Methods and compositions for enhancing in vivo persistence and potency of cell therapy

Abstract

The present disclosure relates to methods and compositions for increasing the in vivo persistence and potency of cell therapies including the administration of CAR cells in a subject.

Description

Attorney Docket No.: POTH-092 / 001WO METHODS AND COMPOSITIONS FOR ENHANCING IN VIVO PERSISTENCE AND POTENCY OF CELL THERAPY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is claims the benefit of U.S. Provisional Patent Applications No.63 / 625,657 filed January 26, 2024, No.63 / 698,868 filed September 25, 2024 and No. 63 / 734,944 filed December 17, 2024, each of which is incorporated herein by reference in its entirety. INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

[0002] The contents of the file named “POTH-092-001WO_SeqList.xml”, which was created on January 14, 2025 and is 126,622 bytes in size are hereby incorporated by reference in their entirety. FIELD

[0003] The disclosure is directed to molecular biology, and more, specifically, to methods and compositions for enhancing in vivo persistence and potency of cell therapies including CAR cells to maintain CAR cells for increased durations to improve clinical outcomes. 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 persistence, potency, and extended treatment duration compared to traditional cell-based therapies. SUMMARY

[0005] In certain aspects, provided are compositions comprising CAR cells engineered to recombinantly express a mutein dihydrofolate reductase (mDHFR), which advantageously results in CAR cells with increased resistance to methotrexate (MTX) or an MTX analog, generating MTX-resistant CAR cells (“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, CD19, CD20, MUC1C, PSMA, CD7, CD70 or c-kit. In certain embodiments, the MTX-CAR cells are T- cells (i.e., MTX-CAR-T cells).Attorney Docket No.: POTH-092 / 001WO

[0006] In certain aspects, provided are method of increasing in vivo persistence and potency of cell therapies. In certain aspects, provided are methods of increasing in vivo persistence of 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 to the subject an effective amount of MTX, or an MTX analog, sufficient to eliminate (reduce by at least 60%) activated T-cells and NK cells targeting the MTX-CAR cells; wherein administration of MTX, or an MTX analog, results in an increase 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, CD19, CD20, MUC1C, PSMA, CD70, CD7, or c-kit. In certain embodiments, the MTX-CAR cells are T-cells (i.e., MTX- CAR-T cells).

[0007] In certain aspects, provided are methods of increasing in vivo persistence and potency of MTX-CAR cells in a subject comprising administering to the subject a therapeutically effective amount of MTX-CAR cells, wherein the MTX-CAR cells comprise a heterologous nucleic acid encoding mutein dihydrofolate reductase (mDHFR) and express mDHFR, subsequently administering to the subject an effective amount of MTX, or an MTX analog, sufficient to eliminate (reduce by at least 60%) activated T-cells and NK cells targeting the MTX-CAR cells; wherein administration of MTX, or an MTX analog, results in 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, CD19, CD20, MUC1C, PSMA, CD70 or c-kit. In certain embodiments, the MTX-CAR cells are T-cells (i.e., MTX-CAR-T cells).

[0008] In certain aspects, provided are methods of increasing in vivo persistence of MTX- CAR cells in a subject in need thereof further comprising administering at a predetermined time an effective dose of MTX, or an MTX analog, 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 five days (Day -5) prior to administration of the MTX-CAR cells. In certain aspects, the administration at a predetermined time an effective dose of MTX post administration of the therapeutically effective dose of the MTX-CAR cells is on Days 1, 3, 6 and 11 days after administration of the MTX-CAR cells.Attorney Docket No.: POTH-092 / 001WO

[0009] 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 cells targeting an oncogenic gene product; and, administering to the subject at a predetermined time post administration of the MTX-CAR cells an effective dose of MTX, or an MTX analog, sufficient to eliminate activated T-cells and NK cells targeting the MTX- CAR cells; wherein the elimination of the activated T-cells and NK cells increases the in vivo persistence and efficacy of the MTX-CAR cells in the subject compared to no MTX administration.

[0010] In certain aspects, provided are methods of treating cancer in a subject in need thereof further comprising administering at a predetermined time an effective dose of MTX, or an MTX analog, prior to administration of the therapeutically effective dose of the MTX-CAR cells. In certain aspects, the administration a predetermined time an effective dose of MTX, or an MTX analog, 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. In certain aspects, the administration at a predetermined time an effective dose of MTX, or an MTX analog, post administration of the therapeutically effective dose of the MTX-CAR cells is on Days 1, 3, 6 and 11 days after administration of the MTX-CAR cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG.1 shows a graph of percent live cells of unstimulated pan T-cells, stimulated pan T-cells and antigen-stimulated, methotrexate resistant CAR-T cells. Pan T-cells were isolated from two donors, transfected with a CAR, and cultured in medium comprising increasing concentrations of methotrexate (“MTX”) (0nM, 5nM, 10nM, 15nM, 20nM, 32.5nM, 250nM and 1,000nM) for 72 hrs.

[0012] FIG.2 shows a graph illustrating the percent of live unstimulated pan T-cells, stimulated pan T-cells, and NK cells isolated from four donors and cultured in medium comprising increasing concentrations of MTX (0nM, 5nM, 10nM, 15nM, 20nM, 32.5nM, 250nM and 1,000nM) for 72 hrs.

[0013] FIGs.3A-3D show graphs of the results of a mixed lymphocyte reaction (MLR) using labeled unedited CAR-T cells or labeled modified CAR-T cells (gene edited to knock out the TRBC locus, the TRBC locus and beta-2-microglobulin gene (B2M), the TRBC locus and CD58 receptor or the TRBC locus and RFX5 gene) as Stimulator cells from Donor A (Fig 3A and 3B) or Donor B (Fig.3C and 3D) and non-labeled PBMCs or non-labeled pooled pan T-Attorney Docket No.: POTH-092 / 001WO cells from four donors as Responder Cells in the presence of cytokines in the absence (Fig. 3A and 3C) or presence of 250nM MTX (Fig 3B and 3D).

[0014] FIGs.4A and 4B show graphs of the percent of unedited CAR-T cells or modified CAR-T cells (gene edited to knock out the TRBC locus, the TRBC locus and beta-2- microglobulin gene (B2M), the TRBC locus and CD58 receptor or the TRBC locus and RFX5 gene) from Donor A or Donor B cultured in the presence of cytokines and in the absence (Fig.4A) or presence of 250nM MTX (Fig.4B).

[0015] FIGs.5A and 5B show graphs showing the precent of live human CAR-T Cells expressing mutein DHFR (mDHFR) compared to human CAR-T cells lacking mDHFR expression after treatment with MTX from two donors. Approximately 50,000 antigen- stimulated or low-level stimulated mDHFR+ CAR-T cells, mDHFR- CAR-T cells or pan T- cells derived from two separate healthy human donors (Donor A - Fig.5A and Donor B - Fig. 5B). Cells were seeded into 96 well plates in 100 µl R10 medium comprising increasing concentrations of MTX (0nM control, 250nM, 750nM or 1,500nM) for 72 hours. Cell viability (% live cells) was determined at 72 hours by flow cytometry.

[0016] FIGs.6A – 6D show graphs showing in vivo persistence of human CAR-T cells expressing mDHFR compared to human CAR-T cells lacking mDHFR expression. Tumor burden was measured weekly by whole-body bioluminescent imaging (BLI) and body weight measurements were taken twice weekly. The study was terminated on Day 28 post-CAR-T.

[0017] FIG.7 shows a graph showing the administration of MTX using an infusion pump. Mice (n = 2 / group) were administered 0.25 mg / kg or 2.5 mg / kg of MTX using an infusion pump (BrainTree Scientific, Inc. #BS-9008) in accordance with the manufacturer’s instructions using a 1 ml syringe (5.78mm syringe diameter), an infusion rate of 0.2 mL / hr (100 µL / 30 min), and a total volume of 0.4 µL infused over a two-hour period.

[0018] FIGs.8A, 8B, and 8C show graphs demonstrating the effect of mDHFR+ and mDHFR- CAR-T cells, in the presence or absence of MTX, on tumor burden (FIG.8A) and CAR-T cell expansion (FIG.8B) in Raji.CBG.GFP bearing NSG mice.0.5 mg / kg MTX was administered via an infusion pump to maintain an MTX serum level in the 500 nM range over the 5 day sampling period (FIG.8C).

[0019] FIGs.9A and 9B show tumor control and CAR-T cell expansion, respectively. In the presence and absence of methotrexate (MTX).Attorney Docket No.: POTH-092 / 001WO DETAILED DESCRIPTION

[0020] The present disclosure relates to methods and compositions for enhancing the in vivo persistence of CAR cells. In particular, the methods relate to enhancing in vivo persistence of methotrexate-resistant CAR cells by post CAR cell administration of methotrexate, or a methotrexate analog, to a subject to eliminate activated T-cells and / or NK cells targeting the methotrexate-resistant CAR cells, thereby increasing persistence and duration of the methotrexate-resistant CAR cells. Compositions

[0021] The CAR cells of the present disclosure may be engineered to recombinantly express dihydrofolate reductase (DHFR), which, without wishing to be bound by theory, is believed to advantageously renders the CAR cells resistant to methotrexate (MTX) and an MTX analogs thereof. 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 CAR cells thereby increasing the in vivo persistence, potency, and efficacy of the MTX-CAR cells in the subject.

[0022] Any cell expressing a CAR may be modified to generate MTX-CAR cell versions of CAR cells. An example of modifying a CAR cell may be to modify the CAR cell comprising a nucleic acid encoding the expressed CAR by introducing a nucleic acid encoding dihydrofolate reductase (DHFR) into the CAR cell and selecting for MTX-resistant cells. Alternatively, MTX-CAR cells may be prepared by co-introducing a nucleic acid encoding the chimeric antigen receptor and a nucleic acid encoding DHFR on a single transposon.

[0023] In some embodiments, the cell expressing a CAR is an immune cell. In some certain embodiments, the immune cell may be a T-cell, a Natural Killer (NK) cell, a Natural Killer (NK)-like cell, a Cytokine Induced Killer (CIK) cell, a hematopoietic progenitor cell, a peripheral blood (PB) derived T cell or an umbilical cord blood (UCB) derived T-cell. Preferably, the immune cell is a T-cell. In some embodiments, the T-cell may be an early memory cell, a stem-like T-cell, a TSCM-like cell, a TSCM or a TCM. In some embodiments, the T-cell may be a TSCM. In some embodiments, the cell may be an artificial antigen presenting cell, which, optionally, may be used to stimulate and expand a modified immune cell or T cell of the disclosure. In some embodiments the cell may be a tumor cell, which, optionally, may be used as an artificial or modified antigen presenting cell.Attorney Docket No.: POTH-092 / 001WO Chimeric Antigen Receptors

[0024] In certain embodiments of the MTX-CAR compositions 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. In certain embodiments, the ectodomain of (a) further comprises a signal peptide. In certain embodiments, the ectodomain of (a) further comprises a hinge between the ligand recognition region and the transmembrane domain. Signal Peptides

[0025] In certain embodiments, the signal peptide is a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR signal peptide. In certain embodiments, the signal peptide is a human CD8α signal peptide or a portion thereof. In certain embodiments, the signal peptide comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 12). In certain embodiments, the signal peptide comprises or consists of the amino acid of SEQ ID NO: 12.

[0026] In certain embodiments, the signal peptide is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence atggcactgccagtcaccgccctgctgctgcctctggctctgctgctgcacgcagctagacca (SEQ ID NO: 3) or atggctctgcctgtgacagctctgcttctgcctctggcactgcttcttcatgcggcgcgccct (SEQ ID NO: 13). In certain embodiments, the signal peptide is encoded by the nucleic acid sequence of SEQ ID NO: 3 or 13. Transmembrane Domains

[0027] In certain embodiments, the transmembrane domain is a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR transmembrane domain. In certain embodiments, the transmembrane domain is a human CD8α transmembrane domain. In certain embodiments, the transmembrane domain comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 4). In certain embodiments, the transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 4.Attorney Docket No.: POTH-092 / 001WO

[0028] In certain embodiments, the transmembrane domain is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence atctacatttgggcaccactggccgggacctgtggagtgctgctgctgagcctggtcatcacactgtactgc (SEQ ID NO: 5) or atctatatctgggcccctctggccggaacatgtggcgttctgctgctcagcctggtcatcaccctgtact gc (SEQ ID NO: 16). In certain embodiments, the transmembrane domain is encoded by the nucleic acid sequence of SEQ ID NO: 5 or 16. Endodomains

[0029] In certain embodiments, the endodomain comprises at least one costimulatory domain. In some embodiments, the endodomain comprises a human CD3ζ endodomain.

[0030] In some embodiments, the CD3ζ intracellular domain comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 52). In some embodiments, the CD3ζ intracellular domain comprises or consists of the amino acid sequence of SEQ ID NO: 52.

[0031] In some embodiments, the CD3ζ intracellular domain is encoded by a polynucleotide comprising or consisting of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence cgcgtgaagtttagtcgatcagcagatgccccagcttacaaacagggacagaaccagctgtataacgagctgaatctgggccgccga gaggaatatgacgtgctggataagcggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaaccctcaggaagg cctgtataacgagctgcagaaggacaaaatggcagaagcctattctgagatcggcatgaagggggagcgacggagaggcaaagg gcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgcaggcactgcctccaagg (SEQ ID NO: 19) or agagtgaagttcagcagatccgccgacgcacccgcctataagcagggacagaatcagctgtacaacgagctgaatctggggcgca gagaagagtacgacgtgctggacaagagaagaggcagggaccctgagatgggcggcaagcccagaagaaagaaccctcaagag ggcctgtataatgagctgcagaaagacaagatggccgaggcctacagcgagatcggaatgaagggcgagcgcagaagaggcaag ggtcacgatggactgtaccagggcctgagcaccgccaccaaggatacctatgatgccctgcacatgcaggccctgcctccaaga SEQ ID NO: 20. Preferably, the CD3ζ intracellular domain is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20.

[0032] In certain embodiments, the at least one costimulatory domain is a human 4-1BB, CD28, CD40, ICOS, MyD88, OX-40 intracellular segment, or any combination thereof. InAttorney Docket No.: POTH-092 / 001WO certain embodiments, the at least one costimulatory domain is a human CD28 and / or a 4-1BB costimulatory domain. In certain embodiments, the CD28 costimulatory domain comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 6). In certain embodiments, the CD28 costimulatory domain comprises or consists of the amino acid sequence of SEQ ID NO: 6.

[0033] In certain embodiments, the CD28 costimulatory domain is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence agaagcaagcggagccggctgctgcacagcgactacatgaacatgacccctagacggcccggacctaccagaaagcactaccag ccttacgctcctcctagagacttcgccgcctaccggtcc (SEQ ID NO: 7). In certain embodiments, the CD28 costimulatory domain is encoded by the nucleic acid sequence of SEQ ID NO: 7.

[0034] In certain embodiments, the 4-1BB costimulatory domain comprises or consist of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO: 8). In certain embodiments, the 4-1BB costimulatory domain comprises or consist of the amino acid sequence of SEQ ID NO: 8.

[0035] In certain embodiments, the 4-1BB costimulatory domain is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence aagagaggcaggaagaaactgctgtatattttcaaacagcccttcatgcgccccgtgcagactacccaggaggaagacgggtgctcc tgtcgattccctgaggaagaggaaggcgggtgtgagctg (SEQ ID NO: 9). In certain embodiments, the 4- 1BB costimulatory domain is encoded by the nucleic acid sequence of SEQ ID NO: 9.

[0036] In certain embodiments, the 4-1BB costimulatory domain is located between the transmembrane domain and the CD28 costimulatory domain. Hinge Domains

[0037] In certain embodiments, the hinge comprises a sequence derived from a human CD8α, IgG4, and / or CD4 sequence. In certain embodiments, the hinge comprises a sequence derived from a human CD8α sequence. In certain embodiments, the hinge comprises or consist of anAttorney Docket No.: POTH-092 / 001WO amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 10). In certain embodiments, the hinge comprises or consist of the amino acid of SEQ ID NO: 10.

[0038] In certain embodiments, the hinge is encoded by a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequenceactaccacaccagcacctagaccaccaactccagctccaaccatcgcgagtcagcccctgagtctgagacctgaggcc tgcaggccagctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 11), accacaacaccggcgcctagacctccaacaccagctcctacaatcgcgagtcagcccctgtctctcagacccgaagcctgcaggcc agctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 14) or acaacaacaccggcgcctcggcctccaacaccagctcctacaattgctagccagccactgtctctgaggcccgaggcttgtagacct gctgctggcggagctgtgcacacaagaggactggatttcgcctgcgac (SEQ ID NO: 15). In certain embodiments, the hinge is encoded by the nucleic acid sequence of SEQ ID NO: 11, 14 or 15. Ligand Recognition Regions

[0039] The chimeric receptors (e.g., CARs) described herein comprise a ligand recognition region. In certain embodiments, the chimeric ligand receptor comprises at least one protein scaffold which specifically binds the ligand.

[0040] In certain embodiments of the MTX-CAR cell compositions of the disclosure, the chimeric ligand receptor comprises (a) an ectodomain comprising a ligand recognition region, wherein the ligand recognition region comprises at least scaffold protein; (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. In certain embodiments, the at least one protein scaffold comprises an antibody, an antibody fragment, a single domain antibody, a single chain antibody, an antibody mimetic, or a Centyrin. In certain embodiments, the ligand recognition region comprises one or more of an antibody, an antibody fragment, a single domain antibody, a single chain antibody, an antibody mimetic, and a Centyrin. In certain embodiments, the single domain antibody comprises or consists of a VHH. In certain embodiments, the antibody mimetic comprises or consists of an affibody, an afflilin, an affimer, an affitin, an alphabody, an anticalin, an avimer, a DARPin, a Fynomer, a Kunitz domain peptide or a monobody. In certain embodiments, the Centyrin comprises or consists of a consensus sequence of at least one fibronectin type III (FN3) domain.

[0041] In certain aspects, the chimeric antigen receptor (CAR) is an scFv CAR.Attorney Docket No.: POTH-092 / 001WO

[0042] In certain aspects, the scFv CAR comprises a heavy chain variable region comprising or consisting of the amino acid sequence QVQLVQSGAEVKKPGSSVKX1SCKTSGYAFSNFWMNWVX2QX3PGQGLEWIGQIYP GDGDTNYNX4KFKGRX5TLTADKSX6STAYMELSSLRSEX7TAVYFCARSYYRSAWF AYWGQGTLVTVSS (SEQ ID NO:1), wherein X1 of SEQ ID NO: 1 is V or I, wherein X2 of SEQ ID NO: 1 is R or K, wherein X3 of SEQ ID NO: 1 is A or R, wherein X4 of SEQ ID NO: 1 is G or A, wherein X5 of SEQ ID NO: 1 is V or A, wherein X6 of SEQ ID NO: 1 is T or S, and wherein X7 of SEQ ID NO: 1 is D or A; and a light chain variable region comprising or consisting of the amino acid sequenceEILLTQSPDFQSVTPKEKVTFTCRASQSIGTSIHWYQQKPNQSPKLLIKYASES ISGVPSRFSGSGSGTDFTLX1INSX2ESEDIAX3YYCQQSNNWPLTFGQGTKLEIK (SEQ ID NO: 2), wherein X1 of SEQ ID NO: 2 is T or S, wherein X2 of SEQ ID NO: 2 is L or V, and wherein X3 of SEQ ID NO: 2 is T or D.

[0043] In certain embodiments, the scFv CAR comprises an ectodomain comprising antigen recognition region, wherein the antigen recognition region comprises at least one anti-MUC1 single chain variable fragment (scFv) of the disclosure; a transmembrane domain, and an endodomain comprising at least one costimulatory domain. The CAR can further comprise a hinge region between the antigen recognition domain and the transmembrane domain. The antigen recognition region can comprise at least two anti-MUC1 scFv. The antigen recognition region can comprise at least three anti-MUC1 scFv. In one aspect, a CAR of the disclosure is a bi-specific CAR comprising at least two scFvs that specifically bind two distinct antigens.

[0044] A composition of the present disclosure (e.g., an anti-MUC1 scFv, CAR comprising an anti-MUC1 scFv) may bind human MUC1 with at least one affinity selected from a KD of less than or equal to 10−9M, less than or equal to 10−10M, less than or equal to 10−11M, less than or equal to 10−12M, less than or equal to 10−13M, less than or equal to 10−14M, and less than or equal to 10−15M. The KDmay be determined by any means, including, but not limited to, surface plasmon resonance.

[0045] In certain aspects, the CAR is a VCAR. In certain embodiments of the VCARs of the disclosure, the VCAR comprises a single domain antibody, VHH, VH or a combination thereof. In some embodiments, the single domain antibody, VHH or VH comprises or consists of a recombinant sequence and / or a chimeric sequence. In some embodiments, the single domain antibody, VHH or VH comprises or consists of a human sequence and / or a humanized sequence.Attorney Docket No.: POTH-092 / 001WO

[0046] 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.

[0047] In certain embodiments, the VCAR comprises an anti-BCMA VHH sequence. In certain embodiments, the VHH comprises or consists of the amino acid sequence:

[0048] MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFSSY AMNWVRQAPGKGLEWVAGIIGSGGSTYYADSVKGRFSISRDNSKNTLDLQMNSLRA EDTAVYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFM RPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREE YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR (VH-A; SEQ ID NO: 21).

[0049] In some embodiments, the VHH is encoded by the nucleic acid sequence:

[0050] atggctctgcctgtgacagctctgctgctgcctctggctctgcttcttcatgcggcgcgccctgaagttcagctgcttgaatct ggcggaggcctggttcaacctggcggatctctgagactgagctgtgccgccagcggcttcacctttagcagctacgccatgaactgg gtccgacaggcccctggcaaaggactggaatgggtggccggaatcatcggcagcggcggcagcacatattacgccgattctgtgaa gggccgcttcagcatcagccgggacaacagcaagaacaccctggacctgcagatgaacagcctgagagccgaggataccgccgt gtactactgcgtgaaggattggaacaccaccatgatcaccgagagaggccagggcacactggtcaccgtgtcctctacaacaacacc ggcgcctcggcctccaacaccagctcctacaatcgcgagtcagcccctgtctctcagacccgaagcctgtagacctgctgctggcgg agctgtgcataccagaggactggatttcgcctgcgacatctacatctgggctcctctggctggcacatgcggagttttgctgctgagcct ggtcatcaccctgtactgtaagagaggcaggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacag gaggaggacggctgctcttgtaggttcccagaggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatg cacctgcatacaagcagggacagaaccagctgtataacgagctgaatctgggccggagagaggagtacgacgtgctggataagag gcggggccgggaccccgagatgggaggcaagcctcggagaaagaacccacaggagggcctgtacaatgagctgcaaaaggaca agatggccgaggcctattctgagatcggcatgaagggagagaggcgccggggcaagggacacgatggcctgtaccagggcctga gcaccgccacaaaggacacctatgatgccctgcacatgcaggccctgccccctagatga (VH-A; SEQ ID NO: 22).

[0051] In certain embodiments, the anti-BCMA VHH comprises or consists of the amino acid sequence:

[0052] MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLTLSCAASGFTFSNY AMNWVRQAPGKGLEWVSGIIGSGATTYYADSVKGRFTISRDNSKNTLNLQMNSLRA EDTAIYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMR PVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEAttorney Docket No.: POTH-092 / 001WO YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR (VH-B; SEQ ID NO: 23).

[0053] In some embodiments, the VHH is encoded by the nucleic acid sequence:

[0054] atggctctgcctgtgacagctctgctgctgcctctggctctgcttcttcatgcggcgcgccctgaagttcagctgcttgaatct ggcggaggcctggttcaacctggcggatctctgacactgagctgtgccgccagcggcttcaccttcagcaactacgccatgaactgg gtccgacaggcccctggcaaaggccttgaatgggtgtccggcatcattggctctggcgccaccacctactacgccgattctgtgaagg gcagattcaccatcagccgggacaacagcaagaacaccctgaacctgcagatgaacagcctgagagccgaggacaccgccatcta ctactgcgtgaaggactggaacaccaccatgatcaccgagagaggccagggcacactggtcaccgtgtcctctacaacaacaccgg cgcctcggcctccaacaccagctcctacaatcgcgagtcagcccctgtctctcagacccgaagcctgtagacctgctgctggcggag ctgtgcataccagaggactggatttcgcctgcgacatctacatctgggctcctctggctggcacatgcggagttttgctgctgagcctgg tcatcaccctgtactgtaagagaggcaggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacagga ggaggacggctgctcttgtaggttcccagaggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatgca cctgcatacaagcagggacagaaccagctgtataacgagctgaatctgggccggagagaggagtacgacgtgctggataagaggc ggggccgggaccccgagatgggaggcaagcctcggagaaagaacccacaggagggcctgtacaatgagctgcaaaaggacaag atggccgaggcctattctgagatcggcatgaagggagagaggcgccggggcaagggacacgatggcctgtaccagggcctgagc accgccacaaaggacacctatgatgccctgcacatgcaggccctgccccctagatga (VH-B; SEQ ID NO: 24).

[0055] In certain embodiments, the anti-BCMA VHH comprises or consists of the amino acid sequence:

[0056] MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGESLRLSCAASGFTFSNY AMNWVRQAPGKGLEWVSGIVGGGGTSYYADSVRGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEAC RPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPF MRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR (VH-C; SEQ ID NO: 25).

[0057] In some embodiments, the VHH is encoded by the nucleic acid sequence:

[0058] atggctctgcctgtgacagctctgctgctgcctctggctctgcttcttcatgcggcgcgccctgaagttcagctgcttgaatct ggcggaggcctggttcagcctggcgaatctctgagactgagctgtgccgccagcggcttcaccttcagcaactacgccatgaactgg gtccgacaggcccctggcaaaggccttgaatgggtgtccggaatcgttggcggcggaggcacaagctactacgccgattctgtgcg gggcagattcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgagagccgaggacaccgccgtg tactactgcgtgaaggactggaacaccaccatgatcaccgagagaggccagggcacactggtcaccgtgtcctctacaacaacacc ggcgcctcggcctccaacaccagctcctacaatcgcgagtcagcccctgtctctcagacccgaagcctgtagacctgctgctggcgg agctgtgcataccagaggactggatttcgcctgcgacatctacatctgggctcctctggctggcacatgcggagttttgctgctgagcct ggtcatcaccctgtactgtaagagaggcaggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacagAttorney Docket No.: POTH-092 / 001WO gaggaggacggctgctcttgtaggttcccagaggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatg cacctgcatacaagcagggacagaaccagctgtataacgagctgaatctgggccggagagaggagtacgacgtgctggataagag gcggggccgggaccccgagatgggaggcaagcctcggagaaagaacccacaggagggcctgtacaatgagctgcaaaaggaca agatggccgaggcctattctgagatcggcatgaagggagagaggcgccggggcaagggacacgatggcctgtaccagggcctga gcaccgccacaaaggacacctatgatgccctgcacatgcaggccctgccccctagatga (VH-C; SEQ ID NO: 26).

[0059] In certain embodiments, the anti-BCMA VHH comprises or consists of the amino acid sequence:

[0060] MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFSNY AMTWIRQAPGKGLEWVSGITGDGGSTFYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFM RPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREE YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR (VH-D; SEQ ID NO: 27).

[0061] In some embodiments, the VHH is encoded by the nucleic acid sequence:

[0062] atggctctgcctgtgacagctctgctgctgcctctggctctgcttcttcatgcggcgcgccctgaagttcagctgcttgaatct ggcggaggcctggttcaacctggcggatctctgagactgagctgtgccgccagcggcttcaccttcagcaattacgccatgacctgga tcagacaggcccctggcaaaggcctggaatgggtgtccggaattacaggcgacggcggcagcaccttttacgccgattctgtgaagg gcagattcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgagagccgaggacaccgccgtgta ctactgcgtgaaggactggaacaccaccatgatcaccgagagaggccagggcacactggtcaccgtgtcctctacaacaacaccgg cgcctcggcctccaacaccagctcctacaatcgcgagtcagcccctgtctctcagacccgaagcctgtagacctgctgctggcggag ctgtgcataccagaggactggatttcgcctgcgacatctacatctgggctcctctggctggcacatgcggagttttgctgctgagcctgg tcatcaccctgtactgtaagagaggcaggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacagga ggaggacggctgctcttgtaggttcccagaggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatgca cctgcatacaagcagggacagaaccagctgtataacgagctgaatctgggccggagagaggagtacgacgtgctggataagaggc ggggccgggaccccgagatgggaggcaagcctcggagaaagaacccacaggagggcctgtacaatgagctgcaaaaggacaag atggccgaggcctattctgagatcggcatgaagggagagaggcgccggggcaagggacacgatggcctgtaccagggcctgagc accgccacaaaggacacctatgatgccctgcacatgcaggccctgccccctagatga (VH-D; SEQ ID NO: 28).

[0063] In certain embodiments, the anti-BCMA VHH comprises or consists of the amino acid sequence:

[0064] MALPVTALLLPLALLLHAARPEVQLLESGGGLAQPGGSLRLSCAASGFTFSSY AMNWIRQAPGKGLEWVSGISGSGGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRA EDTAVYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMAttorney Docket No.: POTH-092 / 001WO RPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREE YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR (VH-E; SEQ ID NO: 29).

[0065] In some embodiments, the VHH is encoded by the nucleic acid sequence:

[0066] atggcactgcctgtgacagccctgctgctgcctctggccctgctgctgcacgcagcacggcccgaggtgcagctgctgga gtccggaggaggcctggcccagcctggcggcagcctgaggctgtcctgcgccgcctctggcttcacctttagctcctacgccatgaa ctggatcagacaggcccctggcaagggcctggagtgggtgtccggcatctccggctctggaggctctacatactatgccgacagcgt gaagggccggttcaccatcagcagagataactccaagaataccgtgtacctccagatgaactctctgcgggccgaggacaccgccgt gtactattgcgtgaaggattggaataccacaatgatcacagagaggggccagggcaccctggtgacagtgtctagcaccacaacccc tgcccccagacctcccacacccgcccctaccatcgcgagtcagccactgtccctgcggcctgaggcctgccggcccgccgccggc ggagcagtgcacacacggggcctggactttgcctgtgacatctacatatgggcaccactggcaggaacctgcggcgtgctgctgctg agcctggtcatcaccctgtactgtaagagaggcaggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagacca cacaggaggaggacggctgctcttgtaggttcccagaggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgc cgatgcacctgcatacaagcagggacagaaccagctgtataacgagctgaatctgggccggagagaggagtacgacgtgctggata agaggcggggccgggaccccgagatgggaggcaagcctcggagaaagaacccacaggagggcctgtacaatgagctgcaaaa ggacaagatggccgaggcctattctgagatcggcatgaagggagagaggcgccggggcaagggacacgatggcctgtaccaggg cctgagcaccgccacaaaggacacctatgatgccctgcacatgcaggccctgccccctagatga (VH-E; SEQ ID NO: 30).

[0067] In certain embodiments, the anti-BCMA VHH comprises or consists of the amino acid sequence:

[0068] MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGRSLRLSCAASGFTFTNY AMNWVRQAPGKGLEWVSGISGGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEAC RPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPF MRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR (VH-F; SEQ ID NO: 31).

[0069] In some embodiments the VHH is encoded nucleic acid sequence:

[0070] atggcactgcctgtgacagccctgctgctgcctctggccctgctgctgcacgcagcacggcccgaggtgcagctgctgga gtctggaggaggcctggtgcagcccggccggtccctgagactgtcttgcgccgccagcggcttcacctttacaaactacgccatgaat tgggtgcggcaggcccctggcaagggcctggagtgggtgtctggcatcagcggaggaggaggcagcacctactatgcagactccg tgaagggcaggttcaccatctcccgcgataactctaagaatacactgtacctccagatgaacagcctgagggcagaggacaccgccg tgtactattgcgtgaaggattggaataccacaatgatcacagagaggggacagggcaccctggtgaccgtgagcagcaccacaacc cctgcccccagacctcccacacccgcccctaccatcgcgagtcagccactgtccctgcggcctgaggcctgccggcccgccgccgAttorney Docket No.: POTH-092 / 001WO gcggagcagtgcacacacggggcctggactttgcctgtgacatctacatatgggcaccactggcaggaacctgcggcgtgctgctgc tgagcctggtcatcaccctgtactgtaagagaggcaggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagac cacacaggaggaggacggctgctcttgtaggttcccagaggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtcc gccgatgcacctgcatacaagcagggacagaaccagctgtataacgagctgaatctgggccggagagaggagtacgacgtgctgg ataagaggcggggccgggaccccgagatgggaggcaagcctcggagaaagaacccacaggagggcctgtacaatgagctgcaa aaggacaagatggccgaggcctattctgagatcggcatgaagggagagaggcgccggggcaagggacacgatggcctgtaccag ggcctgagcaccgccacaaaggacacctatgatgccctgcacatgcaggccctgccccctagatga (VH-F; SEQ ID NO: 32).

[0071] The disclosure further provides a chimeric antigen receptor (CAR) comprising: (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one VH; (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. As used throughout the disclosure, a CAR comprising a VH is referred to as a VCAR. In certain embodiments, the antigen recognition region may comprise two VHs to produce a bi-specific or tandem VCAR. In certain embodiments, the antigen recognition region may comprise three VHs to produce a tri-specific VCAR. In certain embodiments, the ectodomain may further comprise a signal peptide. Alternatively, or in addition, in certain embodiments, the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain. In certain embodiments, the ectodomain may further comprise a signal peptide. Alternatively, or in addition, in certain embodiments, the ectodomain may further comprise a hinge between the antigen recognition region and the transmembrane domain.

[0072] In certain embodiments of the VCARs of the disclosure, including those comprising an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one VH, the VH comprises a human or a humanized sequence.

[0073] In certain embodiments of the VCARs of the disclosure, including those comprising an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one VH, the VH comprises a non-naturally occurring sequence.

[0074] In certain embodiments of the VCARs of the disclosure, including those comprising an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one VH, the VH is not naturally occurring.

[0075] In certain embodiments of the VCARs of the disclosure, including those comprising an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one VH, the VH comprises a recombinant or chimeric sequence.Attorney Docket No.: POTH-092 / 001WO

[0076] In certain embodiments of the VCARs of the disclosure, including those comprising an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one VH, the VH is produced by an in vitro procedure of affinity selection and recombination.

[0077] In certain embodiments of the VCARs of the disclosure, the VH comprises or consists of the amino acid sequence of SEQ ID NO: 21.

[0078] In some embodiments, the VH is encoded by the nucleic acid sequence:

[0079] atggctctgcctgtgacagctctgctgctgcctctggctctgcttcttcatgcggcgcgccctgaagttcagctgcttgaatct ggcggaggcctggttcaacctggcggatctctgagactgagctgtgccgccagcggcttcacctttagcagctacgccatgaactgg gtccgacaggcccctggcaaaggactggaatgggtggccggaatcatcggcagcggcggcagcacatattacgccgattctgtgaa gggccgcttcagcatcagccgggacaacagcaagaacaccctggacctgcagatgaacagcctgagagccgaggataccgccgt gtactactgcgtgaaggattggaacaccaccatgatcaccgagagaggccagggcacactggtcaccgtgtcctctacaacaacacc ggcgcctcggcctccaacaccagctcctacaatcgcgagtcagcccctgtctctcagacccgaagcctgtagacctgctgctggcgg agctgtgcataccagaggactggatttcgcctgcgacatctacatctgggctcctctggctggcacatgcggagttttgctgctgagcct ggtcatcaccctgtactgtaagagaggcaggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacag gaggaggacggctgctcttgtaggttcccagaggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatg cacctgcatacaagcagggacagaaccagctgtataacgagctgaatctgggccggagagaggagtacgacgtgctggataagag gcggggccgggaccccgagatgggaggcaagcctcggagaaagaacccacaggagggcctgtacaatgagctgcaaaaggaca agatggccgaggcctattctgagatcggcatgaagggagagaggcgccggggcaagggacacgatggcctgtaccagggcctga gcaccgccacaaaggacacctatgatgccctgcacatgcaggccctgccccctagatga (VH-A; SEQ ID NO: 22).

[0080] In certain embodiments of the VCARs of the disclosure, the VH comprises or consists of the amino acid sequence:

[0081] MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLTLSCAASGFTFSNY AMNWVRQAPGKGLEWVSGIIGSGATTYYADSVKGRFTISRDNSKNTLNLQMNSLRA EDTAIYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMR PVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREE YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR (VH-B; SEQ ID NO: 23).

[0082] In some embodiments, the VH is encoded by the nucleic acid sequence:

[0083] atggctctgcctgtgacagctctgctgctgcctctggctctgcttcttcatgcggcgcgccctgaagttcagctgcttgaatct ggcggaggcctggttcaacctggcggatctctgacactgagctgtgccgccagcggcttcaccttcagcaactacgccatgaactgg gtccgacaggcccctggcaaaggccttgaatgggtgtccggcatcattggctctggcgccaccacctactacgccgattctgtgaagg gcagattcaccatcagccgggacaacagcaagaacaccctgaacctgcagatgaacagcctgagagccgaggacaccgccatctaAttorney Docket No.: POTH-092 / 001WO ctactgcgtgaaggactggaacaccaccatgatcaccgagagaggccagggcacactggtcaccgtgtcctctacaacaacaccgg cgcctcggcctccaacaccagctcctacaatcgcgagtcagcccctgtctctcagacccgaagcctgtagacctgctgctggcggag ctgtgcataccagaggactggatttcgcctgcgacatctacatctgggctcctctggctggcacatgcggagttttgctgctgagcctgg tcatcaccctgtactgtaagagaggcaggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacagga ggaggacggctgctcttgtaggttcccagaggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatgca cctgcatacaagcagggacagaaccagctgtataacgagctgaatctgggccggagagaggagtacgacgtgctggataagaggc ggggccgggaccccgagatgggaggcaagcctcggagaaagaacccacaggagggcctgtacaatgagctgcaaaaggacaag atggccgaggcctattctgagatcggcatgaagggagagaggcgccggggcaagggacacgatggcctgtaccagggcctgagc accgccacaaaggacacctatgatgccctgcacatgcaggccctgccccctagatga (VH-B; SEQ ID NO: 24).

[0084] In certain embodiments of the VCARs of the disclosure, the VH comprises or consists of the amino acid sequence:

[0085] MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGESLRLSCAASGFTFSNY AMNWVRQAPGKGLEWVSGIVGGGGTSYYADSVRGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEAC RPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPF MRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR (VH-C; SEQ ID NO: 25).

[0086] In some embodiments, VH is encoded by the nucleic acid sequence:

[0087] atggctctgcctgtgacagctctgctgctgcctctggctctgcttcttcatgcggcgcgccctgaagttcagctgcttgaatct ggcggaggcctggttcagcctggcgaatctctgagactgagctgtgccgccagcggcttcaccttcagcaactacgccatgaactgg gtccgacaggcccctggcaaaggccttgaatgggtgtccggaatcgttggcggcggaggcacaagctactacgccgattctgtgcg gggcagattcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgagagccgaggacaccgccgtg tactactgcgtgaaggactggaacaccaccatgatcaccgagagaggccagggcacactggtcaccgtgtcctctacaacaacacc ggcgcctcggcctccaacaccagctcctacaatcgcgagtcagcccctgtctctcagacccgaagcctgtagacctgctgctggcgg agctgtgcataccagaggactggatttcgcctgcgacatctacatctgggctcctctggctggcacatgcggagttttgctgctgagcct ggtcatcaccctgtactgtaagagaggcaggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacag gaggaggacggctgctcttgtaggttcccagaggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatg cacctgcatacaagcagggacagaaccagctgtataacgagctgaatctgggccggagagaggagtacgacgtgctggataagag gcggggccgggaccccgagatgggaggcaagcctcggagaaagaacccacaggagggcctgtacaatgagctgcaaaaggaca agatggccgaggcctattctgagatcggcatgaagggagagaggcgccggggcaagggacacgatggcctgtaccagggcctga gcaccgccacaaaggacacctatgatgccctgcacatgcaggccctgccccctagatga (VH-C; SEQ ID NO: 26).

[0088] In certain embodiments of the VCARs of the disclosure, the VH comprises or consists of the amino acid sequence:Attorney Docket No.: POTH-092 / 001WO

[0089] MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFSNY AMTWIRQAPGKGLEWVSGITGDGGSTFYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFM RPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREE YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR (VH-D; SEQ ID NO: 27).

[0090] In some embodiments, the VH is encoded by the nucleic acid sequence:

[0091] atggctctgcctgtgacagctctgctgctgcctctggctctgcttcttcatgcggcgcgccctgaagttcagctgcttgaatct ggcggaggcctggttcaacctggcggatctctgagactgagctgtgccgccagcggcttcaccttcagcaattacgccatgacctgga tcagacaggcccctggcaaaggcctggaatgggtgtccggaattacaggcgacggcggcagcaccttttacgccgattctgtgaagg gcagattcaccatcagccgggacaacagcaagaacaccctgtacctgcagatgaacagcctgagagccgaggacaccgccgtgta ctactgcgtgaaggactggaacaccaccatgatcaccgagagaggccagggcacactggtcaccgtgtcctctacaacaacaccgg cgcctcggcctccaacaccagctcctacaatcgcgagtcagcccctgtctctcagacccgaagcctgtagacctgctgctggcggag ctgtgcataccagaggactggatttcgcctgcgacatctacatctgggctcctctggctggcacatgcggagttttgctgctgagcctgg tcatcaccctgtactgtaagagaggcaggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagaccacacagga ggaggacggctgctcttgtaggttcccagaggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgccgatgca cctgcatacaagcagggacagaaccagctgtataacgagctgaatctgggccggagagaggagtacgacgtgctggataagaggc ggggccgggaccccgagatgggaggcaagcctcggagaaagaacccacaggagggcctgtacaatgagctgcaaaaggacaag atggccgaggcctattctgagatcggcatgaagggagagaggcgccggggcaagggacacgatggcctgtaccagggcctgagc accgccacaaaggacacctatgatgccctgcacatgcaggccctgccccctagatga (VH-D; SEQ ID NO: 28).

[0092] In certain embodiments of the VCARs of the disclosure, the VH comprises or consists of the amino acid sequence:

[0093] MALPVTALLLPLALLLHAARPEVQLLESGGGLAQPGGSLRLSCAASGFTFSSY AMNWIRQAPGKGLEWVSGISGSGGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRA EDTAVYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFM RPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREE YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR (VH-E; SEQ ID NO: 29).

[0094] In some embodiments, the VH is encoded by the nucleic acid sequence:

[0095] atggcactgcctgtgacagccctgctgctgcctctggccctgctgctgcacgcagcacggcccgaggtgcagctgctgga gtccggaggaggcctggcccagcctggcggcagcctgaggctgtcctgcgccgcctctggcttcacctttagctcctacgccatgaa ctggatcagacaggcccctggcaagggcctggagtgggtgtccggcatctccggctctggaggctctacatactatgccgacagcgtAttorney Docket No.: POTH-092 / 001WO gaagggccggttcaccatcagcagagataactccaagaataccgtgtacctccagatgaactctctgcgggccgaggacaccgccgt gtactattgcgtgaaggattggaataccacaatgatcacagagaggggccagggcaccctggtgacagtgtctagcaccacaacccc tgcccccagacctcccacacccgcccctaccatcgcgagtcagccactgtccctgcggcctgaggcctgccggcccgccgccggc ggagcagtgcacacacggggcctggactttgcctgtgacatctacatatgggcaccactggcaggaacctgcggcgtgctgctgctg agcctggtcatcaccctgtactgtaagagaggcaggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagacca cacaggaggaggacggctgctcttgtaggttcccagaggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtccgc cgatgcacctgcatacaagcagggacagaaccagctgtataacgagctgaatctgggccggagagaggagtacgacgtgctggata agaggcggggccgggaccccgagatgggaggcaagcctcggagaaagaacccacaggagggcctgtacaatgagctgcaaaa ggacaagatggccgaggcctattctgagatcggcatgaagggagagaggcgccggggcaagggacacgatggcctgtaccaggg cctgagcaccgccacaaaggacacctatgatgccctgcacatgcaggccctgccccctagatga (VH-E; SEQ ID NO: 30).

[0096] In certain embodiments of the VCARs of the disclosure, the VH comprises or consists of the amino acid sequence:

[0097] MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGRSLRLSCAASGFTFTNY AMNWVRQAPGKGLEWVSGISGGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCVKDWNTTMITERGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEAC RPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPF MRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALP PR (VH-F; SEQ ID NO: 31).

[0098] In some embodiments, the VH is encoded by the nucleic acid sequence:

[0099] atggcactgcctgtgacagccctgctgctgcctctggccctgctgctgcacgcagcacggcccgaggtgcagctgctgga gtctggaggaggcctggtgcagcccggccggtccctgagactgtcttgcgccgccagcggcttcacctttacaaactacgccatgaat tgggtgcggcaggcccctggcaagggcctggagtgggtgtctggcatcagcggaggaggaggcagcacctactatgcagactccg tgaagggcaggttcaccatctcccgcgataactctaagaatacactgtacctccagatgaacagcctgagggcagaggacaccgccg tgtactattgcgtgaaggattggaataccacaatgatcacagagaggggacagggcaccctggtgaccgtgagcagcaccacaacc cctgcccccagacctcccacacccgcccctaccatcgcgagtcagccactgtccctgcggcctgaggcctgccggcccgccgccg gcggagcagtgcacacacggggcctggactttgcctgtgacatctacatatgggcaccactggcaggaacctgcggcgtgctgctgc tgagcctggtcatcaccctgtactgtaagagaggcaggaagaagctgctgtatatcttcaagcagcccttcatgagacccgtgcagac cacacaggaggaggacggctgctcttgtaggttcccagaggaggaggagggaggatgcgagctgcgcgtgaagtttagccggtcc gccgatgcacctgcatacaagcagggacagaaccagctgtataacgagctgaatctgggccggagagaggagtacgacgtgctgg ataagaggcggggccgggaccccgagatgggaggcaagcctcggagaaagaacccacaggagggcctgtacaatgagctgcaa aaggacaagatggccgaggcctattctgagatcggcatgaagggagagaggcgccggggcaagggacacgatggcctgtaccagAttorney Docket No.: POTH-092 / 001WO ggcctgagcaccgccacaaaggacacctatgatgccctgcacatgcaggccctgccccctagatga (VH-F; SEQ ID NO: 32).

[0100] In certain embodiments of the VCARs of the disclosure, the VH comprises or consists of the amino acid sequence:

[0101] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVAGII GSGGSTYYADSVKGRFSISRDNSKNTLDLQMNSLRAEDTAVYYCVKDWNTTMITER GQGTLVTVSS (SEQ ID NO: 69).

[0102] In certain embodiments of the VCARs of the disclosure, the anti-CD19 VH heavy chain variable region comprises the amino acid sequence:

[0103] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYM SSSGSTIYYADSVKGRFTISRDNAKNSLYLQMTSLRAEDTAVYYCARGGIAATGTWG QGTLVTVSS (SEQ ID NO: 33).

[0104] In certain embodiments of the VCARs of the disclosure, the anti-CD19 VH heavy chain variable region comprises the amino acid sequence:

[0105] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYM SSSGSTIYYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCARGGIAAAGTW GQGTLVTVSS (SEQ ID NO: 34).

[0106] In certain embodiments of the VCARs of the disclosure, the anti-CD19 VH heavy chain variable region comprises the amino acid sequence further comprising a T29D mutation:

[0107] QVQLVESGGGLVKPGGSLRLSCAASGFDFSDYYMSWIRQAPGKGLEWVSYM SSSGSTIYYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCARGGIAAAGTW GQGTLVTVSS (SEQ ID NO: 35).

[0108] In certain embodiments of the VCARs of the disclosure, the anti-CD19 VH heavy chain variable region comprises the amino acid sequence further comprising a S55D mutation:

[0109] QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYM SSDGSTIYYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCARGGIAAAGTW GQGTLVTVSS (SEQ ID NO: 36).

[0110] In certain embodiments of the VCARs of the disclosure, the anti-CD20 VH heavy chain variable region comprises the amino acid sequence:

[0111] EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSSI NWNGGSKGYADSVKGRFTISRDNAKNSLYLQMNSLRVEDTALYQCARERGYRIGH DSFDIWGQGTLVTVSS (SEQ ID NO: 37).Attorney Docket No.: POTH-092 / 001WO

[0112] In certain embodiments of the VCARs of the disclosure, the anti-CD20 VH heavy chain variable region comprises the amino acid sequence:

[0113] QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGNYYWNWIRQPPGKGLEWIGY IYSSGSTKYNPSLKSRVTILVDTSKNQFSLKLSSVTAADTAVYYCARSRLNGDGLFDD RGQGTLVTVSS (SEQ ID NO: 38).

[0114] In certain embodiments of the VCARs of the disclosure, the anti-PSMA VH heavy chain variable region comprises the amino acid sequence:

[0115] EVQLLESGGGVVQPGRSLRLSCAASGFSFSGYGMHWVRQAPGKEREWVAVI SYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDANWGQHP DHTSFDYRGQGTLVTVSS (SEQ ID NO: 43).

[0116] In certain embodiments of the VCARs of the disclosure, the anti-PSMA VH heavy chain variable region comprises the amino acid sequence:

[0117] EVQLLESGGGLVQPGGSLRLSCAASGFTFKFYAMSWVRQAPGKGPEWVSVIS GSGGSTYYADSVKGRFTISRDNSKNTLHLQMNSLRAEDTAVYFCAKEIAEASRGFDY RGQGTLVTVSS (SEQ ID NO: 44).

[0118] In certain embodiments, the anti-MUC1C VH heavy chain variable region comprises the amino acid sequence:

[0119] EVQLVESGGGLVQPGGSLRLSCTASGFAFSGNSMNWVRQAPGKGLEWVAFI TSSGRSIKYADSVKGRFTISRDNAKNSLYLQMNTLRDEDTALYYCATGGTGTSLFDY RGQGTLVTVSS (SEQ ID NO: 45).

[0120] In certain embodiments, the anti-MUC1C VH heavy chain variable region comprises the amino acid sequence:

[0121] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHSMNWVRQAPGKGLEWVSFIS SSSNIKKYADSVKGRFTISRDNAKNSLFLQMNSLRDEDTAVYYCATGGTGITVLDYR GQGTLVTVSS (SEQ ID NO: 46).

[0122] In certain embodiments, the anti-c-kit VH heavy chain variable region comprises the amino acid sequence:

[0123] EVQLLESGGGLVQPGGSLRLSCAASGLTISTYAMSWVRQAPGKGLEWVSAIS TGGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATGYDSSGHYYG GFDYRGQGTLVTVSS (SEQ ID NO: 47).

[0124] In certain embodiments, the anti-c-kit VH heavy chain variable region comprises the amino acid sequence:Attorney Docket No.: POTH-092 / 001WO

[0125] EVQLLESGGGLVQPGGSLRLSCAASGFTFDSYAMSWVRQAPGKGLEWVSAIS VRGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYSCATGYDPSGHYYG GFDYRGQGTLVTVSS (SEQ ID NO: 48).

[0126] In certain embodiments, the anti-c-kit VH heavy chain variable region comprises the amino acid sequence:

[0127] EVQLLESGGGLVQPGGSLRLSCAASGLTISSYAMSWVRQAPGKGLEWVSAIS TGGSRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCATGYDSSGHYYG GFDYRGQGTLVTVSS (SEQ ID NO: 49).

[0128] In certain embodiments, the anti-c-kit VH heavy chain variable region comprises the amino acid sequence:

[0129] EVQLLESGGGLVQPGGSLRLSCAASGFTFDSYAMSWVRQAPGEGLEWVSAIS TGGGSTYYADSVKGRFTISRDNSKNMLFLQMNSLRAEDTAVYSCATGYDSSGYYYG GFDYRGQGTLVTVSS (SEQ ID NO: 50).

[0130] In certain embodiments, the anti-c-kit VH heavy chain variable region comprises the amino acid sequence:

[0131] QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSID YNGGTYNNPSLKSRVTISVDTSKNQFSLKLSYVTAADTAVYYCARQNPRRYASGAS DYRGQGTLVTVSS (SEQ ID NO: 51).

[0132] VHHs and / or VCARs of the disclosure may bind an antigen with at least one affinity selected from a KD of less than or equal to 10-9M, 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.

[0133] In certain embodiments, the disclosure provides an anti-BCMA VCAR. In certain embodiments, the disclosure provides an anti-MUC1C VCAR. In certain embodiments, the disclosure provides an anti-CD19 VCAR. In certain embodiments, the disclosure provides an anti-CD20 VCAR. In certain embodiments, the disclosure provides an anti-PSMA VCAR. In certain embodiments, the disclosure provides an anti-CD70 VCAR. In certain embodiments, the disclosure provides an anti-CD7 VCAR. In certain embodiments, the disclosure provides an anti-c-kit VCAR. Methods of Manufacture

[0134] Amino acids from an scFv or VHH can be altered, added and / or deleted to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity,Attorney Docket No.: POTH-092 / 001WO specificity, half-life, stability, solubility or any other suitable characteristic, as known in the art.

[0135] Optionally, an scFv or VHH can be engineered with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, the scaffold proteins can be optionally prepared by a process of analysis of the parental sequences and various conceptual engineered products using three-dimensional models of the parental and engineered sequences. Three-dimensional models are commonly available and are familiar to those skilled in the art.

[0136] Computer programs are available which illustrate and display probable three- dimensional conformational structures of selected candidate sequences and can measure possible immunogenicity (e.g., Immunofilter program of Xencor, Inc. of Monrovia, Calif.). Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate sequence, i.e., the analysis of residues that influence the ability of the candidate scFv to bind its antigen. In this way, residues can be selected and combined from the parent and reference sequences so that the desired characteristic, such as affinity for the target antigen(s), is achieved. Alternatively, or in addition to, the above procedures, other suitable methods of engineering can be used.

[0137] Screening of an scFv or VHH for specific binding to similar proteins or fragments can be conveniently achieved using nucleotide (DNA or RNA display) or peptide display libraries, for example, in vitro display. This method involves the screening of large collections of peptides for individual members having the desired function or structure. The displayed nucleotide or peptide sequences can be from 3 to 5000 or more nucleotides or amino acids in length, frequently from 5-100 amino acids long, and often from about 8 to 25 amino acids long. In addition to direct chemical synthetic methods for generating peptide libraries, several recombinant DNA methods have been described. One type involves the display of a peptide sequence on the surface of a bacteriophage or cell. Each bacteriophage or cell contains the nucleotide sequence encoding the particular displayed peptide sequence. Such methods are described in PCT Patent Publication Nos. WO 91 / 17271, WO 91 / 18980, WO 91 / 19818, and WO 93 / 08278.

[0138] Other systems for generating libraries of peptides have aspects of both in vitro chemical synthesis and recombinant methods. See, PCT Patent Publication Nos. WO 92 / 05258, WO 92 / 14843, and WO 96 / 19256. See also, U.S. Pat. Nos.5,658,754; and 5,643,768. Peptide display libraries, vector, and screening kits are commercially available from such suppliers as Invitrogen (Carlsbad, Calif.), and Cambridge Antibody TechnologiesAttorney Docket No.: POTH-092 / 001WO (Cambridgeshire, UK). See, e.g., U.S. Pat. Nos.4,704,692, 4,939,666, 4,946,778, 5,260,203, 5,455,030, 5,518,889, 5,534,621, 5,656,730, 5,763,733, 5,767,260, 5856456, assigned to Enzon; 5,223,409, 5,403,484, 5,571,698, 5,837,500, assigned to Dyax, 5,427,908, 5,580,717, assigned to Affymax; 5,885,793, assigned to Cambridge Antibody Technologies; 5,750,373, assigned to Genentech, 5,618,920, 5,595,898, 5,576,195, 5,698,435, 5,693,493, 5,698,417, assigned to Xoma, Colligan, supra; Ausubel, supra; or Sambrook, supra.

[0139] An scFv of the disclosure can bind human or other mammalian proteins with a wide range of affinities (KD). In a preferred aspect, at least one scFv of the present disclosure can optionally bind to a target protein with high affinity, for example, with a KD equal to or less than about 10−7M, such as but not limited to, 0.1-9.9 (or any range or value therein) X 10−8, 10−9, 10−10, 10−11, 10−12, 10−13, 10−14, 10−15or any range or value therein, as determined by surface plasmon resonance or the Kinexa method, as practiced by those of skill in the art.

[0140] The affinity or avidity of a scFv for an antigen can be determined experimentally using any suitable method. (See, for example, Berzofsky, et al., “Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, Janis Immunology, W.H. Freeman and Company: New York, N.Y. (1992); and methods described herein). The measured affinity of a particular scFv-antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH). Thus, measurements of affinity and other antigen-binding parameters (e.g., KD, Kon, Koff) are preferably made with standardized solutions of protein scaffold and antigen, and a standardized buffer, such as the buffer described herein.

[0141] Competitive assays can be performed with the scFv of the disclosure in order to determine what proteins, antibodies, and other antagonists compete for binding to a target protein with the scFv of the present disclosure and / or share the epitope region. These assays as readily known to those of ordinary skill in the art evaluate competition between antagonists or ligands for a limited number of binding sites on a protein. The protein and / or antibody is immobilized or insolubilized before or after the competition and the sample bound to the target protein is separated from the unbound sample, for example, by decanting (where the protein / antibody was pre-insolubilized) or by centrifuging (where the protein / antibody was precipitated after the competitive reaction). Also, the competitive binding may be determined by whether function is altered by the binding or lack of binding of the scFv to the target protein, e.g., whether the scFv molecule inhibits or potentiates the enzymatic activity of, for example, a label. ELISA and other functional assays may be used, as well known in the art.Attorney Docket No.: POTH-092 / 001WO Cells and Modified Cells

[0142] Cells and modified cells of the disclosure can be mammalian cells. Preferably, the cells and modified cells are human cells. Cells and modified cells of the disclosure can be immune cells. The immune cells of the disclosure can comprise 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.

[0143] The immune precursor cells can comprise any cells which can differentiate into one or more types of immune cells. The immune precursor cells can comprise multipotent stem cells that can self-renew and develop into immune cells. The immune precursor cells can comprise hematopoietic stem cells (HSCs) or descendants thereof. The immune precursor cells can comprise precursor cells that can develop into immune cells. The immune precursor cells can comprise hematopoietic progenitor cells (HPCs).

[0144] 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.

[0145] HSCs can be isolated or derived from a primary or cultured stem cell. HSCs can be isolated or derived from an embryonic stem cell, a multipotent stem cell, a pluripotent stem cell, an adult stem cell, or an induced pluripotent stem cell (iPSC).

[0146] Immune precursor cells can comprise an HSC or an HSC descendent cell. 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.

[0147] 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 retain their “stemness” following division and do not differentiate. Consequently, as an adoptive cell therapy, the “primitive” HSCs produced by the disclosed methods not only replenish their numbers, but expand in vivo. “Primitive”Attorney Docket No.: POTH-092 / 001WO HSCs produced by disclosed the methods can be therapeutically-effective when administered as a single dose.

[0148] 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+.

[0149] Primitive HSCs, HSCs, and / or HSC descendent cells can be modified according to the disclosed methods to express DHFR and an exogenous sequence (e.g., a chimeric antigen receptor) to generate HSC MTX-CAR cells. 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.

[0150] The modified MTX-CAR 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).

[0151] The modified MTX-CAR 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.

[0152] Modified MTX-CAR-T cells of the disclosure may be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs. Unlike traditional biologics and chemotherapeutics, the disclosed modified-T cells 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.

[0153] 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, 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) TAttorney Docket No.: POTH-092 / 001WO 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: Naïve T cells (TN) > TSCM > TCM> TEM> TE> TTE, whereby TNis 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 TSCMcells being the most abundant (e.g., TSCM > TCM > TEM > TE > TTE).

[0154] 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 Naïve 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 Naïve T cells (TN), a TSCM, a TCM, a TEM, a TE, or a TTE.

[0155] In some embodiments, methods of the disclosure can modify and / or produce a population of modified MTX-CAR-T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of a plurality of modified T cells in the population expresses one or more cell-surface marker(s) of an early memory T cell. The population of modified early memory T cells comprises a plurality of modified stem cell-like T cells. The population of modified early memory T cells comprises a plurality of modified TSCMcells. The population of modified early memory T cells comprises a plurality of modified TCM cells.

[0156] In some embodiments, the methods of the disclosure can modify and / or produce a population of modified MTX-CAR-T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells in the population expresses one or more cell-surface marker(s) of a stem cell-like T cell. The population of modified stem cell- like T cells comprises a plurality of modified TSCM cells. The population of modified stem cell-like T cells comprises a plurality of modified TCMcells.

[0157] 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 cell-surface markers canAttorney Docket No.: POTH-092 / 001WO comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ. The cell-surface markers can comprise one or more of CD45RA, CD95, IL-2Rβ, CCR7, and CD62L.

[0158] 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 can comprise one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L.

[0159] In some embodiments, the methods of the disclosure can modify and / or produce a population of modified MTX-CAR-T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells in the population expresses one or more cell-surface marker(s) of a naïve T cell (TN). The cell-surface markers can comprise one or more of CD45RA, CCR7 and CD62L.

[0160] In some embodiments, the methods of the disclosure can modify and / or produce a population of modified MTX-CAR-T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells in the population expresses one or more cell-surface marker(s) of an effector T-cell (modified TEFF). The cell-surface markers can comprise one or more of CD45RA, CD95, and IL-2Rβ.

[0161] In some embodiments, the methods of the disclosure can modify and / or produce a population of modified MTX-CAR-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).

[0162] In some embodiments, a plurality of modified MTX-CAR cells of the population comprise a transgene or a sequence encoding the transgene (e.g., a CAR), wherein 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 or the sequenceAttorney Docket No.: POTH-092 / 001WO encoding the transgene, wherein 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 one or more cell-surface marker(s) comprising CD34 or wherein 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 one or more cell-surface marker(s) comprising CD34 (e.g., comprise the cell-surface marker phenotype CD34+).

[0163] In some embodiments, a plurality of modified MTX-CAR cells of the population comprise a transgene or a sequence encoding the transgene (e.g., a CAR), wherein 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 or the sequence encoding the transgene, 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 one or more cell-surface marker(s) comprising CD34 and do not express one or more cell-surface marker(s) comprising CD38, or wherein 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 one or more cell-surface marker(s) comprising CD34 and do not express one or more cell- surface marker(s) comprising CD38 (e.g., comprise the cell-surface marker phenotype CD34+ and CD38-.

[0164] In some embodiments, a plurality of modified MTX-resistant cells of the population comprise a transgene or a sequence encoding the transgene (e.g., a CAR), wherein 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 or the sequence encoding the transgene, wherein 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 modifiedAttorney Docket No.: POTH-092 / 001WO cells express one or more cell-surface marker(s) comprising CD34 and CD90 and do not express one or more cell-surface marker(s) comprising CD38, or wherein 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 one or more cell-surface marker(s) comprising CD34 and CD90 and do not express one or more cell-surface marker(s) comprising CD38 (e.g., comprise the cell-surface marker phenotype CD34+, CD38- and CD90+).

[0165] In some embodiments, a plurality of modified MTX-CAR cells of the population comprise a transgene or a sequence encoding the transgene (e.g., a CAR), wherein 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 or the sequence encoding the transgene, wherein 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 one or more cell-surface marker(s) comprising CD34 and CD90 and do not express one or more cell-surface marker(s) comprising CD38 and CD45RA, or wherein 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 one or more cell-surface marker(s) comprising CD34 and CD90 and do not express one or more cell-surface marker(s) comprising CD38 and CD45RA (e.g., comprise the cell-surface marker phenotype CD34+, CD38-, CD90+, CD45RA-).

[0166] In some embodiments, a plurality of modified MTX-CAR cells of the population comprise a transgene or a sequence encoding the transgene (e.g., a CAR), wherein 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 or the sequence encoding the transgene, wherein 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 leastAttorney Docket No.: POTH-092 / 001WO 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 one or more cell-surface marker(s) comprising CD34, CD90 and CD49f and do not express one or more cell-surface marker(s) comprising CD38 and CD45RA, or wherein 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 one or more cell-surface marker(s) comprising CD34, CD90 and CD49f and do not express one or more cell-surface marker(s) comprising CD38 and CD45RA (e.g., comprise the cell-surface marker phenotype CD34+, CD38-, CD90+, CD45RA- and CD49f+).

[0167] In some embodiments, a plurality of modified MTX-CAR cells of the population comprise a transgene or a sequence encoding the transgene (e.g., a CAR), wherein 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 or the sequence encoding the transgene, wherein 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 one or more cell-surface marker(s) comprising CD34 and CD90 and do not express one or more cell-surface marker(s) comprising CD45RA, or wherein 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 one or more cell-surface marker(s) comprising CD34 and CD90 and do not express one or more cell-surface marker(s) comprising CD45RA (e.g., comprise the cell-surface marker phenotype CD34+, CD90+ and CD45RA-).

[0168] Compositions and methods of producing and / or expanding the immune cells or immune precursor cells (e.g., the disclosed modified MTX-CAR-T-cells) and buffers forAttorney Docket No.: POTH-092 / 001WO 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 modified MTX-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.

[0169] 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. Methods Introducing Nucleic Acids into Cells

[0170] A modified MTX-CAR cell be produced by introducing a nucleic acid encoding DHFR and a nucleic acid encoding a CAR into the cell.

[0171] Introducing a nucleic acid sequence 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 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 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 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.

[0172] Introducing a nucleic acid sequence 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 DHFR and 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. Introducing a nucleic acid sequence into a cell ex vivo, in vivo, in vitro or in situ can comprise a viral vector. The viral vector can be a non-integrating non-chromosomal vector. Non-limiting examples of non-integratingAttorney Docket No.: POTH-092 / 001WO non-chromosomal vectors include adeno-associated virus (AAV), adenovirus, and herpes viruses. The viral vector can be an integrating chromosomal vector. Non-limiting examples of integrating chromosomal vectors include adeno-associated vectors (AAV), Lentiviruses, and gamma-retroviruses.

[0173] Introducing a nucleic acid sequence into a cell ex vivo, in vivo, in vitro or in situ can comprise a combination of vectors. Non-limiting 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.

[0174] Genome modification can comprise introducing a nucleic acid sequence 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 DHFR and / or a CAR.

[0175] Genome modification can comprise introducing a nucleic acid sequence into a cell ex vivo, in vivo, in vitro or in situ to stably integrate a nucleic acid sequence. The stable chromosomal integration can be a random integration, a site-specific integration, or a biased integration. The site-specific integration can be non-assisted or assisted. The assisted site- specific integration is co-delivered with a site-directed nuclease. The site-directed nuclease comprises a transgene with 5’ and 3’ nucleotide sequence extensions that contain a percentage homology to upstream and downstream regions of the site of genomic integration. The transgene with homologous nucleotide extensions enable genomic integration by homologous recombination, microhomology-mediated end joining, or nonhomologous end- joining. The site-specific integration can occur at a safe harbor site. Genomic safe harbor sites are able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements function reliably (for example, are expressed at a therapeutically effective level of expression) and do not cause deleterious alterations to the host genome that cause a risk to the host organism. Non-limiting examples of potential genomic safe harbors include intronic sequences of the human albumin gene, the adeno- associated virus site 1 (AAVS1), a naturally occurring site of integration of AAV virus on chromosome 19, the site of the chemokine (C-C motif) receptor 5 (CCR5) gene and the site of the human ortholog of the mouse Rosa26 locus.Attorney Docket No.: POTH-092 / 001WO

[0176] 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).

[0177] Gene editing compositions, including Cas-CLOVER, and methods of using these compositions for gene editing are described in detail in U.S. Patent Publication Nos. 2017 / 0107541, 2017 / 0114149, 2018 / 0187185 and U.S. Patent No.10,415,024.

[0178] The nuclease or the nuclease domain thereof can comprise a nuclease-inactivated Cas (dCas) protein and an endonuclease. The endonuclease can comprise a Clo051 nuclease or a nuclease domain thereof. The gene editing composition can comprise a fusion protein. The fusion protein can comprise a nuclease-inactivated Cas9 (dCas9) protein and a Clo051 nuclease or a Clo051 nuclease domain. The gene editing composition can further comprise a guide sequence. The guide sequence comprises an RNA sequence.

[0179] An exemplary 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”).

[0180] The NLS-dCas9-Clo051-NLS (“Cas-CLOVER v2”, or “CCv2”, or “dspCas9 Cas- CLOVER”) fusion protein can comprise, consist essentially of, or consist of, the amino acid sequence of SEQ ID NO: 17 or a nucleic acid sequence of SEQ ID NO: 18.

[0181] The disclosure provides compositions comprising a 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 Cas9. A Cas9 construct of the disclosure can comprise an effector comprising a type IIS endonuclease.

[0182] Another means for introducing a nucleic acid or nucleic acids encoding DHFR and a CAR include using a transposon system. The present disclosure provides a transposonAttorney Docket No.: POTH-092 / 001WO comprising a nucleic acid encoding DHFR and a CAR. In a preferred aspect, the transposon is a plasmid DNA transposon comprising a nucleotide sequence encoding DHFR and the CAR (e.g., scFv CAR or 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.

[0183] 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™ (PB) transposase a piggyBac-like (PBL) transposase or a Super piggyBac™ (SPB) transposase. The sequence encoding the SPB transposase is an mRNA sequence.

[0184] 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 transposases that may be used in the methods disclosed herein.

[0185] 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’-TAAA-3’, 5’-TCTC-3’, 5’TGAA-3’, 5’-AAAT-3’, 5’-AATC-3’, 5’-ACAA-3’, 5’-ACAT-3’, 5’-ACTC-3’, 5’-AGTG-3’, 5’-ATAG-3’, 5’-CAAA-3’, 5’- CACA-3’, 5’-CATA-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.

[0186] 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 methods disclosed herein. In some aspects, the PBAttorney Docket No.: POTH-092 / 001WO transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 53. In a preferred aspect, the PB transposase comprises or consists of the amino acid sequence of SEQ ID NO: 53.

[0187] The PB or PBL transposase can comprise or consist of an amino acid sequence having an amino acid substitution at two or more, at three or more or at each of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 53. The transposase can be a SPB transposase that comprises or consists of the amino acid sequence of the sequence of SEQ ID NO: 53 wherein the amino acid substitution at position 30 can be a substitution of a valine (V) for an isoleucine (I), the amino acid substitution at position 165 can be a substitution of a serine (S) for a glycine (G), the amino acid substitution at position 282 can be a substitution of a valine (V) for a methionine (M), and the amino acid substitution at position 538 can be a substitution of a lysine (K) for an asparagine (N).

[0188] In some embodiments, the SPB transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 54. In some embodiments, the SPB transposase comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the SPB transposase is encoded by an nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 66. In some embodiments, the SPB transposase is encoded by the nucleic acid sequence of SEQ ID NO: 66.

[0189] In some embodiments, the SPB transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 67. In some embodiments, the SPB transposase comprises or consists of the amino acid sequence of SEQ ID NO: 67. In some embodiments, the SPB transposase comprises or consists of an nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 68. In some embodiments, the SPB transposase comprises or consists of the nucleic acid sequence of SEQ ID NO: 68.

[0190] In certain aspects wherein the transposase comprises the above-described mutations at positions 30, 165, 282 and / or 538, the PB, PBL and SPB 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: 53 orAttorney Docket No.: POTH-092 / 001WO SEQ ID NO: 54 are described in more detail in PCT Publication No. WO 2019 / 173636 and WO2020051374A1, each of which is incorporated herein by reference in its entirety for examples of mutations that may be introduced into the transposases used in the methods disclosed herein.

[0191] 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 Publication No. WO 2019 / 173636 and WO2020051374A1, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in the methods disclosed herein. In preferred aspects, the PB, PBL or SPB transposases can be isolated or derived from the insect Trichoplusia ni (GenBank Accession No. AAA87375) or Bombyx mori (GenBank Accession No. BAD11135).

[0192] 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 / 173636, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in the methods disclosed herein. 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 that may be used in the methods disclosed herein.

[0193] 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 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 that may be used in the methods disclosed herein.

[0194] 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 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 WOAttorney Docket No.: POTH-092 / 001WO 2019 / 173636, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in the methods disclosed herein.

[0195] 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 aspects, the Sleeping Beauty transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 55. In a preferred aspect, the Sleeping Beauty transposase comprises or consists of the amino acid sequence of SEQ ID NO: 55. In some aspects, the hyperactive Sleeping Beauty (SB100X) transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 56. In a preferred aspect, the hyperactive Sleeping Beauty (SB100X) transposase comprises or consists of the amino acid sequence of SEQ ID NO: 56.

[0196] A transposon of the present disclosure can be a Helraiser transposon. An exemplary Helraiser transposon includes Helibat1, which comprises or consists of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 57. In some embodiments, a Helibat1 comprises or consists of the nucleic acid sequence of SEQ ID NO: 57. In some aspects, when the transposon is a Helraiser transposon, the transposase is a Helitron transposase (for example, as disclosed in WO 2019 / 173636). In some aspects, the Helitron transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 58. In a preferred aspect, Helitron transposase comprises or consists of the amino acid sequence of SEQ ID NO: 58.

[0197] A transposon of the present disclosure can be a Tol2 transposon. An exemplary Tol2 transposon, including inverted repeats, subterminal sequences and the Tol2 transposase, comprises or consists of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 59. 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 a preferred aspect, the Tol2 transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 60. In some embodiments, the Tol2 transposase, comprises or consists of the nucleic acid sequence or amino acid sequence of SEQ ID NO: 59 or 60.Attorney Docket No.: POTH-092 / 001WO

[0198] 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 aspects, a TcBuster transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 61. In a preferred aspect, a TcBuster transposase comprises or consists of the amino acid sequence of SEQ ID NO: 61. 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. In some aspects, a TcBuster transposase is encoded by a polynucleotide comprising or consisting of an nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% (or any percentage in between) identical to SEQ ID NO: 62. In a preferred aspect, a TcBuster transposase is encoded by a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 62.

[0199] 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 Publication No. WO 2019 / 173636 and WO2020051374A1, each of which is incorporated herein by reference in its entirety for examples of transposases that may be used in the methods disclosed herein.

[0200] 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).

[0201] 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, betweenAttorney Docket No.: POTH-092 / 001WO 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.

[0202] 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, 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.

[0203] 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.

[0204] 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.

[0205] 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 ofAttorney Docket No.: POTH-092 / 001WO 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).

[0206] In some aspects of the nanotransposon, the sequence encoding the backbone does not comprise a recombination site, an excision site, a ligation site or a combination thereof. In some aspects, neither the nanotransposon nor the sequence encoding the backbone comprises a product of a recombination site, an excision site, a ligation site or a combination thereof. In some aspects, neither the nanotransposon nor the sequence encoding the backbone is derived from a recombination site, an excision site, a ligation site or a combination thereof.

[0207] 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).

[0208] In some aspects of the nanotransposon, the sequence encoding the backbone does not further comprise a sequence encoding foreign DNA.

[0209] 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 (polyA) 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 (polyA) sequence and a second sequence encoding an insulator.Attorney Docket No.: POTH-092 / 001WO

[0210] Nanotransposons are described in more detail in WO2020132396A1, which is incorporated herein by reference in its entirety for examples of transposons that may be used in the methods disclosed herein.

[0211] 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.

[0212] 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. Methods of Expressing Recombinant DHFR and a Chimeric Antigen Receptor

[0213] In some embodiments, the disclosure provides methods of expressing DHFR conferring MTX resistance and a CAR on the surface of a cell. In some embodiments, the method comprises (a) obtaining a cell population; (b) contacting the cell population to a composition comprising a nucleic acid sequence encoding DHFR and a nucleic acid sequence encoding the CAR, under conditions sufficient to transfer the two nucleic acids across a cell membrane of at least one cell in the cell population, thereby generating a modified cellAttorney Docket No.: POTH-092 / 001WO population; (c) culturing the modified cell population under conditions suitable for integration of the nucleic acid sequences encoding DHFR and the CAR; and (d) selecting for MTX resistance and expanding the at least one cell from the modified cell population that expresses DHFR and the CAR on the cell surface.

[0214] In certain embodiments, the gene encoding DHFR encodes a DHFR mutein enzyme. The DHFR mutein enzyme comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 63. The DHFR mutein enzyme is encoded by a polynucleotide comprising, consisting essential of, or consisting of the nucleic acid sequence of SEQ ID NO: 64 or SEQ ID NO: 65. The amino acid sequence of the DHFR mutein enzyme can further comprise a mutation at one or more of positions 80, 113, or 153. The amino acid sequence of the DHFR mutein enzyme can comprise one or more of a substitution of a Phenylalanine (F) or a Leucine (L) at position 80, a substitution of a Leucine (L) or a Valine (V) at position 113, and a substitution of a Valine (V) or an Aspartic Acid (D) at position 153.

[0215] In some aspects, the cell population can comprise leukocytes and / or CD4+ and CD8+ leukocytes. The cell population can comprise CD4+ and CD8+ leukocytes in an optimized ratio. CD8+ T cells exhibit a potent capacity for tumor cell killing, while CD4+ T cells provide many of the cytokines required to support CD8+ T cell proliferative capacity and function. Because T cells isolated from normal donors are predominantly CD4+, the T-cell product compositions are artificially adjusted in vitro with respect to the CD4+:CD8+ ratio to improve upon the ratio of CD4+ T cells to CD8+ T cells that would otherwise be present in vivo. An optimized ratio may also be used for the ex vivo expansion of the autologous T- cell product composition. In view of the artificially adjusted CD4+:CD8+ ratio of the T-cell product composition, it is important to note that the product compositions of the disclosure may be significantly different and provide significantly greater advantage than any endogenously-occurring population of T-cells.

[0216] The particular ratio at which CD4+ T cells and CD8+ T cells may be reconstituted may depend upon the type and efficacy of expansion technology used, cell medium, and / or growth conditions utilized for expansion of T-cell product compositions. Examples of possible CD4+: CD8+ ratios include, but are not limited to, 50%:50%, 60%:40%, 40%:60% 75%:25% and 25%:75%.

[0217] In some aspects, the conditions sufficient to transfer the nucleic acid sequences encoding DHFR and the CAR, transposon, or vector comprising such 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 supplementalAttorney Docket No.: POTH-092 / 001WO factor(s). In some aspects, the conditions suitable for integration of the sequences encoding DHFR and the CAR comprise at least one of a buffer and one or more supplemental factor(s).

[0218] The buffer can comprise PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, Human T cell nucleofection buffer or any combination thereof. The one or more supplemental factor(s) can comprise (a) a recombinant human cytokine, a chemokine, an interleukin or any combination thereof; (b) a salt, a mineral, a metabolite or any combination thereof; (c) a cell medium; (d) an inhibitor of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathway(s) or combinations thereof; and (e) a reagent that modifies or stabilizes one or more nucleic acids. The recombinant human cytokine, the chemokine, the interleukin or any combination thereof can comprise IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, IL14, IL16, IL17, IL18, IL19, IL20, IL22, IL23, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36, GM-CSF, IFN-gamma, IL-1 alpha / IL-1F1, IL-1 beta / IL-1F2, IL-12 p70, IL-12 / IL-35 p35, IL-13, IL-17 / IL-17A, IL-17A / F Heterodimer, IL-17F, IL-18 / IL- 1F4, IL-23, 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, MgCl2, 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, Poly-vinylpyrrolidone, 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, Caspase1, Pro-IL1B, PI3K, Akt, Wnt3A, inhibitors of glycogen synthase kinase-3β (GSK-3 β) (e.g. TWS119), or any combination thereof. Examples of suchAttorney Docket No.: POTH-092 / 001WO 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.

[0219] 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.

[0220] 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 paramagetic 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.

[0221] In some aspects wherein the transposon or vector comprises an selection gene in addition to DHFR, the selection step comprises contacting at least one cell of the modified cell population with a compound to which the selection gene confers resistance, thereby identifying a cell expressing the selection gene as surviving the selection and identifying a cell failing to express the selection gene as failing to survive the selection step. The selection gene may be used to manufacture of the MTX-CAR cells to ensure a homogenous population of MTX-CAR cells.

[0222] In some embodiments, the disclosure provides a composition comprising the modified, expanded and selected cell population of the methods described herein.

[0223] A more detailed description of methods for expressing a CAR on the surface of a cell is disclosed in PCT Publication No. WO 2019 / 049816 and WO2020051374A1.

[0224] In some embodiments, the present disclosure provides a cell or a population of cells wherein the cell comprises a composition comprising (a) an inducible transgene construct, comprising a sequence encoding an inducible promoter and a sequence encoding a transgene,Attorney Docket No.: POTH-092 / 001WO and (b) a receptor construct, comprising a sequence encoding a constitutive promoter and a sequence encoding an exogenous receptor, such as a CAR, wherein, upon integration of the construct of (a) and the construct of (b) into a genomic sequence of a cell, the exogenous receptor is expressed, and wherein the exogenous receptor, upon binding a ligand or antigen, transduces an intracellular signal that targets directly or indirectly the inducible promoter regulating expression of the inducible transgene (a) to modify gene expression.

[0225] The composition can modify gene expression by decreasing gene expression. The composition can modify gene expression by transiently modifying gene expression (e.g., for the duration of binding of the ligand to the exogenous receptor). The composition can modify gene expression acutely (e.g., the ligand reversibly binds to the exogenous receptor). The composition can modify gene expression chronically (e.g., the ligand irreversibly binds to the exogenous receptor).

[0226] The exogenous receptor can comprise an endogenous receptor with respect to the genomic sequence of the cell. Exemplary receptors include, but are not limited to, intracellular receptors, cell-surface receptors, transmembrane receptors, ligand-gated ion channels, and G-protein coupled receptors.

[0227] The exogenous receptor can comprise a non-naturally occurring receptor. The non- naturally occurring receptor can be a synthetic, modified, recombinant, mutant or chimeric receptor. The non-naturally occurring receptor can comprise one or more sequences isolated or derived from a T-cell receptor (TCR). The non-naturally occurring receptor can comprise one or more sequences isolated or derived from a scaffold protein. In some aspects, including those wherein the non-naturally occurring receptor does not comprise a transmembrane domain, the non-naturally occurring receptor interacts with a second transmembrane, membrane-bound and / or an intracellular receptor that, following contact with the non- naturally occurring receptor, transduces an intracellular signal. The non-naturally occurring receptor can comprise a transmembrane domain. The non-naturally occurring receptor can interact with an intracellular receptor that transduces an intracellular signal. The non- naturally occurring receptor can comprise an intracellular signaling domain. The non- naturally occurring receptor can be a chimeric ligand receptor (CLR). The CLR can be a chimeric antigen receptor (CAR), for example, any CAR or V-CAR described herein.

[0228] The sequence encoding the inducible promoter of comprises a sequence encoding an NFĸB promoter, a sequence encoding an interferon (IFN) promoter or a sequence encoding an interleukin-2 promoter. In some aspects, the IFN promoter is an IFNγ promoter. The inducible promoter can be isolated or derived from the promoter of a cytokine or aAttorney Docket No.: POTH-092 / 001WO chemokine. The cytokine or chemokine can comprise IL2, IL3, IL4, IL5, IL6, IL10, IL12, IL13, IL17A / F, IL21, IL22, IL23, transforming growth factor beta (TGFβ), colony stimulating factor 2 (GM-CSF), interferon gamma (IFNγ), Tumor necrosis factor alpha (TNFα), LTα, perforin, Granzyme C (Gzmc), Granzyme B (Gzmb), C-C motif chemokine ligand 5 (CCL5), C-C motif chemokine ligand 4 (Ccl4), C-C motif chemokine ligand 3 (Ccl3), X-C motif chemokine ligand 1 (Xcl1) or LIF interleukin 6 family cytokine (Lif).

[0229] The inducible promoter can be isolated or derived from the promoter of a gene comprising a surface protein involved in cell differentiation, activation, exhaustion and function. In some aspects, the gene comprises CD69, CD71, CTLA4, PD-1, TIGIT, LAG3, TIM-3, GITR, MHCII, COX-2, FASL or 4-1BB.

[0230] The inducible promoter can be isolated or derived from the promoter of a gene involved in CD metabolism and differentiation. The inducible promoter can be isolated or derived from the promoter of Nr4a1, Nr4a3, Tnfrsf9 (4-1BB), Sema7a, Zfp36l2, Gadd45b, Dusp5, Dusp6 and Neto2.

[0231] In some aspects, the inducible transgene construct comprises or drives expression of a signaling component downstream of an inhibitory checkpoint signal, a transcription factor, a cytokine or a cytokine receptor, a chemokine or a chemokine receptor, a cell death or apoptosis receptor / ligand, a metabolic sensing molecule, a protein conferring sensitivity to a cancer therapy, and an oncogene or a tumor suppressor gene. Non-limiting examples of which are disclosed in PCT Publication No. WO 2019 / 173636 and PCT Application No. WO2020051374A1. Methotrexate and Methotrexate Analogs

[0232] In one aspect, the drug administered to enrich DHFR expressing CAR-T cells, including CAR-T cells expressing a DHFR mutein, is methotrexate. In one aspect, the drug administered to enrich DHFR expressing CAR-T cells, including CAR-T cells expressing a DHFR mutein, is a methotrexate analog.

[0233] Methotrexate (MTX) is a well-known antimetabolite that blocks the activity of dihydrofolate reductase (DHFR), which in turn inhibits cellular synthesis of folic acid. MTX has been used in a clinical setting to treat certain autoimmune disorders and cancer types since the 1950s (e.g., see Wood and Yu Dermatol Clin.2015 October ; 33(4): 747–755. doi:10.1016 / j.det.2015.05.009). For cancer treatment, MTX has been used primarily to treat mycosis fungoides (MF) and Sézary syndrome (SS) and primary cutaneous CD30+Attorney Docket No.: POTH-092 / 001WO lymphoproliferative disorders (LPDs) such as lymphomatoid papulosis (LyP) and anaplastic large cell lymphoma (cALCL).

[0234] Suitable methotrexate analogs for use in the methods disclosed herein include a more potent, recently FDA approved MTX analog, pralatrexate (PDX; 10-propargyl-10- deazaaminopterin). PDX is FDA-approved for refractory or relapsed peripheral T-cell lymphomas (PTCLs). Additional methotrexate analogs suitable for use in the methods disclosed herein include Talotrexin, Leucovorin, Tomudex, Alimta, Trimethoprim, Cycloguanil, Pemetrexed (PMX), Raltitrexed (RTX), Plevitrexed, Piritrexim, Nolatrexed, folic acid (FA), 5-formyl-tetrahydrofolic acid (5FFH4), dihydrofolic acid (FH2), 10-methyl- tetrahydrofolic acid (10MFH4), tetrahydrofolic acid (FH4), aminopterin (AMP), lomexetrol, AQA, multi-target antifolate (MTA), raltitrexed, 10-deaza-aminopterin (10 DAAM), 10- methyl-deaza-aminopterin (10 MDAAM), 10-ethyl-deaza-aminopterin (10 EDAAM), or any combination thereof. Methods of Using the Compositions of the Disclosure

[0235] The disclosure provides the use of a disclosed composition or pharmaceutical composition for the treatment of a cancer in a subject, 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 MTX-CAR cells. In one aspect, the subject is a mammal. Preferably, the subject is human. The terms “subject” and “patient” are used interchangeably herein.

[0236] In some embodiments, 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, 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,Attorney Docket No.: POTH-092 / 001WO testicular cancer, adenocarcinomas, sarcomas, malignant melanoma, hemangioma, metastatic disease, cancer related bone resorption, cancer related bone pain, and the like.

[0237] In some embodiments, the disclosure provides the use of a disclosed composition or pharmaceutical composition for the treatment of an Autoimmune disease in a subject, 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 MTX-CAR cells. In one aspect, the subject is a mammal. Preferably, the subject is human.

[0238] In some embodiments, the disclosure provides a method for modulating or treating an autoimmune disease in a cell, tissue, organ, animal or subject. Non-limiting examples of an autoimmune disease include autoimmune neutropenia, Guillain-Barré syndrome, epilepsy, autoimmune encephalitis, Isaacs' syndrome, nevus syndrome, pemphigus vulgaris, deciduous pemphigus, bullous pemphigoid, acquired epidermolysis bullosa, gestational pemphigoid, mucous membrane pemphigoid, antiphospholipid syndrome, autoimmune anemia, autoimmune Graves' disease, thyroid eye disease (TED), Goodpasture syndrome, myasthenia gravis, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura (ITP), warm autoimmune hemolytic anemia (WAIHA), chronic inflammatory demyelinating polyneuropathy (CIDP), lupus nephritis, IgA nephropathy (Berger’s Disease), dermatomyositis, necrotizing autoimmune myopathy, and membranous nephropathy.

[0239] In some embodiments, 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) or an MTX analog. 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 CAR cells thereby increasing the in vivo persistence, potency, and / or 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 cryopreservation and subsequent thawing. Modified cells can be formulated in a pharmaceutically acceptable carrier for direct administration to a subject from sterile packaging. Modified cells can be formulated in a pharmaceutically acceptable carrier with an indicator of cell viability and / or CAR 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.Attorney Docket No.: POTH-092 / 001WO

[0240] In some embodiments, the methods of the present disclosure comprise administering an effective amount of MTX, or an MTX analog, 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.

[0241] In some embodiments the effective dose of MTX, or an MTX analog, is 5 to 30 mg / m2, 10 to 25 mg / m2, or 15 to 20 mg / m2. In some embodiments the effective dose of MTX, or an MTX analog, is 2 to 35 mg, 5 to 25 mg, or 10 to 20 mg. In some embodiments the effective dose of MTX is administered once per week. In some embodiments, the dose of MTX, or an MTX analog, is administered twice per week. In some embodiments, the MTX, or an MTX analog, is administered orally.

[0242] In some embodiments, the effective dose of MTX, or an MTX analog, is 10 mg / m2once per week or twice per week. In some embodiments, the effective dose of MTX, or an MTX analog, is 20 mg / m2once per week. In some embodiments, the effective dose of MTX, or an MTX analog, is 7.5 mg once per week. In some embodiments the effective dose of MTX, or an MTX analog, is 10-25 mg once per week. In some embodiments, the effective dose of MTX, or an MTX analog, is 25-75 mg one per week.

[0243] The methods can optionally further comprise co-administration or combination therapy for treating 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).

[0244] The methods can optionally further comprise co-administration or combination therapy for treating an autoimmune condition or disease, wherein the administering of any composition or pharmaceutical composition disclosed herein, further comprises administering, before concurrently, and / or after, at least one immunosuppressant or immunostimulatory agent.

[0245] The methods can optionally further comprise co-administration or combination therapy for simultaneously cancer and autoimmune condition or disease, wherein the administering of any composition or pharmaceutical composition disclosed herein, furtherAttorney Docket No.: POTH-092 / 001WO comprises administering, before concurrently, and / or after, at least one immunosuppressant agent, immunostimulatory agent, chemotherapeutic agent, or a combination thereof.

[0246] 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.

[0247] 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 or any number of doses in between that are manufactured simultaneously. In some aspects, where the composition is autologous cells or allogeneic 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.

[0248] In some aspects, the therapeutically effective amount of the MTX-CAR cells in the composition is between 0.5e6 and 5e9 CAR cells. In some aspects, the therapeutically effective amount of the MTX-CAR cells in the composition is between 0.5e7 and 5e8 CAR cells. In some aspects, the therapeutically effective amount of the MTX-CAR cells in the composition is between 5e6 and 15e6 CAR cells. In some aspects, the therapeutically effective amount of the MTX-CAR cells in the composition is between 0.5e6 and 10e6 CAR cells.

[0249] 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 scFv CAR or 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.

[0250] 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), 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 ofAttorney Docket No.: POTH-092 / 001WO 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).

[0251] In certain aspects, provided are methods of increasing in vivo persistence of 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, or an MTX analog, sufficient to eliminate (e.g. reduce by at least 60%) activated T-cells and NK cells targeting the MTX-CAR cells leading to an increased in vivo persistence of the MTX-CAR-T 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, CD19, CD20, MUC1C, PSMA, CD70 or c-kit. In certain embodiments, the MTX- CAR cells are T-cells (i.e., MTX-CAR cells). In certain embodiments, the MTX-CAR cells are T-cells (MTX-CAR-T cells).

[0252] A person of skill in the art will understand that any existing CAR-T cells therapy may be improved by the expression of DHFR as described herein. Examples of CAR-T therapies that may be improved using the methods disclosed tisagenlecleucel (Kymriah®), axicabtagene ciloleucel (Yescarta®), lisocabtagene maraleucel (Breyanzi ®), brexucabtagene autoleucel (Tecartus®), ciltacabtegene autoleucel (Carvykti®) and iIdecabtagene vicleucel (Abecma ®).

[0253] In certain aspects, provided are methods of increasing in vivo persistence and / or potency of CAR-T cells in a subject comprising administering to the subject a therapeutically effective amount of MTX-CAR cells, wherein the MTX-CAR cells comprise a heterologous nucleic acid encoding dihydrofolate reductase (DHFR); and, administering at a predetermined time post administration of the MTX-CAR cells, an effective amount of MTX, or an MTX analog, sufficient to eliminate (reduce by at least 60%) activated T-cells and NK cells targeting the MTX-CAR cells leading to an increased in vivo persistence and / or potency of the 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, CD19, CD20, MUC1C, PSMA, CD70, CD7 or c-kit. In certain embodiments, the MTX-CAR cells are T-cells (i.e., MTX-CAR cells). In certain embodiments, the MTX-CAR cells are T-cells (MTX-CAR-T cells).Attorney Docket No.: POTH-092 / 001WO

[0254] 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 cells; and administering at a predetermined time post administration of the MTX-CAR cells an effective amount of MTX, or an MTX analog, sufficient to eliminate (reduce by at least 60%) activated T-cells and NK cells targeting the CAR cells leading to an increased in vivo persistence and / or potency of the MTX-CAR cells and increased treatment efficacy of the MTX-CAR cells compared to no MTX or MTX analog administration. 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, CD19, CD20, MUC1C, PSMA, CD70, CD7, or c-kit. In certain embodiments, the MTX-CAR cells are T-cells (i.e., MTX-CAR cells). In certain embodiments, the MTX-CAR cells are T-cells (MTX-CAR-T cells).

[0255] In certain embodiments, the administration of a therapeutically effective amount of any composition or pharmaceutical composition disclosed herein and MTX reduces activated T-cells and NK cells targeting the CAR cells by at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% compared to no MTX administrations. In certain embodiments, the administration of a therapeutically effective amount of any composition or pharmaceutical composition disclosed herein and MTX reduces activated T-cells and NK cells targeting the CAR cells by about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% compared to no MTX administration. In certain embodiments, the administration of a therapeutically effective amount of any composition or pharmaceutical composition disclosed herein and MTX reduces the activated T-cells and NK cells targeting CAR cells by 60-99%, 70-95%, or 80-90% compared to no MTX administration.

[0256] In certain embodiments, the administration of a therapeutically effective amount of MTX-CAR cells and MTX reduces activated T-cells and NK cells targeting the CAR cells by at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% compared to no MTX administrations. In certain embodiments, the administration of a therapeutically effective amount of MTX-CAR cells and MTX reduces activated T-cells and NK cells targeting the CAR cells by about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% compared to no MTX administration. In certain embodiments, the administration of a therapeutically effective amount of MTX-CAR cells and MTX reduces the activated T- cells and NK cells targeting CAR cells by 60-99%, 70-95%, or 80-90% compared to no MTX administration.Attorney Docket No.: POTH-092 / 001WO

[0257] In some embodiments, the administration of a therapeutically effective amount of MTX, or an MTX analog, results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% improvement in the persistence of any composition or pharmaceutical composition disclosed herein compared to no MTX or MTX analog administration. In some embodiments, the administration of a therapeutically effective amount of MTX, or an MTX analog, results in an at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% improvement in the persistence of any composition or pharmaceutical composition disclosed herein compared to no MTX or MTX analog administration. In some embodiments, the administration of a therapeutically effective amount of MTX, or an MTX analog, results in a 10-90%, 20-80%, 30-70%, or 40-60% improvement in the persistence of any composition or pharmaceutical composition disclosed herein compared to no MTX or MTX analog administration. In some embodiments, persistence is measured by flow cytometry.

[0258] In some embodiments, the administration of a therapeutically effective amount of MTX, or an MTX analog, results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% improvement in the persistence of MTX-CAR cells compared to no MTX or MTX analog administration. In some embodiments, the administration of a therapeutically effective amount of MTX, or an MTX analog, results in an at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% improvement in the persistence of MTX-CAR cells compared to no MTX or MTX analog administration. In some embodiments, the administration of a therapeutically effective amount of MTX, or an MTX analog, results in a 10-90%, 20-80%, 30-70%, or 40-60% improvement in the persistence of MTX-CAR cells compared to no MTX or MTX analog administration. In some embodiments, persistence is measured by flow cytometry.

[0259] 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.

[0260] 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 theAttorney Docket No.: POTH-092 / 001WO 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 about 8 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.

[0261] 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. In certain embodiments, the predetermined time post administration of the MTX-CARAttorney Docket No.: POTH-092 / 001WO 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.

[0262] In certain embodiments, the 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 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 and 26 post- administration of the MTX-CAR cells.

[0263] In certain aspects, provided are methods of increasing in vivo persistence and / or potency of cell therapies described above further comprising administering at a predetermined time an effective dose of MTX, or an MTX analog, prior to administration of the therapeutically effective dose of the MTX-CAR cells. In certain aspects, provided are methods of increasing in vivo persistence of CAR cells described above further comprising administering at a predetermined time an effective dose of MTX, or an MTX analog, prior to administration of the therapeutically effective dose of the MTX-CAR cells. In certain aspects, provided are methods of increasing in vivo potency of CAR cells described above further comprising administering at a predetermined time an effective dose of MTX, or an MTX analog, 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, or an MTX analog, 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, or an MTX analog, 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, or an MTX analog, 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 aAttorney Docket No.: POTH-092 / 001WO predetermined time an effective dose of MTX, or an MTX analog, 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, or an MTX analog, 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.

[0264] 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, or an MTX analog, 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, or an MTX analog, 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, or an MTX analog, 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, or an MTX analog, 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, or an MTX analog, 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 Acid Molecules

[0265] Nucleic acid molecules of the disclosure can be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including, but not limited to, cDNA and genomic DNA obtained by cloning or produced synthetically, or any combinations thereof. The DNA can be triple-stranded, double-stranded or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also referred to as the anti-sense strand.Attorney Docket No.: POTH-092 / 001WO

[0266] Isolated nucleic acid molecules of the disclosure can include nucleic acid molecules comprising an open reading frame (ORF), optionally, with one or more introns, e.g., but not limited to, at least one specified portion of at least one scFv; nucleic acid molecules comprising the coding sequence for a protein scaffold or loop region that binds to the target protein; and nucleic acid molecules which comprise a nucleotide sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the protein scaffold as described herein and / or as known in the art. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate nucleic acid variants that code for a specific scFv of the present disclosure. See, e.g., Ausubel, et al., supra, and such nucleic acid variants are included in the present disclosure.

[0267] As indicated herein, nucleic acid molecules of the disclosure which comprise a nucleic acid can include, but are not limited to, those encoding the amino acid sequence of a scFv fragment, by itself; the coding sequence for the entire protein scaffold or a portion thereof; the coding sequence for a scFv, fragment or portion, as well as additional sequences, such as the coding sequence of at least one signal leader or fusion peptide, with or without the aforementioned additional coding sequences, such as at least one intron, together with additional, non-coding sequences, including but not limited to, non-coding 5′ and 3′ sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals (for example, ribosome binding and stability of mRNA); an additional coding sequence that codes for additional amino acids, such as those that provide additional functionalities. Thus, the sequence encoding a protein scaffold can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused protein scaffold comprising a protein scaffold fragment or portion.

[0268] The isolated nucleic acids of the disclosure can be made using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and / or (d) combinations thereof, as well-known in the art.

[0269] The nucleic acids can conveniently comprise nucleotide sequences in addition to a polynucleotide of the present disclosure. For example, a multi-cloning site comprising one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in isolation of the polynucleotide. Also, translatable sequences can be inserted to aid in the isolation of the translated polynucleotide of the disclosure. For example, a hexa-histidine marker sequence provides a convenient means to purify the proteins of the disclosure. The nucleic acid of theAttorney Docket No.: POTH-092 / 001WO disclosure, excluding the coding sequence, is optionally a vector, adapter, or linker for cloning and / or expression of a polynucleotide of the disclosure.

[0270] Additional sequences can be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell. Use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra; or Sambrook, supra).

[0271] 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).

[0272] A cDNA or genomic library can be screened using a probe based upon the sequence of a polynucleotide of the disclosure. Probes can be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms. Those of skill in the art will appreciate that various degrees of stringency of hybridization can be employed in the assay; and either the hybridization or the wash medium can be stringent. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for duplex formation to occur. The degree of stringency can be controlled by one or more of temperature, ionic strength, pH and the presence of a partially denaturing solvent, such as formamide. For example, the stringency of hybridization is conveniently varied by changing the polarity of the reactant solution through, for example, manipulation of the concentration of formamide within the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding will vary in accordance with the stringency of the hybridization medium and / or wash medium. The degree of complementarity will optimally be 100%, or 70-100%, or any range or value therein. However, it should be understood that minor sequence variations in the probes and primers can be compensated for by reducing the stringency of the hybridization and / or wash medium.Attorney Docket No.: POTH-092 / 001WO

[0273] 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.

[0274] 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.)

[0275] 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). Commercially 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.

[0276] 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.

[0277] Given the redundancy in the genetic code, a plurality of nucleotide sequences may encode any particular protein. All such nucleotides sequences are contemplated herein.Attorney Docket No.: POTH-092 / 001WO Expression Cassettes

[0278] The disclosure further provides recombinant expression cassettes comprising a nucleic acid of the disclosure. A nucleic acid sequence of the disclosure, for example, a cDNA or a genomic sequence encoding a protein scaffold 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.

[0279] In some aspects, isolated nucleic acids that serve as promoter, enhancer, or other elements can be introduced in the appropriate position (upstream, downstream or in the intron) of a non-heterologous form of a polynucleotide of the disclosure so as to up or down regulate expression of a polynucleotide of the disclosure. For example, endogenous promoters can be altered in vivo or in vitro by mutation, deletion and / or substitution.

[0280] 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 operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, EF-1 Alpha promoter, CAG promoter, SV40 early promoter or SV40 late promoter and the CMV IE promoter.Attorney Docket No.: POTH-092 / 001WO Expression Vectors and Host Cells

[0281] The disclosure also relates to vectors that include isolated nucleic acid molecules of the disclosure, host cells that are genetically engineered with the recombinant vectors, and the production of at least one protein scaffold by recombinant techniques, as is well known in the art. See, e.g., Sambrook, et al., supra; Ausubel, et al., supra, each entirely incorporated herein by reference. As used throughout the disclosure, the term "vector" refers to a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector, and preferably, is a DNA plasmid. A vector may comprise a combination of an amino acid with a DNA sequence, an RNA sequence, or both a DNA and an RNA sequence.

[0282] 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.

[0283] 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.

[0284] In some embodiments, 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 prokaryotics (the above patents are entirely incorporated hereby by reference). Appropriate culture mediums and conditions for the above-described host cells areAttorney Docket No.: POTH-092 / 001WO 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.

[0285] In certain embodiments, expression vectors will preferably but optionally include at least one selectable cell surface marker for isolation of cells modified by the compositions and methods of the disclosure. Selectable cell surface markers of the disclosure comprise surface proteins, glycoproteins, or group of proteins that distinguish a cell or subset of cells from another defined subset of cells. 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).

[0286] In certain embodiments, expression vectors will preferably but optionally include at least one selectable drug resistance marker for isolation of cells modified by the compositions and methods of the disclosure. Selectable drug resistance markers of the disclosure may comprise wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

[0287] At least one protein scaffold of the disclosure can be expressed in a modified form, such as a fusion protein, and can include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of a protein scaffold to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to a protein scaffold of the disclosure to facilitate purification. Such regions can be removed prior to final preparation of a protein scaffold or at least one fragment thereof. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18.

[0288] 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.Attorney Docket No.: POTH-092 / 001WO 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.

[0289] Illustrative of cell cultures useful for the production of the protein scaffolds, specified portions or variants thereof, 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-Ag14, 293 cells, HeLa cells and the like, which are readily available from, for example, American Type Culture Collection, Manassas, Va. (www.atcc.org). Preferred host cells include cells of lymphoid origin, such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC Accession Number CRL-1580) and SP2 / 0-Ag14 cells (ATCC Accession Number CRL-1851). In a preferred aspect, the recombinant cell is a P3X63Ab8.653 or an SP2 / 0-Ag14 cell.

[0290] 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 (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.

[0291] When eukaryotic host cells are employed, polyadenlyation or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript can also be included. An example of a splicing sequence is the VP1Attorney Docket No.: POTH-092 / 001WO 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. Amino Acid Codes

[0292] The amino acids that make up protein scaffolds of the disclosure are often abbreviated. The amino acid designations can be indicated by designating the amino acid by its single letter code, its three letter code, name, or three nucleotide codon(s) as is well understood in the art (see Alberts, B., et al., Molecular Biology of The Cell, Third Ed., Garland Publishing, Inc., New York, 1994). A protein scaffold of the disclosure can include one or more amino acid substitutions, deletions or additions, from spontaneous or mutations and / or human manipulation, as specified herein. Amino acids in a protein scaffold of the disclosure that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as, but not limited to, at least one neutralizing activity. Sites that are critical for protein scaffold binding can also be identified by structural analysis, such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith, et al., J. Mol. Biol.224:899-904 (1992) and de Vos, et al., Science 255:306- 312 (1992)).

[0293] As those of skill will appreciate, the disclosure includes at least one biologically active protein scaffold of the disclosure. Biologically active protein scaffolds have a specific activity at least 20%, 30%, or 40%, and, preferably, at least 50%, 60%, or 70%, and, most preferably, at least 80%, 90%, or 95%-99% or more of the specific activity of the native (non-synthetic), endogenous or related and known protein scaffold. Methods of assaying and quantifying measures of enzymatic activity and substrate specificity are well known to those of skill in the art.

[0294] In another aspect, the disclosure relates to protein scaffolds and fragments, as described herein, which are modified by the covalent attachment of an organic moiety. Such modification can produce a protein scaffold fragment with improved pharmacokinetic properties (e.g., increased in vivo serum half-life). The organic moiety can be a linear or branched hydrophilic polymeric group, fatty acid group, or fatty acid ester group. In particular aspect, the hydrophilic polymeric group can have a molecular weight of about 800 to about 120,000 Daltons and can be a polyalkane glycol (e.g., polyethylene glycol (PEG),Attorney Docket No.: POTH-092 / 001WO polypropylene glycol (PPG)), carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, and the fatty acid or fatty acid ester group can comprise from about eight to about forty carbon atoms.

[0295] The modified protein scaffolds and fragments of the disclosure can comprise one or more organic moieties that are covalently bonded, directly or indirectly, to the antibody. Each organic moiety that is bonded to a protein scaffold or fragment of the disclosure can independently be a hydrophilic polymeric group, a fatty acid group or a fatty acid ester group. As used herein, the term “fatty acid” encompasses mono-carboxylic acids and di-carboxylic acids. A “hydrophilic polymeric group,” as the term is used herein, refers to an organic polymer that is more soluble in water than in octane. For example, polylysine is more soluble in water than in octane. Thus, a protein scaffold modified by the covalent attachment of polylysine is encompassed by the disclosure. Hydrophilic polymers suitable for modifying protein scaffolds of the disclosure can be linear or branched and include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides and the like), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartate and the like), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide and the like) and polyvinyl pyrolidone. Preferably, the hydrophilic polymer that modifies the protein scaffold of the disclosure has a molecular weight of about 800 to about 150,000 Daltons as a separate molecular entity. For example, PEG5000 and PEG20,000, wherein the subscript is the average molecular weight of the polymer in Daltons, can be used. The hydrophilic polymeric group can be substituted with one to about six alkyl, fatty acid or fatty acid ester groups. Hydrophilic polymers that are substituted with a fatty acid or fatty acid ester group can be prepared by employing suitable methods. For example, a polymer comprising an amine group can be coupled to a carboxylate of the fatty acid or fatty acid ester, and an activated carboxylate (e.g., activated with N,N-carbonyl diimidazole) on a fatty acid or fatty acid ester can be coupled to a hydroxyl group on a polymer.

[0296] Fatty acids and fatty acid esters suitable for modifying protein scaffolds of the disclosure can be saturated or can contain one or more units of unsaturation. Fatty acids that are suitable for modifying protein scaffolds of the disclosure include, for example, n- dodecanoate (C12, laurate), n-tetradecanoate (C14, myristate), n-octadecanoate (C18, stearate), n-eicosanoate (C20, arachidate), n-docosanoate (C22, behenate), n-triacontanoate (C30), n-tetracontanoate (C40), cis-Δ9-octadecanoate (C18, oleate), all cis-Δ5,8,11,14- eicosatetraenoate (C20, arachidonate), octanedioic acid, tetradecanedioic acid,Attorney Docket No.: POTH-092 / 001WO octadecanedioic acid, docosanedioic acid, and the like. Suitable fatty acid esters include mono-esters of dicarboxylic acids that comprise a linear or branched lower alkyl group. The lower alkyl group can comprise from one to about twelve, preferably, one to about six, carbon atoms.

[0297] The modified protein scaffolds and fragments can be prepared using suitable methods, such as by reaction with one or more modifying agents. A “modifying agent” as the term is used herein, refers to a suitable organic group (e.g., hydrophilic polymer, a fatty acid, a fatty acid ester) that comprises an activating group. An “activating group” is a chemical moiety or functional group that can, under appropriate conditions, react with a second chemical group thereby forming a covalent bond between the modifying agent and the second chemical group. For example, amine-reactive activating groups include electrophilic groups, such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfides, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehyde functional group can be coupled to amine- or hydrazide-containing molecules, and an azide group can react with a trivalent phosphorous group to form phosphoramidate or phosphorimide linkages. Suitable methods to introduce activating groups into molecules are known in the art (see for example, Hermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)). An activating group can be bonded directly to the organic group (e.g., hydrophilic polymer, fatty acid, fatty acid ester), or through a linker moiety, for example, a divalent C1-C12 group wherein one or more carbon atoms can be replaced by a heteroatom, such as oxygen, nitrogen or sulfur. Suitable linker moieties include, for example, tetraethylene glycol, —(CH2)3—, —NH—(CH2)6—NH—, —(CH2)2—NH— and —CH2—O—CH2—CH2—O—CH2—CH2—O—CH—NH—. Modifying agents that comprise a linker moiety can be produced, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate. The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine that can be coupled to another carboxylate, as described, or can be reacted with maleic anhydride and the resulting product cyclized to produce an activated maleimido derivative of the fatty acid. (See, for example, Thompson, et al., WO 92 / 16221, the entire teachings of which are incorporated herein by reference.)Attorney Docket No.: POTH-092 / 001WO

[0298] The modified protein scaffolds of the disclosure can be produced by reacting a protein scaffold or fragment with a modifying agent. For example, the organic moieties can be bonded to the protein scaffold in a non-site specific manner by employing an amine-reactive modifying agent, for example, an NHS ester of PEG. Modified protein scaffolds and fragments comprising an organic moiety that is bonded to specific sites of a protein scaffold of the disclosure can be prepared using suitable methods, such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci.6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68 (1996); Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)), and the methods described in Hermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996). Definitions

[0299] 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.

[0300] 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. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0301] 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, anAttorney Docket No.: POTH-092 / 001WO "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.

[0302] 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 least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide of the disclosure.

[0303] 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.

[0304] “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.Attorney Docket No.: POTH-092 / 001WO

[0305] “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.

[0306] 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.

[0307] 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.

[0308] The term "scFv" refers to a single-chain variable fragment. scFv is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a linker peptide. The linker peptide may be from about 5 to 40 amino acids or from about 10 to 30 amino acids or about 5, 10, 15, 20, 25, 30, 35, or 40 amino acids in length. Single-chain variable fragments lack the constant Fc region found in complete antibody molecules, and, thus, the common binding sites (e.g., Protein G) used to purify antibodies. The term further includes a scFv that is an intrabody, an antibody that is stable in the cytoplasm of the cell, and which may bind to an intracellular protein.

[0309] The term “single domain antibody” means an antibody fragment having a single monomeric variable antibody domain which is able to bind selectively to a specific antigen. A single-domain antibody generally is a peptide chain of about 110 amino acids long, comprising one variable domain (VH) of a heavy-chain antibody, or of a common IgG, which generally have similar affinity to antigens as whole antibodies, but are more heat-resistant and stable towards detergents and high concentrations of urea. Examples are those derived from camelid or fish antibodies. Alternatively, single-domain antibodies can be made from common murine or human IgG with four chains.

[0310] The terms “specifically bind” and “specific binding” as used herein refer to the ability of an antibody, an antibody fragment or a nanobody to preferentially bind to a particular antigen that is present in a homogeneous mixture of different antigens. In some aspects, a specific binding interaction will discriminate between desirable and undesirable antigens in aAttorney Docket No.: POTH-092 / 001WO sample. In some aspects, more than about ten- to 100-fold or more (e.g., more than about 1000- or 10,000-fold). “Specificity” refers to the ability of an immunoglobulin or an immunoglobulin fragment, such as a nanobody, to bind preferentially to one antigenic target versus a different antigenic target and does not necessarily imply high affinity.

[0311] As used throughout the disclosure, the term "substantially identical" refers to a first and second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical 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 with respect to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.

[0312] As used throughout the disclosure, the term "variant" when used to describe a nucleic acid, refers to (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto.

[0313] As used throughout the disclosure, the term "variant" when used to describe a peptide or polypeptide, refers to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Variant can also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity.

[0314] 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 usefulAttorney Docket No.: POTH-092 / 001WO measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No.4,554,101, incorporated fully herein by reference.

[0315] 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 hyrophobicity 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.

[0316] 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 1. Table 1: Conservative Substitutions I Side chain characteristics Amino Acid

[0317] 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 2.Attorney Docket No.: POTH-092 / 001WO Table 2: Conservative Substitutions II Side Chain Characteristic Amino Acid Non-polar (hydrophobic) Aliphatic: A L I V P, . Table 3: Conservative Substitutions III Original Residue Exemplary SubstitutionAttorney Docket No.: POTH-092 / 001WO Original Residue Exemplary Substitution Ser (S) Thrde 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.

[0320] As used throughout the disclosure, the term “more than one” of the aforementioned amino acid substitutions refers to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more of the recited amino acid substitutions. The term “more than one” may refer to 2, 3, 4, or 5 of the recited amino acid substitutions.

[0321] Polypeptides and proteins of the disclosure, either their entire sequence, or any portion thereof, may be non-naturally occurring. Polypeptides and proteins of the disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not naturally-occur, rendering the entire amino acid sequence non-naturally occurring. Polypeptides and proteins 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 amino acid sequence non-naturally occurring. Polypeptides and proteins of the disclosure may contain modified, artificial, or synthetic amino acids that do not naturally- occur, rendering the entire amino acid sequence non-naturally occurring.

[0322] As used throughout the disclosure, “sequence identity” may be determined by using the stand-alone executable BLAST engine program for blasting two sequences (bl2seq), which can be retrieved from the National Center for Biotechnology Information (NCBI) ftp site, using the default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; which is incorporated herein by reference in its entirety). 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,Attorney Docket No.: POTH-092 / 001WO 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.

[0323] 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.

[0324] 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 is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid, e.g., DNA sequence, or naturally occurring nucleic acid sequence located in a non- naturally occurring genome location.

[0325] 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 Example 1 – General Methods for Preparation of Methotrexate Resistant CAR-T Cells

[0326] Chimeric antigen receptors (CARs) may be prepared having an antigen recognition region comprising a single chain antibody that specifically binds to an epitope of a desired target, e.g., MUC1-C, BMCA, CD19, CD20, PSMA, CD70, or c-kit.Attorney Docket No.: POTH-092 / 001WO

[0327] The following structure was used as an exemplary humanized target-scFv CAR: Signal peptide (CD8α) – Light Chain – Linker – Heavy Chain – Hinge (CD8α) – Transmembrane (CD8α) – Intracellular Signaling (4-1BB) – Intracellular Signaling (CD3ζ).

[0328] Candidate CARs were subcloned into a tricistronic piggyBac transposon (EF1alpha promoter – iC9 Safety Switch – T2A – target CAR – T2A – DHFR selection gene) according to Table 4 below and CAR-T cells were produced using pan T cells from a normal human blood donor as described herein. Table 4 Target Amino Acid Sequence Nucleic Acid Sequence BCMA SEQ ID NO: 72 SEQ ID NO: 73p p ggy confirmed by FACS staining 19 days post-transposon delivery using an His-tagged p62 / p58 target protein, followed by anti-His secondary antibody. Specifically, cells were examined by flow cytometry for surface-expression of CAR on either mock transposed or anti-CD3 / CD28 bead re-activated cells (activation for 48 hours) that received transposon encoding CAR and data are shown as overlaid histograms; numbers represent percentage of cells expressing CAR on cell surface. Example 2 – Antigen-stimulated CAR-T Cells Expressing DHFR and Unstimulated Pan T-Cells are Resistant In Vitro to Therapeutic Levels of MTX

[0330] Pan T-cells were isolated from two donors from either frozen or fresh leukopaks using a CliniMacs Prodigy Instrument (Miltenyi) and cell processing applications for positive selection for CD4+ / CD8+ T-cells. Isolated pan T-cells from one donor (CAR-T Donor 1) were rested overnight and frozen prior to CAR-T cell production.

[0331] CAR-T cells were generated essentially as described in Example 1 using an anti- BCMA chimeric antigen receptor comprising an anti-BCMA VH binder. Anti-BCMA CAR- T cells were stimulated using irradiated K562-BCMA-GFP cells, which express BCMA, at a ratio of 5:1 (1e6 CAR-T cells to 2e5 K562-BCMA-GFP cells) for 4 days at 37 °C.Attorney Docket No.: POTH-092 / 001WO

[0332] Approximately 50,000 unstimulated Pan-T cells, stimulated Pan T-cells or stimulated anti-BCMA CAR-T cells derived from two separate donors were seeded into wells of 96 well plates in 200 µl Immunocult XF medium comprising increasing final concentrations of MTX (0nM control, 5nM, 10nM, 15nM, 32.5nM, 250nM or 1,000nM) for 72 hours. Cell viability (% live cells) was determined at 72 hours by flow cytometry. The results are shown in Fig.1.

[0333] As shown in Fig.1, unstimulated pan T-cells and antigen-stimulated, anti-BCMA CAR-T cells expressing DHFR are relatively resistant to increasing concentrations of MTX showing only a minor reduction in live cells at 1,000 nM, whereas stimulated pan T-cells are eliminated at MTX concentrations of less than 250 n< demonstrating the ability of MTX to selectively target stimulated pan T-cells for elimination while sparing antigen-stimulated CAR-T cells. Example 3 – Low Doses of MTX Selectively Eliminate Stimulated T-cells and NK Cells While Sparing Unstimulated T-cells

[0334] NK cells were isolated from four individual donors using a NK Cell Isolation Kit (STEMCELL Technologies) in accordance with the manufacturer’s instructions. NK cells were cultured for one week in RPMI medium supplemented with 10% fetal bovine serum (FBS) prior to analysis. NK cells were fed 50IU / mL of rhIL-2 twice a week and irradiated artificial antigen-presenting cells (irraAPCs) once a week at a 1:1 E:T ratio.

[0335] Pan T-cells were isolated using a CliniMacs Prodigy Instrument (Miltenyi Biotech) and cell processing applications for positive selection for CD4+ / CD8+ T-cells. Unstimulated T-cells were rested in culture medium post isolation. Stimulated pan T-cells were generated three days prior to MTX plating by stimulating pan T-cells using anti-CD3 / CD28 / CD2 activator beads (STEMCELL Technologies) in accordance with the manufacturer’s instructions.

[0336] Approximately 50,000 unstimulated Pan-T cells, stimulated Pan T-cells or NK cells derived from four separate donors were seeded into 96 well plates in 100 µl R10 medium comprising increasing concentrations of MTX (0 control, 5nM, 10nM, 15nM, 32.5nM, 250nM or 1,000nM) for 72 hours. MTX dilutions were made using R10 medium supplemented with 50 IU / ml of interleukin-2 (IL-2), which resulted in the slight proliferation of unstimulated Pan T-cells. Cell viability (% live cells) was determined at 72 hours by flow cytometry. The results are shown in Fig 2. Cells were stimulated with IL-2in the cell culture medium to improve survival.Attorney Docket No.: POTH-092 / 001WO

[0337] As shown in Fig 2, stimulated Pan T-cells and NK cells derived from four separate donors were highly sensitive to MTX with most viable cells eliminated at a concentration of 32.5 nM whereas low level, IL-2 stimulated Pan T-cells were relatively unaffected, even the highest concentration of MTX. Example 4 - MTX Eliminates Responder Cell Alloreactivity in Mixed Lymphocyte Reactions Stimulator Cells

[0338] Anti-BCMA CAR-T cells were prepared from two donors essentially as described in Example 1. The CAR-T cells from each donor were genetically modified using a Cas- CLOVER gene editing system to knockout the TRBC1 and TRBC2 loci, the TRBC1 and TRBC2 loci and the beta-2-microglobulin (B2M) gene, the TRBC1 and TRBC2 loci and CD58 receptor, the TRBC locus and RFX5 gene, or the TRBC1 and TRBC2 loci and the NLRC5 gene. These genetically modified CAR-T cells were used as Stimulator cells in mixed lymphocyte reactions (MLR), and unedited anti-BCMA CAR-T cells served as controls.

[0339] Stimulator cells were inactivated using Mitomycin C. Briefly, 10e6 Stimulator cells were incubated in 1.5 ml of TCCM medium (RPMI medium supplemented with 10% fetal bovine serum (FBS), 25mM HEPES, 55µM beta-mercaptoethanol (BME), 1mM sodium pyruvate, 1X Glutamax, and 1X NEAA) containing 10 µg / ml Mitomycin C for 1 hr at 37oC. The cells were pelleted, washed in R10 medium twice and then washed in PBS to remove any traces of Mitomycin C. The Mitomycin C inactivated cells were resuspended at a concentration of about 1e6 cells / ml in a CFDA-SE - PBS solution.

[0340] Stimulator cells were labeled with CDFA-SE (ThermoFisher). Briefly, a 10mM stock solution of CDFA-SE was prepared in DMSO and diluted to 1µM concentration in 50 mL of pre-warmed 37oC PBS. Approximately 10e6 Stimulator cells were resuspended in 10 ml of the CDFA-SE-PBS solution and incubated for 1 hr at 37oC. The labeling reaction was stopped by quenching the CDFA-SE-PBS solution by the addition of 40 mL of R10 medium and incubating at room temperature for 5 minutes. Cells (approximately 6e6 total cells) were pelleted, resuspended in 3 mL of TCCM medium and left at room temperature for 10 minutes before co-culturing with Responder Cells. Mitomycin C treated CDFA-SE labeled Stimulator cells do not proliferate after 7 days in culture in the presence or absence of MTX.Attorney Docket No.: POTH-092 / 001WO Responders cells

[0341] Pan T-cells isolated from three donors and PBMCs isolated from a single donor were used as Responder cells. Responder cells were labeled with CTV (ThermoFisher). Briefly, a 5 mM stock of CTV in DMSO was prepared and diluted to 5 µM in pre-warmed 37oC PBS to make a CTV-PBS solution. Responder cells were diluted to a concentration of 1e6 cells / mL using the CTV-PBS solution and incubated for 20 min at 37oC. Four 10 mL aliquots were transferred to 50 mL tubes and 40 mL of R10 medium was added to each tube to quench the labeling reaction. Cells were maintained at room temperature for 5 min. Cells were pelleted, resuspended at a concentration of about 4e6 cells / mL, counted and adjusted to a final concentration of 2e6 cells / mL.

[0342] To initiate the MLR reaction, 1e5 CFDA-SE-labled Stimulator cells and 2e5 CTV- labeled Responder cells were seeded in 96 well flat bottom plates in : a) 100 µL of TCCM medium; b) 100 µL of TCCM medium supplemented with cytokines (100 ng / ml IL-2, 25 ng / ml IL-7 and 25 ng / ml IL-15); c) 100 µL of TCCM medium containing 250 ng / ml MTX; or d) 100 µL of TCCM medium supplemented with cytokines (100 ng / ml IL-2, 25 ng / ml IL-7 and 25 ng / ml IL-15) containing 250 ng / ml MTX. Plates were incubated at 37oC for 7 days. After 7 days, the percentage of CTV negative cells was calculated by flow cytometry. The results are shown in Fig 3A (No MTX) and Fig.3B (+ MTX).

[0343] As shown in Figs.3A and 3B, all Stimulator A cells tested in the absence of MTX and in the presence of cytokines demonstrated robust expansion of both CTV-labeled responder cells; however, in the presence of cytokines and MTX, no expansion CTV-labeled responder cells was detected suggesting MTX-dependent inhibition of proliferation prevented responder cell expansion upon activation. Similar results were observed for Stimulator B cells (Figs. 3C and 3D). Example 5 – Stimulator CAR-T Cells Expressing mDHFR Proliferate in the Presence of Low Doses of MTX During 7 Days of Cell Culture

[0344] The genetically modified and unedited Stimulator CAR-T cells described in Example 4 were labeled with CTV (ThermoFisher). Briefly, a 5 mM stock of CTV in DMSO was prepared and diluted to 5 µM in pre-warmed 37oC PBS to make a CTV-PBS solution. Stimulator CAR-T cells were diluted to a concentration of 1e6 cells / mL using the CTV-PBS solution and incubated for 20 min at 37oC. Four 10 mL aliquots were transferred to 50 mL tubes and 40 mL of R10 medium was added to each tube to quench the labeling reaction andAttorney Docket No.: POTH-092 / 001WO kept at room temperature for 5 min. Cells were pelleted, resuspended at a concentration of about 4e6 cells / mL, counted and adjusted to a final concentration of 2e6 cells / mL.

[0345] CTV-labeled Stimulator cells were cultured in the presence or absence of cytokines (100 ng / ml IL-2, 25 ng / ml IL-7 and 25 ng / ml IL-15) in TCCM medium containing or lacking 250 ng / ml MTX (low dose) for a period of 7 days. After 7 days, the number of CTV positive cells was determined by flow cytometry. The results are shown in Fig.4A (No MTX) and Fig.4B (+MTX).

[0346] As shown in Figs.4A and 4B, CTV-labeled Stimulator cells were capable of proliferating to similar extents in the presence of cytokines in medium containing or lacking MTX. Unlike responder cells in Example 4, Stimulator cells were unaffected by MTX addition during cell expansion and, thus may be manufactured in the presence of 250nM MTX. Example 6 – Human CAR-T Cells Expressing mutein DHFR (mDHFR) Exhibit Increased MTX Resistance Compared to Human CAR-T Cells Lacking mDHFR Expression

[0347] Human CAR-T cells expressing a CD20 CAR (CD20 CAR) were genetically engineered to express either mutein DHFR (mDHFR) or a surrogate human, truncated low- affinity nerve growth factor receptor (tLNGFR). Briefly, the transposon cassette comprising the gene encoding CD20 CAR and an iCasp9 safety switch was modified to further comprise the mDHFR gene or the tLNGFR gene as a control for mDHFR negative CD20 CAR-T cells and act as a surrogate for endogenous (patient) lymphocytes during in vivo experiments. Expression of tLNGFR allows for selection of CD20 mDHFR- CAR-T cells using magnetic beads bound with an anti-LNGFR antibody, and also allows for sorting and detection of mDHFR- CAR-T cells by FACS. The two transposon cassettes were introduced into human T-cells from two healthy donors, selected for tLNGFR+ cells or mDHFR+ cells (using MTX) to generate CD20 CAR-T cells lacking (mDHFR- CAR-T) or expressing mDHFR (mDHFR+ CAR-T), respectively.

[0348] mDHFR+ CAR-T cells or mDHFR- CAR-T cells were divided into two groups: The first group was antigen-stimulated with irradiated CD20-expressing Raji cells at an effector to target ratio of 5 CAR-T cells per Raji cell; and the second group was low-level stimulated using 50 IU / ml interleukin -2 (IL-2). Antigen-stimulated or low-level stimulated pan T-cells were used as a positive control.Attorney Docket No.: POTH-092 / 001WO

[0349] Approximately 50,000 antigen-stimulated or low-level stimulated mDHFR+ CAR-T cells, mDHFR- CAR-T cells or pan T-cells derived from two separate healthy human donors were seeded into 96 well plates in 100 µl R10 medium comprising increasing concentrations of MTX (0nM control, 250nM, 750nM or 1,500nM) for 72 hours. Cell viability (% live cells) was determined at 72 hours by flow cytometry. The results are shown in Fig 5.

[0350] As shown in Figs 5A and 5B, rapidly dividing, antigen-stimulated pan T-cells and mDHFR- CAR-T cells derived from both donors were highly sensitive to MTX addition at a concentration as low as 250nM whereas rapidly dividing antigen-stimulated mDHFR+ CAR- T cells were relatively resistant to levels of 250nM MTX. Low-level stimulation of all CAR- T groups and pan T-cells resulted in increased resistance to lower concentrations of MTX likely as a result of decreased cellular division for low-level IL-2 stimulation. Example 7 –Human CAR-T Cells Expressing mDHFR Exhibit Increased In Vivo Persistence in the Presence of MTX Compared to Human CAR-T Cells Lacking mDHFR Expression

[0351] Single dose IV administration of MTX at various concentrations to mice demonstrated that MTX serum concentrations peak at about 10 – 15 minutes post- administration and a half-life of about 4 hours (data not shown). These data were used to guide timing of MTX administration and dosing for in vivo experiments.

[0352] The in vivo persistence of human CD20 mDHFR+ CAR-T cells and mDHFR- CAR-T cells was assessed using an in vivo Raji.CBG.GFP murine model system. The human Raji, wild type B-cell lymphoma cell line (ATCC Catalog No. CCL-86, Manassas, Virginia) endogenously expresses CD20. The Raji tumor cells were modified to express both click beetle green (CBG) luciferase (luc) and green fluorescent protein (GFP) for tumor growth monitoring and cell tracking (referred to as Raji.CBG.GFP). Cell surface expression CD20 was validated by flow cytometry.

[0353] In vivo efficacy of human CD20 mDHFR+ CAR-T cells and mDHFR- CAR-T cells was evaluated using the Raji B-cell lymphoma model in NSG mice (NOD.Cg- PrkdcscidIl2rgtm1Wjl / Szj; Jackson Laboratory Stock # 005557, Bar Harbor, Maine). To establish tumors, 0.5e6 Raji-CBG-GFP cells were injected intravenously (IV) on Day -5 post- CAR-T into 6- to 7-week-old female NSG mice. Mice were randomized to groups by tumor burden, as measured by bioluminescence imaging (BLI) before treatment.

[0354] On Day 0 post-CAR-T, 5e6 human CD20 mDHFR+ CAR-T cells or mDHFR- CAR- T cells were injected IV into four groups of Raji.CBG.GFP bearing NSG mice (n = 5 / group).Attorney Docket No.: POTH-092 / 001WO On Days 1, 3 and 6 post-CAR-T, mice were injected IV with PBS (negative control), 0.25 mg / kg MTX (Fig.6A) or 10 mg / kg MTX (Fig.6B). Whole blood was collected on Days 7, 13, 21 and 28 post-CAR-T to determine circulating CD4+ / CD8+ T-cell counts. Tumor burden was measured weekly by whole-body bioluminescent imaging (BLI) and body weight measurements were taken twice weekly. The study was terminated on Day 28 post-CAR-T. The results of the study are shown in Table 5 and Figs.6A and 6B. Table 5 D7: CAR-TD7: % Reduction D14: CAR-T Cells D14: % Reduction Cells / uLwith MTX / uL with MTXdecrease in mDHFR- CAR-T cells at Day 7 and an 80% reduction at Day 13 post-CAR-T administration whereas mDHFR+ CAR-T cells were relatively insensitive to MTX administration at Days 7 and 13 demonstrating the ability to selectively eliminate surrogate host T-cells that do not express mDHFR. Example 8 – Administration of MTX Using an Infusion Pump Results in Stable, Therapeutically Effective Levels of MTX for Eliminating mDHFR Negative CAR-T Cells

[0356] Mice (n = 2 / group) were administered 0.25 mg / kg or 2.5 mg / kg of MTX using an infusion pump (BrainTree Scientific, Inc. #BS-9008) in accordance with the manufacturer’s instructions using a 1 ml syringe (5.78mm syringe diameter), an infusion rate of 0.2 mL / hr (100 µL / 30 min), and a total volume of 0.4 µL infused over a two-hour period. Whole-blood samples were drawn at 10, 20, 30, 45, 60, 75, 90, 105 and 120 minutes after initiating MTX infusion to measure MTX serum concentrations over the course of the two-hour infusion period. The results are shown in Fig.7.

[0357] As shown in Fig.7, the administration of 0.25 mg / kg of MTX via infusion pump resulted in relatively stable MTX serum concentrations in both treated mice of about 500nM whereas administration of 2.5 mg / kg of MTX via infusion pump resulted in a high MTXAttorney Docket No.: POTH-092 / 001WO serum concentration of about 3,500 nM in one treated mouse and intermediate MTX serum concentrations about 1,500 nM in the second treated mouse. These data suggest that use of an infusion pump can produce MTX serum concentrations at levels sufficient to eliminate NK cells and activated CAR-T-cells lacking mDHFR expression in vitro (>250 nM; Example 3) and sufficient to maintain efficacious mDHFR+ CAR-T cell in vivo (Example 7). Example 9 – Administration of Therapeutically Effective Levels of MTX Do Not Affect mDHFR+ CAR-T Expansion or In Vivo Efficacy, While mDHFR- CAR-T (A Surrogate For Alloreactive Patient T Cells) Exhibit Delayed Expansion and Tumor Control

[0358] In vivo efficacy of human CD20 mDHFR+ CAR-T cells and mDHFR- CAR-T cells was evaluated using the Raji B-cell lymphoma model in NSG mice (NOD.Cg- PrkdcscidIl2rgtm1Wjl / Szj; Jackson Laboratory Stock # 005557, Bar Harbor, Maine). To establish tumors, 0.5e6 Raji-CBG-GFP cells were injected intravenously (IV) on Day -5 post- CAR-T administration, into 10-week-old female NSG mice. Mice were randomized to groups by tumor burden, as measured by bioluminescence imaging (BLI) before treatment.

[0359] On Day 0 post-CAR-T administration, 1.8e6 human CD20 mDHFR+ CAR-T cells or mDHFR- CAR-T cells (TCR+ / B2M+) were injected IV into four groups of Raji.CBG.GFP bearing NSG mice (n = 5 / group). Mice were administered PBS intraperitoneally as needed throughout the study.

[0360] On Day 1 post-CAR-T administration, mice in Groups 4 and 5 were administered 0.5 mg / kg of MTX for three days using an infusion pump (Alzet Osmotic Pumps, #1003D) in accordance with the manufacturer’s instructions, an infusion rate of 1.0 µL / hour, and a total volume of 72 µL infused over a 72-hour period. Whole-blood samples were drawn on Days 9, 14, 21, and 28 post-CAR-T administration. BLI measurements were taken on Days -1, 8, 15, 22, and 27 post-CAR-T administration. The BLI results are shown in FIG.8A.

[0361] As shown in FIG.8A, mDHFR+ CAR-T cells were able to robustly control tumor growth in the absence (Group 2) or the presence of ~500 nM (FIG.8C) in vivo MTX concentration (Group 4) whereas mDHFR- CAR-T cells were able to control tumor growth in the absence of MTX with notable delayed expansion (Group 3) but not in the presence of MTX (Group 5). This demonstrates that mDHFR+ CAR-T cells retain similar in vivo cytotoxicity in the absence or presence of MTX, whereas mDHFR- CAR-T cells, a surrogate of host T-cells, are eliminated and unable to control tumor growth.Attorney Docket No.: POTH-092 / 001WO

[0362] Circulating CD4+ T-cells and CD8+ T-cells were enumerated from whole blood samples taken on Days 9, 14, 21, and 28 by flow cytometry, using Countbright Beads (Thermo Fisher, #C36950). The total counts of human CD45+, CD4+ and CD8+ cells are shown in FIG.8B.

[0363] As shown in FIG.8B, mDHFR+ CAR-T cell counts increased in the absence (Group 2) or the presence of ~500 nM in vivo MTX concentration (Group 4) over the course of the study, whereas mDHFR- CAR-T cells (Group 3) exhibited delayed expansion compared to mDHFR+ CAR-T cells. mDHFR- CAR-T cells plus 0.5 mg / kg MTX (Group 5) failed to appreciably expand until well after MTX administration had ceased, demonstrating the ability of MTX to reduce non-mDHFR expressing T cells.

[0364] As shown in FIG.8C, MTX serum levels were consistently in the ~500 nM range over the 5 day sampling period, a clinically-relevant concentration for patients receiving therapeutic MTX doses.

[0365] Without wishing to be bound by theory, these results collectively demonstrate that mDHFR+ CAR-T cells were able to effectively expand and control tumor growth in the absence (group 2) or presence (group 4) of clinically-relevant MTX concentrations. In contrast, mDHFR- CAR-T cells, used as a surrogate for alloreactive host T cells, exhibited delayed expansion and tumor control in the presence of MTX. These results suggest that mDHFR+ CAR-T cells could be dosed in combination with MTX therapy in patients to prevent the rebound of potentially alloreactive patient T cell populations, thereby increasing the persistence of mDHFR+ CAR-T cells. Example 10 – Human anti-MUC1C+ CAR-T Cells Expressing mDHFR Exhibit In Vivo Efficacy in the Presence of or Absence of MTX

[0366] In vivo efficacy of human anti-MUC1C+ mDHFR+ CAR-T cells was evaluated using MDA-M8-468 (L6) model in NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl / Szj; Jackson Laboratory Stock # 005557, Bar Harbor, Maine). To establish tumors, MDA-M8-468 (L6) cells in 50% Matrigel were implanted in the right hind limb subcutaneously (SC) on Day 0 into 6- to 7-week-old female NSG mice. Mice were randomized into four groups by tumor burden (~240mm3), as determined by caliper measurements prior to treatment.

[0367] On study day 73, PBS vehicle control or 2.5e6 human anti-MUC1C mDHFR+ CAR- T cells were injected intravenously (IV) into four groups of MDA-M8-468 (L6) bearing NSG mice (2 groups each; n = 3 or 7 / group). On day 1 post CAR-T administration, one control group and one CAR-T treated group of mice were administered PBS-containing AlzetAttorney Docket No.: POTH-092 / 001WO infusion pumps (vehicle), or one control group and one CAR-T treated group of mice were administered 0.25 mg / kg MTX-containing Alzet infusion pumps that delivered vehicle or MTX over a 7-day period. Whole blood was collected on days 7, 14, 21, 28 and 35 post CAR-T infusion to determine circulating CD4+ / CD8+ T-cell counts. Tumor burden was measured twice weekly by caliper measurements and body weights were measured twice weekly. The study was terminated on day 35 post CAR-T administration. The results of the study are shown in Fig.9A.

[0368] As shown in Fig.9A, anti-MUC1C mDHFR+ CAR-T cells were capable of similarly controlling tumor growth in the absence or presence of therapeutically relevant doses of MTX.

[0369] Anti-MUC1C mDHFR+ CAR-T expansion was determined for whole blood samples collected over the course of the treatment and the results are shown on Fig.9B.

[0370] As shown in Fig.9B, anti-MUC1C mDHFR+ CAR-T cell expansion was observed with peak expansion occurring between Day 14 and Day 21 post-CAR-T infusion. No CAR- T cells were detected in PBS groups, so 0.1 units were added to visualize on log 10 scale.

[0371] These results collectively demonstrate that anti-MUC1C mDHFR+ CAR-T cells are capable in vivo of expanding and controlling MUC1C+ tumors in the absence or presence of therapeutic levels of MTX.

Claims

Attorney Docket No.: POTH-092 / 001WO CLAIMS What is claimed is:

1. A method of enhancing the persistence of cells expressing a chimeric antigen receptor (CAR cells) in a subject, comprising: a) administering to the subject a therapeutically effective amount of a composition comprising the CAR cells, wherein the CAR cells comprise a chimeric antigen receptor and a recombinant mutein dihydrofolate reductase (mDHFR); and b) administering to the subject after step a), an effective amount of methotrexate (MTX), or an MTX analog, sufficient to eliminate activated T-cells, B-cells, and NK cells in the subject.

2. The method of claim 1, wherein the CAR targets an oncogenic gene product comprising BCMA, CD19, CD20, MUC1C, PSMA, CD70, CD7, or c-kit.

3. The method of claims 1, wherein a nucleic acid encoding the CAR and a nucleic acid encoding the mDHFR are introduced into the cell.

4. The method of claim 3, wherein the nucleic acids are introduced via a transposon or virus.

5. The method of claim 4, wherein the transposon further comprises a nucleic acid encoding an iCasp9 safety switch.

6. The method of claim 1, wherein the CAR cells are lymphocytes.

7. The method of claim 6, wherein the lymphocytes are B-cells and / or T-cells.

8. The method of any one of the preceding claims, wherein the effective amount of MTX, or the MTX analog, is sufficient to obtain an MTX serum concentration between 32 nM and 250 nM.

9. The method of any one of the preceding claims, wherein the therapeutically effective amount of CAR cells in the composition is between about 5e6 to 15e6 CAR cells.Attorney Docket No.: POTH-092 / 001WO 10. The method of any one of the preceding claims, wherein the therapeutically effective amount of CAR cells in the composition is between about 0.5e6 to 10e6 CAR cells.

11. The method of claim 1, wherein the MTX, or the MTX analog, is administered as a single dose on the third day after administration of the composition comprising CAR cells.

12. The method of claim 1, wherein the MTX, or the MTX analog, is administered as a single dose on the sixth day after administration of the composition comprising CAR cells.

13. The method of claim 1, wherein the MTX, or the MTX analog, is administered as a multi-dose regiment on the third and sixth day after administration of the composition comprising CAR cells.

14. The method of claim 1, wherein the MTX, or the MTX analog, is administered as a multi-dose regiment on the first, third, and sixth days after the administration of the composition comprising CAR cells.

15. The method of claim 1, wherein the MTX, or the MTX analog, is administered as a multi-dose regiment on the first, third, sixth, and eleventh days after the administration of the composition comprising CAR cells.

16. The method of claim 1, wherein the MTX, or the MTX analog, is administered as a multi-dose regiment on the first, third, sixth, eleventh, fourteenth, eighteenth, twenty second, and twenty sixth day after the administration of the composition comprising CAR cells.

17. The method of claim 1, wherein the MTX, or the MTX analog, is administered as a weekly dose starting on the first day after the administration of the composition comprising CAR cells.

18. The method of any one of claims 1-17, further comprising, prior to step a), administering an effective dose of MTX or an MTX analog.

19. The method of claim 18, wherein the administration of the effective dose of MTX, or the MTX analog, is administered 5 days prior to step a).Attorney Docket No.: POTH-092 / 001WO 20. The method of any one of claims 1-19, wherein the method results in at least a 10% increase in persistence of the CAR cells in the blood of the subject compared a subject that does not receive MTX or an MTX analog.

21. A method of treating cancer in a subject in need thereof, comprising: a) administering to the subject a therapeutically effective amount of a composition comprising cells expressing a chimeric antigen receptor (CAR cells), wherein the CAR cells comprise a chimeric antigen receptor (CAR) and a recombinant mutein dihydrofolate reductase (mDHFR); and b) administering to the subject after step a), an effective amount of amount of methotrexate (MTX), or an MTX analog.

22. The method of claim 21, wherein the CAR targets an oncogenic gene product comprising BCMA, CD7, CD19, CD20, MUC1C, PSMA, CD70 or c-kit.

23. The method of claims 21, wherein a nucleic acid encoding the CAR and a nucleic acid encoding the mDHFR are introduced into the cell.

24. The method of claim 23, wherein the nucleic acids are introduced via a transposon or virus.

25. The method of claim 24, wherein the transposon further comprises a nucleic acid encoding an iCasp9 safety switch.

26. The method of claim 21, wherein the CAR cells are lymphocytes.

27. The method of claim 26, wherein the lymphocytes are T-cells or b-cells.

28. The method of claim 21, wherein the MTX analog is pralatrexate (PDX), Talotrexin, Leucovorin, Tomudex, Alimta, Trimethoprim, Cycloguanil, Pemetrexed (PMX), Raltitrexed (RTX), Plevitrexed, Piritrexim, Nolatrexed, folic acid (FA), 5-formyl-tetrahydrofolic acid (5FFH4), dihydrofolic acid (FH2), 10-methyl-tetrahydrofolic acid (10MFH4), tetrahydrofolic acid (FH4), aminopterin (AMP), lomexetrol, AQA, multi-target antifolate (MTA), raltitrexed, 10-deaza-aminopterin (10 DAAM), 10-methyl-deaza-aminopterin (10 MDAAM), or 10- ethyl-deaza-aminopterin (10 EDAAM)Attorney Docket No.: POTH-092 / 001WO 29. The method of claim 21, wherein the MTX analog is pralatrexate (PDX).

30. A method of treating an autoimmune disease in a subject in need thereof, comprising: a) administering to the subject a therapeutically effective amount of a composition comprising cells expressing a chimeric antigen receptor (CAR cells), wherein the CAR cells comprise a chimeric antigen receptor (CAR) and a recombinant mutein dihydrofolate reductase (mDHFR); and b) administering to the subject after step a), an effective amount of methotrexate (MTX), or an MTX analog.

31. The method of claim 30, wherein the autoimmune disease is selected from the group consisting of: autoimmune neutropenia, Guillain-Barré syndrome, epilepsy, autoimmune encephalitis, Isaacs' syndrome, nevus syndrome, pemphigus vulgaris, deciduous pemphigus, bullous pemphigoid, acquired epidermolysis bullosa, gestational pemphigoid, mucous membrane pemphigoid, antiphospholipid syndrome, autoimmune anemia, myasthenia gravis, autoimmune Graves' disease, thyroid eye disease (TED), Goodpasture syndrome, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura (ITP), warm autoimmune hemolytic anemia (WAIHA), chronic inflammatory demyelinating polyneuropathy (CIDP), lupus nephritis, and membranous nephropathy.

32. The method of claim 1, claim 21, or claim 30, wherein the MTX, or the MTX analog, is administered orally or intravenously.

33. The method of claim 1, claim 21, or claim 30, wherein the MTX, or the MTX analog, is administered once per week or twice per week.

34. The method of any one of the preceding claims, wherein the administration of the MTX, or the MTX analog, leads to an at least 25% decrease in activated host T-cells.