Compositions and methods for use in car cell therapies

Abstract

Provided herein compositions and method of using chimeric antigen receptor (CAR) cell therapies for treating diseases or disorders, such as cancer or autoimmune disorders, including dual CARs that binds to both B-cell maturation antigen (BCMA) and CD19. Polynucleotides, vectors, host cells and methods for producing such dual CARs and cell therapies comprising the dual CARs are also provided herein.

Description

Attorney Docket No.: 000218-0154-WO1COMPOSITIONS AND METHODS FOR USE IN CAR CELL THERAPIESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 704,154, filed October 7, 2024, and U.S. Provisional Patent Application No. 63 / 786,525, filed April 10, 2025, the contents of each of which are herein incorporated by reference in their entireties.FIELD

[0002] The disclosure is directed to molecular biology, and more specifically, to compositions and methods for preparing and using genetically modified immune cells that express a chimeric antigen receptor (CAR).INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety.Said XML copy, created on October 6, 2025, is named 000218-0154-WOl-SL.xml and is 127,664 bytes in size.BACKGROUND

[0004] The discovery of cell-based therapies capable of recognizing and binding to a specific target protein with high affinity and avidity has been a focus of the biopharmaceutical industry. There remains a need for more efficacious cell-based therapies that demonstrate enhanced in vivo activity, persistence and extended treatment duration compared to traditional cell-based therapies.SUMMARY

[0005] In one aspect, provided herein is a dual CAR comprising (a) a first ectodomain comprising a first antigen recognition region that binds to BCMA; (b) a first transmembrane domain, (c) a first endodomain comprising at least one costimulatory domain; (d) a second ectodomain comprising a second antigen recognition region that binds to CD 19; (e) a second transmembrane domain, (f) a second endodomain comprising at least one costimulatory domain; wherein the first antigen recognition region comprises the amino acid sequence ofAttorney Docket No.: 000218-0154-WO1EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVAGIIGS GGSTYYADSVKGRFSISRDNSKNTLDLQMNSLRAEDTAVYYCVKDWNTTMITE RGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFT FSNYAMTWIRQAPGKGLEWVSGITGDGGSTFYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCVKDWNTTMITERGQGTLVTVSS (SEQ ID NO: 51). In some embodiments, the second antigen recognition region comprises the amino acid sequence of QVQLVESGGGLVKPGGSLRLSCAASGFDFSDYYMSWIRQAPGKGLEWVSYMSS SGSTIYYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCARGGIAAAGTW GQGTLVTVSS (SEQ ID NO: 57).

[0006] In some embodiments, the first ectodomain and / or second ectodomain further comprises a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1.

[0007] In some embodiments, the dual CAR further comprises a first hinge region between the first antigen recognition region and the first transmembrane domain and / a second hinge region between the second antigen recognition region and the second transmembrane domain. In some embodiments, the first hinge region and / or the second hinge region comprises the amino acid sequence of SEQ ID NO: 9.

[0008] In some embodiments, the first transmembrane domain and / or the second transmembrane domain comprises a CD8 transmembrane domain. In some embodiments, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 3.

[0009] In some embodiments, the first endodomain comprises a CD3(^ costimulatory domain and a 4-1BB costimulatory domain, and wherein the 4-1BB costimulatory domain is located between the first transmembrane domain and the CD3(^ costimulatory domain. In some embodiments, the 4- IBB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the second endodomain comprises a CD3(^ costimulatory domain and a TACI costimulatory domain, and wherein the TACI costimulatory domain is located between the second transmembrane domain and the CD3(^ costimulatory domain. In some embodiments, the TACI costimulatory domain comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the CD3(^ costimulatory domain comprises the amino acid sequence of SEQ ID NO: 5.

[0010] In another aspect, provided herein is a dual CAR comprising (a) the amino acid sequencesAttorney Docket No.: 000218-0154-WO1MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYA MNWVRQAPGKGLEWVAGIIGSGGSTYYADSVKGRFSISRDNSKNTLDLQMNSL RAEDTAVYYCVKDWNTTMITERGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLE SGGGLVQPGGSLRLSCAASGFTFSNYAMTWIRQAPGKGLEWVSGITGDGGSTFY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKDWNTTMITERGQGTL VTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAP LAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR (SEQ ID NO: 53) and (b) the amino acid sequence MALPVTALLLPLALLLHAARPQVQLVESGGGLVKPGGSLRLSCAASGFDFSDYY MSWIRQAPGKGLEWVSYMSSSGSTIYYADSVKGRFTISRDNAKKSLYLQMNSLR AEDTAVYYCARGGIAAAGTWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPE ACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKKRGDPCSCQ PRSRPRQSPAKSSQDHAMEAGSPVSTSPEPVETCSFCFPECRAPTQESAVTPGTPD PTCAGRWGCHTRTTVLQPCPHIPDSGLGIVCVPAQEGGPGARVKFSRSADAPAY KQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 61)

[0011] In another aspect, provided herein is a polynucleotide comprising a nucleic acid sequence encoding a dual CAR disclosed herein.

[0012] In another aspect, provided herein is a transposon comprising a nucleic acid sequence encoding a dual CAR disclosed herein. In some embodiments, comprises the nucleic acid sequence of SEQ ID NO: 64. In some embodiments, the transposon further comprises a nucleic acid encoding an inducible caspase polypeptide, a nucleic acid encoding a chimeric stimulatory receptor, a nucleic acid encoding a selection gene, a nucleic acid encoding a therapeutic agent, or a combination thereof. In some embodiments, the selection gene comprises a DHFR resistance gene. In some embodiments, the transposon is a piggyBac transposon. In some embodiments, the transposon comprises a nucleic acid sequence of SEQ ID NO: 66.

[0013] In another aspect, provided herein is a vector comprising a polynucleotide or a transposon described herein.

[0014] In another aspect, provided herein is a cell comprising a dual CAR or a transposon described herein.Attorney Docket No.: 000218-0154-WO1

[0015] In another aspect, provided herein is a population of cells comprising a dual CAR described herein. In some embodiments, the cells are immune cells. In some embodiments, the cells are T-cells. In some embodiments, a portion of the immune cells comprises a genetic modification which reduces or inhibits expression of a T-cell receptor or a major histocompatibility complex (MHC). In some embodiments, the genetic modification is an insertion of a sequence encoding a P-2 microglobulin (P2M) and wherein the genetic modification reduces or inhibits expression of a MHC I. In some embodiments, the genetic modification is an insertion of a sequence encoding an a chain (TCRa), a P chain (TCRP), or a combination thereof and wherein the genetic modification reduces or inhibits expression of a TCR. In some embodiments, 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 95%, at least 96%, at least 97%, at least 98%, at least 99% of cells express the dual CAR.

[0016] In another aspect, provided herein is a composition comprising a dual CAR, a cell or a population of cells described herein.

[0017] In another aspect, provided herein is a pharmaceutical composition comprising a composition of described herein and a pharmaceutically acceptable carrier.

[0018] In another aspect, provided herein is a method of treating cancer in a subject in need thereof comprising administering a therapeutically In some embodiments, the cancer is a primary tumor, a metastatic cancer, a multiply resistant cancer, a progressive tumor or recurrent cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a lymphoma, a leukemia, a myeloma, a malignant immunoproliferative disease, a lung cancer, a brain cancer, a head and neck cancer, a breast cancer, a skin cancer, a liver cancer, a pancreatic cancer, a stomach cancer, a colon cancer, a rectal cancer, a uterine cancer, a cervical cancer, an ovarian cancer, a prostate cancer, a testicular cancer, a skin cancer or an esophageal cancer. In some embodiments, the cancer is diffuse large B cell lymphoma or multiple myeloma.

[0019] In another aspect, provided herein is a method of treating an autoimmune disorder in a subject in need thereof comprising administering a therapeutically effective amount of a composition or a pharmaceutical composition described herein. In some embodiments, the autoimmune disorder is selected from: autoimmune neutropenia, Guillain-Barre syndrome, epilepsy, autoimmune encephalitis, Isaacs' syndrome, nevus syndrome, pemphigus vulgaris, deciduous pemphigus, bullous pemphigoid, acquiredAttorney Docket No.: 000218-0154-WO1 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, or membranous nephropathy.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIGs. 1A and IB show a pair of graphs showing in vivo efficacy of BCMA binders in MMl.S xenograft model NOD.CgPrkdcscldI12rgtmlw-’1 / SzJ (NSG) immunodeficient mice. Tumor burden was measured by bioluminescence (BLI) at the indicated timepoints (FIG. 1 A) and survival was assessed during the entire duration of the study (90 days post CAR-T injection, FIG. IB).

[0021] FIG. 2 is a pair of graphs showing in vivo efficacy of CD19 binders in Raji lymphoma subcutaneous xenograft model in NOD.CgPrkdcscldI12rgtmlwj1 / SzJ (NSG) immunodeficient mice. Tumor burden was measured weekly by bioluminescence (BLI). Area under the curve (AUC) for tumor burden and T cell expansion in the peripheral blood was calculated for the entire duration of the study (35 days post CAR-T injection).

[0022] FIGs. 3A-3C are graphs showing in vivo efficacy of BCMA binders in RPMI- 8226 or Pfeiffer subcutaneous xenograft model in NOD.CgPrkdcscldI12rgtmlwj1 / SzJ (NSG) immunodeficient mice. Tumor burden was measured weekly by caliper for RPMI-8226 model (Exp 1 and 2, FIGS. 3A and 3B) and by bioluminescence (BLI) forPfeiffer. Luc. GFP (Exp 3, FIG. 3C). Area under the curve (AUC) was calculated for the study duration of 41 days post CAR-T injection for Expl (FIG. 3 A), 35 days for Exp2 (FIG. 3B), 21 days for Exp3 (FIG. 3C).

[0023] FIG. 4 is a schematic showing construction of BCMA, CD 19 and BCMA / CD19 dual CAR-T cells. These CAR-T constructs contained 4-1BB co-stimulatory and CD3(^ signaling domains.

[0024] FIG. 5A and 5B are a pair of graphs showing in vivo efficacy of BCMA, CD 19, and BCMA / CD19 dual CAR-T cells in RPMI-8226 CD 19+ subcutaneous xenograft model in NOD.CgPrkdcscldI12rgtmlwj1 / SzJ (NSG) immunodeficient mice. Tumor burden was measured weekly by caliper (FIG. 5 A) and T cell expansion (FIG. 5B) in the peripheral blood collected from the tail vein on days 7, 14, 21, 28, and 35 post CAR-TAttorney Docket No.: 000218-0154-WO1 injection. The VH523 / VH34.28D dual CAR-T demonstrates superior in vivo activity over VH34.28D / VH523 dual CAR & single CAR-T cells.

[0025] FIGs. 6A and 6B is a pair of graphs showing in vivo efficacy of BCMA, CD 19, and BCMA / CD19 dual CAR-T cells in RPMI-8226 CD 19+ or Pfeiffer subcutaneous xenograft model in NOD.CgPrkdcscldI12rgtmlw-’1 / SzJ (NSG) immunodeficient mice. Tumor burden was measured by caliper for RPMI-8226 CD 19+ model (FIG. 6 A) and weekly by bioluminescence (BLI) for Pfeiffer.Luc.GFP (FIG. 6B). Data demonstrate similar in vivo activity across dual CAR-T cells.

[0026] FIG. 7 is a graph showing in vivo efficacy of BCMA / CD19 dual CAR-T cells (T202.523 / VH034.28D and VH034.28D / T202.523) in RPMI-8226 CD19+ subcutaneous xenograft model in NOD.CgPrkdcscldI12rgtmlwj1 / SzJ (NSG) immunodeficient mice. Tumor burden was measured by caliper at the indicated timepoints. Dual CAR with T202.523 / VH034.28D orientation demonstrates more durable tumor control over VH034.28D / T202.523 orientation.

[0027] FIGs. 8A and 8B are a pair of graphs showing in vitro cytotoxicity and exhaustion of CD 19 CAR-T cells (VH034.28D), BCMA CAR-T cells (VH523) and BCMA / CD19 dual CAR-T cells (T202.523 / VH034.28D) in a restimulation assay against RPMI-8226 CD19+ tumor cells. T202.523 / VH034.28D dual CAR-T cells demonstrates long-term killing capacity in in vitro serial re-stimulation assay and expresses less exhaustion markers after 5th challenge.

[0028] FIG. 9 is a series of graphs showing antigen-specific reactivity of CD 19 CAR-T cells (VH034.28D), BCMA CAR-T cells (VH523) and BCMA / CD19 dual CAR-T cells (T202.523 / VH034.28D) in RPMI-8226 models expressing both BCMA and CD19 (BCMA+CD19+), BCMA only (BCMA+CD19-), CD 19 only (BCMA-CD19+) and neither (BCMA-CD19-). Area under the curve (AUC) for tumor growth is shown.

[0029] FIGs. 10A-10D are a graph and a tabular presentation of the in vivo efficacy of BCMA / CD19 dual CAR-T cells with different ICD combinations assessed using RPMI- 8226 CD19+ subcutaneous xenograft model in NOD.CgPrkdcscldI12rgtmlwj1 / SzJ (NSG) immunodeficient mice.

[0030] FIGs. 11A and 11B are a pair of graphs showing in vivo efficacy of BCMA / CD19 dual CAR-T cells with 4-1BB / 4-1BB or 4-1BB / TACI ICD combination were assessed using RPMI-8226 CD 19+ (A) or Pfeiffer (B) subcutaneous xenograft model in NOD.CgPrkdcscldI12rgtmlwj1 / SzJ (NSG) immunodeficient mice.Attorney Docket No.: 000218-0154-WO1

[0031] FIGs. 12A and 12B are a pair of schematic diagrams depicting a piggyBac transposon cassette comprising the nucleic acid sequences encoding a dual CAR having a BCMA recognition region and CD19 recognition region. FIG. 12A is a schematic map of the BCMA and CD 19 CARs in the context of a candidate tandem anti -BCMA anti-CD19 CAR-T transposon. FIG. 12B is a schematic map of a candidate tandem anti-BCMA anti- CD19 CAR-Transposon in the nanoplasmid backbone (non-integrating region). As shown in FIG. 12, the transposon cassette comprises a left end ITR, an EFla promoter operably associated with the nucleic acids encoding: an iCas9 safety switch, a CAR-Targeting BCMA, a CAR-Targeting CD 19, a DHFR mutein selectable marker, a poly A sequence; and a right end ITR.

[0032] FIGS. 13A-13D are graphs showing the in vitro killing of dual and single target antigen-positive tumor cell lines by a candidate tandem anti-BCMA anti-CD19 CAR-T. Cytolytic activity of the candidate tandem anti-BCMA anti-CD19 CAR-T cells against GFP-positive tumor cell lines expressing target antigens was measured using Incucyte after co-culture at different E:T ratios for 72 hours: 7(A) RPMI-8226 double positive (DP) cells, 7 (B) RPMI-8226 BCMA single positive (SP), 7(C) RPMI-8226 CD 19 SP, 7(D) RPMI-8226 double negative (DN) cells. Non-transposed donor-matched mock T cells were used as controls.

[0033] FIGs. 14A-14D are graphs showing the in vitro killing activity of tandem anti- BCMA anti-CD19 CAR-T, anti-BCMA CAR-T and anti-CD19 CAR-T. Cytolytic activity of the tandem anti-BCMA anti-CD19 CAR-T, anti-BCMA CAR-T, and antiCD 19 CAR-T cells manufactured from donor C against GFP-positive tumor cell lines expressing target antigens was measured using Incucyte after co-culture at different E:T ratios for 72 hours: A) RPMI-8226 double positive (DP) cells, B) RPMI-8226 BCMA single positive (SP), C) RPMI-8226 CD 19 SP, D) RPMI-8226 double negative (DN) cells. Non-transposed, donor-matched mock T cells were used as controls.

[0034] FIG. 15 is a series of graphs showing tumor burden reduction with tandem anti- BCMA anti-CD19 CAR-T cells. Tumor burden was measured by calipers in mice bearing RPMI-8226 double positive (DP) tumors after infusion with 3 dose levels of the tandem anti-BCMA anti-CD19 CAR-T cell research lots manufactured from Donor A, Donor B, or Donor C. Mice in PBS control group received no CAR-T cells. Data points represent the mean and error bars denote standard error of the mean (SEM). N = 5 mice / group for all groups except for donor A which had N = 4 mice / group at 1.2 * 106and 2.4 * 106non-apoptotic CAR-T cells / mouse.Attorney Docket No.: 000218-0154-WO1

[0035] FIG. 16 is a series of graphs showing a candidate tandem anti-BCMA anti-CD19 CAR-T expansion kinetics in peripheral blood. Total T cells in blood was measured at study time points by flow cytometric TruCount staining for human CD45-positive cells per pL blood for all animals in the studies. Data points represent the mean and error bars denote standard error of the mean (SEM). N= 5 mice per group.

[0036] FIG. 17 is a series of charts showing the phenotype of circulating cells after administration of tandem anti-BCMA anti-CD19 CAR-T cells in the RPMI-8226 DP xenograft model. Frequency of CD8-positive T cell memory subsets in blood of the animals treated with 1.2 x 106(donor A) or 2.4 x 106(donors B and C) non-apoptotic CAR-T (NAC) cells, assessed by flow cytometry. Error bars represent standard error of the mean (SEM). Samples from animals with fewer than 100 hCD45-positive cells / pL blood were pooled prior to evaluation of T cell subsets. TSCM: CD45RA-positive / CD62L- positive, TCM: CD45RO-positive / CD62L-positive, TEM: CD45RO-positive / CD62L- negative, TEFF: CD45RA-positive / CD62L-negative.

[0037] FIGs. 18A-18C shows the in vivo efficacy of a tandem anti-BCMA anti-CD19 CAR-T in Pfeiffer Xenograft Model. FIG. 18A is a graph showing tumor burden measurements by bioluminescent imaging (BLI) of mice bearing Pfeiffer tumors and infused with 3 dose levels of tandem anti-BCMA anti-CD19 dual CAR-T cells manufactured from Donor A. Mice in PBS control group received no CAR-T cells. FIG. 18B is a graph showing total T cells in blood measured at study time points by flow cytometric TruCount staining for human CD45-positive cells per pL blood for all animals in the studies. FIG. 18C is a chart showing frequency of CD8-positive T cell memory subsets in blood of the animals treated with 2.4 x 106non-apoptotic tandem anti-BCMA anti-CD19 CAR-T cells (NAC), assessed by flow cytometry. Samples from animals with fewer than 100 hCD45-positive cells / pL blood were pooled prior to evaluation of T cell memory subsets. TSCM: CD45RA-positive / CD62L-positive, TCM: CD45RO- positive / CD62L-positive, TEM: CD45RO-positive / CD62L-negative, TEFF: CD45RA- positive / CD62L-negative. Data points represent the mean and error bars denote SEM. N= 5 mice per group.

[0038] FIG. 19 is a series of graphs showing reactivity (degranulation) BCMA CAR-T cells using T202.523, VH523 and VH202 BCMA binders to BCMA WT and four known BCMA escape mutants (R27P, P33Del, P33S, P30Del). Significant degranulation was seen for VH523 RNA CAR-T against all escape mutants (WT, R27P, P33Del, P33S and S30Del), however degranulation against R27P was -20-40% reduced vs WT. SimilarAttorney Docket No.: 000218-0154-WO1 results were observed for VH202. Tandem (T202.523) RNA CAR-T showed equivalent degranulation against all (WT, R27P, P33Del, P33S and S30Del) and no reduction in degranulation against R27P.

[0039] FIG. 20A-20C are a series of figures showing binding of T202.523, VH523 and VH202 BCMA binders to BCMA WT and four known BCMA escape mutants (R27P, P33Del, P33S, S30Del). Percent bound is shown in FIG. 20A, EC50 is shown in FIG. 20B and representative flow analysis is shown in FIG. 20C. Significant binding is seen for VH523-Fc to all BCMA proteins (WT, R27P, P33Del, P33S and S30Del), however binding of R27P was -50% reduced vs WT. Similar results were observed for VH202. The tandem fusion protein (T202.523) had equivalent binding to all BCMA proteins (WT, R27P, P33Del, P33S and S30Del) and no reduction in binding to R27P was observed.

[0040] FIG. 21 is a series of graphs showing cytotoxicity of a single BCMA (VH523) and tandem BCMA (T202.523) sPB-generated CAR-T cells against K562 cells transfected with BCMA WT mRNA and four escape mutants (WT, R27P, P33Del, P33S and S30Del).

[0041] FIG. 22 is a series of graphs showing B cell specific killing by tandem anti- BCMA anti-CD19 CAR-T cells in Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), and Multiple Sclerosis (MS) patient samples.

[0042] FIGs. 23A-23E show elimination of CD81+CD19+ progenitor cells and colonyforming units from bone marrow MM patient samples by P-BCMACD19-ALLO1. FIG. 23A shows a distinct CD81+CD19+ subset indicative of a less differentiated population identified in three representative MM bone marrow primary samples. FIG. 23B shows that CD81+CD19+ subset has no to very negligible BCMA expression. FIG. 23C shows a representative flow plot of surviving CD81+CD19+ cells for anti-BCMA CAR-T, anti- CD19 CAR-T and P-BCMACD19-ALLO1, illustrating the cytolytic activity of P- BCMACD19-ALLO1 cells against primary CD81+CD19+ cells. FIG. 23D shows the quantification of CD81+CD19+ cell killing across 4 patient samples. FIG. 23E shows the percent killing of colony-forming units (CFUs) across 4 patient samples.

[0043] All documents cited herein, including any cross referenced or related patent or application are hereby incorporated herein by reference in its entirety for all purposes, unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches,Attorney Docket No.: 000218-0154-WO1 suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.DETAILED DESCRIPTIONChimeric Antigen ReceptorsIn one aspect, provided herein are chimeric antigen receptors (CARs) and cells comprising the same. In certain embodiments of the disclosure, the chimeric antigen receptor comprises (a) an ectodomain comprising a ligand recognition region; (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain.

[0044] In some aspects, a CAR disclosed herein comprises: (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. As used throughout the disclosure, a CAR comprising a VH as the antigen recognition is referred to as a VCAR.

[0045] In some aspects, a CAR disclosed herein comprises a transmembrane domain. In certain embodiments, the transmembrane domain may comprise a sequence encoding a human CD2, CD35, CD3s, CD3y, CD3< CD4, CD8a, CD19, CD28, 4-1BB or GM- CSFR transmembrane domain. In certain embodiments, the transmembrane domain may comprise a sequence encoding a human CD8a transmembrane domain. The CD8a transmembrane domain may comprise the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 3) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 3). The CD8a transmembrane domain may be encoded by the nucleic acid comprising the sequence atctacatttgggcaccactggccgggacctgtggagtgctgctgctgagcctggtcatcacactgtactgc (SEQ ID NO: 4).

[0046] In some embodiments, a CAR provided herein comprises an endodomain comprising one or more co-stimulation domains. In certain embodiments, the endodomain may comprise a human CD3(^ endodomain.Attorney Docket No.: 000218-0154-WO1

[0047] The CD3(^ costimulatory domain may comprise the amino acid sequence: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR (SEQ ID NO: 5) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR (SEQ ID NO: 5).

[0048] The CD3(^ costimulatory domain may be encoded by a nucleic acid comprising the sequence: cgcgtgaagtttagtcgatcagcagatgccccagcttacaaacagggacagaaccagctgtataacgagctgaatctgggccg ccgagaggaatatgacgtgctggataagcggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaaccctc aggaaggcctgtataacgagctgcagaaggacaaaatggcagaagcctattctgagatcggcatgaagggggagcgacgga gaggcaaagggcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgcaggcact gcctccaagg (SEQ ID NO: 6).

[0049] In certain embodiments, the at least one costimulatory domain may comprise a human 4-1BB intracellular domain, a TACI intracellular domain, a CD28 intracellular domain, a CD40 intracellular domain, an ICOS intracellular domain, a MyD88 intracellular domain, or an OX-40 intracellular domain, or any combination thereof. In certain embodiments, the endodomain comprises a CD28 co-stimulatory domain and a 4- 1BB costimulatory domain. In certain embodiments, the endodomain comprises a CD3(^ co-stimulatory domain and a 4-1BB costimulatory domain. In certain embodiments, the endodomain comprises a CD3(^ co-stimulatory domain and a TACI costimulatory domain.

[0050] The 4-1BB costimulatory domain may comprise the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 7) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 7). The 4-1BB costimulatory domain may be encoded by a nucleic acid comprising the sequence

[0051] aagagaggcaggaagaaactgctgtatattttcaaacagcccttcatgcgccccgtgcagactacccaggaggaaga cgggtgctcctgtcgattccctgaggaagaggaaggcgggtgtgagctg (SEQ ID NO: 8). The 4-1BB costimulatory domain may be located between the transmembrane domain and the CD28 costimulatory domain. The CD28 costimulatory domain comprises an amino acidAttorney Docket No.: 000218-0154-WO1 sequence comprising:RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 11).

[0052] In certain embodiments, the CD28 costimulatory domain is encoded by a nucleic acid sequence comprising: agaagcaagcggagccggctgctgcacagcgactacatgaacatgacccctagacggcccggacctaccagaaagcactac cagccttacgctcctcctagagacttcgccgcctaccggtcc (SEQ ID NO: 12).

[0053] The TACI costimulatory domain may comprise the amino acid sequence KKRGDPC SCQPRSRPRQSPAKS SQDHAMEAGSP VSTSPEPVETC SFCFPECRAPTQ ESAVTPGTPDPTCAGRWGCHTRTTVLQPCPHIPDSGLGIVCVPAQEGGPGA (SEQ ID NO: 13) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequenceKKRGDPC SCQPRSRPRQSPAKS SQDHAMEAGSP VSTSPEPVETC SFCFPECRAPTQ ESAVTPGTPDPTCAGRWGCHTRTTVLQPCPHIPDSGLGIVCVPAQEGGPGA (SEQ ID NO: 13).

[0054] In certain embodiments, the TACI costimulatory domain is encoded by a nucleic acid comprising the sequence aaaaagcggggcgatccttgctcctgccagccaagatcaagacccagacagtctcctgccaagtcctctcaggaccacgccat ggaagctggcagccctgtgtctacatctcccgagccagtggaaacatgctccttttgtttccctgagtgtcgggccccaactcaag aaagcgcagtgacaccaggcactccagatccaacctgcgctggccgatggggatgccatacaagaacaaccgtgctccaacc atgtccacacattcccgatagcggactgggaattgtgtgtgtccccgcacaagaaggtggcccaggggcc (SEQ ID NO:14). The TACI costimulatory domain may be located between the transmembrane domain and the CD28 costimulatory domain.

[0055] In some aspects, a CAR disclosed herein comprises a hinge region between the transmembrane domain and the ectodomain. In certain embodiments, the hinge region 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 region may comprise a sequence derived from a human CD8a sequence. The hinge region may comprise the amino acid sequenceTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 9) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 9). The human CD8a hinge region may be encoded by a nucleic acid comprising the sequenceAttorney Docket No.: 000218-0154-WO1 actaccacaccagcacctagaccaccaactccagctccaaccatcgcgagtcagcccctgagtctgagacctgaggcctgcag gccagctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 10).

[0056] In some aspects, a CAR provided herein further comprises a signal peptide. In certain embodiments, the signal peptide may comprise a sequence encoding a human CD2 signal peptide, a human CD35 signal peptide, a human CD3s signal peptide, a human CD3y signal peptide, a human CD3(^ signal peptide, a human CD4 signal peptide, a human CD8a signal peptide, a human CD 19 signal peptide, a human CD28 signal peptide, a human 4-1BB signal peptide or a human GM-CSFR signal peptide. In certain embodiments of the VCARs of the disclosure, the signal peptide may comprise a human CD8a signal peptide. The human CD8a signal peptide may comprise the amino acid sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 1) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 1). The human CD8a signal peptide may be encoded by a nucleic acid comprising the sequence atggcactgccagtcaccgccctgctgctgcctctggctctgctgctgcacgcagctagacca (SEQ ID NO: 2).

[0057] VCARs of the disclosure may bind an antigen an affinity of 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 10'13M, less than or equal to 10'14M, and less than or equal to 10'15M. The KD may be determined by surface plasmon resonance.

[0058] In certain embodiments, a CAR as disclosed comprises an ectodomain comprising an antigen recognition region that binds to BCMA. In certain embodiments, a CAR as disclosed comprises an ectodomain comprising an antigen recognition region that binds to CD 19. In certain embodiments, a CAR as disclosed comprises an ectodomain comprising an antigen recognition region that binds to BCMA and an antigen recognition region that binds to CD 19.

[0059] In certain embodiments of the VCARs of the disclosure, the VCAR comprises a single domain antibody, a VHH (also known as a nanobody), a VH, or a combination thereof. In some embodiments, the single domain antibody, VHH or VH comprises or consists of a recombinant sequence. In some embodiments, the single domain antibody, VHH or VH comprises or consists of a chimeric sequence. In some embodiments, the single domain antibody, VHH or VH comprises or consists of a human sequence. In some embodiments, the single domain antibody, VHH or VH comprises or consists of a humanized sequence.Attorney Docket No.: 000218-0154-WO1

[0060] In certain embodiments, an antigen recognition region may comprise two VHs to produce a bi-specific or tandem VCAR. The two VHs present in a tandem VCAR may bind to the same antigen or to different antigens. In some embodiments, the two VHs present in a tandem VCAR disclosed herein comprise two VHs with different sequences, both of which bind to BCMA.

[0061] In certain embodiments, the antigen recognition region may comprise three VHs to produce a tri-specific VCAR. The three VHs present in a tri-specific VCAR may bind to the same antigen or to different antigens. In some embodiments, the three VHs present in a tri-specific VCAR disclosed herein comprise two VHs with different sequences, both of which bind to BCMA and a third VH which binds to an antigen other than BCMA. In certain embodiments, the third VH binds CD 19.

[0062] In certain embodiments of the VCARs of the disclosure, the VCAR comprises a single domain antibody. In some embodiments, the single domain antibody is a VHH or a VH antibody. In some embodiments, the VH antibody is a UniDab antibody. In some embodiments, VH antibody is not a fragment of a naturally occurring monoclonal antibody.In certain embodiments, the VH comprises a human or a humanized sequence. In certain embodiments, the VH comprises a non-naturally occurring sequence. In certain embodiments, the VH is not naturally occurring. In certain embodiments, the VH comprises a recombinant or chimeric sequence. In certain embodiments, the VH is produced by an in vitro procedure of affinity selection and recombination.BCMA Sequences

[0063] In some aspects, CAR disclosed herein is a VCAR comprising VH sequence that binds to BCMA. In certain embodiments, the VCAR comprises a VH that binds to BCMA comprising or consisting of the amino acid sequence: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVAGIIGS GGSTYYADSVKGRFSISRDNSKNTLDLQMNSLRAEDTAVYYCVKDWNTTMITE RGQGTLVTVSS (VH202, SEQ ID NO: 48).

[0064] In some embodiments, the VH that binds to BCMA is encoded by a polynucleotide comprising or consisting of the nucleic acid sequence gaagtgcaactgctggaaagtggcggcggacttgttcagccaggcggaagccttagactgtcttgtgccgcctctggctttacct tctcctcctatgccatgaattgggttcgacaagcacccggcaagggactcgaatgggtggccggaattatcggctctggcggcaAttorney Docket No.: 000218-0154-WO1 gcacctactatgccgacagcgtgaaaggacggttcagcatctcccgggacaactccaagaatactctggatctccagatgaaca gcctgcgggccgaagatacagccgtgtattattgcgtgaaggactggaacaccaccatgatcaccgagagaggacagggaac cctcgtgaccgtgtcctcc (VH202; SEQ ID NO: 67).

[0065] In certain embodiments, the VCAR comprises a VH that binds to BCMA comprising or consisting of the amino acid sequenceEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMTWIRQAPGKGLEWVSGITGD GGSTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKDWNTTMITE RGQGTLVTVSS (VH523, SEQ ID NO: 49).

[0066] In some embodiments, the VH that binds to BCMA is encoded by a polynucleotide comprising or consisting of the nucleic acid sequence gaagtgcaactgctggaaagtggcggcggacttgttcagccaggcggaagccttagactgtcttgtgccgcctctggctttacct tctccaactatgccatgacctggatcaggcaggcacccggaaaaggattggagtgggtgtccggaattacaggcgacggcgg cagcaccttttacgccgatagtgtgaaaggccggtttaccatcagccgggataactccaagaatacgctgtatctccaaatgaac agcctgcgcgccgaagatacagccgtgtattattgcgtgaaggactggaacaccaccatgatcaccgagagaggacagggaa ccctcgtgaccgtgtcctcc (VH523 SEQ ID NO: 68).

[0067] In certain embodiments, the VH is a tandem VH that binds to BCMA. In certain embodiments, the tandem VH that binds to BCMA is a VH202.523 sequence, i.e., a sequence comprising, in N-terminal to C-terminal direction, the VH202 and the VH523 sequences. In certain embodiments, the tandem VH that binds to BCMA has a linker connecting two VH sequences in tandem. In certain embodiments, the linker comprises or consists of the amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 50) In certain embodiments, the tandem VH that binds to BCMA comprises or consists of the amino acid sequence:

[0068] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVA GIIGSGGSTYYADSVKGRFSISRDNSKNTLDLQMNSLRAEDTAVYYCVKDWNTT MITERGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAA SGFTFSNYAMTWIRQAPGKGLEWVSGITGDGGSTFYADSVKGRFTISRDNSKNT LYLQMNSLRAEDTAVYYCVKDWNTTMITERGQGTLVTVSS (VH202.523, SEQ ID NO: 51).

[0069] In some embodiments, the tandem VH that binds to BCMA is encoded by a polynucleotide comprising or consisting of the nucleic acid sequence gaggtgcagctgttggaatctggcggtggactggttcagcctggcggatctctgagactgtcttgtgccgccagcggcttcacct ttagcagctacgccatgaactgggtccgacaggcccctggcaaaggactggaatgggtggccggaatcatcggctcaggcgg cagcacatattacgccgactctgtgaagggccgcttcagcatcagcagagacaacagcaagaacaccctggacctgcagatgaAttorney Docket No.: 000218-0154-WO1 actccctgagagccgaggacacagccgtgtactactgcgtgaaggattggaacaccaccatgatcaccgagagaggacaggg caccctcgtgacagtttcttctggcggaggcggcagcggaggcggaggttcaggtggtggtggatctgaagtccagctgctgg aaagtggtggcggacttgtgcaacctggtggctcactgagactgagctgtgccgcttccggctttaccttctccaactatgccatg acctggatcaggcaggctccaggcaagggacttgagtgggtttccggcattacaggcgacggcggaagcaccttctatgccga cagtgtgaaaggccggtttaccatcagccgggataactccaagaatacgctgtatctccagatgaacagcctccgcgccgaaga taccgctgtgtattattgtgtgaaagactggaatacgacgatgattacggaacgcggccagggaaccctggtcaccgttagctct (VH202.523, SEQ ID NO: 52).

[0070] In certain embodiments, the VH that binds to BCMA is a tandem VH523.202 sequence (i.e., a sequence comprising, in N-terminal to C-terminal order, the VH523 and the VH202 sequences). In certain embodiments, the tandem VH that binds to BCMA has a linker connecting two VH sequences in tandem. In certain embodiments, the linker comprises or consists of the amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 50) In certain embodiments, the tandem VH that binds to BCMA comprises or consists of the amino acid sequence

[0071] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMTWIRQAPGKGLEWVSG ITGDGGSTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKDWNTT MITERGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAA SGFTFSSYAMNWVRQAPGKGLEWVAGIIGSGGSTYYADSVKGRFSISRDNSKNT LDLQMNSLRAEDTAVYYCVKDWNTTMITERGQGTLVTVSS (VH 523.202, SEQ ID NO: 55)

[0072] In some embodiments, the tandem VH that binds to BCMA is encoded by a polynucleotide comprising or consisting of the nucleic acid sequence gaagttcagctgttggaatctggcggtggcctggttcaacctggcggatctctgagactgagctgtgccgccagcggcttcacct tcagcaattacgccatgacctggatcagacaggcccctggcaaaggcctggaatgggtgtccggaattacaggcgacggcgg cagcaccttttacgccgattctgtgaagggcagattcaccatcagcagggacaacagcaagaacaccctgtacctgcagatgaa ctccctgagagccgaggacaccgccgtgtactactgcgtgaaggactggaacaccaccatgatcaccgagagaggccaggg cacactggtcacagtttctagcggaggcggaggtagtggtggcggaggttcaggtggcggtggatctgaagtgcaactgctgg aaagtggcggcggacttgttcagccaggcggaagccttagactgtcttgtgccgcctctggctttaccttctcctcctatgccatga attgggttcgacaggctcccggcaagggacttgagtgggtggcaggcattatcggctctggcggctctacctactacgccgaca gtgtgaaaggccggttcagcatctcccgggacaactctaagaataccctggatctccaaatgaacagcctgcgcgccgaagata cagccgtgtattattgtgtcaaggattggaatacgacgatgattacggaacgcggccagggaaccctcgtgaccgttagctct (CH523.202, SEQ ID NO: 69).

[0073] In certain embodiments, the VCAR comprises an ectodomain comprising a VH that binds to BCMA, a signal peptide, a hinge region, a transmembrane domain, and anAttorney Docket No.: 000218-0154-WO1 endodomain comprising two costimulatory domains. In certain embodiments of the VCARs of the disclosure, the VCAR comprises a CD8a signal peptide, a tandem VH VH202.523 that binds to BCMA antigen binding region, a CD8a Hinge, a CD8a transmembrane, a 41BB costimulatory domain, and a CD3(^ costimulatory domain. In some embodiments, a VCAR comprises or consists of the sequenceMALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYA MNWVRQAPGKGLEWVAGIIGSGGSTYYADSVKGRFSISRDNSKNTLDLQMNSL RAEDTAVYYCVKDWNTTMITERGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLE SGGGLVQPGGSLRLSCAASGFTFSNYAMTWIRQAPGKGLEWVSGITGDGGSTFY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKDWNTTMITERGQGTL VTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAP LAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR ( SEQ ID NO: 53).

[0074] In some embodiments, the VCAR is encoded by a polynucleotide comprising or consisting of the nucleic acid sequence atggctctgcctgtgacagctctgcttctgcctctggcactgcttcttcatgcggcgcgccctgaagttcagctgttggaatctggc ggtggcctggttcaacctggcggatctctgagactgagctgtgccgccagcggcttcacctttagcagctacgccatgaactgg gtccgacaggcccctggcaaaggactggaatgggtggccggaatcatcggctcaggcggcagcacatattacgccgactctg tgaagggccgcttcagcatcagcagagacaacagcaagaacaccctggacctgcagatgaactccctgagagccgaggatac cgccgtgtactactgcgtgaaggattggaacaccaccatgatcaccgagagaggccagggcacactggtcacagtttctagcg gaggcggaggtagtggtggcggaggttcaggtggcggtggatctgaagtgcaactgctggaaagtggcggcggacttgttca gccaggcggaagccttagactgtcttgtgccgcctctggctttaccttctccaactatgccatgacctggatcaggcaggctccag gcaagggacttgagtgggtttccggcattacaggcgacggcggctctaccttttacgccgatagcgtgaagggcagattcacca tctctcgggacaactccaagaataccctgtatctccaaatgaacagcctgcgcgccgaggacacagcagtgtattattgtgtgaa agactggaatacgacgatgattacggaacgcggccagggaaccctcgtgaccgttagctctacaacaacaccggcgcctcgg cctccaacaccagctcctacaattgctagccagccactgtctctgaggcccgaggcttgtagacctgctgctggcggagctgtgc acacaagaggactggatttcgcctgcgacatctatatctgggcccctctggctggcacatgtggcgttctgctgctcagcctggtc atcaccctgtactgcaagcggggcagaaagaagctgctgtacatctttaagcagcccttcatgaggcccgtgcagaccacacaa gaagaggacggctgctcctgccgcttccccgaggaagaagaaggcggttgcgaactgagagtgaagttctccagaagcgcc gacgcacccgcctataagcagggacagaaccagctgtacaacgagctgaacctggggagaagagaagagtacgacgtgctg gacaagcggagaggcagagatcctgagatgggcggcaagcccagacggaagaatcctcaagagggcctgtataatgagctg cagaaagacaagatggccgaggcctacagcgagatcggaatgaagggcgagcgcagaagaggcaagggacacgatggacAttorney Docket No.: 000218-0154-WO1 tgtaccagggcctgagcaccgccaccaaggatacctatgatgccctgcacatgcaggccctgcctccaaga (SEQ ID NO: 54).

[0075] The VCARs provided herein are useful in targeting both wildtype and mutant forms of BCMA. Recent studies have identified several key BCMA mutations, including R27P, P33Del, P33S, and P30Del, that have been implicated in the pathogenesis of relapsed disease. These mutations can affect the binding affinity of CAR-T therapies and monoclonal antibodies, potentially leading to decreased therapeutic effectiveness. The R27P mutation is particularly notable as it results in a substitution of a proline for arginine at position 27, which may alter the protein's structural stability and its interaction with therapeutic agents. Similarly, the P33Del mutation, characterized by a deletion of proline at position 33, can disrupt the normal function of BCMA, affecting its role in cell signaling pathways crucial for plasma cell survival. The P33S mutation introduces a serine at position 33, which may also impact BCMA's interaction with ligands and therapeutic agents. Lastly, the P30Del mutation, which involves the deletion of proline at position 30, poses similar challenges, potentially affecting both the protein's function and the immune response it elicits.

[0076] Moreover, the presence of BCMA mutations such as R27P, P33Del, P33S, and P30Del poses challenges to the success of CAR-T treatments. For instance, mutations can affect the CAR-T cells’ ability to recognize and bind to the mutated BCMA, potentially leading to antigen escape. Studies have demonstrated that BCMA mutations can lead to reduced surface expression of the antigen, thereby diminishing the efficacy of CAR-T cells designed to target BCMA.

[0077] Advantageously, the VCARs described herein bind to mutant forms of BCMA and can therefore be used to target wildtype as well as mutant BCMA. In some embodiments, a VCAR described herein binds to a BCMA R27P mutant and wildtype BCMA with comparable affinity. In some embodiments, a VCAR described herein binds to a BCMA P33Del mutant and wildtype BCMA with comparable affinity. In some embodiments, a VCAR described herein binds to a BCMA P33S mutant and wildtype BCMA with comparable affinity. In some embodiments, a VCAR described herein binds to a BCMA P30del mutant and wildtype BCMA with comparable affinity. “Comparable affinity” in this context means that the binding affinity of the VCAR for the mutant form of BCMA is within 10% of the binding affinity of the VCAR for wildtype BCMA.Attorney Docket No.: 000218-0154-WO1CD 19 Sequences

[0078] In some aspects, a VCAR disclosed herein comprises an ectodomain comprising a VH that binds to CD 19. In certain embodiments, the VH that binds to CD 19 comprises the amino acid sequenceQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYMSS SGSTIYYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCARGGIAAAGTW GQGTLVTVSS (VH034, SEQ ID NO: 56).

[0079] In some embodiments, the VH that binds to CD 19 is encoded by a polynucleotide comprising or consisting of the nucleic acid sequence caagtgcagctggtggaatctggcggcggacttgtgaaacctggcggctctctgagactgagctgtgccgcttccggcttcacct tctccgactactacatgagctggatcagacaggcccctggcaaaggcctggaatgggtgtcctatatgagcagcgacggctcca ccatctactacgccgatagcgtgaaaggccggttcaccatctccagagacaacgccaagaagtccctgtacctgcagatgaact ccctgagagccgaggacaccgccgtgtactattgtgctagaggcggaattgccgccgctggcacatggggacagggaacact ggttaccgtgtccagc (VH034, SEQ ID NO: 70).

[0080] In certain embodiments, the VH that binds to CD 19 comprises an amino acid sequence further comprising a T28D mutation relative to the sequence set forth in SEQ ID NO: 56 (a VH034 T28D). In some embodiments, the VH that binds to CD19 comprises the amino acid sequence

[0081] QVQLVESGGGLVKPGGSLRLSCAASGFDFSDYYMSWIRQAPGKGLEWVS YMSSSGSTIYYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCARGGIAA AGTWGQGTLVTVSS (VH034 T28D, SEQ ID NO: 57).

[0082] In some embodiments, the VH that binds to CD 19 is encoded by a polynucleotide comprising or consisting of the nucleic acid sequence caagtgcagctggtggaatctggcggcggacttgtgaaacctggcggctctctgagactgagctgtgccgcttccggcttcgac ttctccgactactacatgagctggatcagacaggcccctggcaaaggcctggaatgggtgtcctatatgagcagcagcggctcc accatctactacgccgatagcgtgaaaggccggttcaccatctccagagacaacgccaagaagtccctgtacctgcagatgaac tccctgagagccgaggacaccgccgtgtactattgtgctagaggcggaattgccgccgctggcacatggggacagggaacac tggttaccgtgtccagc

[0083] (VH034 T28D, SEQ ID NO: 71).

[0084] In certain embodiments, the VH that binds to CD 19 comprises an amino acid sequence further comprising a S54D mutation relative to SEQ ID NO: 56 (a VH034 S54D). In some embodiments, the anti- VH that binds to CD19 comprises the amino acid sequenceAttorney Docket No.: 000218-0154-WO1QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYMSS DGSTIYYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCARGGIAAAGT WGQGTLVTVSS (VH034 S54D, SEQ ID NO: 58).

[0085] In some embodiments, the VH that binds to CD 19 is encoded by a polynucleotide comprising or consisting of the nucleic acid sequence caagtgcagctggtggaatctggcggcggacttgtgaaacctggcggctctctgagactgagctgtgccgcttccggcttcacct tctccgactactacatgagctggatcagacaggcccctggcaaaggcctggaatgggtgtcctatatgagcagcgacggctcca ccatctactacgccgatagcgtgaaaggccggttcaccatctccagagacaacgccaagaagtccctgtacctgcagatgaact ccctgagagccgaggacaccgccgtgtactattgtgctagaggcggaattgccgccgctggcacatggggacagggaacact ggttaccgtgtccagcaccaca

[0086] (VH034 S54D, SEQ ID NO: 72).

[0087] In certain embodiments, the VH that binds to CD 19 comprises an amino acid sequence further comprising a A103S mutation relative to SEQ ID NO: 56 (a VH034 A103S). In some embodiments, the VH that binds to CD 19 comprises the amino acid sequenceQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYMSS SGSTIYYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCARGGIASAGTW GQGTLVTVSS (VH034 A103S, SEQ ID NO: 59).

[0088] In some embodiments, the VH that binds to CD 19 is encoded by a polynucleotide comprising or consisting of the nucleic acid sequence caagtgcagctggtggaatctggcggcggacttgtgaaacctggcggctctctgagactgagctgtgccgcttccggcttcacct tctccgactactacatgagctggatcagacaggcccctggcaaaggcctggaatgggtgtcctatatgagcagcagcggctcca ccatctactacgccgatagcgtgaaaggccggttcaccatctccagagacaacgccaagaagtccctgtacctgcagatgaact ccctgagagccgaggacaccgccgtgtactattgtgctagaggcggaattgccagcgctggcacatggggacagggaacact ggttaccgtgtccagc (VH034 A103S, SEQ ID NO: 73).

[0089] In certain embodiments, the VH that binds to CD 19 comprises an amino acid sequence further comprising a A104G mutation relative to SEQ ID NO: 56 (a VH034 A104G). In some embodiments, the VH that binds to CD 19 comprises the amino acid sequenceQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYMSS SGSTIYYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCARGGIAAGGTW GQGTLVTVSS (VH034 A104, SEQ ID NO: 60).

[0090] In some embodiments, the VH that binds to CD 19 is encoded by a polynucleotide comprising or consisting of the nucleic acid sequenceAttorney Docket No.: 000218-0154-WO1 caagtgcagctggtggaatctggcggcggacttgtgaaacctggcggctctctgagactgagctgtgccgcttccggcttcacct tctccgactactacatgagctggatcagacaggcccctggcaaaggcctggaatgggtgtcctatatgagcagcagcggctcca ccatctactacgccgatagcgtgaaaggccggttcaccatctccagagacaacgccaagaagtccctgtacctgcagatgaact ccctgagagccgaggacaccgccgtgtactattgtgctagaggcggaattgccgccggcggcacatggggacagggaacac tggttaccgtgtccagc (VH034 A104G, SEQ ID NO: 74).

[0091] In certain embodiments, a VCAR comprises an ectodomain comprising a VH that binds to CD 19 signal peptide, a hinge region, a transmembrane domain, and an endodomain comprising two costimulatory domains. In certain embodiments, the VCAR comprises a CD8a signal peptide, a CD 19 VH034 T28D, a CD8a Hinge, a CD8a transmembrane domain, a TACI costimulatory domain, and a CD3^ costimulatory domain. In some embodiments, a VCAR of the disclosure comprises the amino acid sequenceMALPVTALLLPLALLLHAARPQVQLVESGGGLVKPGGSLRLSCAASGFDFSDYY MSWIRQAPGKGLEWVSYMSSSGSTIYYADSVKGRFTISRDNAKKSLYLQMNSLR AEDTAVYYCARGGIAAAGTWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPE ACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKKRGDPCSCQ PRSRPRQSPAKSSQDHAMEAGSPVSTSPEPVETCSFCFPECRAPTQESAVTPGTPD PTCAGRWGCHTRTTVLQPCPHIPDSGLGIVCVPAQEGGPGARVKFSRSADAPAY KQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 61).

[0092] In some embodiments, the VCAR is encoded by a polynucleotide comprising or consisting of the nucleic acid sequence atggcacttccggtgaccgctttgctgctgcctcttgcactgctgctgcatgccgctagacctcaagtgcagctggtggaatctgg cggcggacttgtgaaacctggcggctctctgagactgagctgtgccgcttccggcttcgacttctccgactactacatgagctgg atcagacaggcccctggcaaaggcctggaatgggtgtcctatatgagcagcagcggctccaccatctactacgccgatagcgt gaaaggccggttcaccatctccagagacaacgccaagaagtccctgtacctgcagatgaactccctgagagccgaggacacc gccgtgtactattgtgctagaggcggaattgccgccgctggcacatggggacagggaacactggttaccgtgtccagcacaac aacaccggcgcctcggccacctacaccagcaccaactatcgcaagccaacctctcagcctgcggccagaagcctgtagaccc gcagcaggcggtgccgttcatacaagaggcctcgactttgcctgtgatatctacatttgggcacccctggccggaacctgcgga gttcttctgctgtccctcgtgatcaccctgtattgcaaaagaggccggaagaaactcctgtatatcttcaaacagccgtttatgaggc cggtccagacaactcaagaggaagatggttgcagctgtcggttccctgaagaggaagaaggcggctgcgagctgcgcgtgaa attctccagatctgccgacgctccagcttacaaacagggccagaatcagctctataatgaactgaacctcggcaggcgcgagga atatgatgtgctggataagcggaggggcagagatcccgagatgggaggaaaacccagacggaaaaatccccaagaaggcctAttorney Docket No.: 000218-0154-WO1 ttacaacgaactgcaaaaggataagatggctgaagcttactccgagattggcatgaagggcgaacgtcggagaggaaaagga cacgacggcctgtaccagggactgtctacagccacaaaggacacatacgacgcactccatatgcaggctctcccacctaga (SEQ ID NO: 62).Dual CARs

[0093] In another aspect, provided herein are dual CARs comprising (a) a first ectodomain comprising a first antigen recognition region that binds to a first antigen, (b) a first transmembrane domain, (c) a first endodomain comprising at least one costimulatory domain; (d) a second ectodomain comprising a second antigen recognition region that binds to a second antigen; (e) a second transmembrane domain, (f) a second endodomain comprising at least one costimulatory domain.

[0094] In some embodiments, a dual CAR comprises (a) a first ectodomain comprising a first antigen recognition region that binds to BCMA, (b) a first transmembrane domain, (c) a first endodomain comprising at least one costimulatory domain; (d) a second ectodomain comprising a second antigen recognition region that binds to an antigen other than BCMA; (e) a second transmembrane domain, (f) a second endodomain comprising at least one costimulatory domain.

[0095] In some embodiments, a dual CAR comprises (a) a first ectodomain comprising a first antigen recognition region that binds to BCMA, (b) a first transmembrane domain, (c) a first endodomain comprising at least one costimulatory domain; (d) a second ectodomain comprising a second antigen recognition region that binds to CD 19; (e) a second transmembrane domain, (f) a second endodomain comprising at least one costimulatory domain.

[0096] In some embodiments, the first ectodomain comprises any of the antigen recognition regions that bind to BCMA described herein and the second ectodomain comprises any of the antigen recognition regions that bind to CD 19 described herein.

[0097] Any of the antigen recognition regions that bind to BCMA disclosed herein may be used in the first ectodomain of the dual CARs disclosed herein. In some embodiments, the first ectodomain comprises a VH that binds to BCMA. In some embodiments, the first ectodomain comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 48 or 49. In some embodiments, the first ectodomain comprises a tandem VH that binds to BCMA. In some embodiments, the first ectodomain comprises a tandem VH comprising the sequence set forth in any one of SEQ ID NOs: 51, 53, or 55.Attorney Docket No.: 000218-0154-WO1

[0098] Any of the antigen recognition regions that bind to CD 19 disclosed herein may be used in the second ectodomain of the dual CAR disclosed herein. In some embodiments, the second ectodomain comprises a VH that binds to CD 19. In some embodiments, the second ectodomain comprises a VH comprising the amino acid sequence set forth in any one of SEQ ID NOs: 56-60.

[0099] The first transmembrane domain and the second transmembrane domain of the dual CARs disclosed herein may be the same or different. In some embodiments, the first transmembrane is a CD8a transmembrane domain. In some embodiments, the second transmembrane is a CD8a transmembrane domain. In some embodiments, the first transmembrane is a CD8a transmembrane domain and the second transmembrane is a CD8a transmembrane domain. An illustrative sequence of the CD8a transmembrane domain is set forth in SEQ ID NO: 3.

[0100] The first endodomain and the second endodomain of the dual CARs described herein may be the same or different. Thus, the first endodomain and the second endodomain may each comprise the same combination of costimulatory domains, or the first endodomain and the second endodomain may comprise different combinations of costimulatory domains.

[0101] In some embodiments, the first endodomain comprises a CD3(^ costimulatory domain. In some embodiments, the second endodomain comprises a CD3(^ costimulatory domain. In some embodiments, the first endodomain comprises a CD3(^ costimulatory domain and the second endodomain comprises a CD3(^ costimulatory domain. An illustrative sequence of the CD3(^ costimulatory domain is set forth in SEQ ID NOL 5.

[0102] In some embodiments, the first endodomain comprises a CD28 costimulatory domain. In some embodiments, the second endodomain comprises a CD28 costimulatory domain. In some embodiments, the first endodomain comprises a CD28 costimulatory domain and the second endodomain comprises a CD28 costimulatory domain. An illustrative sequence of the CD28 costimulatory domain is set forth in SEQ ID NO: 11.

[0103] In some embodiments, the first endodomain comprises a 4-1BB costimulatory domain. In some embodiments, the second endodomain comprises a 4-1BB costimulatory domain. In some embodiments, the first endodomain comprises a 4- IBB costimulatory domain and the second endodomain comprises a 4- IBB costimulatory domain. AN illustrative sequence of the 4-1BB costimulatory domain is set forth in SEQ ID NO: 8.

[0104] In some embodiments, the first endodomain comprises a TACI costimulatory domain. In some embodiments, the second endodomain comprises a TACI costimulatoryAttorney Docket No.: 000218-0154-WO1 domain. In some embodiments, the first endodomain comprises a TACI costimulatory domain and the second endodomain comprises a TACI costimulatory domain. An illustrative sequence of the TACI costimulatory domain is set forth in SEQ ID NO: 13.

[0105] In some embodiments, the first endodomain comprises a CD3(^ costimulatory domain and a 4- IBB costimulatory domain, and the second endodomain comprises a CD3(^ costimulatory domain and a TACI costimulatory domain, some embodiments, the first endodomain comprises a CD3(^ costimulatory domain and a 4- IBB costimulatory domain, and the second endodomain comprises a CD3(^ costimulatory domain and a 4- 1BB costimulatory domain.

[0106] In some aspects, the first ectodomain and / or the second ectodomain of the dual CARs described herein further comprises a signal peptide. In some embodiments, the first ectodomain comprises a CD8a signal peptide. In some embodiments, the second ectodomain comprises a CD8a signal peptide. In some embodiments, the first ectodomain comprises a CD8a signal peptide and the second ectodomain comprises a CD8a signal peptide. An illustrative sequence of the CD8a signal peptide is set forth in SEQ ID NO: 1.

[0107] In some aspects, a dual CAR disclosed herein further comprises a first hinge region between the first antigen recognition region and the first transmembrane domain. In some aspects, a dual CAR disclosed herein further comprises a second hinge region between the second antigen recognition region and the second transmembrane domain. In some embodiments, a dual CAR disclosed herein further comprises (a) a first hinge region between the first antigen recognition region and the first transmembrane domain and (b) a second hinge region between the second antigen recognition region and the second transmembrane domain.

[0108] The first and second hinge regions may be the same or they may be different. In some embodiments, the first hinge region is a CD8a hinge region. In some embodiments, the second hinge region is a CD8a hinge region. In some embodiments, the first hinge region is a CD8a hinge region and the second hinge region is a CD8a hinge region. An illustrative sequence of the CD8a hinge region is set forth in SEQ ID NO: 9.

[0109] In some embodiments, a dual CAR comprises the sequence of SEQ ID NO: 63 shown below with the CD8a signal italicized, the CD8a transmembrane domain underlined, the CD3 costimulatory domains in bold, the TACI costimulatory domain in bold-italics, the 4-1BB costimulatory domain in bold-underline, and the CD8a hinge domain shown in underlined italics. The tandem V 11 that binds to BCM AAttorney Docket No.: 000218-0154-WO1VII 202.523) is shown in double underlined bold letters, and the VH that binds toCD 19 (VHP 34 T28D) is shown in double underlined italics.GCSCRFPEEEEGGCELRVKFSRSADAPAYKQGONOLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGEGRGSLLTCGDVEENPGPAZ4ZPVTALLLPLALLLHAARPQVQLVESGGGLVKPGGSLRLSCAASGFDFSDYYMSWIRQAPGKGLEWVSYMSSSGSTIYYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCARGGIAAAGTWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD l A( D\ \\\ \\A. \G\ G\\.\.\E\.\\ \ \ KKR(iI)PCSC()PRSRPR()SPAKSS()I)IlAMEAGSPVSTSPEPVETCSFCFPECRAPTQESAVTPGTPDPTCAGRWGCHTRTTV ZgPCPH / PDAGZG / PCPPAgEGGPGARVKFSRSADAPAYKQGQNQLYNELNLG RREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0110] In some embodiments, a dual CAR disclosed herein is encoded by the nucleic acid sequence atggcacttccggtgaccgctttgctgctgcctcttgcactgctgctgcatgccgctagacctgaggtgcagctgttggaatctgg cggtggactggttcagcctggcggatctctgagactgtcttgtgccgccagcggcttcacctttagcagctacgccatgaactgg gtccgacaggcccctggcaaaggactggaatgggtggccggaatcatcggctcaggcggcagcacatattacgccgactctg tgaagggccgcttcagcatcagcagagacaacagcaagaacaccctggacctgcagatgaactccctgagagccgaggaca cagccgtgtactactgcgtgaaggattggaacaccaccatgatcaccgagagaggacagggcaccctcgtgacagtttcttctg gcggaggcggcagcggaggcggaggttcaggtggtggtggatctgaagtccagctgctggaaagtggtggcggacttgtgc aacctggtggctcactgagactgagctgtgccgcttccggctttaccttctccaactatgccatgacctggatcaggcaggctcca ggcaagggacttgagtgggtttccggcattacaggcgacggcggaagcaccttctatgccgacagtgtgaaaggccggtttac catcagccgggataactccaagaatacgctgtatctccagatgaacagcctccgcgccgaagataccgctgtgtattattgtgtga aagactggaatacgacgatgattacggaacgcggccagggaaccctggtcaccgttagctctacaacaacaccagctccgcg gccgccaactcctgctcctacaattgctagccagccactgtctctgaggcccgaggcttgtagaccagctgctggcggagctgt gcacacaagaggactggatttcgcctgcgacatctatatctgggcccctctggctggcacatgtggcgttctgctgctcagcctg gtcatcacactgtactgcaagcggggcagaaagaagctgctgtacatctttaagcagcccttcatgcggcccgtgcagaccaca caagaagaggacggctgctcctgcagattccccgaggaagaagaaggcggttgcgaactgagagtgaagttcagcagatccg ccgacgcacccgcctataagcagggacagaaccagctgtacaacgagctgaatctggggcgcagagaagagtacgacgtgc tggacaagagaagaggcagggaccctgagatgggaggcaagcccagacggaagaatcctcaagagggcctgtataatgag ctgcagaaagacaaaatggccgaggcctacagcgagatcggaatgaagggcgagcgcagaagaggaaagggacacgacg gactgtatcagggcctgagcaccgccaccaaggatacctatgatgccctgcacatgcaggccctgccacctagaggaagcggAttorney Docket No.: 000218-0154-WO1 agaaggacgaggatcactgctgacctgtggcgacgtcgaagaaaatcctggtccaatggctctgcccgtgacagccctgttgct tcctctggctctccttctgcatgcggcgcgcccacaagtgcagctggtggaatctggcggcggacttgtgaaacctggcggctct ctgagactgagctgtgccgcttccggcttcgacttctccgactactacatgagctggatcagacaggcccctggcaaaggcctg gaatgggtgtcctatatgagcagcagcggctccaccatctactacgccgatagcgtgaaaggccggttcaccatctccagagac aacgccaagaagtccctgtacctgcagatgaactccctgagagccgaggacaccgccgtgtactattgtgctagaggcggaatt gccgccgctggcacatggggacagggaacactggttaccgtgtccagcacaacaacaccggcgcctcggccacctacacca gcaccaactatcgcaagccaacctctcagcctgcggccagaagcctgtagacccgcagcaggcggtgccgttcatacaagag gcctcgactttgcctgtgatatctacatttgggcacccctggccggaacctgcggagttcttctgctgtccctcgtgatcaccctgta ttgcaaaaagcggggcgatccttgctcctgccagccaagatcaagacccagacagtctcctgccaagtcctctcaggaccacg ccatggaagctggcagccctgtgtctacatctcccgagccagtggaaacatgctccttttgtttccctgagtgtcgggccccaact caagaaagcgcagtgacaccaggcactccagatccaacctgcgctggccgatggggatgccatacaagaacaaccgtgctcc aaccatgtccacacattcccgatagcggactgggaattgtgtgtgtccccgcacaagaaggtggcccaggggcccgcgtgaaa ttctccagatctgccgacgctccagcttacaaacagggccagaatcagctctataatgaactgaacctcggcaggcgcgaggaa tatgatgtgctggataagcggaggggcagagatcccgagatgggaggaaaacccagacggaaaaatccccaagaaggccttt acaacgaactgcaaaaggataagatggctgaagcttactccgagattggcatgaagggcgaacgtcggagaggaaaaggac acgacggcctgtaccagggactgtctacagccacaaaggacacatacgacgcactccatatgcaggctctcccacctaga (SEQ ID NO: 64).

[0111] As described above, the BCMA binding domains provided herein are useful in targeting both wildtype and mutant forms of BCMA, including R27P, P33Del, P33S, and P30Del, that have been implicated in the pathogenesis of relapsed disease and antigen escape from CAR-T cell therapies. In some embodiments, a dual CAR described herein binds to a BCMA R27P mutant and wildtype BCMA with comparable affinity. In some embodiments, a dual CAR described herein binds to a BCMA P33Del mutant and wildtype BCMA with comparable affinity. In some embodiments, a dual CAR described herein binds to a BCMA P33S mutant and wildtype BCMA with comparable affinity. In some embodiments, a dual CAR described herein binds to a BCMA P30del mutant and wildtype BCMA with comparable affinity. “Comparable affinity” in this context means that the binding affinity of the dual CAR for the mutant form of BCMA is within 10% of the binding affinity of the dual CAR for wildtype BCMA.

[0112] The dual CARs herein may be used to target cells expressing either target antigen (i.e., cells expressing BCMA or CD19). Thus, in some embodiments, a dual CAR described herein binds to a cell expressing CD 19 but not BCMA (a BCMA- / CD19+ cell). In some embodiments, a dual CAR described herein binds to a cell expressing BCMA but not CD 19 (a BCMA+ / CD19- cell). In some embodiments, a dual CAR described hereinAttorney Docket No.: 000218-0154-WO1 targets a cell expressing both BCMA and CD 19 (a BCMA+ / CD19+ cell). In some embodiments, the cell that the dual CAR described herein binds to is a cell that is a precursor to a tumor cell (e.g., a pre-cancerous cell or a progenitor cell). In certain embodiments, the pre-cancerous cell or progenitor cell expresses CD 19 but not BCMA (BCMA- / CD19+). In certain embodiments, the pre-cancerous cell or progenitor cell is correlated with worse outcomes or relapse. In certain embodiments, the dual CAR described herein is more effective in killing pre-cancerous cells or progenitor cells than either the BCMA CAR or CD 19 CAR alone.Cells and Modified Cells

[0113] In another aspect, provided herein are cells modified to comprise the CARs (e.g., VCAR or dual CARs) provided herein. Cells and modified cells of the disclosure can be mammalian cells. In some embodiments, the cells and modified cells are human cells.

[0114] 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.

[0115] In some embodiments, 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 selfrenew 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.

[0116] In some embodiments, the immune precursor cells are hematopoietic progenitor cells (HPCs). Hematopoietic stem cells (HSCs) are multipotent, self-renewing cells. All differentiated blood cells from the lymphoid and myeloid lineages arise from HSCs. HSCs can be found in adult bone marrow, peripheral blood, mobilized peripheral blood, peritoneal dialysis effluent and umbilical cord blood.

[0117] 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).Attorney Docket No.: 000218-0154-WO1

[0118] Immune precursor cells can be HSCs or HSC descendent cells. Non-limiting examples of 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.

[0119] 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. For example, the “primitive” HSCs produced by the disclosed methods may retain their “sternness” following division and may not differentiate. Consequently, as an adoptive cell therapy, the “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.

[0120] 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+.

[0121] 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.

[0122] 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. In some aspects, non-activated NK cells are derived from CD3- depleted leukapheresis (containing CD14 / CD19 / CD56+ cells).

[0123] 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.Attorney Docket No.: 000218-0154-WO1

[0124] Modified T cells of the disclosure may be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs. Unlike traditional biologies 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.

[0125] In some embodiments, 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. In some embodiments, an inducible proapoptotic polypeptide operably linked to a ligand binding region that may be optimized to bind a chemical inducer of dimerization. When the ligand binding region specifically binds the induction agent, 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 (iCas9) polypeptides. In some embodiments, 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.

[0126] In some embodiments, 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. In certain embodiments, the non-human sequence comprises a restriction site. In certain embodiments, the ligand binding region may be a multimeric ligand binding region. Inducible safety switches such as iCas9 are further described in international patent application publication No. WO 2018 / 068022, which is incorporated herein by reference in its entirety for examples of inducible safety switches that may be used in the cells described herein. An illustrative amino acid sequence of an iCas9 safety switch is set forth in SEQ ID NO: 46. In some embodiments, the iCas9 safety switch is encoded by the nucleic acid sequence set forth in SEQ ID NO: 43.

[0127] Intensive efforts have been focused on the development of antigen receptor molecules that do not cause T cell exhaustion through antigen-independent (tonic) signaling, as well as of a modified-T cell product containing early memory T cells,Attorney Docket No.: 000218-0154-WO1 especially stem cell memory (TSCM) or stem cell-like T cells. Stem cell-like modified-T cells of the disclosure exhibit the greatest capacity for self-renewal and multipotent capacity to derive central memory (TCM) T cells or TCM like cells, effector memory (TEM) and effector T cells (TE), thereby producing better tumor eradication and long-term modified-T cell engraftment. A linear pathway of differentiation may be responsible for generating these cells: Naive T cells (TN) > TSCM > TCM > TEM > TE > TTE, whereby TN is the parent precursor cell that directly gives rise to TSCM, 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).

[0128] 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 Naive 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 Naive T cells (TN), a TSCM, a TCM, a TEM, a TE, or a TTE.

[0129] 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 cellsurface marker(s) of an early memory T cell. In some embodiments, at least 20% of cells in the population express one or more cell-surface marker(s) of an early memory T cell. In some embodiments, at least 25% of cells in the population express one or more cellsurface marker(s) of an early memory T cell. In some embodiments, at least 50% of cells in the population express one or more cell-surface marker(s) of an early memory T cell. In some embodiments, at least 75% of cells in the population express one or more cellsurface marker(s) of an early memory T cell. Examples of cell surface markers of early memory T cells include CD95, CD45RA, CCR7, CD62L, CD28, CD27, CDl la, CD58, CD122 (IL-2R ), CXCR3, and CD127.

[0130] In some embodiments, 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). In some embodiments, the population of modified early memory T cells comprises a plurality of modified TCM cells.Attorney Docket No.: 000218-0154-WO1

[0131] 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 cellsurface marker(s) of a stem cell-like T cell. The population of modified stem cell-like T cells comprises a plurality of modified TSCM cells. The population of modified stem celllike T cells comprises a plurality of modified TCM cells.

[0132] In some aspects, 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 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% 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 memory T cell (TSCM) or a TscM-like cell; and wherein the one or more cell-surface marker(s) comprise CD45RA and CD62L. The cellsurface 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-2Rp. The cell-surface markers of a TSCM or a TscM-like cell can also comprise one or more of CD45RA, CD95, IL-2RP, CCR7, and CD62L.

[0133] In some aspects, 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 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the plurality of modified T cells in the population expresses one or more cell-surface marker(s) of a central memory T cell (TCM) or a TcM-like cell; and wherein the one or more cell-surface marker(s) comprise CD45RO and CD62L. The cell-surface markers of a TCM or a TCM- like cell can also comprise one or more of CD45RO, CD95, IL-2RP, CCR7, and CD62L.

[0134] 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 cellsurface marker(s) of a naive T cell (TN). The cell-surface markers of a TN cell can comprise one or more of CD45RA, CCR7 and CD62L.

[0135] 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%,Attorney Docket No.: 000218-0154-WO145%, 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 cellsurface marker(s) of an effector T-cell (modified TEFF). The cell-surface markers of a modified TEFF can comprise one or more of CD45RA, CD95, and IL-2Rp.

[0136] 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 (TSCM) or a central memory T cell (TCM).

[0137] The modified cells of the population may comprise a transgene. The transgene may be, for example, a gene encoding a CAR. The populations of cells disclosed herein may be characterized by their expression of certain cell surface markers, for example, CD34, CD90, CD38, CD45RA and CD49f.

[0138] 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 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 cellsurface marker phenotype CD34+). 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 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+).In some embodiments, 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. In some embodiments, the transgene encodes a CAR.

[0139] 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 leastAttorney Docket No.: 000218-0154-WO198%, 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 99.9% or 100% of the population of modified cells express CD34 and do not CD38 (i.e., the cells have the cell-surface marker phenotype CD34+ and CD38-). 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 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-). In some embodiments, 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. In some embodiments, the transgene encodes a CAR.

[0140] 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 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 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 and CD90 and do not express CD38 (i.e., the cells have the cell-surface marker phenotype CD34+, CD38- and CD90+).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 the transgene and at least about 0.2% to about 40%, about 0.2% toAttorney Docket No.: 000218-0154-WO1 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 (i.e., the cells have the cellsurface marker phenotype CD34+, CD38- and CD90+). In some embodiments, 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. In some embodiments, the transgene encodes a CAR.

[0141] 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 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 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 and CD90 and do not express CD38 or CD45RA (i.e., the cells have the cell-surface marker phenotype CD34+, CD38-, CD90+, and CD45RA-). 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 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-). In some embodiments, 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. In some embodiments, the transgene encodes a CAR.Attorney Docket No.: 000218-0154-WO1

[0142] 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 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 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 99.9% or 100% of the population of modified cells express CD34, CD90 and CD49f and do not express CD38 or CD45RA (z.e.., the cells have the cell-surface marker phenotype CD34+, CD38-, CD90+, CD45RA- and CD49f+). 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 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+). In some embodiments, 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. In some embodiments, the transgene encodes a CAR.

[0143] 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 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%,Attorney Docket No.: 000218-0154-WO1 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 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 CD34 and CD90 and do not express CD45RA (i.e., the cells have the cellsurface marker phenotype CD34+, CD90+ and CD45RA-). 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 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-). In some embodiments, 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. In some embodiments, the transgene encodes a CAR.

[0144] Compositions and methods of producing and / or expanding the immune cells or immune precursor cells (e.g., the disclosed CAR-T-cells) and buffers for maintaining or enhancing a level of cell viability and / or a stem-like phenotype of the immune cells or immune precursor cells (e.g., the disclosed CAR-T-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.

[0145] 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 be 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.Attorney Docket No.: 000218-0154-WO1Methods of Introducing Nucleic Acids into Cells

[0146] A CAR cell can be produced by introducing a nucleic acid encoding a CAR or dual CAR into the cell. In some embodiments, a transgene and / or genomic editing construct are also introduced into the cell.

[0147] 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.

[0148] 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 protonati on-deprotonation. In particular, at certain pHs, 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. In particular, 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. In one example, site-specific cleavage of the double stranded DNA is enabled by delivery of a nuclease using the poly(histidine)-based micelles. Without wishing to be bound by a particular theory, it is believed that believed that in the micelles that are formed by the various triblock copolymers, 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.Attorney Docket No.: 000218-0154-WO1

[0149] In an aspect, the disclosure provides triblock copolymers made of a hydrophilic block, a hydrophobic block, and a charged block. In some aspects, the hydrophilic block may be polyethylene oxide) (PEO), and 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.

[0150] Diblock copolymers that can be used as intermediates for making triblock copolymers can have hydrophilic biocompatible polyethylene 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(s- caprolactone) (PCL), and poly (trimethylene carbonate) (PTMC). Polymeric micelles comprised of 100% PEGylated surfaces possess improved in vitro chemical stability, augmented in vivo bioavailability, 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.

[0151] 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), DoggyBone™ 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, such as a piggyBac transposon.

[0152] 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 nonintegrating non-chromosomal vectors include adeno-associated virus (AAV), adenovirus, and herpes viruses. The viral vector can be an integrating chromosomal vector. Non-Attorney Docket No.: 000218-0154-WO1 limiting examples of integrating chromosomal vectors include adeno-associated vectors (AAV), Lentiviruses, and gamma-retroviruses.

[0153] 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. Nonlimiting examples of vector combinations include viral and non-viral vectors, a plurality of non-viral vectors, or a plurality of viral vectors. Non-limiting examples of 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.

[0154] 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.

[0155] The nucleic acid sequence or transgene can be about 1 kb to about 15 kb in size. The nucleic acid sequence or transgene can be at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, 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.

[0156] 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. In a preferred aspect, the transposon is a plasmid DNA transposon comprising a nucleotide sequence encoding the CAR (e.g., VCAR) as disclosed herein flanked by two cis-regulatory insulator elements. The present disclosure also provides a composition comprising a transposon. In a preferred aspect, 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. Preferably, the sequence encoding the transposase is an mRNA sequence.

[0157] A transposon of the present disclosure can be a piggyBac™ (PB) transposon. In some aspects when the transposon is a PB transposon, the transposase is a piggyBac™Attorney Docket No.: 000218-0154-WO1(PB) transposase a piggyBac-like (PBL) transposase or a Super piggyBac™ (SPB) transposase. The sequence encoding the SPB transposase is an mRNA sequence.

[0158] Non-limiting examples of 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. 8,399,643 and PCT Publication No. WO 2010 / 099296, each of which is incorporated herein by reference in its entirety for examples of transposon systems that may be used in the methods described herein.

[0159] 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). 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 ’-T AAA-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 ’-AT AG-3’, 5 ’-C AAA-3’, 5’-CACA-3’, 5 ’-C ATA-3’, 5’- CCAG-3’, 5’-CCCA-3’, 5’-CGTA-3’, 5’-GTCC-3’, 5’-TAAG-3’, 5’-TCTA-3’, 5’- TGAG-3’, 5’-TGTT-3’, 5’-TTCA-3’5’-TTCT-3’ and 5’-TTTT-3’. The PB or PBL transposon system has no payload limit for the genes of interest that can be included between the ITRs.

[0160] 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. Illustrative sequences of transposases and transposons that may be used in methods described herein are also shown in Table 1 below. In some embodiments, 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: 15: MGSSLDDEHILSALLQSDDELVGEDSDSEISDHVSEDDVQSDTEEAFIDEVHEVQP TSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSALN IVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTGATFRD TNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCL RMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFAttorney Docket No.: 000218-0154-WO1RMYIPNKPSKYGIKILMMCDSGYKYMINGMPYLGRGTQTNGVPLGEYYVKELS KPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRP VGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQT KGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKV QSRKKFMRNLYMSLTSSFMRKRLEAPTLKRYLRDNISNILPNEVPGTSDDSTEEP VMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF (SEQ ID NO:15).

[0161] In some embodiments, the PB transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15 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: 15.

[0162] The PB or PBL transposase can also comprise or consist of the amino acid sequence of SEQ ID NO: 15 with amino acid substitution at positions 30, 165, 282, and / or 538 of the sequence of SEQ ID NO: 15. The transposase can comprises or consists of the amino acid sequence of the sequence of SEQ ID NO: 15 comprising one, two, three or all of the following amino acid substitutions: BOV, G165S, M282V, N538K.

[0163] In a some embodiments, 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: 16: MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQ PTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSRVSAL NIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFR DTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRC LRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCP FRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELS KPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRP VGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQT KGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKV QSRKKFMRNLYMSLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEP VMKKRTYCTYCPSKIRRKANASCKKCKKVICREHNIDMCQSCF (SEQ ID NO:16).

[0164] In some embodiments, the SPB transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16 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: 16.

[0165] In certain aspects wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and / or 538, 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: 15Attorney Docket No.: 000218-0154-WO1 or SEQ ID NO: 16. Such mutations are 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.

[0166] 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. In preferred aspects, the PB, PBL or SPB transposases is be isolated or derived from the insect Trichoplusia ni (GenBank Accession No. AAA87375) or Bombyx mori (GenBank Accession No. BAD11135).

[0167] A hyperactive PB or PBL transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In a preferred aspect, 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.

[0168] In some aspects, 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. A list of integration deficient amino acid substitutions is disclosed in US 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.

[0169] In some aspects, the PB or PBL transposase is fused to a nuclear localization signal. Examples of PB or PBL transposases fused to a nuclear localization signal areAttorney Docket No.: 000218-0154-WO1 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.

[0170] A transposon of the present disclosure can be a Sleeping Beauty transposon. In some aspects, 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. In some embodiments, 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: 17. In some embodiments, the Sleeping Beauty transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17 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: 17. 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: 18. In some embodiments, the SB100X transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18 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: 18.

[0171] A transposon of the present disclosure can be a Helraiser transposon. An exemplary Helraiser transposon includes Helibatl. In some embodiments, the Helibatl 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: 19. In some embodiments, the Helibatl transposon comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 19. In some aspects, when the transposon is a Helraiser transposon (e.g., a Helibatl transposon), the transposase is a Helitron transposase (for example, as disclosed in WO 2019 / 173636). in some embodiments, 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%, orAttorney Docket No.: 000218-0154-WO1 at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 61. In some embodiments, the Helitron transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20 with one, two, three, four or five conservative amino acid substitutions. In some embodiments, the Helitron transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20.

[0172] A transposon of the present disclosure can be a Tol2 transposon. A Tol2 transposon may include inverted repeats, subterminal sequences. In some embodiments, 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: 21. In some embodiments, the Tol2 transposon comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 21. In some aspects, when the transposon is a Tol2 transposon, the transposase is a Tol2 transposase (for example, as disclosed in WO 2019 / 173636). In some embodiments, 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: 22. In some embodiments, the Tol2 transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 with one, two, three, four or five conservative amino acid substitutions. In some embodiments, the Tol2 transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22.

[0173] A transposon of the present disclosure can be a TcBuster transposon. In some aspects, 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. In some embodiments, 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: 23. In some embodiments, the TcBuster transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 with one, two, three, four or five conservative amino acid substitutions. In some embodiments, the TcBuster transposase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23. 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. InAttorney Docket No.: 000218-0154-WO1 some embodiments, 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: 24. In some embodiments, the TcBuster transposase is encoded by the nucleic acid sequence set forth in SEQ ID NO: 24.

[0174] In some aspects, 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.

[0175] 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. In some aspects, the inter-ITR sequence of (c) comprises the sequence of (b). In some aspects, the intra-ITR sequence of (a) comprises the sequence of (b).

[0176] The sequence encoding the backbone can comprise between 1 and 600 nucleotides, inclusive of the endpoints. In some aspects, 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.

[0177] The inter-ITR sequence can comprise between 1 and 1000 nucleotides, inclusive of the endpoints. In some aspects, 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,Attorney Docket No.: 000218-0154-WO1 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 nucleotides, each range inclusive of the endpoints.

[0178] 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.

[0179] 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.

[0180] 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. In some aspects, 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).

[0181] In some aspects of the nanotransposon, 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, neitherAttorney Docket No.: 000218-0154-WO1 the nanotransposon nor the sequence encoding the backbone is derived from a recombination site, an excision site, and / or a ligation site.

[0182] In some aspects of the nanotransposon, a recombination site comprises a sequence resulting from a recombination event. In some aspects, a recombination site comprises a sequence that is a product of a recombination event. In some aspects, the recombination event comprises an activity of a recombinase (e.g., a recombinase site).

[0183] In some aspects of the nanotransposon, the sequence encoding the backbone does not further comprise a sequence encoding foreign DNA.

[0184] In some aspects of the nanotransposon, the inter-ITR sequence does not comprise a recombination site, an excision site, a ligation site or a combination thereof. In some aspects, 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. 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 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 (poly A) sequence 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, at least one exogenous sequence, a polyadenosine (poly A) sequence and a second sequence encoding an insulator.

[0185] In certain aspects, a transposon cassette comprising a nucleic acid sequence encoding a dual CAR as illustrated in FIG. 12. In certain aspects, as shown in FIG. 12, the transposon cassette comprises a left end ITR, an EFla promoter operably associated with the nucleic acids encoding: an iCas9 safety switch, a CAR targeting BCMA, a CAR targeting CD 19, a DHFR mutein selectable marker, a poly A sequence; and a right end ITR. In certain aspects, a transposon cassette is constructed via Gibson assembly.Attorney Docket No.: 000218-0154-WO1

[0186] 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.

[0187] Furthermore, 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). Exemplary adeno- associated viruses and recombinant adeno-associated viruses include, but are not limited to, rAAV-LK03.

[0188] A vector of the present disclose can be a nanoparticle. Non-limiting examples of 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. A nanoparticle vector can be passively or actively transported across a cell membrane.Table 1: Illustrative Transposon and Transposase SequencesAttorney Docket No.: 000218-0154-WO1Attorney Docket No.: 000218-0154-WO1Attorney Docket No.: 000218-0154-WO1Attorney Docket No.: 000218-0154-WO1Attorney Docket No.: 000218-0154-WO1Attorney Docket No.: 000218-0154-WO1Attorney Docket No.: 000218-0154-WO1Attorney Docket No.: 000218-0154-WO1Attorney Docket No.: 000218-0154-WO1Gene Editing Compositions and Methods for Targeted Genome Editing at Selected Locus

[0189] 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 codelivered with a site-directed nuclease. The site-directed nuclease comprises a transgene with 5’ and 3’ nucleotide sequence extensions that contain a percentage homology toAttorney Docket No.: 000218-0154-WO1 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.

[0190] 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-rej ection.

[0191] 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 sitespecific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.

[0192] 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-CLOVER™, 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). Nonlimiting examples of break-inducing enzymes are described in International Patent Application Publications No. WO 2016 / 205554, No. WO 2019 / 126578, and No. WO 2018 / 064681, each of which are incorporated by reference in their entirety for examples of gene editing compositions that may be used in the methods disclosed herein.Attorney Docket No.: 000218-0154-WO1Exemplary mutant Cas-CLOVER break-inducing enzymes of the disclosure are also described below.

[0193] The site-specific transgene integration can be controlled by a vector-mediated integration site bias.

[0194] 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. In one aspect, 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.

[0195] 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.

[0196] The genome modification can be a non-stable chromosomal integration of a transgene. The integrated transgene can become silenced, removed, excised, or further modified.

[0197] 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).

[0198] 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 oneAttorney Docket No.: 000218-0154-WO1 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. In one aspect, the transgene can comprise a sequence for a CAR. In one aspect, the transgene comprising a CAR that specifically binds to BCMA, or CD 19 or a combination thereof. 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.

[0199] In gene editing systems that involve inserting new or existing nucleotides / nucleic acids, 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). 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.

[0200] 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. dCas9-Clo051 (Cas-CLOVER) fusion proteins

[0201] Examples of fusion proteins that may be used for the gene editing methods disclosed herein include fusion proteins comprising a nuclease-inactivated Cas (dCas)Attorney Docket No.: 000218-0154-WO1 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. Illustrative sequences for gene editing system components are also shown in Table 2 below.

[0202] 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. In some embodiments, the fusion protein comprises a Staphylococcus aureus Cas9 with an active catalytic site comprising the amino acid sequence of SEQ ID NO: 25.

[0203] 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. In some embodiments, the fusion protein comprises a dSaCas9 comprising the amino acid sequence of SEQ ID NO: 26, which includes a D10A and a N580A mutation to inactivate the catalytic site.

[0204] 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.

[0205] The dCas9 can be isolated or derived from Streptococcus pyogenes. The dCas9 can comprise the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 28. The dCas9 can also comprise amino acid substitutions at amino acid positions 10 and 840 of SEQ ID NO: 27 or 28, which inactivate the catalytic site. In some aspects, these substitutions are DIOA and H840A.

[0206] An illustrative Clo051 nuclease domain comprises, consists essentially of or consists of, the amino acid sequence of SEQ ID NO: 29. 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 someAttorney Docket No.: 000218-0154-WO1 aspects, the amino acid substitution is at position 35, 37, 60, 98, 100 or 146 of SEQ ID NO: 29.

[0207] An illustrative dCas9-Clo051 (Cas-CLOVER) fusion protein can comprise, consist essentially of, or consist of, the amino acid sequence of SEQ ID NO: 30. 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: 31. The nucleic acid encoding the dCas9-Clo051 fusion protein can be DNA or RNA.

[0208] An illustrative dCas9-Clo051 (Cas-CLOVER) fusion protein can comprise, consist essentially of, or consist of, the amino acid sequence of SEQ ID NO: 32. 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: 33. The nucleic acid encoding the dCas9-Clo051 fusion protein can be DNA or RNA.

[0209] A dCas9-Clo051 fusion (Cas-CLOVER) fusion protein of the disclosure may further comprise at least one nuclear localization sequence (NLS). In some embodiments, the dCas9-Clo051 fusion protein of the disclosure comprises at least two nuclear localization sequences. In some embodiments, the NLS is on the N-terminal end of the dCas9-Clo051 fusion protein (NLS-dCas9-Clo051). In some embodiments, the NLS is on the C-terminal end of the dCas9-Clo051 fusion protein (dCas9-Clo051-NLS). In some embodiments, 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”).

[0210] 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: 30.

[0211] NLS-dCas9-Clo051-NLS amino acid sequence (NLS amino acid sequence is bolded and underlined): MAPKKKRKVEGIKSNISLLKDELRGQISHISHEYLSLIDLAFDSKQNRLFEMKVL ELLVNEYGFKGRHLGGSRKPDGIVYSTTLEDNFGIIVDTKAYSEGYSLPISQADEM ERYVRENSNRDEEVNPNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRRLSMTT GVNGSAVNVVNLLLGAEKIRSGEMTIEELERAMFNNSEFILKYGGGGSDKKYSIG LAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATR LKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDL NPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALV RQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLN REDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEAttorney Docket No.: 000218-0154-WO1KVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKV TVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGI RDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIA NLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRE RMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS DYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLL NAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTK YDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTA LIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFS KESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSI<RVILADANLDI<VLSAYNI<HRDI<PIREQAENIIHLFTLTNLGAPAAFI<YF DTTIDRKRYTSTKEVLDATLIHOSITGLYETRIDLSOLGGDGSPKKKRKVSS (SEQ ID NO: 30).

[0212] In some embodiments, 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: 30. In some embodiments, the wildtypeCas-CLOVER fusion protein comprises or consists of the amino acid sequence of SEQID NO: 30 with one, two, three, four or five conservative amino acid substitutions.

[0213] The nucleic acid encoding the NLS-dCas9-Clo051-NLS (“wildtype Cas-CLOVER”) fusion protein can be DNA or RNA. In some embodiments, 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: 34.

[0214] NLS-dCas9-Clo051-NLS mRNA sequence (NLS amino acid sequence is bolded and underlined): atggcaccaaagaagaaaagaaaagtggagggcatcaagtcaaacatcagcctgctgaaagacgaactgcggggacaga ttagtcacatcagtcacgagtacctgtcactgattgatctggccttcgacagcaagcagaatagactgtttgagatgaaagtgctg gaactgctggtcaacgagtatggcttcaagggcagacatctgggcgggtctaggaaacctgacggcatcgtgtacagtaccac actggaagacaacttcggaatcattgtcgataccaaggcttattccgagggctactctctgccaattagtcaggcagatgagatgg aaaggtacgtgcgcgaaaactcaaatagggacgaggaagtcaaccccaataagtggtgggagaatttcagcgaggaagtgaa gaaatactacttcgtctttatctcaggcagcttcaaagggaagtttgaggaacagctgcggagactgtccatgactaccggggtg aacggatctgctgtcaacgtggtcaatctgctgctgggcgcagaaaagatcaggtccggggagatgacaattgaggaactgga acgcgccatgttcaacaattctgagtttatcctgaagtatggaggcgggggaagcgataagaaatactccatcggactggccatt ggcaccaattccgtgggctgggctgtcatcacagacgagtacaaggtgccaagcaagaagttcaaggtcctggggaacaccg atcgccacagtatcaagaaaaatctgattggagccctgctgttcgactcaggcgagactgctgaagcaacccgactgaagcgg actgctaggcgccgatatacccggagaaaaaatcggatctgctacctgcaggaaattttcagcaacgagatggccaaggtgga cgatagtttctttcaccgcctggaggaatcattcctggtggaggaagataagaaacacgagcggcatcccatctttggcaacattg tggacgaagtcgcttatcacgagaagtaccctactatctatcatctgaggaagaaactggtggactccaccgataaggcagacct gcgcctgatctatctggccctggctcacatgatcaagttccgggggcattttctgatcgagggagatctgaaccctgacaattctgAttorney Docket No.: 000218-0154-WO1 atgtggacaagctgttcatccagctggtccagacatacaatcagctgtttgaggaaaacccaattaatgcctcaggcgtggacgc aaaggccatcctgagcgccagactgtccaaatctaggcgcctggaaaacctgatcgctcagctgccaggagagaagaaaaac ggcctgtttgggaatctgattgcactgtccctgggcctgacacccaacttcaagtctaattttgatctggccgaggacgctaagctg cagctgtccaaagacacttatgacgatgacctggataacctgctggctcagatcggcgatcagtacgcagacctgttcctggccg ctaagaatctgagtgacgccatcctgctgtcagatattctgcgcgtgaacacagagattactaaggccccactgagtgcttcaatg atcaaaagatatgacgagcaccatcaggatctgaccctgctgaaggctctggtgaggcagcagctgcccgagaaatacaagga aatcttctttgatcagagcaagaatggatacgccggctatattgacggcggggcttcccaggaggagttctacaagttcatcaagc ccattctggaaaagatggacggcaccgaggaactgctggtgaagctgaatcgggaggacctgctgagaaaacagaggacatt tgataacggaagcatccctcaccagattcatctgggcgaactgcacgccatcctgcgacggcaggaggacttctacccatttctg aaggataaccgcgagaaaatcgaaaagatcctgaccttcagaatcccctactatgtggggcctctggcacggggaaatagtag atttgcctggatgacaagaaagtcagaggaaactatcaccccctggaacttcgaggaagtggtcgataaaggcgctagcgcac agtccttcattgaaaggatgacaaattttgacaagaacctgccaaatgagaaggtgctgcccaaacacagcctgctgtacgaata tttcacagtgtataacgagctgactaaagtgaagtacgtcaccgaagggatgcgcaagcccgcattcctgtccggagagcagaa gaaagccatcgtggacctgctgtttaagacaaatcggaaagtgactgtcaaacagctgaaggaagactatttcaagaaaattgag tgtttcgattcagtggaaatcagcggcgtcgaggacaggtttaacgcctccctggggacctaccacgatctgctgaagatcatca aggataaggacttcctggacaacgaggaaaatgaggacatcctggaggacattgtgctgacactgactctgtttgaggatcgcg aaatgatcgaggaacgactgaagacttatgcccatctgttcgatgacaaagtgatgaagcagctgaaaagaaggcgctacacc ggatggggacgcctgagccgaaaactgatcaatgggattagagacaagcagagcggaaaaactatcctggactttctgaagtc cgatggcttcgccaacaggaacttcatgcagctgattcacgatgactctctgaccttcaaggaggacatccagaaagcacaggt gtctggccagggggacagtctgcacgagcatatcgcaaacctggccggcagccccgccatcaagaaagggattctgcagacc gtgaaggtggtggacgaactggtcaaggtcatgggacgacacaaacctgagaacatcgtgattgagatggcccgcgaaaatc agacaactcagaagggccagaaaaacagtcgagaacggatgaagagaatcgaggaaggcatcaaggagctggggtcacag atcctgaaggagcatcctgtggaaaacactcagctgcagaatgagaaactgtatctgtactatctgcagaatggacgggatatgt acgtggaccaggagctggatattaacagactgagtgattatgacgtggatgccatcgtccctcagagcttcctgaaggatgactc cattgacaacaaggtgctgaccaggtccgacaagaaccgcggcaaatcagataatgtgccaagcgaggaagtggtcaagaaa atgaagaactactggaggcagctgctgaatgccaagctgatcacacagcggaaatttgataacctgactaaggcagaaagagg aggcctgtctgagctggacaaggccggcttcatcaagcggcagctggtggagacaagacagatcactaagcacgtcgctcag attctggatagcagaatgaacacaaagtacgatgaaaacgacaagctgatcagggaggtgaaagtcattactctgaaatccaag ctggtgtctgactttagaaaggatttccagttttataaagtcagggagatcaacaactaccaccatgctcatgacgcatacctgaac gcagtggtcgggaccgccctgattaagaaataccccaagctggagtccgagttcgtgtacggagactataaagtgtacgatgtc cggaagatgatcgccaaatctgagcaggaaattggcaaggccaccgctaagtatttcttttacagtaacatcatgaatttctttaag accgaaatcacactggcaaatggggagatcagaaaaaggcctctgattgagaccaacggggagacaggagaaatcgtgtgg gacaagggaagggattttgctaccgtgcgcaaagtcctgtccatgccccaagtgaatattgtcaagaaaactgaagtgcagacc gggggattctctaaggagagtattctgcctaagcgaaactctgataaactgatcgcccggaagaaagactgggaccccaagaa gtatggcgggttcgactctccaacagtggcttacagtgtcctggtggtcgcaaaggtggaaaaggggaagtccaagaaactga agtctgtcaaagagctgctgggaatcactattatggaacgcagctccttcgagaagaatcctatcgattttctggaagccaagggc tataaagaggtgaagaaagacctgatcattaagctgccaaaatactcactgtttgagctggaaaacggacgaaagcgaatgctg gcaagcgccggagaactgcagaagggcaatgagctggccctgccctccaaatacgtgaacttcctgtatctggctagccacta cgagaaactgaaggggtcccctgaggataacgaacagaagcagctgtttgtggagcagcacaaacattatctggacgagatca ttgaacagatttcagagttcagcaagagagtgatcctggctgacgcaaatctggataaagtcctgagcgcatacaacaagcacc gagacaaaccaatccgggagcaggccgaaaatatcattcatctgttcaccctgacaaacctgggcgcccctgcagccttcaagt attttgacaccacaatcgatcggaagagatacacttctaccaaagaggtgctggatgctaccctgatccaccagagtattaccgg cctgtatgagacacgcatcgacctgtcacagctgggaggcgatgggagccccaagaaaaagcggaaggtgtctagttaatg a (SEQ ID NO: 34). gRNAs

[0215] In some aspects, 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 sufficientAttorney Docket No.: 000218-0154-WO1 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. The terms “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.

[0216] 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.

[0217] The guide RNA can comprise a sequence complementary to at least one target sequence on a transposon, plasmid or vector. In some aspects, the complementary sequence to the guide RNA on the transposon, plasmid or vector is located within the transgene for targeted nucleic acid insertion. In some aspects, the complementary sequence to the guide RNA on the transposon, plasmid or vector is located within the transgene for targeted nucleic acid insertion. In some aspects, 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.

[0218] The term “target region”, “target sequence” or “protospacer” as used interchangeably herein 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. For example, the S. pyogenes Type II system uses an “NGG” sequence, where “N” can be any nucleotide.Attorney Docket No.: 000218-0154-WO1

[0219] 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). In some embodiments, 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. In certain embodiments, 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.

[0220] In certain embodiments, 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. In certain embodiments, 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.

[0221] In some embodiments, 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.

[0222] In some embodiments, the sequence of the guide molecule (direct repeat and / or spacer) 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). 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).

[0223] As described above, the Cas-Clover system and the CRISPR / Cas9 system utilizes targeting gRNA and a shuttling gRNA that provides the targeting of the Cas-CloverAttorney Docket No.: 000218-0154-WO1 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.

[0224] In some embodiment, 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. In some embodiments, 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. In some embodiments, 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.

[0225] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0226] 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. Methods for designing such target binding regions are known in the art, see, e.g., Doench et al., Nat Biotechnol. (2014) 32: 1262-7; and Doench et al., Nat Biotechnol. (2016) 34: 184-91, incorporated by reference herein in their entirety. Design tools are available at, e.g., Feng Zhang lab's target Finder, Michael Boutros lab's Target Finder (E-CRISP), RGEN Tools (Cas-OF Finder), CasFinder, and CRISPR Optimal Target Finder. In certainAttorney Docket No.: 000218-0154-WO1 embodiments, 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.

[0227] In one embodiment, the target binding region is complementary, e.g., completely complementary, to the target region in the target gene. In one embodiment, 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. As used throughout the disclosure, 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.

[0228] 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 comprise, consist essentially of or consists of the sequences as shown in Table 3.

[0229] In certain aspects, the cells of the disclosure are modified cells. The cells of the disclosure can be modified by the methods of targeted genome editing described herein or known in the art.

[0230] 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. A reduction of a level of expression or activity of a gene is when at least 50%, at least 75%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the expression of the gene in a cell or the functional activity of the gene in a cell is reduced when compared to the naturally occurring wild-type counterpart of the cell.

[0231] In certain aspects, the cells of the disclosure comprise a modification of an endogenous sequence encoding Beta-2 -Microglobulin (B2M). In certain aspects, the modification reduces or eliminates a level of expression or activity of a majorAttorney Docket No.: 000218-0154-WO1 histocompatibility complex (MHC) class I (MHC-I). In certain aspects, the cells of the disclosure comprise a modification of an endogenous sequence encoding a T-cell Receptor (TCR). In certain aspects, the modification reduces or eliminates a level of expression or activity of the TCR. In certain embodiments, the TCR is TCRb. In certain aspects, the cells are modified using a Cas-Clover system and sgRNA in Table 2.Table 2: Illustrative Sequences for Gene EditingAttorney Docket No.: 000218-0154-WO1Attorney Docket No.: 000218-0154-WO1Attorney Docket No.: 000218-0154-WO1Attorney Docket No.: 000218-0154-WO1Attorney Docket No.: 000218-0154-WO1Attorney Docket No.: 000218-0154-WO1Attorney Docket No.: 000218-0154-WO1Attorney Docket No.: 000218-0154-WO1Attorney Docket No.: 000218-0154-WO1Attorney Docket No.: 000218-0154-WO1Attorney Docket No.: 000218-0154-WO1

[0232] Gene editing compositions, including Cas-CLOVER, and methods of using these compositions for gene editing are described in detail in PCT Application Numbers PCT / US2024 / 016659, PCT / US2016 / 037922, PCT / US2018 / 066941, PCT / US2017 / 054799, U.S. Patent Publication Nos. 2017 / 0107541, 2017 / 0114149,2018 / 0187185 and U.S. Patent No. 10,415,024, each of which are incorporated herein byAttorney Docket No.: 000218-0154-WO1 reference in its entirety. Exemplary gene editing compositions including mutant Cas- CLOVER and methods of using these compositions for gene editing are described herein.Methods of Expressing a CAR

[0233] The disclosure provides methods of expressing a CAR or dual CAR on the surface of a cell. In some aspects, the method comprises (a) obtaining a cell population; (b) contacting the cell population with a composition comprising a CAR or dual CAR, or with a sequence encoding the CAR or dual CAR, under conditions sufficient to transfer the CAR or dual 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 or dual CAR; and (d) expanding and / or selecting at least one cell from the modified cell population that express the CAR or dual CAR on the cell surface.

[0234] In some aspects, 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. In some embodiments, the optimized ratio of CD4+ to CD8+ leukocytes does not naturally occur in vivo. The cell population can also comprise a tumor cell.

[0235] In some aspects, 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). In some aspects, 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).

[0236] 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,Attorney Docket No.: 000218-0154-WO1IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36, GM-CSF, IFN- gamma, IL-1 alpha / IL-lFl, IL-1 beta / IL-lF2, 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, IL-24, IL-32, IL-32 beta, IL-32 gamma, IL-33, LAP (TGF-beta 1), Lymphotoxin-alpha / TNF-beta, TGF-beta, TNF-alpha, TRANCE / TNFSF11 / RANK L or any combination thereof. 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, MgCh, Na2HPO4, NAH2PO4, Sodium lactobionate, Mannitol, Sodium succinate, Sodium Chloride, CINa, Glucose, Ca(NO3)2, Tris / HCl, K2HPO4, KH2PO4, Polyethylenimine, Poly-ethylene-glycol, Poloxamer 188, Poloxamer 181, Poloxamer 407, Polyvinylpyrrolidone, Pop313, Crown-5, or any combination thereof. 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, Caspasel, Pro-ILIB, PI3K, Akt, Wnt3A, inhibitors of glycogen synthase kinase-3p (GSK-3 P) (e.g. TWS119), or any combination thereof. Examples of such 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.

[0237] 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.Attorney Docket No.: 000218-0154-WO1

[0238] 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.

[0239] In some aspects, wherein the transposon or vector comprises a selection gene, 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.

[0240] The disclosure provides a composition comprising the modified, expanded and selected cell population of the methods described herein.

[0241] A more detailed description of methods for expressing a CAR on the surface of a cell is disclosed in PCT Publication No. WO 2020 / 051374, which is incorporated herein by reference in its entirety for examples of methods that may be used to modify the cells disclosed herein.Methods of Use

[0242] The disclosure also provides 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 or dual CAR). In one aspect, the subject is a mammal. Preferably, the subject is human. The terms “subject” and “patient” are used interchangeably herein.

[0243] The disclosure provides a method for modulating or treating cancer in a cell, tissue, organ, animal or subject. Non-limiting examples of a cancer include leukemia,Attorney Docket No.: 000218-0154-WO1 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 malignancy, solid tumors, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head cancer, neck cancer, hereditary nonpolyposis cancer, Hodgkin's lymphoma, liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, testicular cancer, adenocarcinomas, sarcomas, malignant melanoma, hemangioma, metastatic disease, cancer related bone resorption, cancer related bone pain, and the like.

[0244] In certain aspects, provided herein are methods of treating multiple myeloma (MM). MM is a hematological malignancy characterized by the uncontrolled proliferation of monoclonal plasma cells in the bone marrow. Despite advancements in therapeutic strategies, including the use of immunomodulatory agents, proteasome inhibitors, and monoclonal antibodies, relapsed and refractory multiple myeloma (RRMM) remains a significant clinical challenge. One of the most promising targets in the treatment of MM is B-cell maturation antigen (BCMA), a protein that is highly expressed on malignant plasma cells. However, mutations in the BCMA gene, particularly in patients with RRMM, can lead to treatment resistance, limiting the efficacy of BCMA-targeted therapies such as CAR-T cells, monoclonal antibodies, and small molecules.

[0245] Chimeric antigen receptor T-cell (CAR-T) therapy has emerged as a groundbreaking treatment modality for RRMM, particularly with the development of BCMA-targeted CAR-T cells. These therapies have shown remarkable efficacy in clinical trials, providing significant response rates even in heavily pre-treated patients. However, the presence of BCMA mutations such as R27P, P33Del, P33S, and P30Del (described above) poses challenges to the success of CAR-T treatments. For instance, mutations can affect the CAR-T cells’ ability to recognize and bind to the mutated BCMA, potentially leading to antigen escape. Studies have demonstrated that BCMA mutations can lead to reduced surface expression of the antigen, thereby diminishing the efficacy of CAR-T cells designed to target BCMA.Attorney Docket No.: 000218-0154-WO1

[0246] Furthermore, the heterogeneity of BCMA expression in the tumor microenvironment can complicate the effectiveness of CAR-T therapy. In cases where BCMA is mutated or lost, the CAR-T cells may become ineffective, underscoring the need for strategies that either target multiple antigens or enhance the persistence and functionality of CAR-T cells in the presence of BCMA mutations. Some researchers are exploring the combination of BCMA-targeted CAR-T therapy with other modalities, such as checkpoint inhibitors, to enhance anti-tumor responses and overcome resistance associated with BCMA mutations.

[0247] Without wishing to be bound by theory, it is hypothesized that the CARs, VCAR and dual CARs with antigen binding domains that bind to BCMA can effectively bind to both wildtype BCMA as well as mutant BCMA. Therefore, the CAR cells disclosed herein are believed to be effective in treating cancers in which BCMA has mutated (such as MM). In particular, the CAR cells disclosed herein were shown to target CD81+CD19+ progenitor cells, which are comparatively less differentiated cells that are believed to drive MM progression.

[0248] In some embodiments, a CAR cell described herein binds to a cell expressing a BCMA R27P mutant and a cell expressing wildtype BCMA with comparable affinity. In some embodiments, a CAR cell described herein binds to a cell expressing a BCMA P33Del mutant and a cell expressing wildtype BCMA with comparable affinity. In some embodiments, a CAR cell described herein binds to a cell expressing a BCMA P33S mutant and a cell expressing wildtype BCMA with comparable affinity. In some embodiments, a CAR cell described herein binds to a cell expressing a BCMA P30del mutant and a cell expressing wildtype BCMA with comparable affinity. “Comparable affinity” in this context means that the binding affinity of the a CAR cell for a cell expressing the mutant form of BCMA is within 10% of the binding affinity of the a CAR cell for a cell expressing wildtype BCMA.

[0249] A cell expressing a dual CAR disclosed (a dual CAR cell) herein can be used to treat a cancer that expresses one or both of the antigens targeted by the dual CAR. For example, in some embodiments, a dual CAR cell disclosed herein can be used to treat a cancer expressing BCMA but not CD 19. In some embodiments, a dual CAR cell disclosed herein can be used to treat a cancer expressing CD 19 but not BCMA. In some embodiments, a dual CAR cell disclosed herein can be used to treat a cancer expressing both BCMA and CD 19. In another aspect, the dual CAR cell disclosed herein can be used to target multiple myeloma precursor cells which express CD 19 but not BCMA.Attorney Docket No.: 000218-0154-WO1

[0250] In certain aspects, the CAR cells of the present disclosure are modified to recombinantly express dihydrofolate reductase (DHFR), which advantageously renders the CAR cells resistant to methotrexate (MTX). The MTX resistant CAR cells (“MTX- CAR cells”) may be used in methods of treating a subject in need thereof in combination with subsequent MTX administration to eliminate activated T-cells and NK cells targeting the MTX-CAR cells thereby increasing the in vivo persistence and efficacy of the MTX-CAR cells in the subject. Modified cells can be formulated for storage at any temperature including room temperature and body temperature. Modified cells can be formulated for cry opreservation 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 expression level to ensure a minimal level of cell function and CAR 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 CAR may comprise a wildtype form of the DHFR gene or a mutein thereof. An illustrative amino acid sequence of a DHFR mutein is set forth in SEQ ID NOs: 44 and 47.

[0251] An illustrative sequence of a dual CAR comprising a tandem VH that binds toBCMA, a VH that binds to CD 19, and iCas9 safety switch and a DHFR gene is set forth in SEQ ID NO: 65MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGK QEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE GGGGSGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSN IDC EKLRRRF S SLHFMVEVKGDLTAKKMVLALLEL AQQDHGALDCC VVVILSHG CQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKD HGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGF VSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCF NFLRKKLFFKTSGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAARPE VQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVAGIIGSG GSTYYADSVKGRFSISRDNSKNTLDLQMNSLRAEDTAVYYCVKDWNTTMITER GQGTLVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTF SNYAMTWIRQAPGKGLEWVSGITGDGGSTFYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPL SLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKK LLYIFKQPFMRP VQTTQEEDGC SCRFPEEEEGGCELRVKF SRS ADAP AYKQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGEGRGSLLTC GDVEENPGPMALPVTALLLPLALLLHAARPQVQLVESGGGLVKPGGSLRLSCAA SGFDF SD YYMSWIRQAPGKGLEWVS YMS S SGSTIYYADS VKGRFTISRDNAKKS LYLQMNSLRAEDTAVYYCARGGIAAAGTWGQGTLVTVSSTTTPAPRPPTPAPTIAttorney Docket No.: 000218-0154-WO1ASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCK KRGDPCSCQPRSRPRQSPAKSSQDHAMEAGSPVSTSPEPVETCSFCFPECRAPTQE SAVTPGTPDPTCAGRWGCHTRTTVLQPCPHIPDSGLGIVCVPAQEGGPGARVKFS RSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPRGSGEGRGSLLTCGDVEENPGPMVGSLNCIVAVSQNMGIGKNGDFPWPPLRN ESRYFQRMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVLSRELKEPPQ GAHFLSRSLDDALKLTEQPELANKVDMVWIVGGSSVYKEAMNHPGHLKLFVTRI MQDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND (SEQ ID NO: 65)

[0252] 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 cells. As used herein as it relates to the methods herein, “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 cells but spares the MTX-CAR 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. As disclosed herein, the MTX-CAR cells of the present disclosure are resistant to MTX levels well in excess of 200 nM.

[0253] The methods can optionally further comprise co-admini strati on 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).

[0254] In some aspects, the subject does not develop graft vs. host (GvH) and / or host vs. graft (HvG) following administration. In one aspect, the administration is systemic. Systemic administration can be any means known in the art and described in detail herein. Preferably, systemic administration is by an intravenous injection or an intravenous infusion. In one aspect, the administration is local. Local administration can be any means known in the art and described in detail herein. Preferably, 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.

[0255] In some aspects, the therapeutically effective dose of the MTX-CAR cells is a single dose. In some aspects, 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. In some aspects, where the composition is autologous cells or allogeneicAttorney Docket No.: 000218-0154-WO1 cells, the dose is an amount sufficient for the cells to engraft and / or persist for a sufficient time to treat the disease or disorder.

[0256] In one example, the disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a composition comprising a VCAR that specifically binds to an antigen on a tumor cell. In aspects where the composition comprises a modified cell or cell population, the cell or cell population may be autologous or allogeneic.

[0257] In some aspects of the methods of treatment described herein, the treatment can be modified or terminated. Specifically, in aspects where the 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. In some aspects, 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).

[0258] In certain aspects, provided are methods of increasing in vivo persistence of MTX- CAR cells in a subject comprising administering to the subject a therapeutically effective amount of MTX-CAR cells; and administering at a predetermined time post administration of the MTX-CAR cells an effective amount of MTX sufficient to eliminate (reduce by at least 25%) activated T-cells and NK cells targeting the MTX- CAR cells leading to an increased in vivo persistence of the MTX-CAR cells. In certain embodiments, the MTX-CAR cells express at least one chimeric antigen receptor (CAR) targeting an oncogenic gene product. In certain embodiments, the oncogenic gene product is BCMA, CD 19 or a combination thereof. In certain embodiments, the MTX- CAR cells are T-cells (i.e., MTX-CAR-T cells).

[0259] In certain aspects, provided are methods of increasing in vivo persistence of MTX- CAR-T -T cells in a subject comprising administering to the subject a therapeutically effective amount of MTX-CAR-T cells, wherein the MTX-CAR-T cells comprise a heterologous nucleic acid encoding dihydrofolate reductase (DHFR); and, administering at a predetermined time post administration of the MTX-CAR-T cells, an effective amount of MTX sufficient to eliminate (reduce by X) activated T-cells and NK cellsAttorney Docket No.: 000218-0154-WO1 targeting the MTX-CAR-T cells leading to an increased in vivo persistence of the MTX- CAR cells. In certain embodiments, the MTX-CAR-T cells express at least one chimeric antigen receptor (CAR) targeting an oncogenic gene product. In certain embodiments, the oncogenic gene product is BCMA, CD19 or a combination thereof. In certain embodiments, the MTX-CAR cells are T-cells (i.e., MTX-CAR-T cells).

[0260] In certain aspects, provided are methods of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of MTX-CAR-T cells; and administering at a predetermined time post administration of the MTX-CAR-T 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 cells and increased treatment efficacy of the MTX-CAR- T cells compared to no MTX administration. In certain embodiments, the MTX-CAR-T cells express at least one chimeric antigen receptor (CAR) targeting an oncogenic gene product. In certain embodiments, the oncogenic gene product is BCMA, CD 19 or a combination thereof. In certain embodiments, the MTX-CAR cells are T-cells (i.e., MTX-CAR-T cells).

[0261] In certain embodiments, the predetermined time post administration of the MTX- CAR cells is about 10 minutes. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 15 minutes. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 20 minutes. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 30 minutes. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 40 minutes. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 50 minutes.

[0262] In certain embodiments, the predetermined time post administration of the MTX- CAR cells is about 1 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 2 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 3 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 4 hr. In certain embodiments, the predetermined time post administration of the MTX- CAR cells is about 5 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 6 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 7 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is aboutAttorney Docket No.: 000218-0154-WO18 hr. In certain embodiments, the predetermined time post administration of the MTX- CAR cells is about 9 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 10 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 11 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 12 hr. In certain embodiments, the predetermined time post administration of the MTX- CAR cells is about 13 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 14 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 15 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 16 hr. In certain embodiments, the predetermined time post administration of the MTX- CAR cells is about 17 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 18 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 19 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 20 hr. In certain embodiments, the predetermined time post administration of the MTX- CAR cells is about 21 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 22 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 23 hr. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 24 hr.

[0263] In certain embodiments, the predetermined time post administration of the MTX- CAR cells is about 2 days. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 3 days. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 4 days. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 5 days. In certain embodiments, the predetermined time post administration of the MTX- CAR cells is about 6 days.

[0264] In certain embodiments, the predetermined time post administration of the MTX- CAR cells is about 1 week. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 2 weeks. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 3 weeks. In certain embodiments, the predetermined time post administration of the MTX-CAR cells is about 1 month.Attorney Docket No.: 000218-0154-WO1

[0265] In certain embodiments, the effective amount of MTX at a predetermined time post administration of the MTX-CAR cells is a single dose. In certain embodiments, the single dose is administered on Day 3 post-administration of the MTX-CAR cells. In certain embodiments, the single dose is administered on Day 6 post-administration of the MTX-CAR cells. In certain embodiments, the effective amount of MTX at a predetermined time post administration of the MTX-CAR cells is a multi-dose regiment. In certain embodiments, two doses are administered on Days 3 and 6 post-administration of the MTX-CAR cells. In certain embodiments, three doses are administered on Days 1, 3, and 6 post-administration of the MTX-CAR cells. In certain embodiments, four doses are administered on Days 1, 3, 6 and 11 post-administration of the MTX-CAR cells. In certain embodiments, eight doses are administered on Days 1, 3, 6, 11, 14, 18, 22 & 26 post-administration of the MTX-CAR cells.

[0266] In certain aspects, provided are methods of 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 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 cells is one day (Day -1) prior to administration of the MTX-CAR 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 cells is two days (Day -2) prior to administration of the MTX-CAR 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 cells is three days (Day -3) prior to administration of the MTX-CAR 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 cells is four days (Day -4) prior to administration of the MTX-CAR 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 cells is five days (Day -5) prior to administration of the MTX-CAR cells.Nucleic Acids and Vectors

[0267] In another aspect, provided herein are nucleic acids and vectors comprising the nucleic acids provided herein. 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 orAttorney Docket No.: 000218-0154-WO1RNA 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.

[0268] 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. In some aspects, 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).

[0269] 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.

[0270] Known methods of DNA or RNA amplification include, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188, to Mullis, et al.; 4,795,699 and 4,921,794 to Tabor, et al; 5,142,033 to Innis; 5,122,464 to Wilson, et al.; 5,091,310 to Innis; 5,066,584 to Gyllensten, et al; 4,889,818 to Gelfand, et al; 4,994,370 to Silver, et al; 4,766,067 to Biswas; 4,656,134 to Ringold) and RNA mediated amplification that uses anti-sense RNA to the target sequence as a template for double-stranded DNA synthesis (U.S. Pat. No. 5,130,238 to Malek, et al, with the tradename NASBA), the entire contents of which references are incorporated herein by reference. (See, e.g., Ausubel, supra, or Sambrook, supra

[0271] For instance, polymerase chain reaction (PCR) technology can be used to amplify the sequences of polynucleotides of the disclosure and related genes directly from genomic DNA or cDNA libraries. PCR and other 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. Examples of techniques sufficient to direct persons of skill through in vitro amplification methods are found in Berger, supra, Sambrook, supra, and Ausubel, supra, as well as Mullis, et al., U.S. Pat. No. 4,683,202 (1987); and Innis, et al., PCR Protocols A Guide to Methods and Applications, Eds., Academic Press Inc., San Diego, Calif. (1990). CommerciallyAttorney Docket No.: 000218-0154-WO1 available kits for genomic PCR amplification are known in the art. See, e.g., Advantage- GC Genomic PCR Kit (Clontech). Additionally, e.g., the T4 gene 32 protein (Boehringer Mannheim) can be used to improve yield of long PCR products.

[0272] 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. 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

[0273] The disclosure further provides recombinant expression cassettes comprising a nucleic acid or polynucleotide of the disclosure. A nucleic acid or polynucleotide 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.

[0274] In some aspects, isolated nucleic acid or polynucleotide sequences 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. For example, endogenous promoters can be altered in vivo or in vitro by mutation, deletion and / or substitution.Expression Vectors and Host Cells

[0275] The disclosure also relates to vectors that include isolated nucleic acid or polynucleotide molecules of the disclosure, host cells that are genetically engineered with the recombinant vectors, and the production of at least one protein scaffold byAttorney Docket No.: 000218-0154-WO1 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.

[0276] The polynucleotides can optionally be joined to a vector containing a selectable marker for propagation in a host. Generally, 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.

[0277] 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.

[0278] 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. 5,122,464; 5,770,359; 5,827,739), 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 prokaryotes (the above patents are entirely incorporated hereby by reference). Appropriate culture mediums and conditions for the abovedescribed host cells are known in the art. Suitable vectors will be readily apparent to the skilled artisan. Introduction of a vector construct into a host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid- mediated transfection, electroporation, transduction, infection or other known methods. Such methods are described in the art, such as Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, 16.

[0279] 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,Attorney Docket No.: 000218-0154-WO1 glycoproteins, or group of proteins that distinguish a cell or subset of cells from another defined subset of cells. Preferably 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).

[0280] 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 wildtype or mutant Neo, DHFR, TYMS, FRANCE, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

[0281] 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. Alternatively, 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. Such methods are well known in the art, e.g., as described in U.S. Pat. Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirely incorporated herein by reference.

[0282] Illustrative of cell cultures useful for the production of proteins disclosed herein are bacterial, yeast, and mammalian cells as known in the art. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions or bioreactors can also be used. A number of suitable host cell lines capable of expressing intact glycosylated proteins have been developed in the art, and include the 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) and BSC-1 (e.g., ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2 / 0-Agl4, 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-Agl4 cellsAttorney Docket No.: 000218-0154-WO1(ATCC Accession Number CRL-1851). In a preferred aspect, the recombinant cell is a P3X63Ab8.653 or an SP2 / 0-Agl4 cell.

[0283] 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. 5,168,062; 5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alpha promoter (EFla 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. Other cells useful for production of 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. An illustrative sequence encoding the EFla promoter is set forth in SEQ ID NO: 42.

[0284] When eukaryotic host cells are employed, polyadenylation or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is the polyadenylation 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)). Additionally, gene sequences to control replication in the host cell can be incorporated into the vector, as known in the art.

[0285] In certain aspects, provided are nucleic acids or polynucleotides comprising in the 5’ to 3’ direction: a nucleic acid sequence encoding iCas9 safety switch; a nucleic acid sequence encoding a chimeric antigen receptor (CAR) as disclosed herein; and a nucleic acid encoding a selectable marker.

[0286] In certain aspects, provided are nucleic acid or polynucleotides comprising in the 5’ to 3’ direction: a promoter; a nucleic acid sequence encoding iCas9 safety switch; a nucleic acid sequence encoding one or more chimeric antigen receptor (CAR) as disclosed herein; and a nucleic acid encoding a selectable marker.

[0287] In certain aspects, the nucleic acid sequence encoding iCas9 safety switch 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.Attorney Docket No.: 000218-0154-WO1

[0288] In certain aspects, the promoter is the EFla promoter. In certain embodiments, the EFla promoter comprises the nucleic acid sequence set forth in SEQ ID No: 42. In certain aspects, the iCas9 safety switch comprises the nucleic acid sequence set forth in SEQ ID No: 43. In certain aspects, the iCas9 safety switch comprises the amino acid sequence set forth in SEQ ID No: 46. In certain aspects, the selectable marker is a recombinant dihydrofolate reductase mutein gene (mDHFR). In certain aspects, the mDHFR comprises the nucleic acid sequence set forth in SEQ ID NO: 44. In certain aspects, the mDHFR comprises the amino acid sequence set forth in SEQ ID NO: 47. In certain aspects, the one or more CAR targets a gene product comprising BCM A, CD 19, or both BCMA and CD 19 as provided herein.

[0289] In certain aspects, provided are transposons comprising any of the polynucleotides of the present disclosure. In one embodiment, the transposon is a piggyBac transposon. In one embodiment, the piggyBac left end ITR of the piggyBac transposon comprises the nucleic acid sequence set forth in SEQ ID NO: 41. In one embodiment, the piggyBac right end ITR of the piggyBac transposon comprises the nucleic acid sequence set forth in SEQ ID NO: 45.

[0290] In certain aspects, provided are vectors comprising any of the polynucleotides of the present disclosure. In one embodiment, the vector comprises any of the transposons of the present disclosure.

[0291] In certain aspects, provided are cells comprising the polynucleotides, transposons or vectors of the present disclosure. In one embodiment, the cell expresses an iCas9 safety switch, one or more functional CAR as disclosed herein, and a selectable marker.

[0292] In certain aspects, provided are compositions comprising a T-cell population isolated from at least one healthy human donor; wherein the T-cell population expresses one or more chimeric antigen receptor as disclosed herein, and a selectable marker.

[0293] In certain aspects, provided are compositions comprising a T-cell population isolated from a patient having a disease or disorder; wherein the T-cell population expresses one or more chimeric antigen receptor as disclosed herein and mDHFR.

[0294] Illustrative sequences for the proteins that may be used in the constructs and methods described herein are set forth in Table 3.Attorney Docket No.: 000218-0154-WO1Table 3: Illustrative Nucleic Acid and Related Protein for use in the Polynucleotides, Transposons, Vectors and Cells of the Present DisclosureAttorney Docket No.: 000218-0154-WO1Certain Embodiments

[0295] Provided are certain non-limiting aspects of the disclosure.

[0296] Embodiment 1. A tandem BCMA heavy chain variable region (HV) comprising the amino acid sequence ofEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVAGIIGSGGSTYYADSVKGRFSISRDNSKNTLDLQMNSLRAEDTAVYYCVKDWNTTMITERGQGTLVTVSS (SEQ ID NO: 48) andEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMTWIRQAPGKGLEWVSGITGD GGSTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKDWNTTMITE RGQGTLVTVSS (SEQ ID NO: 49) joined by a linker.

[0297] Embodiment 2. The tandem BCMA HV of Embodiment 1, wherein the linker comprises the amino acid sequence ofAttorney Docket No.: 000218-0154-WO1GGGGSGGGGSGGGGS (SEQ ID NO: 50).

[0298] Embodiment 3. A tandem BCMA heavy chain variable region (VH) comprising the amino acid sequence ofEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVAGIIGS GGSTYYADSVKGRFSISRDNSKNTLDLQMNSLRAEDTAVYYCVKDWNTTMITE RGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFT FSNYAMTWIRQAPGKGLEWVSGITGDGGSTFYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCVKDWNTTMITERGQGTLVTVSS (SEQ ID NO: 51.

[0299] Embodiment 4. A chimeric antigen receptor (CAR) comprising (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least two BCMA heavy chain variable regions (VH); (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain; wherein the two BCMA HV regions comprising the amino acid sequences ofEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVAGIIGS GGSTYYADSVKGRFSISRDNSKNTLDLQMNSLRAEDTAVYYCVKDWNTTMITE RGQGTLVTVSS (SEQ ID NO: 48) andEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMTWIRQAPGKGLEWVSGITGD GGSTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKDWNTTMITE RGQGTLVTVSS (SEQ ID NO: 49).

[0300] Embodiment 5. The CAR of Embodiment 4, wherein the two BCMA HV regions are joined by a linker.

[0301] Embodiment 6. The CAR of Embodiment 5, wherein the linker comprises the amino acid sequence ofGGGGSGGGGSGGGGS (SEQ ID NO: 50).

[0302] Embodiment 7. The CAR of Embodiment 4, wherein the ectodomain further comprises a signal peptide.

[0303] Embodiment 8. The CAR of Embodiment 7, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 1.

[0304] Embodiment 9. The CAR of any one of Embodiments 4-7, wherein CAR further comprises a hinge region between the antigen recognition region and the transmembrane domain.

[0305] Embodiment 10. The CAR of Embodiment 9, wherein the hinge region comprises the amino acid sequence of SEQ ID NO: 9.Attorney Docket No.: 000218-0154-WO1

[0306] Embodiment 11. The CAR of Embodiment 4, wherein the transmembrane domain comprises a CD8 transmembrane domain.

[0307] Embodiment 12. The CAR of Embodiment 11, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 3.

[0308] Embodiment 13. The CAR of Embodiment 4, wherein the at least one costimulatory domain comprises a CD3(^ costimulatory domain, a 4-1BB costimulatory domain, a TACI costimulatory domain, or a combination thereof.

[0309] Embodiment 14. The CAR of Embodiment 13, wherein the at least one costimulatory domain comprises a CD3(^ costimulatory domain and a 4-1BB costimulatory domain, and wherein the 4-1BB costimulatory domain is located between the transmembrane domain and the CD3(^ costimulatory domain.

[0310] Embodiment 15. The CAR of Embodiment 14, wherein the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 7.

[0311] Embodiment 16. The CAR of Embodiment 14, wherein the CD3(^ costimulatory domain comprises the amino acid sequence of SEQ ID NO: 5.

[0312] Embodiment 17. A tandem BCMA chimeric antigen receptor (CAR) comprising the amino acid sequence ofMALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYA MNWVRQAPGKGLEWVAGIIGSGGSTYYADSVKGRFSISRDNSKNTLDLQMNSL RAEDTAVYYCVKDWNTTMITERGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLE SGGGLVQPGGSLRLSCAASGFTFSNYAMTWIRQAPGKGLEWVSGITGDGGSTFY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKDWNTTMITERGQGTL VTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAP LAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR (SEQ ID NO: 53)

[0313] Embodiment 18. A dual chimeric antigen receptor ( dual CAR) comprising (a) a first ectodomain comprising a first antigen recognition region, wherein the first antigen recognition region comprises at least one BCMA heavy chain variable (VH) region; (b) a first transmembrane domain, (c) a first endodomain comprising at least one costimulatory domain; (d) a second ectodomain comprising a second antigen recognition region; (e) a second transmembrane domain, (f) a second endodomain comprising at least oneAttorney Docket No.: 000218-0154-WO1 costimulatory domain; wherein the second antigen recognition region comprises a CD 19 VH region.

[0314] Embodiment 19. The dual CAR of Embodiment 18, wherein the first antigen recognition region comprises the BCMA VH amino acid sequence of

[0315] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVAGIIGSGGSTYYADSVKGRFSISRDNSKNTLDLQMNSLRAEDTAVYYCVKDWNTT MITERGQGTLVTVSS (SEQ ID NO: 48) orEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMTWIRQAPGKGLEWVSGITGD GGSTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKDWNTTMITE RGQGTLVTVSS (SEQ ID NO: 49) or a combination thereof.

[0316] Embodiment 20. The dual CAR of Embodiment 19, wherein the first antigen recognition region comprises the BCMA VH amino acid sequence of

[0317] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVAGIIGSGGSTYYADSVKGRFSISRDNSKNTLDLQMNSLRAEDTAVYYCVKDWNTT MITERGQGTLVTVSS (SEQ ID NO: 48) andEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMTWIRQAPGKGLEWVSGITGD GGSTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKDWNTTMITE RGQGTLVTVSS (SEQ ID NO: 49)

[0318] Embodiment 21. The CAR of Embodiment 20, wherein the two BCMA HV regions are joined by a linker.

[0319] Embodiment 22. The CAR of Embodiment 21, wherein the linker comprises the amino acid sequence of

[0320] GGGGSGGGGSGGGGS (SEQ ID NO: 50).

[0321] Embodiment 23. The dual CAR of Embodiment 18, wherein the second antigen recognition region comprises the CD19 VH amino acid sequence ofQVQLVESGGGLVKPGGSLRLSCAASGFDFSDYYMSWIRQAPGKGLEWVSYMSSSGSTIYYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCARGGIAAAGTW GQGTLVTVSS (SEQ ID NO: 57)

[0322] Embodiment 24. The dual CAR of Embodiment 18, wherein the first and second ectodomain further comprises a signal peptide.

[0323] Embodiment 25. The dual CAR of Embodiment 24, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 1.Attorney Docket No.: 000218-0154-WO1

[0324] Embodiment 26. The dual CAR of Embodiment 18, wherein dual CAR further comprises a first hinge region between the first antigen recognition region and the first transmembrane domain.

[0325] Embodiment 27. The dual CAR of Embodiment 18, wherein dual CAR further comprises a second hinge region between the second antigen recognition region and the second transmembrane domain.

[0326] Embodiment 28. The CAR of Embodiment 26 or 27, wherein the hinge region comprise the amino acid sequence of SEQ ID NO: 9.

[0327] Embodiment 29. The dual CAR of Embodiment 18, wherein the first or second transmembrane domain or both the first and second transmembrane domain comprise a CD8 transmembrane domain.

[0328] Embodiment 30. The dual CAR of Embodiment 29, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 3.

[0329] Embodiment 31. The CAR of Embodiment 18, wherein the at least one costimulatory domain of the first and / or second endodomain comprises a CD3(^ costimulatory domain, a 4-1BB costimulatory domain, a TACI costimulatory domain, or a combination thereof.

[0330] Embodiment 32. The CAR of Embodiment 31, wherein the at least one costimulatory domain of the first endodomain comprises a CD3(^ costimulatory domain and a 4-1BB costimulatory domain, and wherein the 4-1BB costimulatory domain is located between the first transmembrane domain and the CD3(^ costimulatory domain.

[0331] Embodiment 33. The CAR of Embodiment 32, wherein the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 7.

[0332] Embodiment 34. The CAR of Embodiment 32, wherein the CD3(^ costimulatory domain comprises the amino acid sequence of SEQ ID NO: 5.

[0333] Embodiment 35. The CAR of Embodiment 31, wherein the at least one costimulatory domain of the second endodomain comprises a CD3(^ costimulatory domain and a TACI costimulatory domain, and wherein the TACI costimulatory domain is located between the second transmembrane domain and the CD3(^ costimulatory domain.

[0334] Embodiment 36. The CAR of Embodiment 35, wherein the TACI costimulatory domain comprises the amino acid sequence of SEQ ID NO: 13.

[0335] Embodiment 37. The CAR of Embodiment 35, wherein the CD3(^ costimulatory domain comprises the amino acid sequence of SEQ ID NO: 5.Attorney Docket No.: 000218-0154-WO1

[0336] Embodiment 38. A dual chimeric antigen receptor (dual CAR) comprising the amino acid sequence ofMALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYA MNWVRQAPGKGLEWVAGIIGSGGSTYYADSVKGRFSISRDNSKNTLDLQMNSL RAEDTAVYYCVKDWNTTMITERGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLE SGGGLVQPGGSLRLSCAASGFTFSNYAMTWIRQAPGKGLEWVSGITGDGGSTFY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKDWNTTMITERGQGTL VTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAP LAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR (SEQ ID NO: 53) andMALPVTALLLPLALLLHAARPQVQLVESGGGLVKPGGSLRLSCAASGFDFSDYY MSWIRQAPGKGLEWVSYMSSSGSTIYYADSVKGRFTISRDNAKKSLYLQMNSLR AEDTAVYYCARGGIAAAGTWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPE ACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKKRGDPCSCQ PRSRPRQSPAKSSQDHAMEAGSPVSTSPEPVETCSFCFPECRAPTQESAVTPGTPD PTCAGRWGCHTRTTVLQPCPHIPDSGLGIVCVPAQEGGPGARVKFSRSADAPAY KQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 61)

[0337] Embodiment 39. A polynucleotide comprising a nucleic acid sequence encoding the tandem BCMA heavy chain variable region (HV) of Embodiment 3.

[0338] Embodiment 40. A polynucleotide comprising a nucleic acid sequence encoding the CAR of Embodiment 17 or dual car of Embodiment 38.

[0339] Embodiment 41. A transposon comprising a nucleic acid sequence encoding the CAR of Embodiment 17.

[0340] Embodiment 42. The transposon of Embodiment 41, wherein the transposon comprises the nucleic acid sequence of SEQ ID NO: 54.

[0341] Embodiment 43. A transposon comprising a nucleic acid sequence encoding the dual CAR of Embodiment 38.

[0342] Embodiment 44. The transposon of Embodiment 43, wherein the transposon comprises the nucleic acid sequence of SEQ ID NO: 64.Attorney Docket No.: 000218-0154-WO1

[0343] Embodiment 45. The transposon of Embodiment 41 or 43, wherein the transposon further comprises a nucleic acid encoding an inducible caspase polypeptide, a nucleic acid encoding a chimeric stimulatory receptor, a nucleic acid encoding a selection gene, a nucleic acid encoding a therapeutic agent, or a combination thereof.

[0344] Embodiment 46. The transposon of Embodiment 45, wherein the selection gene comprises a DHFR resistance gene.

[0345] Embodiment 47. The transposon of Embodiment 45, wherein the transposon is a piggyBac transposon.

[0346] Embodiment 48. The transposon of Embodiment 45, wherein the transposon comprises a nucleic acid sequence of SEQ ID NO: 66.

[0347] Embodiment 49. A vector comprising the polynucleotide of Embodiment 39 or 40.

[0348] Embodiment 50. A vector comprising the transposon of any one of Embodiments 41-44.

[0349] Embodiment 51. A cell comprising the BCMA VH of Embodiment 1.

[0350] Embodiment 52. A cell comprising the CAR of Embodiment 17.

[0351] Embodiment 53. A cell comprising the CAR of Embodiment 38.

[0352] Embodiment 54. A cell comprising the transposon of any one of Embodiment 41- 44.

[0353] Embodiment 55. A population of cells, wherein a plurality of the population of cells are modified to express the CAR of Embodiment 17.

[0354] Embodiment 56. The population of cells of Embodiment 55, wherein the plurality of modified cells is a plurality of modified immune cells.

[0355] Embodiment 57. The population of cells of Embodiment 56, wherein a portion of the immune cells of the plurality of immune cells comprises a genetic modification and wherein the genetic modification reduces or inhibits expression of a T-cell receptor or a major histocompatibility complex (MHC).

[0356] Embodiment 58. The population of cells of Embodiment 57, wherein the genetic modification comprises a sequence encoding a P-2 microglobulin (P2M) and wherein the genetic modification reduces or inhibits expression of a MHC I.

[0357] Embodiment 59. The population of cells of Embodiment 56, wherein the genetic modification comprises a sequence encoding an a chain (TCRa), a P chain (TCRP), or a combination thereof and wherein the genetic modification reduces or inhibits expression of a TCR.Attorney Docket No.: 000218-0154-WO1

[0358] Embodiment 60. The population of cells of Embodiment 55, wherein the plurality of modified cells is a plurality of modified T-cells.

[0359] Embodiment 61. The population of cells of Embodiment 60, wherein the plurality of the population of cells comprises 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 95%, at least 96%, at least 97%, at least 98%, at least 99% of cells that express the CAR of Embodiment 17.

[0360] Embodiment 62. A population of cells, wherein a plurality of the population of cells are modified to express the CAR of Embodiment 38.

[0361] Embodiment 63. The population of cells of Embodiment 62, wherein the plurality of modified cells is a plurality of modified immune cells.

[0362] Embodiment 64. The population of cells of Embodiment 63, wherein a portion of the immune cells of the plurality of immune cells comprises a genetic modification and wherein the genetic modification reduces or inhibits expression of a T-cell receptor or a major histocompatibility complex (MHC).

[0363] Embodiment 65. The population of cells of Embodiment 64, wherein the genetic modification comprises a sequence encoding a P-2 microglobulin (P2M) and wherein the genetic modification reduces or inhibits expression of a MHC I.

[0364] Embodiment 66. The population of cells of Embodiment 64, wherein the genetic modification comprises a sequence encoding an a chain (TCRa), a P chain (TCRP), or a combination thereof and wherein the genetic modification reduces or inhibits expression of a TCR.

[0365] Embodiment 67. The population of cells of Embodiment 62, wherein the plurality of modified cells is a plurality of modified T-cells.

[0366] Embodiment 68. The population of cells of Embodiment 67, wherein the plurality of the population of cells comprises 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 95%, at least 96%, at least 97%, at least 98%, at least 99% of cells that express the CAR of Embodiment 38.

[0367] Embodiment 69. A composition comprising the BCMA VH of Embodiment 1.

[0368] Embodiment 70. A composition comprising the CAR of Embodiment 17.

[0369] Embodiment 71. A composition comprising the CAR of Embodiment 38.Attorney Docket No.: 000218-0154-WO1

[0370] Embodiment 72. A composition comprising the cell of any one of Embodiments 51-53.

[0371] Embodiment 73. A composition comprising the population of cells of Embodiment 55 or 62.

[0372] Embodiment 74. A pharmaceutical composition comprising the composition of any one of Embodiments 69-73 and a pharmaceutically acceptable carrier.

[0373] Embodiment 75. A method of treating cancer in a subject in need thereof comprising administering a therapeutically effective amount of the composition of any one of Embodiments 69-73.

[0374] Embodiment 76. A method of treating cancer in a subject in need thereof comprising administering a therapeutically effective amount of the pharmaceutical composition of Embodiment 74.

[0375] Embodiment 77. The method of Embodiment 75, wherein the cancer is a primary tumor, a metastatic cancer, a multiply resistant cancer, a progressive tumor or recurrent cancer.

[0376] Embodiment 78. The method of Embodiment 75, wherein the cancer is a solid tumor.

[0377] Embodiment 79. The method of Embodiment 75, wherein the cancer is a lymphoma, a leukemia, a myeloma, a malignant immunoproliferative disease, a lung cancer, a brain cancer, a head and neck cancer, a breast cancer, a skin cancer, a liver cancer, a pancreatic cancer, a stomach cancer, a colon cancer, a rectal cancer, a uterine cancer, a cervical cancer, an ovarian cancer, a prostate cancer, a testicular cancer, a skin cancer or an esophageal cancer.

[0378] Embodiment 80. A method of treating an autoimmune disorder in a subject in need thereof comprising administering a therapeutically effective amount of the composition of any one of Embodiments 69-73.

[0379] Embodiment 81. A method of treating an autoimmune disorder in a subject in need thereof comprising administering a therapeutically effective amount of the pharmaceutical composition of Embodiment 74.

[0380] Embodiment 82. The method of Embodiment 80, wherein the autoimmune disorder is selected from: autoimmune neutropenia, Guillain-Barre syndrome, epilepsy, autoimmune encephalitis, Isaacs' syndrome, nevus syndrome, pemphigus vulgaris, deciduous pemphigus, bullous pemphigoid, acquired epidermolysis bullosa, gestational pemphigoid, mucous membrane pemphigoid, antiphospholipid syndrome, autoimmuneAttorney Docket No.: 000218-0154-WO1 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 (WAH4A), chronic inflammatory demyelinating polyneuropathy (CIDP), lupus nephritis, or membranous nephropathy.

[0381] Embodiment 83. A composition of any of the above embodiments for use in therapy.

[0382] Embodiment 84. A composition of any of the above embodiments for use in the preparation of a medicament.

[0383] Embodiment 85. The medicament of embodiment 84 for use in treating cancer.

[0384] Embodiment 86. The medicament of embodiment 85 for use in treating a lymphoma, a leukemia, a myeloma, a malignant immunoproliferative disease, a lung cancer, a brain cancer, a head and neck cancer, a breast cancer, a skin cancer, a liver cancer, a pancreatic cancer, a stomach cancer, a colon cancer, a rectal cancer, a uterine cancer, a cervical cancer, an ovarian cancer, a prostate cancer, a testicular cancer, a skin cancer or an esophageal cancer.

[0385] Embodiment 87. The medicament of embodiment 84 for use in treating an autoimmune disorder.

[0386] Embodiment 88. The medicament of embodiment 87 for use in treating autoimmune neutropenia, Guillain-Barre 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, or membranous nephropathy.Definitions

[0387] As used throughout the disclosure, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.Attorney Docket No.: 000218-0154-WO1

[0388] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more standard deviations. Alternatively, “about” can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value.

[0389] 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. Thus, 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. Optimally, 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. For example, in various aspects, 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. When the protein of the disclosure or biologically active portion thereof is recombinantly produced, 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.

[0390] The disclosure provides fragments and variants of the disclosed DNA sequences and proteins encoded by these DNA sequences. As used throughout the disclosure, the term "fragment" refers to a portion of the DNA sequence or a portion of the amino acid sequence and hence protein encoded thereby. 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. Alternatively, 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. Thus, fragments of a DNA sequence may range from at leastAttorney Docket No.: 000218-0154-WO1 about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full- length polynucleotide of the disclosure.

[0391] As used herein, "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.

[0392] “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.

[0393] The term “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.

[0394] 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. The ability of non-covalently linked components, such as proteins, to associate and dissociate enables a functional association only or primarily under circumstances where such association is needed for the desired activity. The linkage may be of duration sufficient to allow the desired effect.

[0395] 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.

[0396] 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 aminoAttorney Docket No.: 000218-0154-WO1 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.

[0397] 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.

[0398] 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.

[0399] 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.

[0400] Given the redundancy in the genetic code, a plurality of nucleotide sequences may encode any particular protein. All such nucleotides sequences are contemplated herein.

[0401] As used throughout the disclosure, 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 theAttorney Docket No.: 000218-0154-WO1 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.

[0402] As used throughout the disclosure, the term "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. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operatorpromoter, 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.

[0403] A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the 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. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, incorporated fully herein by reference.Attorney Docket No.: 000218-0154-WO1

[0404] Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity. Substitutions can be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, 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.

[0405] As used herein, “conservative” amino acid substitutions may be defined as set out in Tables A, B, or C below. In some aspects, 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 5.Table 5: Conservative Amino Acid Substitutions I

[0406] 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 6.Attorney Docket No.: 000218-0154-WO1Table 6: Conservative Amino Acid Substitutions II

[0407] Alternately, exemplary conservative substitutions are set out in Table 7.Table 7: Conservative Amino Acid Substitutions IIIAttorney Docket No.: 000218-0154-WO1

[0408] It should be understood that the 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. As used throughout the disclosure, “sequence identity” may be determined by using the standalone 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). The terms "identical" or "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. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0409] As used throughout the disclosure, 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.

[0410] As used throughout the disclosure, the term "exogenous" refers to nucleic acid or protein sequence not naturally associated with a target gene or a host cell into which it isAttorney Docket No.: 000218-0154-WO1 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.

[0411] The disclosure provides methods of introducing a polynucleotide construct comprising a DNA sequence into a host cell. By "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.EXAMPLES

[0412] The Examples in this section are provided for illustration and are not intended to limit the invention.Example 1: In Vivo Tumor Control by BCMA Binders

[0413] In vivo efficacy of BCMA binders were assessed using MMES xenograft model in NOD.CgPrkdcscldI12rgtmlw-’1 / SzJ (NSG) immunodeficient mice. MMES (pMMP-Luc- Neo) cells were injected intravenously via tail vein into each NSG mouse at 5 x 106tumor cells per mouse. 7 days after tumor injection, MMl.S-bearing NSG mice were treated with 4 x 106CAR-T cells via tail vein intravenous injection (N = 5 mice per group). Tumor burden was measured by bioluminescence (BLI) at the indicated timepoints (FIG. 1 A) and survival was assessed during the entire duration of the study (90 days post CAR-T injection, FIG. IB). VCAR.523 and VCAR202 demonstrated the best tumor control and survival.Example 2: In Vivo Tumor Control by CD19 Binders

[0414] In vivo efficacy of CD19 binders (FMC63, VH034, VH034.28D, VH034.54D, VH034.103S, VH034.104G, tandem T034.034 and VH034.28D54D) were assessed using Raji lymphoma subcutaneous xenograft model in NOD.CgPrkdcscldI12rgtmlwj1 / SzJ (NSG) immunodeficient mice (Jackson Laboratory). 5 x io5Raji-CBG-GFP cells were injected intravenously via the tail vein into each NSG mouse. 4 days post tumor injection, Raji- bearing NSG mice were treated with either 10 x 106T cells (Exp 1 and 2) or 5 x 106(ExpAttorney Docket No.: 000218-0154-WO13) via tail vein injection (N = 5 mice per group). The control group received the same volume of PBS as in the CAR-T treated groups. Tumor burden was measured weekly by bioluminescence (BLI) and blood samples were collected from the tail vein on days 7, 14, 21, 28, and 35 post CAR-T injection. Area under the curve (AUC) for tumor burden and T cell expansion in the peripheral blood was calculated for the entire duration of the study 35 days post CAR-T cell injection. VH034.28D was superior compared to VH03455D, VH03428D55D, and benchmark FMC63 (the CD19 binder used in FDA- approved CAR-T therapies Kymriah, Yescarta, Tecartus, and Breyanzi).Example 3: In Vivo Tumor Control by Single and Tandem BCMA Binders

[0415] In vivo efficacy of BCMA binders (VH523, and tandem constructs VH202.523 (“T202.523”) and VH523.202 (“T523.202”)) were assessed using RPMI-8226 multiple myeloma or Pfeiffer diffuse large B cell lymphoma subcutaneous xenograft model in NOD.CgPrkdcscldI12rgtmlwj1 / SzJ (NSG) immunodeficient mice (Jackson Laboratory). RPMI8226 parental cells (BCMA+CD19-, Experiment (Exp) 1), RPMI-8226 CD19+ (BCMA+CD19+, Exp2), and Pfeiffer.Luc.GFP (BCMA+CD19+, Exp3) were injected subcutaneously into each NSG mouse at 10 x 106tumor cells per mouse. 10 days after tumor implantation, mice were treated with 2.5 x 106or 5 x 106CAR-T cells via tail vein intravenous injection (N = 5 mice per group). The control group received the same volume of PBS as in the CAR-T cell treated groups. Tumor burden was measured weekly by caliper for RPMI-8226 model (Exp 1 and 2, FIG. 3 A and 3B) and by bioluminescence (BLI) for Pfeiffer.Luc.GFP (Exp 3, FIG. 3C). Area under the curve (AUC) was calculated based on 41 days post CAR-T injection for Expl (FIG. 3 A), 35 days for Exp2 (FIG. 3B), 21 days for Exp3 (FIG. 3C). The tandem construct T202.523 performed similar to binder VH523 against RPMI-8226 parental and engineered RPMI-8226 CD 19+ and Pfeiffer. Both T202.523 and VH523 were advanced to dual CAR screening.Example 4: Impact of Dual CAR Orientation on Activity

[0416] In vivo efficacy of CD 19 CAR-T cells (VH34.28D), BCMA CAR-T cells (VH523) and BCMA / CD19 dual CAR-T cells (VH523 / VH34.28D or VH34.28D / VH523) was assessed using RPMI-8226 CD 19+ subcutaneous xenograft model in NOD.CgPrkdcscldI12rgtmlwj1 / SzJ (NSG) immunodeficient mice (Jackson Laboratory). All CAR constructs tested contained 4-1BB co-stimulatory and CD3(^ signaling domains and are shown in FIG. 4. RPMI8226 RPMI-8226 CD 19+ cells were injected subcutaneouslyAttorney Docket No.: 000218-0154-WO1 into each NSG mouse at 10 x 106tumor cells per mouse. 10 days after tumor implantation, mice were treated with 2.5 x 106CAR-T cells via tail vein intravenous injection (N = 5 mice per group). The control group received the same volume of PBS as in the CAR-T cell treated groups (N = 5 mice per group). Tumor burden was measured weekly by caliper (FIG. 5A) and T cell expansion (FIG. 5B) in peripheral blood collected from the tail vein on days 7, 14, 21, 28, and 35 post CAR-T injection. VH523 / VH34.28D dual CAR-T cells demonstrates superior in vivo activity over the dual CAR in VH34.28D / VH523 orientation as well as over single CAR-T cells (FIGs. 5A and 5B).Example 5: Dual CAR vs Single CAR Comparisons

[0417] In vivo efficacy of CD 19 CAR-T cells (VH034.28D), BCMA CAR-T cells (VH523 or T202.523) and BCMA / CD19 dual CAR-T cells (T202.523 / VH034.28D, VH034.28D / T202.523, or VH523 / VH034.28D) was assessed using RPMI-8226 CD19+ or Pfeiffer subcutaneous xenograft model in NOD.CgPrkdcscldI12rgtmlwj1 / SzJ (NSG) immunodeficient mice (Jackson Laboratory). RPMI8226 RPMI-8226 CD 19+ cells and Pfeiffer. Luc. GFP were injected subcutaneously into each NSG mouse at 10 x 106tumor cells per mouse. 10 days after tumor implantation, mice were treated with 2.5 x 106or 5 x 106CAR-T cells via tail vein intravenous injection (N = 5 mice per group). The control group received the same volume of PBS as in the CAR-T cell treated groups (N = 5 mice per group). Tumor burden was measured by caliper for RPMI-8226 CD 19+ model (FIG. 6A) and weekly by bioluminescence (BLI) for Pfeiffer.Luc.GFP (FIG. 6B).

[0418] BCMA / CD19 dual CAR-T cells expressing either VH523 or T202.523 BCMA binder in combination with the VH034.28D CD 19 binder demonstrate similar activity (FIGs. 6A and 6B).Example 6: Impact of Dual CARs Orientation on Durable Tumor Control

[0419] In vivo efficacy of BCMA / CD19 dual CAR-T cells (T202.523 / VH034.28D and VH034.28D / T202.523) were assessed using RPMI-8226 CD19+ subcutaneous xenograft model in NOD.CgPrkdcscldI12rgtmlw-’1 / SzJ (NSG) immunodeficient mice (Jackson Laboratory). RPMI8226 RPMI-8226 CD 19+ cells and were injected subcutaneously into each NSG mouse at 10 x 106tumor cells per mouse. 10 days after tumor implantation, mice were treated with 5 x 106or 5 x 106CAR-T cells via tail vein intravenous injection (N = 5 mice per group). The control group received the same volume of PBS as in the CAR-T cell treated groups (N = 5 mice per group). Tumor burden was measured byAttorney Docket No.: 000218-0154-WO1 caliper at the indicated timepoints. Dual CAR-T cells expressing T202.523 / VH034.28D demonstrated more durable tumor control over VH034.28D / T202.523 orientation (FIG. 7).Example 7: Durability of Response Following Serial Tumor Challenge

[0420] In vitro cytotoxicity of CD19 CAR-T cells (VH034.28D), BCMA CAR-T cells (VH523) and BCMA / CD19 dual CAR-T cells (T202.523 / VH034.28D) were assessed against RPMI-8226 CD 19+ tumor cells using Incucyte Serial Killing Assay. The initial challenge was set at 10: 1 effector-to-target cell ratio and re-challenged with 1 x 104of RPMI-8226 CD 19+ cells at the indicated timepoint (FIG. 8 A). At the end of the last challenge, CAR-T cells were immunophenotyped for PD1, TIGIT and TIM3 exhaustion marker expression (FIG. 8B).

[0421] T202.523 / VH034.28D dual CAR-T cells demonstrated long-term killing capacity in in vitro serial re-stimulation assay and expresses less exhaustion markers after 5th challenge (FIG. 8).Example 8: Antigen Specific Killing

[0422] Antigen-specific reactivity of CD 19 CAR-T cells (VH034.28D), BCMA CAR-T cells (VH523) and BCMA / CD19 dual CAR-T cells (T202.523 / VH034.28D) were assessed against RPMI-8226 tumor models expressing both BCMA and CD19 (BCMA+CD19+), BCMA only (BCMA+CD19-), CD 19 only (BCMA-CD19+) and neither (BCMA-CD19-). Incucyte serial killing assay was performed at 2.5: 1 effector-to- target cell ratio for 16 days. Area under the curve (AUC) for tumor growth is shown. Dual CAR-T demonstrates good tumor control in single and double antigen RPMI-8226 tumor cells but not their double KO derivative. Single BCMA and CD 19 CAR-T cells kill double antigen to a lesser extent and either the BCMA or CD 19 single antigen tumor cells, respectively. Similar results were seen with Pfeiffer DLBCL model (data not shown).Example 9: Evaluation of alternative Intracellular Domains (ICDs) for BCMA / CD19 dual CAR-T

[0423] In vivo efficacy of BCMA / CD19 dual CAR-T cells with different ICD combinations (see FIG. 10 A) were assessed using RPMI-8226 CD 19+ subcutaneous xenograft model in NOD.CgPrkdcscldI12rgtmlw-’1 / SzJ (NSG) immunodeficient mice (Jackson Laboratory). Immunophenotype for all of BCMA / CD19 dual CAR-T cellsAttorney Docket No.: 000218-0154-WO1 combinations was similar. RPMI8226 RPMI-8226 CD 19+ cells were injected subcutaneously into each NSG mouse at 10 x 106tumor cells per mouse. 10 days after tumor implantation, mice were treated with 5 x 106CAR-T cells via tail vein intravenous injection (N = 5 mice per group). The control group received the same volume of PBS as in the CAR-T cell treated groups (N = 5 mice per group). Tumor burden was measured by caliper (FIG. 10B) and T cell expansion (FIG. 10C) in peripheral blood collected from the tail vein on days 7, 14, 21, 28, and 35 post CAR-T injection. Tabulation of in vivo results in FIG. 10B are presented in FIG. 10D.

[0424] In vitro cytotoxicity was assessed against RPMI-8226 models expressing both BCMA and CD 19 (BCMA+CD19+), BCMA only (BCMA+CD19-) or CD 19 only (BCMA-CD19+). Incucyte killing was performed at 2.5: 1 effector-to-target cell ratio for 16 days. Percent tumor control is shown (FIG. 10D). 4-1BB / 4-1BB and 4-1BB / TACI ICD combinations ranked the best based on in vitro and in vivo data.Example 10: Superior In Vivo Activity of anti-BCMA anti-CD19 CAR Construct with 4-1BB / TACI ICD compared to 4-1BB / 4-1BB ICD Combination

[0425] In vivo efficacy of BCMA / CD19 dual CAR-T cells expressing dual CARs with 4- 1BB / 4-1BB or 4-1BB / TACI ICD combinations were assessed using RPMI-8226 CD19+ (FIG. 11 A) or Pfeiffer (FIG. 1 IB) subcutaneous xenograft model in NOD.CgPrkdcscldI12rgtmlwj1 / SzJ (NSG) immunodeficient mice (Jackson Laboratory).RPMI8226 RPMI-8226 CD 19+ cells and Pfeiffer.Luc.GFP were injected subcutaneously into each NSG mouse at 10 x 106tumor cells per mouse. 10 days after tumor implantation, mice were treated with 2.5 x 106or 5 x 106CAR-T cells via tail vein intravenous injection (N = 5 mice per group). The control group received the same volume of PBS as in the CAR-T cell treated groups (N = 5 mice per group). Tumor burden was measured by caliper for RPMI-8226 CD 19+ model (FIG. 11 A) and weekly by bioluminescence (BLI) for Pfeiffer.Luc.GFP (FIG. 1 IB). Immunophenotype of CAR- T cells was assessed 14 days post CAR-T cell injection. The BCMA / CD19 dual CAR-T cells containing 4- IBB costimulatory domain in the BCMA CAR and TACI in the CD 19 CAR (T202.523-4-1BB / VH034 28D-TACI) demonstrated superior in vivo tumor control at a stress dose of 2.5 x io6CAR-T cells per mouse compared to cells containing the dual CAR containing 4- IBB costimulatory domain in both the BCMA and CD 19 CARs (T202.523 -4- 1 BB / VH034_28D-4- IBB).Attorney Docket No.: 000218-0154-WO1Example 11: In Vitro Killing of a Candidate Tandem Anti-BCMA Anti-CD19 CAR- T Against Target Positive and Negative Cell Lines

[0426] The cytotoxic function of BCMA CAR and CD 19 CAR in a candidate tandem anti-BCMA anti-CD19 CAR-T cell in three different research lots was assessed in vitro following co-culture with BCMA and CD 19 dual or single antigen expressing RPMI- 8226 cells, as well as BCMA / CD19 target-negative RPMI-8226 cells (FIG. 13). Cytolytic activity of the candidate tandem anti-BCMA anti-CD19 CAR-T cells against GFP- positive tumor cell lines expressing target antigens was measured using Incucyte after coculture at different E:T ratios for 72 hours: 13(A) RPMI-8226 double positive (DP) cells, 13 (B) RPMI-8226 BCMA single positive (SP), 13(C) RPMI-8226 CD19 SP, 13(D) RPMI-8226 double negative (DN) cells. Non-transposed donor-matched mock T cells were used as controls. All 3 research lots displayed cytolytic activity against single and double antigen expressing RPMI-8226 cells at the range of effector-to-target (E:T) ratios tested, but not against target antigen-negative RPMI-8226 DN cells. Moreover, both individual CARs utilized in the candidate tandem anti-BCMA anti-CD19 CAR-T cells, BCMA CAR-T cells and CD 19 CAR-T cells, were functional, as demonstrated by specific killing of RPMI-8226 BCMA SP and RPMI-8226 CD19 SP cells, respectively. In contrast, non-transposed mock control cells did not display cytotoxicity against these cell lines at any of the E:T ratios tested, indicating CAR-specific cytolytic function of the candidate tandem anti-BCMA anti-CD19 CAR-T cells.

[0427] As a comparison, anti-BCMA CAR-T cells and anti-CD19 CAR-T cells were generated from donor C, using VH523 and VH034 28D binders, respectively, with a 4- 1BB costimulatory domain. These research lots were tested in the same study shown in FIG. 13. Unlike the candidate tandem anti-BCMA anti-CD19 CAR-T cells which demonstrates robust activity against single and double target antigen-positive tumor cells (RPMI-8226 DP, BCMA SP and CD19 SP cells), anti-BCMA CAR-T cells demonstrate cytotoxicity only against RPMI-8226 DP and BCMA SP cells but were ineffective against RPMI-8226 CD19 SP (FIG. 14). The background activity of anti-BCMA CAR-T cells against RPMI-8226 CD 19 SP is likely due to low levels of residual BCMA expression remaining after generation and sorting of RPMI-8226 BCMA-KO cells (data not shown). In contrast, anti-CD19 CAR-T cells exhibits robust activity against RPMI- 8226 DP and CD19 SP cells but not against RPMI-8226 BCMA SP. Both anti-BCMA CAR-T and anti-CD19 CAR-T cells do not exhibit cytotoxicity against RPMI-8226 DN cells. Together, these results demonstrate the ability of the candidate tandem anti-BCMAAttorney Docket No.: 000218-0154-WO1 anti-CD19 CAR-T to recognize tumor cells expressing BCMA, CD 19 or both antigens while anti -BCMA CAR-T and anti-CD19 CAR-T only recognize tumor cells that are BCMA and CD 19 positive, respectively.Example 12: In Vivo Efficacy of Tandem Anti-BCMA Anti-CD19 CAR-T in RPMI- 8226 DP MM Xenograft Model

[0428] In vivo efficacy of candidate tandem anti -BCMA anti-CD19 CAR-T cell research lots was assessed using RPMI-8226 DP cells as a BCMA / CD19 dual target antigen expressing subcutaneous xenograft model in NOD.Cg-PrkdcscldI12rgtmlwj1 / SzJ (NSG) immunodeficient mice. A dose response study was conducted based on the number of non-apoptotic BCMA / CD19 CAR-T cells measured by Annexin / Sytox staining 24hr post-thaw (research lots contained 21.1%, 15.1% and 24.4%, non-apoptotic CAR-T cells [NAC] manufactured from donors A, B and C, respectively).

[0429] To generate tumor-bearing NSG mice, 1 x 107RPMI-8226 DP cells were implanted subcutaneously. Once tumor volume reached 200 mm3(8 days post implant), candidate tandem anti -BCMA anti-CD19 CAR-T cells were injected at 3 dose levels of 0.3 x 106, 0.6 x 106, and 1.2 x 106NAC cells per mouse for donor A, and 0.6 x 106, 1.2 x 106, and 2.4 x 106NAC cells per mouse for donors B and C. Donor A did not receive the highest 2.4 x io6NAC cell dose as complete tumor control was already achieved with 1.2 x 106NAC cell dose (FIG. 15). Anti -turn or activity of candidate tandem anti -BCMA anti-CD19 CAR-T cell research lots was evaluated by measuring tumor volume by caliper; T cell expansion and persistence in peripheral blood was measured by flow cytometry once a week.

[0430] An anti -tumor response was observed for all 3 candidate research lots (FIG. 15) with tumor control at the dose level of 1.2 x 106(donor A) or 2.4 x 106(donors B and C) NAC cells / mouse. As expected, all 3 research lots had no-to-partial tumor control at the lowest dose levels of 0.3 x io6and 0.6 x io6NAC cells per mouse.

[0431] Consistent with observed anti -tumor activity, robust expansion of all 3 candidate research lots was observed at the dose levels of 1.2 x io6(donor A) and 2.4 x io6(donors B and C) NAC cells per mouse (FIG. 16). T cell expansion peaked between Day 7 and Day 14 followed by a retraction after Day 14 post CAR-T cell infusion. The candidate tandem anti -BCMA anti-CD19 CAR-T research lots contained a high percentage of desirable TSCM phenotype cells prior to dosing animals. After administration of candidate cells to tumor-bearing animals, the CAR-T cells differentiated to include more centralAttorney Docket No.: 000218-0154-WO1 memory T cells (TCM), effector memory T cells (TEM), and effector T cells (TEFF), presumably to facilitate anti-tumor activity, which then contracted back to lower frequencies following tumor regression (FIG. 17). TSCM cells persisted at detectable levels for the duration of studies in all groups treated with candidate cells.Example 13: In Vivo Efficacy Against Pfeiffer DLBCL Xenograft Model

[0432] In vivo efficacy of tandem anti-BCMA anti-CD19 CAR-T cell research lots was assessed using the Pfeiffer DLBCL model. A dose response study was conducted based on the number of non-apoptotic CAR-T cells (NAC) measured by Annexin / Sytox 24 hours post-thaw as described in Example 12. Anti-tumor activity of research lot manufactured from donor A was evaluated by measuring luminescence of Pfeiffer. Luc. GFP (Pfeiffer) cells (FIG. 18A); T cell expansion and persistence in peripheral blood was measured by flow cytometry (FIG. 18B).

[0433] Robust anti -tumor activity was observed at 1.2 * 106and 2.4 * 106NAC dose levels while only partial control was observed at a stress dose of 0.6 * 106NAC cells per mouse. Minimal T cell expansion was seen in the peripheral blood at the highest dose level of 2.4 * 106NAC cells per mouse with peak T cell expansion between Day 7 and Day 14 followed by a decrease in the circulating T cell numbers after Day 14 post-CAR- T-cell infusion. Similar to the RPMI-8226 DP MM model, the phenotype of candidate cells shifted from TscM-rich phenotype pre-infusion to a more differentiated phenotype after administration to the tumor-bearing mice that included TCM, TEM, and TEFF cells (FIG. 18C).Example 14: Activity and Binding of Single and Dual BCMA Binders to BCMA WT and Escape Mutants

[0434] The activity and binding of tandem VH202.523, as well as single VH523 and VH202 BCMA binders to wildtype BCMA (BCMA WT) and four known BCMA escape mutants (R27P, P33Del, P33S, P30Del) was tested. These mutations can affect the binding affinity of CAR-T therapies and monoclonal antibodies, potentially leading to decreased therapeutic effectiveness. The R27P mutation is particularly notable as it results in a substitution of a proline for arginine at position 27, which may alter the protein's structural stability and its interaction with therapeutic agents. Similarly, the P33Del mutation, characterized by a deletion of proline at position 33, can disrupt the normal function of BCMA, affecting its role in cell signaling pathways crucial for plasmaAttorney Docket No.: 000218-0154-WO1 cell survival. The P33S mutation introduces a serine at position 33, which may also impact BCMA's interaction with ligands and therapeutic agents. Lastly, the P30Del mutation, which involves the deletion of proline at position 30, poses similar challenges, potentially affecting both the protein's function and the immune response it elicits.

[0435] To assess reactivity (degranulation) and binding, 2.5* 106bead-stimulated T cells were electroporated with 2pg BCMA CAR mRNA (VHT202.523, VH523, VH202, MOCK) and 5* 106K562 cells were electroporated with 5 pg mRNA encoding BCMA WT or each of the mutants. For the degranulation assay, BCMA CAR mRNA transfected T cells were cocultured with BCMA mutant mRNA transfected K562 cells at E:T=1 :2 or 1 :4 in the presence of monensin and CD 107a antibody for 4 hours. FIG. 19 shows significant degranulation for VH523 RNA CAR-T cells against all escape mutants ( R27P, P33Del, P33S and S30Del) as well as wildtype, however degranulation against R27P was reduced by approximately 20-40% compared to wildtype. Similar results were observed for VH202. Tandem (T202.523) RNA CAR-T cells showed equivalent degranulation against all BCMA proteins (BCMA WT, R27P, P33Del, P33S and S30Del) and no reduction in degranulation against R27P.

[0436] For binding assay, K562 cells were blocked, incubated with VH-Fc fusion proteins (VHT202.523, VH523, VH202, mouse anti-human BCMA antibody clone 19F2 (Biolegend Cat: 357502), Anti-CD19 VH034T28D as negative control) for 30mins on ice, washed, and followed by secondary antibody staining for 30min on ice, washed, then analyzed by flow cytometer. Percent bound is shown in FIG. 20A, EC50 is shown in FIG. 20B and representative flow analysis is shown in FIG. 20C. Significant binding is seen for VH523-Fc to all BCMA proteins (WT, R27P, P33Del, P33S and S30Del), however binding of R27P was reduced by approximately 50% compared to wildtype. Similar results were observed for VH202. The tandem fusion protein (T202.523) showed equivalent binding to all BCMA proteins (WT, R27P, P33Del, P33S and S30Del) and no reduction in binding to R27P was observed.Example 15: Cytotoxicity of single BCMA (VH523) and tandem BCMA (T202.523) sPB-Generated CAR-T Cells Against K562 Cells Transfected with BCMA WT mRNA and Four Escape Mutants

[0437] In vitro cytotoxicity of VH523 and T202.523 CAR-T cells were evaluated against K562 cells transfected with BCMA WT mRNA and each of the BCMA escape mutants (R27P, P33Del, P33S, and S30Del) using Incucyte serial killing assay. CAR-T cells andAttorney Docket No.: 000218-0154-WO1K562 cells were co-incubated at 10: 1 and 2.5: 1 effector-to-target (E:T) ratios. Better activity was seen with T202.523 CAR-T compared to VH523 CAR-T (FIG. 21).Example 16: B Cell Specific Killing of Diseased PBMCs by CD19 CAR-T cells

[0438] The cytotoxic activity of the three research lots (Donors 1-3) of BCMACD19 CAR-T cells used in this study was assessed by flow cytometry. For this assay, 200,000 cryopreserved PBMC from healthy donors (HD) and patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS) as well as isolated T or B cells from healthy donors were plated in a 48-well non-treated in RPMI 1640 supplemented with 10% FBS and co-cultured in duplicates with BCMACD19 CAR-T cells for 48 hours at two effector-to-target (E:T) ratios of 1 : 1 and 5 : 1. At the end of the co-culture, all cells were collected and supernatant was stored for subsequent cytokine analysis. Cells were then stained for 20 minutes at 4°C in the dark with a viability stain (LIVE / DEAD™ Fixable Aqua, Invitrogen) and fluorophores conjugated mAbs specific for CD3, CD5, CD19, CD20, CD56 and CD14 (Biolegend, San Diego, CA) at dilutions recommended by manufacturer. To prevent nonspecific binding of the mAbs and reduce nonspecific fluorophores interactions with monocytes, cells were pre-incubated with Human TruStain FcX™ and True-Stain Monocyte Blocker™ solution (Biolegend, San Diego, CA) prior to addition of mAbs, according to the manufacturer’s instructions. After staining, cells were washed twice, CountBright™ Absolute Counting Beads (Invitrogen, Carlsbad, CA) were added to allow for quantification of each population and all samples were analyzed on a LSRFortessa™ X-20 cell analyzer (BD Biosciences, San Jose, CA). For each population, remaining cell numbers at the end of the co-culture were then normalized to the average cell numbers of the target sample alone (without CAR-T cells) to express data as % of surviving cells after treatment with CAR-T. FIG. 22 shows B Cell specific killing at both E:Ts with near complete depletion at 5: 1 ET in RA, SLE and MS samples. HD data similarly showed higher killing at 5: 1 ET and Mock T cell controls showed little to no targeting as expected (data not shown).Example 17: Elimination of CD81+CD19+progenitor cells and colony-forming units from bone marrow MM patient samples.

[0439] Cytolytic activity of BCMA CAR-T, CD 19 CAR-T, and P-BCMACD19-ALLO1 cells against primary CD81+CD19+cells were assessed by co-incubation of CAR-T cells with primary patient samples (Discovery Life Sciences Huntsville, AL) at 3 : 1 E:T ratio.Attorney Docket No.: 000218-0154-WO1After 24 hours of co-incubation, T cells were depleted by CD4 / CD8 magnetic beads (Thermo Fisher Life Technologies, San Diego, CA) and the remaining cells were stained for 20 minutes at 4 °C in the dark with a viability stain (LIVE / DEAD™ Fixable Aqua, Invitrogen) and fluorophores conjugated mAbs specific for CD4, CD8, CD 19, CD81 (Biolegend, San Diego, CA). To prevent non-specific binding of the mAbs, cells were pre-incubated with Human TruStain FcX™ (Biolegend, San Diego, CA) prior to addition of mAbs, according to the manufacturer’s instructions. The surviving CD81+CD19+cells were quantified by flow cytometry.

[0440] Representative flow plots of surviving CD81+CD19+cells are shown for BCMA CAR-T, CD19 CAR-T and P-BCMACD19-ALLO1 (FIGs. 23A-23C). Quantification of CD81+CD19+cell killing across 4 patient samples is shown in FIG. 23D. Anti-CD19 CAR-T and P-BCMACD19-ALLO1 were able to deplete CD81+CD19+subset whereas anti-BCMA CAR-T was ineffective.

[0441] Surviving progenitor cells following co-incubation with indicated CAR-T cells were further evaluated for their colony-forming capabilities. Surviving cells were grown at 37°C with 5% CO2 in MethoCult media (StemCell Technologies, Cambridge, MA) for 14 days. Colonies were visualized under a bright-phase microscope and enumerated. Percent killing of colony -forming units (CFUs) across 4 patient samples is shown in FIG. 23E. Anti-CD19 CAR-T and P-BCMACD19-ALLO1 demonstrate increased activity against CFUs versus anti-BCMA CAR-T.

Claims

Attorney Docket No.: 000218-0154-WO1CLAIMSWe claim:

1. A dual CAR comprising (a) a first ectodomain comprising a first antigen recognition region that binds to BCMA; (b) a first transmembrane domain, (c) a first endodomain comprising at least one costimulatory domain; (d) a second ectodomain comprising a second antigen recognition region that binds to CD 19; (e) a second transmembrane domain, (f) a second endodomain comprising at least one costimulatory domain; wherein the first antigen recognition region comprises the amino acid sequence ofEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVAGIIGS GGSTYYADSVKGRFSISRDNSKNTLDLQMNSLRAEDTAVYYCVKDWNTTMITE RGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFT FSNYAMTWIRQAPGKGLEWVSGITGDGGSTFYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCVKDWNTTMITERGQGTLVTVSS (SEQ ID NO: 51) .

2. The dual CAR of claim 1, wherein the second antigen recognition region comprises the amino acid sequence of QVQLVESGGGLVKPGGSLRLSCAASGFDFSDYYMSWIRQAPGKGLEWVSYMSS SGSTIYYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCARGGIAAAGTW GQGTLVTVSS (SEQ ID NO: 57)3. The dual CAR of claim 1 or 2, wherein the first ectodomain and / or second ectodomain further comprises a signal peptide.

4. The dual CAR of claim 3, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 1.

5. The dual CAR of any one of claims 1-4, further comprising a first hinge region between the first antigen recognition region and the first transmembrane domain and / a second hinge region between the second antigen recognition region and the second transmembrane domain.

6. The CAR of claim 5, wherein the first hinge region and / or the second hinge region comprises the amino acid sequence of SEQ ID NO: 9.

7. The dual CAR of any one of claims 1-6, wherein the first transmembrane domain and / or the second transmembrane domain comprises a CD8 transmembrane domain.Attorney Docket No.: 000218-0154-WO18. The dual CAR of claim 7, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 3.

9. The dual CAR of any one of claims 1-8, wherein the first endodomain comprises a CD3(^ costimulatory domain and a 4-1BB costimulatory domain, and wherein the 4- 1BB costimulatory domain is located between the first transmembrane domain and the CD3(^ costimulatory domain.

10. The dual CAR of claim 9, wherein the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 7.

11. The dual CAR of any one of claims 1-10, wherein the second endodomain comprises a CD3(^ costimulatory domain and a TACI costimulatory domain, and wherein the TACI costimulatory domain is located between the second transmembrane domain and the CD3(^ costimulatory domain.

12. The dual CAR of claim 11, wherein the TACI costimulatory domain comprises the amino acid sequence of SEQ ID NO: 13.

13. The dual CAR of any one of claims 9-12, wherein the CD3(^ costimulatory domain comprises the amino acid sequence of SEQ ID NO: 5.

14. A dual CAR comprising (a) the amino acid sequencesMALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYA MNWVRQAPGKGLEWVAGIIGSGGSTYYADSVKGRFSISRDNSKNTLDLQMNSL RAEDTAVYYCVKDWNTTMITERGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLE SGGGLVQPGGSLRLSCAASGFTFSNYAMTWIRQAPGKGLEWVSGITGDGGSTFY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKDWNTTMITERGQGTL VTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAP LAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR (SEQ ID NO: 53) and (b) the amino acid sequenceMALPVTALLLPLALLLHAARPQVQLVESGGGLVKPGGSLRLSCAASGFDFSDYY MSWIRQAPGKGLEWVSYMSSSGSTIYYADSVKGRFTISRDNAKKSLYLQMNSLR AEDTAVYYCARGGIAAAGTWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPE ACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKKRGDPCSCQ PRSRPRQSPAKSSQDHAMEAGSPVSTSPEPVETCSFCFPECRAPTQESAVTPGTPDAttorney Docket No.: 000218-0154-WO1PTCAGRWGCHTRTTVLQPCPHIPDSGLGIVCVPAQEGGPGARVKFSRSADAPAY KQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 61)15. A polynucleotide comprising a nucleic acid sequence encoding the dual car of any one of claims 1-14.

16. A transposon comprising a nucleic acid sequence encoding the dual CAR of any one of claims 1-15.

17. The transposon of claim 16, wherein the transposon comprises the nucleic acid sequence of SEQ ID NO: 64.

18. The transposon of claim 16 or 17, wherein the transposon further comprises a nucleic acid encoding an inducible caspase polypeptide, a nucleic acid encoding a chimeric stimulatory receptor, a nucleic acid encoding a selection gene, a nucleic acid encoding a therapeutic agent, or a combination thereof.

19. The transposon of claim 18, wherein the selection gene comprises a DHFR resistance gene.

20. The transposon of any one of claims 16-19, wherein the transposon is a piggyBac transposon.

21. The transposon of any one of claims 16-20, wherein the transposon comprises a nucleic acid sequence of SEQ ID NO: 66.

22. A vector comprising the polynucleotide of claim 15.

23. A vector comprising the transposon of any one of claims 16-21.

24. A cell comprising the dual CAR of any one of claims 1-14.

25. A cell comprising the transposon of any one of claim 16-21.

26. A population of cells comprising the dual CAR of any one of claims 1-14.

27. The population of cells of claim 26, wherein the cells are immune cells.

28. The population of cells of claim 26 or 27, wherein cells are T-cells.Attorney Docket No.: 000218-0154-WO129. The population of cells of any one of claims 26-28, wherein a portion of the immune cells comprises a genetic modification which reduces or inhibits expression of a T-cell receptor or a major histocompatibility complex (MHC).

30. The population of cells of claim 29, wherein the genetic modification is an insertion of a sequence encoding a P-2 microglobulin (P2M) and wherein the genetic modification reduces or inhibits expression of a MHC I.

31. The population of cells of claim 29, wherein the genetic modification is an insertion of a sequence encoding an a chain (TCRa), a P chain (TCRP), or a combination thereof and wherein the genetic modification reduces or inhibits expression of a TCR.

32. The population of cells of any one of claims 26-31, wherein at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of cells express the dual CAR.

33. A composition comprising the dual CAR of any one of claims 1-14.

34. A composition comprising the cell of claim 24 or 25.

35. A composition comprising the population of cells of any one of claims 26-32.

36. A pharmaceutical composition comprising the composition of any one of claims 33-35 and a pharmaceutically acceptable carrier.

37. A method of treating cancer in a subject in need thereof comprising administering a therapeutically effective amount of the composition of any one of claims 33-35. or the pharmaceutical composition of claim 3638. The method of claim 37, wherein the cancer is a primary tumor, a metastatic cancer, a multiply resistant cancer, a progressive tumor or recurrent cancer.

39. The method of claim 37, wherein the cancer is a solid tumor.

40. The method of claim 37, wherein the cancer is a lymphoma, a leukemia, a myeloma, a malignant immunoproliferative disease, a lung cancer, a brain cancer, a head and neck cancer, a breast cancer, a skin cancer, a liver cancer, a pancreatic cancer, aAttorney Docket No.: 000218-0154-WO1 stomach cancer, a colon cancer, a rectal cancer, a uterine cancer, a cervical cancer, an ovarian cancer, a prostate cancer, a testicular cancer, a skin cancer or an esophageal cancer.

41. The method of claim 37, wherein the cancer is diffuse large B cell lymphoma or multiple myeloma.

42. A method of treating an autoimmune disorder in a subject in need thereof comprising administering a therapeutically effective amount of the composition of any one of claims 33-35 or the pharmaceutical composition of claim 36.

43. The method of claim 42, wherein the autoimmune disorder is selected from: autoimmune neutropenia, Guillain-Barre 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, or membranous nephropathy.

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