Publications No. WO 2016/205554, No. WO 2019/126578, and No.
  • the site-specific transgene integration can be controlled by a vector-mediated integration site bias.
  • the site-specific transgene integration site can be a non-stable chromosomal insertion.
  • the integrated transgene can be become silenced, removed, excised, or further modified.
  • the genome modification can be a non-stable integration of a transgene.
  • the non-stable integration can be a transient non-chromosomal integration, a semi-stable non chromosomal integration, a semi-persistent non-chromosomal insertion, or a non-stable chromosomal insertion.
  • the transient non-chromosomal insertion can be epi-chromosomal or cytoplasmic.
  • the transient non-chromosomal insertion of a transgene does not integrate into a chromosome and the modified genetic material is not replicated during cell division.
  • the genome modification can be a semi-stable or persistent non-chromosomal integration of a transgene.
  • a DNA vector encodes a Scaffold/matrix attachment region (S- MAR) module that binds to nuclear matrix proteins for episomal retention of a non-viral vector allowing for autonomous replication in the nucleus of dividing cells.
  • the genome modification can be a non-stable chromosomal integration of a transgene.
  • the integrated transgene can become silenced, removed, excised, or further modified.
  • the transgene can comprise a sequence encoding for a therapeutic agent.
  • the therapeutic agent can be a protein or an RNA that provides a therapeutic benefit when administered to a cell or a subject.
  • the therapeutic agent can be a therapeutic protein or a therapeutic RNA.
  • the therapeutic agent can be human beta-globin (HBB), T87Q human beta- globin (HBB T87Q), BAF chromatin remodeling complex subunit (BCL11A) shRNA, insulin like growth factor 2 binding protein 1 (IGF2BP1), interleukin 2 receptor gamma (IL2RG), alpha galactosidase A (GLA), alpha-L-idurondase (IDUA), iduronate 2-sulfatase (IDS), cystinosin lysosomal cysteine transporter (CTNS).
  • the transgene can comprise a sequence of Factor VIII or Factor IX.
  • the transgene can comprise a sequence encoding a chimeric antigen receptor (CAR).
  • the transgene can comprise a sequence encoding a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an ectodomain comprising a activation component, wherein the activation component is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain comprising at least one signal transduction domain, wherein the at least one signal transduction domain is isolated or derived from a second protein; wherein the first protein and the second protein are not identical.
  • the transgene can comprise a sequence for a CAR and a sequence for a CSR.
  • the transgene comprising a CAR or a CSR specifically binds to BCMA, PSMA, MUC1-C, CD133, c-KIT, CD19 or CD20.
  • the transgene can comprise a sequence encoding for an inducible proapoptotic polypeptide comprising (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible proapoptotic polypeptide does not comprise a non-human sequence.
  • the transgene can be integrated into the genome of the HSC. The integration can be stable or transient.
  • insertion tools e.g., DNA template vectors, transposable elements (transposons or retrotransposons) must be delivered to the cell in addition to the cutting enzyme (e.g., a nuclease, recombinase, integrase or transposase).
  • the cutting enzyme e.g., a nuclease, recombinase, integrase or transposase.
  • Examples of such insertion tools for a recombinase may include a DNA vector.
  • Other gene editing systems require the delivery of an integrase along with an insertion vector, a transposase along with a transposon/retrotransposon, etc.
  • An example recombinase that may be used as a cutting enzyme is the CRE recombinase.
  • Non-limiting examples of integrases that may be used in insertion tools include viral based enzymes taken from any of a number of viruses including AAV, gamma retrovirus, and lentivirus. Examples transposons/retrotransposons that may be used in insertion tools are described in more detail herein.
  • the present disclosure provides methods of targeted genome editing comprising introducing a gene editing composition and/or a cell comprising the gene editing composition.
  • the gene editing composition can comprise a sequence encoding a DNA binding domain and a sequence encoding a nuclease protein or a nuclease domain thereof.
  • the sequence encoding a nuclease protein or the sequence encoding a nuclease domain thereof can comprise a DNA sequence, an RNA sequence, or a combination thereof.
  • the nuclease or the nuclease domain thereof can comprise one or more of a CRISPR/Cas protein, a Transcription Activator-Like Effector Nuclease (TALEN), a Zinc Finger Nuclease (ZFN), and an endonuclease.
  • TALEN Transcription Activator-Like Effector Nuclease
  • ZFN Zinc Finger Nuclease
  • fusion proteins that may be used for the gene editing methods disclosed herein include fusion proteins comprising a nuclease-inactivated Cas (dCas) protein and an endonuclease.
  • the endonuclease can comprise a Clo051 nuclease or a nuclease domain thereof.
  • the gene editing composition can further comprise a guide sequence.
  • the guide sequence may comprise an RNA sequence.
  • the disclosure provides compositions comprising a small, Cas9 (Cas9) operatively- linked to an effector.
  • the disclosure provides a fusion protein comprising, consisting essentially of or consisting of a DNA localization component and an effector molecule, wherein the effector comprises a small, Cas9 (Cas9).
  • a small Cas9 construct of the disclosure can further comprise an effector comprising a type IIS endonuclease.
  • the fusion protein comprises a Staphylococcus aureus Cas9 with an active catalytic site comprising the amino acid sequence of SEQ ID NO: 66.
  • the disclosure provides compositions comprising an inactivated, small, Cas9 (dSaCas9) operatively-linked to an effector.
  • the disclosure provides a fusion protein comprising, consisting essentially of or consisting of a DNA localization component and an effector molecule, wherein the effector comprises a small, inactivated Cas9 (dSaCas9).
  • a small, inactivated Cas9 (dSaCas9) construct of the disclosure can further comprise an effector comprising a type IIS endonuclease.
  • the fusion protein comprises a dSaCas9 comprising the amino acid sequence of SEQ ID NO: 67, which includes a D10A and a N580A mutation to inactivate the catalytic site.
  • the disclosure provides compositions comprising an inactivated Cas9 (dCas9) operatively-linked to an effector.
  • the disclosure provides a fusion protein comprising, consisting essentially of or consisting of a DNA localization component and an effector molecule, wherein the effector comprises an inactivated Cas9 (dCas9).
  • An inactivated Cas9 (dCas9) construct of the disclosure can further comprise an effector comprising a type IIS endonuclease.
  • the dCas9 can be isolated or derived from Streptococcus pyogenes.
  • the dCas9 can comprise the amino acid sequence of SEQ ID NO: 68 or SEQ ID NO: 69.
  • the dCas9 can also comprise amino acid substitutions at amino acid positions 10 and 840 of SEQ ID NO: 68 or 69, which inactivate the catalytic site. In some aspects, these substitutions are D10A and H840A.
  • An illustrative Clo051 nuclease domain comprises, consists essentially of or consists of, the amino acid sequence of SEQ ID NO: 70. In some aspects, the Clo051 nuclease domain comprises at least one amino acid substitution. In some aspects, the amino acid substitution is in the alpha-helix-loop domain of the Clo051 nuclease. In some aspects, the amino acid substitution is at position 35, 37, 60, 98, 100 or 146 of SEQ ID NO: 70.
  • An illustrative dCas9-Clo051 (Cas-CLOVER) fusion protein can comprise, consist essentially of, or consist of, the amino acid sequence of SEQ ID NO: 71.
  • the illustrative dCas9- Clo051 fusion protein can be encoded by a polynucleotide which comprises, consists essentially of, or consists of, the nucleic acid sequence of SEQ ID NO: 72.
  • the nucleic acid encoding the dCas9-Clo051 fusion protein can be DNA or RNA.
  • An illustrative dCas9-Clo051 (Cas-CLOVER) fusion protein can comprise, consist essentially of, or consist of, the amino acid sequence of SEQ ID NO: 73.
  • the illustrative dCas9- Clo051 fusion protein can be encoded by a polynucleotide which comprises, consists essentially of, or consists of, the nucleic acid sequence of SEQ ID NO: 74.
  • the nucleic acid encoding the dCas9-Clo051 fusion protein can be DNA or RNA.
  • a dCas9-Clo051 fusion (Cas-CLOVER) fusion protein of the disclosure may further comprise at least one nuclear localization sequence (NLS).
  • the dCas9- Clo051 fusion protein of the disclosure comprises at least two nuclear localization sequences.
  • the NLS is on the N’terminal end of the dCas9-Clo051 fusion protein (NLS-dCas9-Clo051).
  • the NLS is on the C-terminal end of the dCas9- Clo051 fusion protein (dCas9-Clo051-NLS).
  • the NLS is on the N’terminal end and at the C’terminal end of the dCas9-Clo051 fusion protein (“NLS-dCas9- Clo051-NLS” or “wildtype Cas-CLOVER”).
  • the NLS-dCas9-Clo051-NLS (“wildtype Cas-CLOVER”) fusion protein can comprise, consist essentially of, or consist of, the amino acid sequence of SEQ ID NO: 71.
  • NLS-dCas9-Clo051-NLS amino acid sequence (NLS amino acid sequence is bolded and underlined): MAPKKKRKVEGIKSNISLLKDELRGQISHISHEYLSLIDLAFDSKQNRLFEMKVLELL VNEYGFKGRHLGGSRKPDGIVYSTTLEDNFGIIVDTKAYSEGYSLPISQADEMERYVR ENSNRDEEVNPNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRRLSMTTGVNGSAV NVVNLLLGAEKIRSGEMTIEELERAMFNNSEFILKYGGGGSDKKYSIGLAIGTNSVG WAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTR RKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY PTIYHLRKKLVDSTDKADLRLIYLALAHMI
  • the wildtype Cas-CLOVER fusion protein comprises or consists of an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 71.
  • the wildtype Cas-CLOVER fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 71 with one, two, three, four or five conservative amino acid substitutions.
  • the nucleic acid encoding the NLS-dCas9-Clo051-NLS (“wildtype Cas-CLOVER”) fusion protein can be DNA or RNA.
  • a dCas9-Clo051 fusion protein comprising two NLS regions is encoded by an mRNA sequence comprising, consisting essentially of or consisting of SEQ ID NO: 75.
  • NLS-dCas9-Clo051-NLS mRNA sequence (NLS amino acid sequence is bolded and underlined): atggcaccaaagaagaaaagaaaagtggagggcatcaagtcaaacatcagcctgctgaaagacgaactgcggggacagattagt cacatcagtcacgagtacctgtcactgattgatctggccttcgacagcagaatagactgtttgagatgaaagtgctggaactgctg gtcaacgtcaacagaatagactgtttgagatgaaagtgctggaactgctg gtcaacgacgagt
  • the Cas-CLOVER fusion proteins described above are used in conjunction with a guide sequence.
  • a guide sequence in the context of a Cas-Clover system or a CRISPR-Cas9 system can be any polynucleotide sequence having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of a nucleic acid-targeting complex to the target nucleic acid sequence.
  • the guide sequence may form a duplex with a target sequence.
  • the duplex may be a DNA duplex, an RNA duplex, or a RNA/DNA duplex.
  • guide molecule and “guide RNA” and “single guide RNA” are used interchangeably herein to refer to RNA-based molecules that are capable of forming a complex with a Cas-Clover or a CRISPR-Cas protein and comprises a guide sequence.
  • the guide molecule or guide RNA may encompass RNA-based molecules having one or more chemically modifications (e.g., by chemical linking two ribonucleotides or by replacement of one or more ribonucleotides with one or more deoxyribonucleotides), as described herein.
  • the guide RNA can comprise a sequence complementary to a target sequence within a genomic DNA sequence.
  • the target sequence within a genomic DNA sequence can be a target sequence within a safe harbor site of a genomic DNA sequence.
  • exemplary target sequences include but are not limited to HBB, TRAC, B2M, TCRb, GAPDH or SOX17.
  • the guide RNA can comprise a sequence complementary to at least one target sequence on a transposon, plasmid or vector.
  • the complementary sequence to the guide RNA on the transposon, plasmid or vector is located within the transgene for targeted nucleic acid insertion.
  • the complementary sequence to the guide RNA on the transposon, plasmid or vector is located within the transgene for targeted nucleic acid insertion.
  • the complementary sequence on the transposon, plasmid or vector facilitates binding of a gRNA which is bound to an effector molecule, thereby tethering all components.
  • target region refers to the region of the target gene to which the Cas-Clover system or the CRISPR/Cas9-based system targets.
  • the Cas-Clover or the CRISPR/Cas9-based system may include more than one gRNA, wherein the gRNAs target different DNA sequences.
  • the target DNA sequences may be overlapping.
  • the Cas-Clover system may include at least two gRNAs, wherein the gRNAs target different DNA sequences.
  • the target sequence or protospacer is followed by a PAM sequence at the 3' end of the protospacer.
  • Different Type II systems have differing PAM requirements.
  • the S. pyogenes Type II system uses an “NGG” sequence, where “N” can be any nucleotide.
  • N can be any nucleotide.
  • the guide RNA or the guide RNA of a Cas-Clover protein or a CRISPR-Cas protein may comprise a tracr-mate sequence (encompassing a “direct repeat” in the context of an endogenous CRISPR system) and a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system).
  • the Cas-Clover or the CRISPR-Cas system or complex as described herein does not comprise and/or does not rely on the presence of a tracr sequence.
  • the guide molecule may comprise, consist essentially of, or consist of a direct repeat sequence fused or linked to a guide sequence or spacer sequence.
  • the guide sequence or spacer length of the guide molecules is 15 to 50 nucleotides in length. In certain embodiments, the spacer length of the guide RNA is at least 15 nucleotides in length.
  • the spacer length is from 15 to 17 nucleotides in length, from 17 to 20 nucleotides in length, from 20 to 24 nucleotides in length, from 23 to 25 nucleotides in length, from 24 to 27 nucleotides in length, from 27-30 nucleotides in length, from 30-35 nucleotides in length, or greater than 35 nucleotides in length.
  • the guide sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length.
  • the sequence of the guide molecule is selected to reduce the degree secondary structure within the guide molecule. In some embodiments, about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the nucleic acid-targeting guide RNA participate in self- complementary base pairing when optimally folded. Optimal folding may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res.9 (1981), 133-148).
  • RNAfold Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g., A.R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151- 62).
  • the Cas-Clover system and the CRISPR/Cas9 system utilizes targeting gRNA and a shuttling gRNA that provides the targeting of the Cas-Clover system and the CRISPR/Cas9-based system.
  • the gRNA may be a fusion of two noncoding RNAs: a crRNA and a tracrRNA.
  • the sgRNA may target any desired DNA sequence by exchanging the sequence encoding a 20 bp protospacer which confers targeting specificity through complementary base pairing with the desired DNA target.
  • gRNA mimics the naturally occurring crRNA: tracrRNA duplex involved in the Type II Effector system. This duplex, which may include, for example, a 42-nucleotide crRNA and a 75-nucleotide tracrRNA, acts as a guide for the Cas9 to cleave the target nucleic acid.
  • the gRNA targets a region upstream of the target gene (e.g., B2M gene locus), e.g., between 0-1000 bp upstream of a target gene.
  • the gRNA targets a region between 0-50 bp, 0-100 bp, 0-150 bp, 0-200 bp, 0-250 bp, 0-300 bp, 0- 350 bp, 0-400 bp, 0-450 bp, 0-500 bp, 0-550 bp, 0-600 bp, 0-650 bp, 0-700 bp, 0-750 bp, 0- 800 bp, 0-850 bp, 0-900 bp, 0-950 bp or 0-1000 bp upstream of the transcription start site of the target gene.
  • the gRNA targets a region within about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp or about 1500 bp upstream of the target gene.
  • the gRNA targets a region downstream of a target gene (e.g., B2M gene locus), e.g., between 0-1000 bp downstream of a target gene.
  • the gRNA targets a region between 0-50 bp, 0-100 bp, 0-150 bp, 0-200 bp, 0-250 bp, 0-300 bp, 0-350 bp, 0-400 bp, 0-450 bp, 0-500 bp, 0-550 bp, 0-600 bp, 0-650 bp, 0-700 bp, 0-750 bp, 0- 800 bp, 0-850 bp, 0-900 bp, 0-950 bp or 0-1000 bp downstream of the target gene.
  • the gRNA targets a region within about 100 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp, about 1200 bp, about 1300 bp, about 1400 bp or about 1500 bp downstream of the target gene.
  • gRNA can be divided into a target binding region and a Cas9 binding region. The target binding region hybridizes with a target region in a target gene.
  • the target binding region can be between about 15 and about 50 nucleotides in length (about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 nucleotides in length). In certain embodiments, the target binding region can be between about 19 and about 21 nucleotides in length. In one embodiment, the target binding region is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. [0248] In one embodiment, the target binding region is complementary, e.g., completely complementary, to the target region in the target gene.
  • the target binding region is substantially complementary to the target region in the target gene. In one embodiment, the target binding region comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides that are not complementary to the target region in the target gene.
  • Exemplary sgRNAs of the disclosure include but are not limited to sequences for targeting HBB, B2M, TRAC or GAPDH gene locus. Exemplary sgRNAs of the disclosure also include but are not limited to sequences for targeting hemoglobin, albumin, TTR, APOC3, PCSK9 and KLKB1. Exemplary sgRNAs of the disclosure comprise, consist essentially of or consists of the sequences as shown in Table 1.
  • Methods of Expressing a CAR and/or a gamma delta T-cell receptor (gdTCR) or an alpha beta T-cell receptor (abTCR) [0251]
  • the disclosure provides methods of expressing a CAR on the surface of a cell.
  • the method comprises (a) obtaining a cell population; (b) contacting the cell population to a composition comprising a CAR or a sequence encoding the CAR, under conditions sufficient to transfer the CAR across a cell membrane of at least one cell in the cell population, thereby generating a modified cell population; (c) culturing the modified cell population under conditions suitable for integration of the sequence encoding the CAR; and (d) expanding and/or selecting at least one cell from the modified cell population that express the CAR on the cell surface.
  • the cell population can comprise leukocytes, including, for example, CD4+ and/or CD8+ leukocytes.
  • the cell population can comprise CD4+ and CD8+ leukocytes in an optimized ratio.
  • the optimized ratio of CD4+ to CD8+ leukocytes does not naturally occur in vivo.
  • the cell population can also comprise a tumor cell.
  • the conditions sufficient to transfer the CAR or the sequence encoding the CAR, transposon, or vector across a cell membrane of at least one cell in the cell population comprises at least one of an application of one or more pulses of electricity at a specified voltage, a buffer, and one or more supplemental factor(s).
  • the conditions suitable for integration of the sequence encoding the CAR comprise at least one of a buffer and one or more supplemental factor(s).
  • the buffer can comprise PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, Human T cell nucleofection buffer or any combination thereof.
  • the one or more supplemental factor(s) can comprise (a) a recombinant human cytokine, a chemokine, an interleukin or any combination thereof; (b) a salt, a mineral, a metabolite or any combination thereof; (c) a cell medium; (d) an inhibitor of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathway(s) or combinations thereof; and (e) a reagent that modifies or stabilizes one or more nucleic acids.
  • the recombinant human cytokine, the chemokine, the interleukin or any combination thereof can comprise IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, IL14, IL16, IL17, IL18, IL19, IL20, IL22, IL23, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36, GM-CSF, IFN-gamma, IL-1 alpha/IL-1F1, IL-1 beta/IL-1F2, IL-12 p70, IL-12/IL-35 p35, IL- 13, IL-17/IL-17A, IL-17A/F Heterodimer, IL-17F, IL-18/IL-1F4, IL-23
  • the salt, the mineral, the metabolite or any combination thereof can comprise HEPES, Nicotinamide, Heparin, Sodium Pyruvate, L-Glutamine, MEM Non-Essential Amino Acid Solution, Ascorbic Acid, Nucleosides, FBS/FCS, Human serum, serum-substitute, antibiotics, pH adjusters, Earle’s Salts, 2-Mercaptoethanol, Human transferrin, Recombinant human insulin, Human serum albumin, Nucleofector PLUS Supplement, KCL, MgCl2, Na2HPO4, NAH2PO4, Sodium lactobionate, Mannitol, Sodium succinate, Sodium Chloride, CINa, Glucose, Ca(NO 3 ) 2 , Tris/HCl, K2HPO4, KH2PO4, Polyethylenimine, Poly-ethylene-glycol, Poloxamer 188, Poloxamer 181, Poloxamer 407, Poly-vinylpyrrolidone, Pop
  • the cell medium can comprise PBS, HBSS, OptiMEM, DMEM, RPMI 1640, AIM-V, X-VIVO 15, CellGro DC Medium, CTS OpTimizer T Cell Expansion SFM, TexMACS Medium, PRIME-XV T Cell Expansion Medium, ImmunoCult-XF T Cell Expansion Medium or any combination thereof.
  • the inhibitor of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathway(s) or combinations thereof comprise inhibitors of TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, Type 1 Interferons, pro- inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, Caspase1, Pro-IL1B, PI3K, Akt, Wnt3A, inhibitors of glycogen synthase kinase-3 (GSK-3 ) (e.g. TWS119), or any combination thereof.
  • TLR9 TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, Type 1 Interferons, pro- inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA
  • inhibitors can include Bafilomycin, Chloroquine, Quinacrine, AC-YVAD-CMK, Z- VAD-FMK, Z-IETD-FMK or any combination thereof.
  • the reagent that modifies or stabilizes one or more nucleic acids comprises a pH modifier, a DNA-binding protein, a lipid, a phospholipid, CaPO4, a net neutral charge DNA binding peptide with or without a NLS sequence, a TREX1 enzyme or any combination thereof.
  • the expansion and selection steps can occur concurrently or sequentially. The expansion can occur prior to selection. The expansion can occur following selection, and, optionally, a further (i.e. second) selection can occur following expansion. Concurrent expansion and selection can be simultaneous.
  • the expansion and/or selection steps can proceed for a period of 10 to 14 days, inclusive of the endpoints.
  • the expansion can comprise contacting at least one cell of the modified cell population with an antigen to stimulate the at least one cell through the CAR, thereby generating an expanded cell population.
  • the antigen can be presented on the surface of a substrate.
  • the substrate can have any form, including, but not limited to a surface, a well, a bead or a plurality thereof, and a matrix.
  • the substrate can further comprise a paramagnetic or magnetic component.
  • the antigen can be presented on the surface of a substrate, wherein the substrate is a magnetic bead, and wherein a magnet can be used to remove or separate the magnetic beads from the modified and expanded cell population.
  • the antigen can be presented on the surface of a cell or an artificial antigen presenting cell.
  • Artificial antigen presenting cells can include, but are not limited to, tumor cells and stem cells.
  • the selection step comprises contacting at least one cell of the modified cell population with a compound to which the selection gene confers resistance, thereby identifying a cell expressing the selection gene as surviving the selection and identifying a cell failing to express the selection gene as failing to survive the selection step.
  • the disclosure provides a composition comprising the modified, expanded and selected cell population of the methods described herein.
  • a more detailed description of methods for expressing a CAR on the surface of a cell is disclosed in PCT Publications No.
  • the present disclosure also provides a cell or a population of cells wherein the cell comprises a composition comprising (a) an inducible transgene construct, comprising a sequence encoding an inducible promoter and a sequence encoding a transgene, and (b) a receptor construct, comprising a sequence encoding a constitutive promoter and a sequence encoding an exogenous receptor, such as a CAR, wherein, upon integration of the construct of (a) and the construct of (b) into a genomic sequence of a cell, the exogenous receptor is expressed, and wherein the exogenous receptor, upon binding a ligand or antigen, transduces an intracellular signal that targets directly or indirectly the inducible promoter regulating expression of the inducible transgene to modify gene expression.
  • a composition comprising (a) an inducible transgene construct, comprising a sequence encoding an inducible promoter and a sequence encoding a transgene
  • a receptor construct comprising a sequence
  • the composition can modify gene expression by decreasing gene expression.
  • the composition can modify the gene expression by increasing the gene expression.
  • the composition can modify gene expression by transiently modifying gene expression (e.g., for the duration of binding of the ligand to the exogenous receptor).
  • the composition can modify gene expression acutely (e.g., the ligand reversibly binds to the exogenous receptor).
  • the composition can modify gene expression chronically (e.g., the ligand irreversibly binds to the exogenous receptor).
  • the exogenous receptor can comprise an endogenous receptor with respect to the genomic sequence of the cell.
  • Exemplary receptors include, but are not limited to, intracellular receptors, cell-surface receptors, transmembrane receptors, ligand-gated ion channels, and G- protein coupled receptors.
  • the exogenous receptor can comprise a non-naturally occurring receptor.
  • the non-naturally occurring receptor can be a synthetic, modified, recombinant, mutant or chimeric receptor.
  • the non-naturally occurring receptor can comprise one or more sequences isolated or derived from a T-cell receptor (TCR).
  • TCR T-cell receptor
  • the non-naturally occurring receptor can comprise one or more sequences isolated or derived from a scaffold protein.
  • the non-naturally occurring receptor interacts with a second transmembrane, membrane-bound and/or an intracellular receptor that, following contact with the non-naturally occurring receptor, transduces an intracellular signal.
  • the non-naturally occurring receptor can comprise a transmembrane domain.
  • the non-naturally occurring receptor can interact with an intracellular receptor that transduces an intracellular signal.
  • the non-naturally occurring receptor can comprise an intracellular signaling domain.
  • the non-naturally occurring receptor can be a chimeric ligand receptor (CLR).
  • the CLR can be a chimeric antigen receptor (CAR).
  • the sequence encoding the inducible promoter of comprises a sequence encoding an NF B promoter, a sequence encoding an interferon (IFN) promoter or a sequence encoding an interleukin-2 promoter.
  • the IFN promoter is an IFN promoter.
  • the inducible promoter can be isolated or derived from the promoter of a cytokine or a chemokine.
  • the cytokine or chemokine can comprise IL2, IL3, IL4, IL5, IL6, IL10, IL12, IL13, IL17A/F, IL21, IL22, IL23, transforming growth factor beta (TGF ), colony stimulating factor 2 (GM-CSF), interferon gamma (IFN ), Tumor necrosis factor alpha (TNF ), LT , perforin, Granzyme C (Gzmc), Granzyme B (Gzmb), C-C motif chemokine ligand 5 (CCL5), C-C motif chemokine ligand 4 (Ccl4), C-C motif chemokine ligand 3 (Ccl3), X-C motif chemokine ligand 1 (Xcl1) or LIF interleukin 6 family cytokine (Lif).
  • TGF transforming growth factor beta
  • GM-CSF colony stimulating factor 2
  • IFN interferon gamma
  • the inducible promoter can be isolated or derived from the promoter of a gene comprising a surface protein involved in cell differentiation, activation, exhaustion and function.
  • the gene comprises CD69, CD71, CTLA4, PD-1, TIGIT, LAG3, TIM-3, GITR, MHCII, COX-2, FASL or 4-1BB.
  • the inducible promoter can be isolated or derived from the promoter of a gene involved in CD metabolism and differentiation.
  • the inducible promoter can be isolated or derived from the promoter of Nr4a1, Nr4a3, Tnfrsf9 (4-1BB), Sema7a, Zfp36l2, Gadd45b, Dusp5, Dusp6 and Neto2.
  • the inducible transgene construct comprises or drives expression of a signaling component downstream of an inhibitory checkpoint signal, a transcription factor, a cytokine or a cytokine receptor, a chemokine or a chemokine receptor, a cell death or apoptosis receptor/ligand, a metabolic sensing molecule, a protein conferring sensitivity to a cancer therapy, and an oncogene or a tumor suppressor gene.
  • proteins that may be controlled by the inducible transgene constructs disclosed herein are disclosed in PCT Publications No. WO 2019/173636 and No. WO 2020/051374, each of which is incorporated herein by reference in its entirety.
  • the inducible transgene construct comprises or drives the expression of the gdTCR or abTCR.
  • the inducible promoter is activated upon T-cell activation.
  • the T-cells are activated through the simulation of the CAR on the cell.
  • the CAR stimulation transduces an intracellular signal that targets the inducible promoter to modify the expression of the gene encoding the gdTCR or the abTCR.
  • the disclosure provides methods of expressing a CAR and a gdTCR or an abTCR on the surface of a cell.
  • the method comprises (a) obtaining a cell population; (b) contacting the cell population with a composition comprising a CAR or a sequence encoding the CAR and a gdTCR or abTCR, or a sequence encoding the gdTCR or abTCR, under conditions sufficient to transfer the CAR and gdTCR or abTCR, or nucleic acids encoding same, across a cell membrane of at least one cell in the cell population, thereby generating a modified cell population; (c) culturing the modified cell population under conditions suitable for integration of the nucleic acid sequence encoding the CAR and the gdTCR or abTCR; and (d) expanding and/or selecting at least one cell from the modified cell population that express the CAR and gdTCR or abTCR on the cell surface.
  • gdTCRs and abTCRs are set forth above.
  • the coding sequences of the safety switch, CAR, gdTCR or abTCR, and/or selectable marker are gene optimized for maximum expression in the desired cell type.
  • gene optimization may include, but not limited to, optimization at: a) the transcriptional level (e.g., GC content, removal of consensus and cryptic splice sites, splice donor sequences, and TATA boxes); b) the mRNA level (e.g., removal of RNA instability sites, ribosome entry sites and repetitive sequences) and/or c) the translational level (e.g., codon usage, removal of premature polyA sequences, ribosomal entry sites and undesired secondary structures).
  • the transcriptional level e.g., GC content, removal of consensus and cryptic splice sites, splice donor sequences, and TATA boxes
  • mRNA level e.g., removal of RNA instability sites, ribosome entry sites and repetitive sequences
  • translational level e.g., codon usage, removal of premature polyA sequences, ribosomal entry sites and undesired secondary structures.
  • compositions of the Disclosure provide the use of a disclosed composition or pharmaceutical composition for the treatment of a disease or disorder in a subject, e.g., cancer or an autoimmune disorder, as known in the art or as described herein, using the disclosed compositions and pharmaceutical compositions, e.g., administering to the subject a therapeutic effective amount of the composition or pharmaceutical composition comprising cells expressing a CAR and gdTCR (CAR/gdTCR cells) or cells expressing a CAR and abTCR (CAR/abTCR cells).
  • the subject is a mammal.
  • the subject is human.
  • subject and patient are used interchangeably herein.
  • the disclosure provides a method for modulating or treating cancer in a cell, tissue, organ, animal or subject.
  • a cancer include leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), acute myelogenous leukemia, chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodyplastic syndrome (MDS), a lymphoma, Hodgkin's disease, a malignant lymphoma, non-Hodgkin’s lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal carcinoma, pancreatic carcinoma, nasopharyngeal carcinoma, malignant histiocytosis, paraneoplastic syndrome/hypercalcemia of malign
  • ALL acute lympho
  • the CAR/gdTCR cells or CAR/abTCR cells of the present disclosure are modified to recombinantly express dihydrofolate reductase (DHFR), which advantageously renders the CAR/gdTCR cells or CAR/abTCR cells resistant to methotrexate (MTX).
  • DHFR dihydrofolate reductase
  • MTX-CAR/gdTCR cells MTX-CAR/gdTCR cells
  • MTX-CAR/abTCR cells MTX resistant CAR/abTCR cells
  • Modified cells can be formulated for storage at any temperature including room temperature and body temperature.
  • Modified cells can be formulated for cryopreservation and subsequent thawing. Modified cells can be formulated in a pharmaceutically acceptable carrier for direct administration to a subject from sterile packaging. Modified cells can be formulated in a pharmaceutically acceptable carrier with an indicator of cell viability and/or CAR, gdTCR or abTCR expression level to ensure a minimal level of cell function and CAR/gdTCR or CAR/abTCR expression. Modified cells can be formulated in a pharmaceutically acceptable carrier at a prescribed density with one or more reagents to inhibit further expansion and/or prevent cell death.
  • the methods of the present disclosure comprise administering an effective amount of MTX to a subject at a predetermined time post administration of the MTX CAR/gdTCR cells or MTX-CAR/abTCR cells.
  • an effective dose of MTX is a dose that is sufficient to eliminate the subject’s activated T-cells and NK cells targeting the previously administered MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells but spares the MTX-CAR/TCR cells.
  • An exemplary effective amount of MTX is an MTX serum level in a subject of about 200 nM, which equates to serum levels calculated for low dose MTX therapies for autoimmune disorder patients.
  • the MTX- CAR/gdTCR cells of the present disclosure are resistant to MTX levels well in excess of 200 nM.
  • the methods can optionally further comprise co-administration or combination therapy for treating such cancer, wherein the administering of any composition or pharmaceutical composition disclosed herein, further comprises administering, before concurrently, and/or after, at least one chemotherapeutic agent (e.g., an alkylating agent, an a mitotic inhibitor, a radiopharmaceutical).
  • at least one chemotherapeutic agent e.g., an alkylating agent, an a mitotic inhibitor, a radiopharmaceutical.
  • the subject does not develop graft vs. host (GvH) and/or host vs. graft (HvG) following administration.
  • the administration is systemic.
  • Systemic administration can be any means known in the art and described in detail herein.
  • systemic administration is by an intravenous injection or an intravenous infusion.
  • the administration is local.
  • Local administration can be any means known in the art and described in detail herein.
  • local administration is by intra-tumoral injection or infusion, intraspinal injection or infusion, intracerebroventricular injection or infusion, intraocular injection or infusion, or intraosseous injection or infusion.
  • the therapeutically effective dose of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is a single dose.
  • the single dose is one of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 doses that are manufactured simultaneously.
  • the dose is an amount sufficient for the cells to engraft and/or persist for a sufficient time to treat the disease or disorder.
  • the disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a composition comprising a scFv CAR or a VCAR that specifically binds to an antigen on a tumor cell and a gdTCR or abTCR.
  • a composition comprising a scFv CAR or a VCAR that specifically binds to an antigen on a tumor cell and a gdTCR or abTCR.
  • the composition comprises a modified cell or cell population
  • the cell or cell population may be autologous or allogeneic.
  • the treatment can be modified or terminated.
  • composition used for treatment comprises an inducible proapoptotic polypeptide (iCASp9 or iC9)
  • apoptosis may be selectively induced in the cell by contacting the cell with an induction agent.
  • a treatment may be modified or terminated in response to, for example, a sign of recovery or a sign of decreasing disease severity/progression, a sign of disease remission/cessation, and/or the occurrence of an adverse event.
  • the method comprises the step of administering an inhibitor of the induction agent to inhibit modification of the cell therapy, thereby restoring the function and/or efficacy of the cell therapy (for example, when a sign or symptom of the disease reappear or increase in severity and/or an adverse event is resolved).
  • methods of increasing in vivo persistence of MTX- CAR/gdTCR cells or MTX-CAR/abTCR cells in a subject comprising administering to the subject a therapeutically effective amount of MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells; and administering at a predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells an effective amount of MTX sufficient to eliminate (reduce by at least 25%) activated T-cells and NK cells targeting the MTX-CAR/TCR cells leading to an increased in vivo persistence of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells.
  • the MTX-CAR/gdTCR cells or or MTX-CAR/abTCR cells express at least one chimeric antigen receptor (CAR) targeting an oncogenic gene product.
  • the oncogenic gene product is BCMA, CD19, CD20, MUC1C, PSMA, CD70, mesothelin or c-kit.
  • the MTX-CAR/gdTCR cells or MTX- CAR/abTCR cells are T-cells (i.e., MTX-CAR-T cells).
  • the MTX- CAR/gdTCR cells or MTX-CAR/abTCR cells are T-cells (MTX-CAR-T/gdTCR cells or MTX-CAR-T/abTCR cells).
  • MTX- CAR-T/gdTCR cells or MTX-CAR-T/abTCR cells comprising administering to the subject a therapeutically effective amount of MTX-CAR-T/gdTCR cells or or MTX-CAR- T/abTCR cells cells, wherein the MTX-CAR-T/gdTCR cells or MTX-CAR-T/abTCR cells comprise a heterologous nucleic acid encoding dihydrofolate reductase (DHFR); and, administering at a predetermined time post administration of the MTX-CAR-T/gdTCR cells or MTX-CAR/abTCR cells, an effective amount of MTX sufficient to eliminate (reduce by X) activated T-cells and NK cells targeting the MTX-CAR-T/gdTCR cells or or MTX-CAR- T/ab
  • DHFR dihydrofolate reductase
  • the MTX-CAR-T/gdTCR cells or MTX-CAR-T/abTCR cells express at least one chimeric antigen receptor (CAR) targeting an oncogenic gene product.
  • the oncogenic gene product is BCMA, CD19, CD20, MUC1C, PSMA, CD70, mesothelin or c-kit.
  • the MTX-CAR/gdTCR cells are T-cells (i.e., MTX-CAR-T/gdTCR cells).
  • the MTX-CAR/abTCR cells are T-cells (MTX-CAR-T/abTCR cells).
  • methods of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of MTX-CAR- T/gdTCR cells or MTX-CAR-T/abTCR cells; and administering at a predetermined time post administration of the MTX-CAR-T/gdTCR cells or MTX-CAR-T/abTCR cells an effective amount of MTX sufficient to eliminate (reduce by X) activated T-cells and NK cells targeting the CAR cells leading to an increased in vivo persistence of the MTX-CAR-T/gdTCR cells or MTX-CAR-T/abTCR cells and increased treatment efficacy of the MTX-CAR-T/gdTCR cells or MTX-CAR-T/abTCR cells compared to no MTX administration.
  • the MTX-CAR-T/gdTCR cells express at least one chimeric antigen receptor (CAR) targeting an oncogenic gene product.
  • the oncogenic gene product is BCMA, CD19, CD20, MUC1C, PSMA, CD70, mesothelin or c-kit.
  • the MTX- CAR/gdTCR cells are T-cells (i.e., MTX-CAR-T/gdTCR cells).
  • the or MTX-CAR/abTCR cells are T-cells (MTX-CAR-T/abTCR cells).
  • the predetermined time post administration of the MTX- CAR/gdTCR cells or MTX-CAR/abTCR cells is about 10 minutes. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX- CAR/abTCR cells is about 15 minutes. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR-T/abTCR cells is about 20 minutes. In certain embodiments, the predetermined time post administration of the MTX- CAR/gdTCR cells or MTX-CAR/abTCR cells is about 30 minutes.
  • the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX- CAR/abTCR cells is about 40 minutes. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 50 minutes. [0284] In certain embodiments, the predetermined time post administration of the MTX- CAR/gdTCR cells or MTX-CAR/abTCR cells is about 1 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 2 hr.
  • the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 3 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX- CAR/abTCR cells is about 4 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 5 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 6 hr.
  • the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 7 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 8 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 9 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 10 hr.
  • the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 11 hr. In certain embodiments, the predetermined time post administration of the MTX- CAR/gdTCR cells or MTX-CAR/abTCR cells is about 12 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells is about 13 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 14 hr.
  • the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 15 hr. In certain embodiments, the predetermined time post administration of the MTX- CAR/gdTCR cells or MTX-CAR/abTCR cells is about 16 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 17 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 18 hr.
  • the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX- CAR/abTCR cells is about 19 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 20 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 21 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 22 hr.
  • the predetermined time post administration of the MTX- CAR/gdTCR cells or MTX-CAR/abTCR cells is about 23 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 24 hr. [0285] In certain embodiments, the predetermined time post administration of the MTX- CAR/gdTCR cells or MTX-CAR/abTCR cells is about 2 days. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 3 days.
  • the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 4 days. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 5 days. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 6 days. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells is about 1 week.
  • the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 2 weeks. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 3 weeks. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is about 1 month. [0286] In certain embodiments, the predetermined time post administration of the MTX- CAR/gdTCR cells or MTX-CAR/abTCR cells is a single dose.
  • the single dose is administered on Day 3 post-administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells. In certain embodiments, the single dose is administered on Day 6 post-administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells. In certain embodiments, the predetermined time post administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is a multi-dose regiment. In certain embodiments, two doses are administered on Days 3 and 6 post-administration of the MTX-CAR/gdTCR cells or MTX- CAR/abTCR cells.
  • three doses are administered on Days 1, 3, and 6 post-administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells. In certain embodiments, four doses are administered on Days 1, 3, 6 and 11 post-administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells. In certain embodiments, eight doses are administered on Days 1, 3, 6, 11, 14, 18, 22 & 26 post-administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells.
  • kits for treating cancer in a subject in need thereof as described above further comprising administering at a predetermined time an effective dose of MTX prior to administration of the therapeutically effective dose of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells.
  • the administration at a predetermined time an effective dose of MTX prior to administration of the therapeutically effective dose of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is one day (Day -1) prior to administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells.
  • the administration at a predetermined time an effective dose of MTX prior to administration of the therapeutically effective dose of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is two days (Day -2) prior to administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells.
  • the administration at a predetermined time an effective dose of MTX prior to administration of the therapeutically effective dose of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is three days (Day -3) prior to administration of the MTX- CAR/gdTCR cells or MTX-CAR/abTCR cells.
  • the administration at a predetermined time an effective dose of MTX prior to administration of the therapeutically effective dose of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells is four days (Day - 4) prior to administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells. In certain aspects, the administration at a predetermined time an effective dose of MTX prior to administration of the therapeutically effective dose of the MTX-CAR/gdTCR cells or MTX- CAR/abTCR cells is five days (Day -5) prior to administration of the MTX-CAR/gdTCR cells or MTX-CAR/abTCR cells.
  • a disease or disorder in a subject in need thereof comprising: a) administering to the subject a therapeutically effective amount of a composition comprising cells expressing a chimeric antigen receptor and a recombinant T cell receptor (CAR/gdTCR cells or CAR/abTCR cells), wherein the CAR/gdTCR cells or MTX- CAR/abTCR cells comprise a chimeric antigen receptor (CAR) and a recombinant gamma delta T-cell receptor (gdTCR) or a recombinant alpha beta T-cell receptor (abTCR), and wherein the CAR/gdTCR cells or CAR/abTCR cells exhibit enhanced cytolytic activity compared to CAR-T cells; b) monitoring the subject for disease progression; and c) upon disease progression, administering to the subject an effective amount of a bispecific antibody, wherein the bispecific antibody binds to the same target cells as the CAR/gdTCR cells or CAR/
  • a disease or disorder in a subject in need thereof comprising: a) administering to the subject a therapeutically effective amount of a composition comprising cells expressing a chimeric antigen receptor and a recombinant T cell receptor (CAR/gdTCR cells or CAR/abTCR cells), wherein the CAR/gdTCR cells or MTX- CAR/abTCR cells comprise a chimeric antigen receptor (CAR) and a recombinant gamma delta T-cell receptor (gdTCR) or a recombinant alpha beta T-cell receptor (abTCR), and wherein the CAR/gdTCR cells or CAR/abTCR cells exhibit enhanced cytolytic activity compared to CAR-T cells; b) monitoring the subject for disease progression; and c) upon disease progression, administering to the subject an effective amount of a bispecific antibody, wherein the bispecific antibody binds to different target cells than the CAR
  • the time of monitoring of the subject prior to administration of the bispecific antibody is one month. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is two months. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is three months. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is four months. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is five months. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is six months. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is seven months. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is eight months.
  • the time of monitoring of the subject prior to administration of the bispecific antibody is nine months. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is ten months. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is eleven months. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is twelve months. [0291] In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is two years. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is two and one half years. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is three years.
  • the time of monitoring of the subject prior to administration of the bispecific antibody is three and one-half years. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is four years. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is four and one-half years. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is five years. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is five and one-half years. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is six years. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is six and one-half years.
  • the time of monitoring of the subject prior to administration of the bispecific antibody is seven years. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is seven and one-half years. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is eight years. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is eight and one-half years. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is nine years. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is nine and one-half years. In one embodiment, the time of monitoring of the subject prior to administration of the bispecific antibody is ten years.
  • Exemplary bispecific antibodies useful in the methods disclosed herein include RG6189, RG6353, RG6156 (RO7428731), RG7828 (Lunsumio, mosunetuzumab), RG7802 (RO6958688, cibisatamab), RG6007 (RO7283420), RG6129 (RO7444973), IMCC-103C (RG6290), simlukafusp alfa (RG7461, RO6874281), lomvastomig (RO7121661, RG7769), RG6232 (RO7293583), fazpilodemab (RG7992), RG6296 (AFM26, RO7297089), Vabysmo (faricimab), RG7386 (RO6874813), vanucizumab (RO5520985), XGFR2, XGFR4, Hemlibra (emicizumab), Columvi (glofitamab,
  • Methods of re-engaging CAR-T cells comprising monitoring the treated subject for disease reoccurrence and upon reoccurrence administering to the subject a bispecific antibody targeting an oncology target and a CD3 binding domain, wherein the bispecific antibody interaction with the autologous CAR-T cells results in increased cell expansion of the autologous CAR-T cells.
  • provided are methods of re-engaging autologous CAR T-cells in a subject treated therewith comprising monitoring the treated subject for disease reoccurrence and upon reoccurrence administering to the subject a bispecific antibody targeting an oncology target and a CD3 binding domain, wherein the bispecific antibody interaction with the autologous CAR-T cells results in a therapeutically effective expansion of the autologous CAR-T cells and reduced tumor burden in the subject.
  • the bispecific antibody interacting with the allogenic CAR-T cells results in increased cell expansion of the allogenic CAR-T cells, compared to the cell expansion that is observed in the absence of the bispecific antibody.
  • the administration of the bispecific antibody increases CAR-T cell expansions by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% compared to the expansion of the CAR-T cells in a subject that is not administered the bispecific antibody. Expansion of CAR-T cells may be determined using any suitable method known in the art or described herein, such as counting the number of CAR-T cells in the blood over time.
  • the method of re-engaging CAR-T cells in a subject results in reduced tumor burden in the subject.
  • the tumor burden may be reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% compared to the reduction in tumor burden achieved with a CAR-T cell therapy in a subject that is not administered the bispecific antibody.
  • the tumor burden may be reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% compared to the tumor burden in the subject prior to administration of the administration of the bispecific antibody.
  • Tumor burden may be determined using any suitable method known in the art or described herein, for example, imaging such as PET or MRI.
  • the bispecific antibody binds the same oncology target as the CAR-T cells being reactivated and CD3. In other methods of reactivating CAR-T cells, the bispecific antibody binds different oncology targets than the CAR-T cells being reactivated and CD3. In some embodiments, the tumor cells targeted by the reactivation of the CAR-T cells do not express the CAR-targeting antigen. In certain embodiments, the oncology target is BCMA, CD19, CD20, MUC1C, PSMA, CD70, mesothelin or c-kit.
  • Bispecific antibodies that may be used to reactivate CAR-T cells include RG6189, RG6353, RG6156, RO7428731, RG7828, Lunsumio, mosunetuzumab, RG7802, RO6958688, cibisatamab, RG6007, RO7283420, RG6129, RO7444973, IMCC-103C, RG6290, simlukafusp alfa, RG7461, RO6874281, lomvastomig, RO7121661, RG7769, RG6232, RO7293583, fazpilodemab, RG7992, RG6296, AFM26, RO7297089, Vabysmo, faricimab, RG7386, RO6874813, vanucizumab, RO5520985, XGFR2, XGFR4, Hemlibra, emicizumab, Columvi, glofit
  • the bispecific antibody targets CD3 and CD70.
  • the bispecific antibody may be administered at a dose and at the frequency specified in the prescribing information approved by the regulatory authorities.
  • the CAR-T cells being reactivated by the methods disclosed herein may be allogeneic or autologous to the subject.
  • Nucleic Acids and Vectors [0302] In another aspect, provided herein are nucleic acids and vectors comprising the nucleic acids provided herein. [0303]
  • the isolated nucleic acid compositions of this disclosure such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methodologies known to those of skill in the art.
  • oligonucleotide probes that selectively hybridize, under stringent conditions, to the polynucleotides of the present disclosure are used to identify the desired sequence in a cDNA or genomic DNA library.
  • the isolation of RNA, and construction of cDNA and genomic libraries are well known to those of ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook, supra).
  • Methods of amplification of RNA or DNA are well known in the art and can be used according to the disclosure without undue experimentation, based on the teaching and guidance presented herein.
  • RNA mediated amplification that uses anti-sense RNA to the target sequence as a template for double-stranded DNA synthesis
  • PCR polymerase chain reaction
  • in vitro amplification methods can also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes.
  • the isolated nucleic acids of the disclosure can also be prepared by direct chemical synthesis by known methods (see, e.g., Ausubel, et al., supra). Chemical synthesis generally produces a single-stranded oligonucleotide, which can be converted into double-stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template.
  • a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.
  • One of skill in the art will recognize that while chemical synthesis of DNA can be limited to sequences of about 100 or more bases, longer sequences can be obtained by the ligation of shorter sequences.
  • Recombinant Expression Cassettes [0308] The disclosure further provides recombinant expression cassettes comprising a nucleic acid of the disclosure.
  • a nucleic acid sequence of the disclosure can be used to construct a recombinant expression cassette that can be introduced into at least one desired host cell.
  • a recombinant expression cassette will typically comprise a polynucleotide of the disclosure operably linked to transcriptional initiation regulatory sequences that will direct the transcription of the polynucleotide in the intended host cell.
  • Both heterologous and non- heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the disclosure.
  • isolated nucleic acids that serve as promoter, enhancer, or other elements can be introduced in the appropriate position (upstream, downstream or in the intron) of a non-heterologous form of a polynucleotide of the disclosure so as to up or down regulate expression of a polynucleotide of the disclosure.
  • endogenous promoters can be altered in vivo or in vitro by mutation, deletion and/or substitution.
  • the disclosure also relates to vectors that include isolated nucleic acid molecules of the disclosure, host cells that are genetically engineered with the recombinant vectors, and the production of at least one protein scaffold by recombinant techniques, as is well known in the art. See, e.g., Sambrook, et al., supra; Ausubel, et al., supra, each entirely incorporated herein by reference.
  • the polynucleotides can optionally be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells. [0312]
  • the DNA insert should be operatively linked to an appropriate promoter.
  • the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (e.g., UAA, UGA or UAG) appropriately positioned at the end of the mRNA to be translated, with UAA and UAG preferred for mammalian or eukaryotic cell expression.
  • a termination codon e.g., UAA, UGA or UAG
  • Expression vectors will preferably but optionally include at least one selectable marker.
  • Such markers include, e.g., but are not limited to, ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418/Geneticin (neo gene), DHFR (encoding Dihydrofolate Reductase and conferring resistance to Methotrexate), mycophenolic acid, or glutamine synthetase (GS, U.S. Pat. Nos.
  • blasticidin bsd gene
  • resistance genes for eukaryotic cell culture as well as ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418/Geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline resistance genes for culturing in E. coli and other bacteria or prokaryotics (the above patents are entirely incorporated hereby by reference). Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • Expression vectors will preferably but optionally include at least one selectable cell surface marker for isolation of cells modified by the compositions and methods of the disclosure. Selectable cell surface markers of the disclosure comprise surface proteins, glycoproteins, or group of proteins that distinguish a cell or subset of cells from another defined subset of cells.
  • the selectable cell surface marker distinguishes those cells modified by a composition or method of the disclosure from those cells that are not modified by a composition or method of the disclosure.
  • Such cell surface markers include, e.g., but are not limited to, “cluster of designation” or “classification determinant” proteins (often abbreviated as “CD”) such as a truncated or full length form of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof.
  • Cell surface markers further include the suicide gene marker RQR8 (Philip B et al. Blood.2014 Aug 21; 124(8):1277-87).
  • Expression vectors will preferably but optionally include at least one selectable drug resistance marker for isolation of cells modified by the compositions and methods of the disclosure.
  • Selectable drug resistance markers of the disclosure may comprise wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.
  • Those of ordinary skill in the art are knowledgeable in the numerous expression systems available for expression of a nucleic acid encoding a protein of the disclosure.
  • nucleic acids of the disclosure can be expressed in a host cell by turning on (by manipulation) in a host cell that contains endogenous DNA encoding a protein scaffold of the disclosure.
  • COS-1 e.g., ATCC CRL 1650
  • COS- 7 e.g., ATCC CRL-1651
  • HEK293, BHK21 e.g., ATCC CRL-10
  • CHO e.g., ATCC CRL 1610
  • BSC-1 e.g., ATCC CRL-26 cell lines
  • Cos-7 cells CHO cells
  • hep G2 cells hep G2 cells
  • P3X63Ag8.653, SP2/0-Ag14 293 cells
  • HeLa cells and the like, which are readily available from, for example, American Type Culture Collection, Manassas, Va. (www.atcc.org).
  • Preferred host cells include cells of lymphoid origin, such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC Accession Number CRL- 1580) and SP2/0-Ag14 cells (ATCC Accession Number CRL-1851). In a preferred aspect, the recombinant cell is a P3X63Ab8.653 or an SP2/0-Ag14 cell.
  • Expression vectors for these cells can include one or more of the following expression control sequences, such as, but not limited to, an origin of replication; a promoter (e.g., late or early SV40 promoters, the CMV promoter (U.S. Pat. Nos.
  • an HSV tk promoter an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alpha promoter (U.S. Pat. No. 5,266,491), at least one human promoter; an enhancer, and/or processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences. See, e.g., Ausubel et al., supra; Sambrook, et al., supra.
  • nucleic acids or proteins of the present disclosure are known and/or available, for instance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (www.atcc.org) or other known or commercial sources.
  • polyadenlyation or transcription terminator sequences are typically incorporated into the vector.
  • An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript can also be included.
  • An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al., J. Virol.45:773-781 (1983)).
  • gene sequences to control replication in the host cell can be incorporated into the vector, as known in the art.
  • the disclosure provides isolated or substantially purified polynucleotide or protein compositions.
  • An "isolated” or “purified” polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment.
  • an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • an "isolated" polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived.
  • the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived.
  • a protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein.
  • optimally culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.
  • Fragments of a DNA sequence comprising coding sequences may encode protein fragments that retain biological activity of the native protein and hence DNA recognition or binding activity to a target DNA sequence as herein described.
  • fragments of a DNA sequence that are useful as hybridization probes generally do not encode proteins that retain biological activity or do not retain promoter activity.
  • fragments of a DNA sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide of the disclosure.
  • expression refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. [0325] “Gene expression” refers to the conversion of the information, contained in a gene, into a gene product.
  • a gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, micro RNA, structural RNA or any other type of RNA) or a protein produced by translation of an mRNA.
  • Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.
  • “Modulation” or “regulation” of gene expression refers to a change in the activity of a gene.
  • Modulation of expression can include, but is not limited to, gene activation and gene repression.
  • operatively linked or its equivalents (e.g., “linked operatively”) means two or more molecules are positioned with respect to each other such that they are capable of interacting to affect a function attributable to one or both molecules or a combination thereof.
  • Non-covalently linked components and methods of making and using non-covalently linked components are disclosed. The various components may take a variety of different forms as described herein. For example, non-covalently linked (i.e., operatively linked) proteins may be used to allow temporary interactions that avoid one or more problems in the art.
  • a method for directing proteins to a specific locus in a genome of an organism is disclosed.
  • the method may comprise the steps of providing a DNA localization component and providing an effector molecule, wherein the DNA localization component and the effector molecule are capable of operatively linking via a non-covalent linkage.
  • the term "scFv" refers to a single-chain variable fragment.
  • scFv is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a linker peptide.
  • the linker peptide may be from about 5 to 40 amino acids or from about 10 to 30 amino acids or about 5, 10, 15, 20, 25, 30, 35, or 40 amino acids in length.
  • Single- chain variable fragments lack the constant Fc region found in complete antibody molecules, and, thus, the common binding sites (e.g., Protein G) used to purify antibodies.
  • the term further includes a scFv that is an intrabody, an antibody that is stable in the cytoplasm of the cell, and which may bind to an intracellular protein.
  • a “target site” or “target sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist.
  • Nucleic acids of the disclosure may be single- or double-stranded. Nucleic acids of the disclosure may contain double-stranded sequences even when the majority of the molecule is single-stranded. Nucleic acids of the disclosure may contain single-stranded sequences even when the majority of the molecule is double-stranded. Nucleic acids of the disclosure may include genomic DNA, cDNA, RNA, or a hybrid thereof. Nucleic acids of the disclosure may contain combinations of deoxyribo- and ribo-nucleotides.
  • Nucleic acids of the disclosure may contain combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids of the disclosure may be synthesized to comprise non-natural amino acid modifications. Nucleic acids of the disclosure may be obtained by chemical synthesis methods or by recombinant methods. [0333] Nucleic acids of the disclosure, either their entire sequence, or any portion thereof, may be non-naturally occurring. Nucleic acids of the disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not naturally-occur, rendering the entire nucleic acid sequence non-naturally occurring.
  • Nucleic acids of the disclosure may contain one or more duplicated, inverted or repeated sequences, the resultant sequence of which does not naturally- occur, rendering the entire nucleic acid sequence non-naturally occurring.
  • Nucleic acids of the disclosure may contain modified, artificial, or synthetic nucleotides that do not naturally-occur, rendering the entire nucleic acid sequence non-naturally occurring.
  • a plurality of nucleotide sequences may encode any particular protein. All such nucleotides sequences are contemplated herein.
  • the term "operably linked” refers to the expression of a gene that is under the control of a promoter with which it is spatially connected.
  • a promoter can be positioned 5' (upstream) or 3' (downstream) of a gene under its control.
  • the distance between a promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. Variation in the distance between a promoter and a gene can be accommodated without loss of promoter function.
  • promoter refers to a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell.
  • a promoter can comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same.
  • a promoter can also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
  • a promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals.
  • a promoter can regulate the expression of a gene component constitutively or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents.
  • promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, EF-1 Alpha promoter, CAG promoter, or SV40 late promoter and the CMV IE promoter.
  • the term “substantially complementary” refers to a first sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.
  • the term "vector” refers to a nucleic acid sequence containing an origin of replication.
  • a vector can be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome.
  • a vector can be a DNA or RNA vector.
  • a vector can be a self-replicating extrachromosomal vector, and preferably, is a DNA plasmid.
  • a vector may comprise a combination of an amino acid with a DNA sequence, an RNA sequence, or both a DNA and an RNA sequence.
  • hydropathic index of amino acids As understood in the art. Kyte et al., J. Mol. Biol.157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. Amino acids of similar hydropathic indexes can be substituted and still retain protein function. In an aspect, amino acids having hydropathic indexes of ⁇ 2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function.
  • amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.
  • “conservative” amino acid substitutions may be defined as set out in Tables A, B, or C below.
  • fusion polypeptides and/or nucleic acids encoding such fusion polypeptides include conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the disclosure. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure.
  • a conservative substitution is a substitution of one amino acid for another amino acid that has similar properties.
  • Exemplary conservative substitutions are set out in Table 2.
  • Table 2 Conservative Amino Acid Substitutions I [0342] Alternately, conservative amino acids can be grouped as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp.71-77) as set forth in Table 3.
  • Table 3 Conservative Amino Acid Substitutions II [0343] Alternately, exemplary conservative substitutions are set out in Table 4.
  • polypeptides of the disclosure are intended to include polypeptides bearing one or more insertions, deletions, or substitutions, or any combination thereof, of amino acid residues as well as modifications other than insertions, deletions, or substitutions of amino acid residues.
  • Polypeptides or nucleic acids of the disclosure may contain one or more conservative substitution.
  • sequence identity may be determined by using the stand-alone executable BLAST engine program for blasting two sequences (bl2seq), which can be retrieved from the National Center for Biotechnology Information (NCBI) ftp site, using the default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; which is incorporated herein by reference in its entirety).
  • NCBI National Center for Biotechnology Information
  • identity when used in the context of two or more nucleic acids or polypeptide sequences, refer to a specified percentage of residues that are the same over a specified region of each of the sequences.
  • the percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity.
  • the residues of single sequence are included in the denominator but not the numerator of the calculation.
  • the term “endogenous” refers to nucleic acid or protein sequence naturally associated with a target gene or a host cell into which it is introduced.
  • the term “exogenous” refers to nucleic acid or protein sequence not naturally associated with a target gene or a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid, e.g., DNA sequence, or naturally occurring nucleic acid sequence located in a non- naturally occurring genome location.
  • the disclosure provides methods of introducing a polynucleotide construct comprising a DNA sequence into a host cell.
  • introducing is intended presenting to the cell the polynucleotide construct in such a manner that the construct gains access to the interior of the host cell.
  • the methods of the disclosure do not depend on a particular method for introducing a polynucleotide construct into a host cell, only that the polynucleotide construct gains access to the interior of one cell of the host.
  • Methods for introducing polynucleotide constructs into bacteria, plants, fungi and animals are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
  • Example 1 Construction of PiggyBac Transposon CAR-T/TCR-T Polynucleotides
  • a piggyBac transposon cassette comprising the nucleic acid sequences encoding a CAR and gamma delta TCR chains is illustrated in Fig.1.
  • the transposon cassette comprising a piggyBac left end ITR (SEQ ID NO: 82), an EF1a promoter (SEQ ID NO: 83) operably associated with the nucleic acids encoding: an iCas9 safety switch (SEQ ID NO: 84) a CAR targeting MUC1C (SEQ ID NO: 85); a gene optimized G115 TCR gamma chain (SEQ ID NO: 49), a gene optimized, G115 TCR delta chain (SEQ ID NO: 51), a DHFR mutein selectable marker (SEQ ID NO: 86); a polyA sequence (SEQ ID NO: 87); and a piggyBac right end ITR (SEQ ID NO: 88) was constructed via Gibson assembly to generate full-length transposon cassette polynucleotide.
  • Control piggyBac transposon cassettes identical to that illustrated in Fig. 1 were constructed lacking either the nucleic acid encoding the gamma delta TCR G115 or the nucleic acid encoding the CAR for use as comparators expressing only the CAR or gamma delta TCR G115 (gdTCR).
  • Example 2 Generation of T-cell Populations Lacking an Endogenous TCR that Express a CAR Targeting MUC1C and Heterologous gdTCR
  • Pan T-cells were isolated from healthy donors and used to generate T-cell populations useful for allogeneic cell therapies.
  • pan T-cells were subject to electroporation to introduce: 10 ⁇ g of an mRNA encoding a Cas-CLOVER fusion protein v2.0 (SEQ ID NO: 90), 1 ⁇ g each of a gRNA pair targeting beta-2-microglobulin (B2M; SEQ ID NOs: 76 and 77), 1 ⁇ g each of a gRNA pair targeting the endogenous TCRb (SEQ ID NOs: 78 and 79) and TRAC (SEQ ID Nos: 80 and 81) to knockout the B2M gene and the endogenous TCR, respectively, 20 ⁇ g of an mRNA encoding Super piggyBac transposase v2.0 (SEQ ID NO: 91), 10 ⁇ g of an mRNA encoding a chimeric stimulatory receptor (SEQ ID NO: 92) as well as the transposon cassette or control transposon cassettes described in Example 1.
  • SEQ ID NO: 90 10 ⁇ g of an mRNA encoding
  • T-cells were selected for by culturing cells in a medium comprising 250nM methotrexate (MTX).
  • MTX resistant cells were harvested using MultiMACS, (Miltenyi) and residual endogenous abTCR positive cells were removed using a -biotin labeled anti-TCRa antibody (ClinMacs) and anti-biotin microbeads.
  • T-cell populations are endogenous abTCR negative, B2M edited, DHFR positive, express a CAR targeting MUC1C and the heterologous gdTCR, whereas the control T-cells express only the CAR or only the gdTCR.
  • Example 3 T-cell Populations Lacking an Endogenous abTCR that Express a CAR Targeting MUC1C and Heterologous gdTCR Exhibit Increased Proliferation upon Stimulation Compared to CAR only Expressing Cells.
  • MUC1C CAR T-cells and MUC1C CAR + gdTCR T-cells were seeded at 1-410e6 cells/ml in 25 ml T-flasks comprising prewarmed R10 culture medium and allowed to rest overnight at 37 °C, 0.5% CO2.
  • approximately 5,000 GFP+A375.MUC1C target cells in 100 ⁇ l of prewarmed R10 culture medium were plated in designated wells in a 3595 flat bottom 96 well plate and allowed to rest overnight at 37 °C, 0.5% CO2.
  • MUC1C CAR T-cells, gdTCR T-cells, and MUC1C CAR + gdTCR T-cells were centrifuged @ 500g x 6 min and stained for FACS sorting using the primary and secondary markers shown in Table 5: Table 5 [0359] After staining, T cells were washed with 14mL of FACS buffer and resuspended in 1mL of FACS buffer. T cells were then sorted, into 15mL tubes containing 5mL of R10 media, based on antigen-receptor expression as follows: MUC1C CAR+, gdTCR+ or CAR+gdTCR+T-cells using a SH800S cell sorter (Sony).
  • Sorted T-cells were resuspended at a concentration of 15,000 cells/100 ⁇ l of R10 medium. Approximately 15,000 sorted MUC1C CAR T-cells, gdTCR T-cells, and MUC1C CAR + gdTCR T-cells were mixed with 5,000 GFP+A375.MUC1C cells (ratio of 3:1) in a 96- well flat-bottom plate. [0361] On Days 2, 5, 7, 9 and 13, T cells from each well were harvested and transferred to a 96 V-bottom plate. To each well, 5 ⁇ l of TrueCount beads (Beckton Dickinson) was added and the plate subject to centrifugation at 2,000 rpm for 2 minutes.
  • TrueCount beads Beckton Dickinson
  • MUC1C CAR T-cells, gdTCR T-cells, and MUC1C CAR + gdTCR T-cells were stimulated with GFP+A375-MUC1 cells:on Day 2 by removing 100 ⁇ l of culture medium from each well and adding a 100 ⁇ l aliquout comprising 20,000 cells GFP+A375.MUC1C cells on Day 5 by removing 100 ⁇ l of culture medium from each well and adding a 100 ⁇ l aliquout comprising 40,000 GFP+A375.MUC1C cells.
  • MUC1C CAR T-cells and MUC1C CAR+ gdTCR T-cells were harvested and transferred to 24 well plates and restimulated using 440,000 cells GFP+A375.MUC1C cells.
  • the results of the proliferation assay are shown in Fig.2.
  • T-cells expressing the CAR targeting MUC1C and gdTCR exhibited robust cell expansion with an expansion peak at about Day 9 (fourth restimulation) whereas T-cells expressing only a CAR or only gdTCR exhibited little to no observable proliferation throughout the time course of the restimulation assay.
  • Example 4 T-cell Populations Lacking an Endogenous TCR that Express a CAR Targeting MUC1C and Heterologous TCR gamma and delta chains (gdTCR) Exhibit Increased Cytotoxicity Compared to CAR only Expressing T-Cells.
  • the cytotoxicity of T-cell populations expressing a CAR targeting MUC1C or T-cell populations expressing a CAR targeting MUC1C and the TCR gamma and delta chains (gdTCR) was compared in restimulation assays.
  • On Day -2 approximately 5 million GFP+A375.MUC1C cells were subjected to centrifugation at 4,000 kg for 4 minutes, and the supernatants were discarded.
  • MUC1C CAR T-cells, gdTCR T-cells, MUC1C CAR + gdTCR T-cells were seeded at 1-410e6 cells/ml in 25 ml T-flasks comprising prewarmed R10 culture medium and allowed to rest overnight at 37 °C, 0.5% CO2.
  • GFP+A375.MUC1C target cells in 100 ⁇ l of prewarmed R10 culture medium were plated in designated wells in a 3595 flat bottom 96 well plate, and allowed to rest overnight at 37 °C, 0.5% CO2.
  • cells were FACS sorted as described on Example 3.
  • Sorted T cells were resuspended at a concentration of 15,000 cells/100 ⁇ l of (R10).
  • MUC1C CAR T-cells Approximately, 15,000 MUC1C CAR T-cells, gdTCR T-cells, and MUC1C CAR + gdTCR T- cells were mixed with 5,000 GFP+A375.MUC1C cells (ratio of 3:1), transferred to an Incucyte plate, and the plate was placed in the Incucyte reader for real time monitoring of GFP expression.
  • T-cells expressing the CAR targeting MUC1C or the CAR targeting MUC1C and gdTCR each demonstrated cytolytic activity upon restimulation; however, CAR+gdTCR positive T-cells exhibited enhanced target cell killing resulting in near complete elimination of target cells compared to CAR+ control T-cells.
  • Example 5 T-cell Populations Lacking an Endogenous TCR that Express a CAR Targeting MUC1C and Recombinant TCR alpha and beta chains (abTCR) Dually and Concurrently Target CAR and abTCR antigens [0372]
  • the cytotoxicity of T-cell populations expressing a CAR targeting MUC1C or T-cell populations expressing a CAR targeting MUC1C and the TCR alpha and beta chains targeting HLA-A*02:01-NY-ESO-1 (NYESO1:abTCR) were probed against single TCR- Antigen(Ag)+CAR-Ag–, Single TCR-Ag–CAR-Ag+ , double TCR-Ag+CAR-Ag+ and double TCR-Ag–CAR-Ag– A375 cells.
  • the pelleted cells were resuspended in 20 ml of prewarmed R10 medium, seeded into T75 flasks and allowed to rest overnight at 37 °C, 0.5% CO2 [0374] On Day -1, NYESO1:abTCR, MUC-1-CAR+NYESO1:abTCR and Mock transposed T-cells were seeded at 1-410e6 cells/ml in 25 ml T-flasks comprising prewarmed R10 culture medium and allowed to rest overnight at 37 °C, 0.5% CO2.
  • TCR-Ag+CAR-Ag–, TCR-Ag–CAR-Ag+, TCR-Ag+CAR-Ag+ and TCR-Ag–CAR- Ag– A375 target cells in 100 ⁇ l of prewarmed R10 culture medium were plated in designated wells in a 3595 flat bottom 96 well plate, and allowed to rest overnight at 37 °C, 0.5% CO 2 . [0375] On Day 0, T cells were resuspended at a concentration of 25,000 cells/100 ⁇ l of (R10).
  • T-cells expressing the CAR targeting MUC1C or the CAR targeting MUC1C and abTCR each demonstrated cytolytic activity upon restimulation; however, only CAR+abTCR positive T-cells exhibited the capacity to kill double TCR-Ag+CAR-Ag+ as well as single TCR-Ag+CAR-Ag–, TCR-Ag–CAR-Ag+ A375 tumor cells. Control samples mock, exhibited no activity throughout the course of the assay.
  • Example 6 Very Late Lymphocytosis and Anti-Myeloma Activity of a T SCM Rich CAR- T When Activated by a Bispecific T-cell Engager (TCE)
  • TCE Bispecific T-cell Engager
  • a 57-year-old woman was treated with P-BCMA-101 (described in detail in PCT Publication Number WO 2019/126574), a T SCM rich autologous CAR-T manufactured using the piggyBac DNA delivery system, on a phase 1 study for relapsed refractory multiple myeloma (RRMM).
  • the final CAR-T product had a mean 1.97 copies of the CAR transgene per cell.
  • WBC count was 7.1 X 10 9 cells/L and lymphocyte count was 4.1 X 10 9 cells/ L.
  • Quantitative polymerase chain reaction on the peripheral blood was evaluated for the presence of the CAR-T by QPCR, revealing 685,016 copies/ug DNA of the CAR transgene consistent with the presence of CAR T cells accounting for the majority of circulating white blood cells.
  • Integration site analysis was performed using LM-PCR and Illumina NGS on 2 samples, each with 6 replicates, on isolated PBMCs drawn from the patient. Over 11,500 CAR transgene insertion sites were detected. The top 4 most abundant insertions mapped to introns or proximal promoters of 4 genes.
  • Example 7 T-cell Populations Lacking an Endogenous TCR that Express a CAR Targeting MUC1C and a Heterologous TCR (abTCR or gdTCR) are Reactivated In Vivo Upon Subsequent Administration of a CD3-comprising T-cell Engager (TCE) [0008] Pre-existing MUC1C expressing CAR-T+abTCR/gdTCR cells may be reactivated in vivo to eliminate tumors expressing a different tumor antigen targeted by the MUC1C CAR by administration of a bispecific CD3-comprising TCE.
  • TCE T-cell Engager
  • mice On Day 0, 6–7-week-old female NSG mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl /Szj) (Jackson Laboratories Stock # 005557) were implanted with 2.5 X 10 6 GFP+A375.MUC1C cells subcutaneously (“S.C.”) in 50% Matrigel and tumor growth was monitored by caliper measurements (FIG.5A).
  • mice were re-challenged with 2.5 X 10 6 b2M –/– GFP+A375 cells (CD70+, TCR-Antigen – MUC1C-) in 50% Matrigel implanted subcutaneously (on the side opposite the primary challenge), with an average tumor volume of about 100 mm 3 .
  • mice in Groups 3, 6, and 8 were administered 0.8 mg/kg of a bispecific CD70/CD3 TCE (Creative BioLabs, Cat # XS-0722-ZP3892) and mice were followed until Day 24 after rechallenge/Day 62 after the primary challenge.
  • the volume of the primary tumor (FIG.5A) and the secondary tumor (FIG.5B) were measured through Day 24 after rechallenge.
  • b2M –/– GFP+A375 cells to activate the MUC1C CAR-T/TCR cells to eliminate CD70 positive tumors, thereby redirecting the CAR-T/TCR cells to eliminate tumors expressing a different tumor antigen than that targeted by the CAR-T/TCR cells (e.g. GFP+A375.MUC1C cells).
  • Example 8 T-Cell Populations Lacking an Endogenous TCR that Express a CAR Targeting MUC1C and a Heterologous TCR [abTCR] Demonstrated Moderate CD25 level and Enriched in Tscm Featured Gene-set Expression Post-stimulation by Antigen- positive Tumor Cells [0378]
  • This Example illustrates that CAR+TCR-T cells targeting HLA*0201:NY-ESO-1 157-165 (TCR-Ag) and Mucin glycoprotein MUC1 (CAR-Ag) may be activated by Dual antigen positive tumor cells ( MUC1+/ HLA*0201:NY-ESO-1 157-165 +) to express T cell activation marker CD25 and T cell differentiation genes.
  • CAR+TCR-T cells targeting HLA*0201-NY-ESO-1 157-165 TCR- Ag
  • CAR-Ag Mucin glycoprotein MUC1
  • FACS Flow Cytometry
  • CAR+TCR-T cells exhibited intermediate levels of CD25 expression, significantly lower than CAR-T cells but higher than TCR-T cells.
  • the results demonstrate CAR+TCR-T cells are moderately activated by antigen-stimulation compared to CAR-Ts, which could promote cell persistence by limiting T-cell terminal-differentiation and exhaustion.
  • CAR+TCR-T cells targeting HLA*0201-NY-ESO-1 157-165 (TCR-Ag) and Mucin glycoprotein MUC1 (CAR-Ag) were co-cultured with Dual-Ag+ (CAR- Ag+/TCR-Ag+) tumor cells for 48hrs at E:T ration 1:1.
  • TCR+CAR-T cells were sorted and cDNA library was prepared for bulk RNAseq (Illumina # 20040536).
  • the sequencing data were aligned to the Human genome for transcriptome profiling using STAR essentially as previously described (e.g., Dobin, A., et al., (2013). Bioinformatics (Oxford, England), 29(1), 15–21.) and gene differentiation expression was analyzed by DEseq2 essentially as previously described (Love, M. I., et al., (2014). Genome biology, 15(12), 550).

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Abstract

L'invention concerne des compositions et un méthodes d'utilisation de thérapies cellulaires CAR/TCR pour le traitement de maladies ou de troubles, tels que le cancer ou des troubles auto-immuns.
PCT/US2025/024771 2024-04-16 2025-04-15 Compositions et méthodes destinées à être utilisées dans des thérapies cellulaires car/tcr Pending WO2025221789A1 (fr)

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