Nano-transposon composition and method for using the same

Abstract

To provide a nano-transposon composition including non-naturally originated structural improvement in a vector holding a transposon sequence in order to use particularly for a human cell as a method for modifying a cell for gene therapy, a method for manufacturing and a usage method of the composition.SOLUTION: Disclosed is a composition including: a first nucleic acid sequence, which includes (a) a first inverted terminal repeat sequence (ITR), (b) a second ITR, and (c) an inter-ITR sequence, in which the inter-ITR sequence includes a transposon sequence; and a second nucleic acid sequence including an inter-ITR sequence, where the length of the inter-ITR sequence is 1 to 600 nucleotides including both ends. Preferably, the composition is nano-transposon.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Patent Application No. 62 / 783,133, filed December 20, 2018, U.S. Patent Application No. 62 / 815,335, filed March 7, 2019, and U.S. Patent Application No. 62 / 815,845, filed March 8, 2019, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Incorporating an array list by reference The contents of the file entitled "POTH-047_001WO Seq Listing_ST25.txt", created on December 19, 2019 and having a size of 295 MB, are hereby incorporated by reference in their entirety.

[0003] Field of the Disclosure FIELD OF THE DISCLOSURE This disclosure relates to molecular biology, and more specifically to nanotransposons, cellular compositions containing nanotransposons, methods of making and using same. [Background technology]

[0004] There is a long felt and unmet need in the art for improved gene transfer compositions and methods for use in gene therapy. The present disclosure provides nanotransposon compositions that contain non-naturally occurring structural modifications to vectors carrying transposon sequences, methods of making and using these compositions, for use in particular in human cells as a method of modifying cells for gene therapy. Summary of the Invention

[0005] SUMMARY OF THE PRESENT APPLICATION The present disclosure provides a composition comprising: a first nucleic acid sequence, which comprises (a) a first inverted terminal repeat (ITR), (b) a second ITR, and (c) an intra-ITR sequence, the intra-ITR sequence comprising a transposon sequence; and a second nucleic acid sequence comprising an inter-ITR sequence having a length of 1-600 nucleotides inclusive. In a preferred embodiment, the length of the inter-ITR sequence is 1-100 nucleotides inclusive. The composition may be a transposon or may be a nanotransposon. In a preferred embodiment, the transposon is a piggyBac transposon.

[0006] The first nucleic acid sequence and / or the second nucleic acid sequence may further comprise an origin of replication sequence. The length of the origin of replication sequence may be 1 to 450 nucleotides. The origin of replication sequence may comprise an R6K origin of replication.

[0007] The first nucleic acid sequence and / or the second nucleic acid sequence may further comprise a sequence encoding a first selection marker. The length of the first selection marker may be 1 to 200 nucleotides. The first selection marker may be a sucrose selection marker. In a preferred embodiment, the sucrose selection marker is an RNA-OUT selection marker.

[0008] The first and / or second nucleic acid sequences may not include recombination sites, excision sites, ligation sites, or combinations thereof. The first and / or second nucleic acid sequences may not include sequences encoding foreign DNA.

[0009] The first nucleic acid sequence may further comprise at least one exogenous sequence and a sequence encoding a promoter capable of expressing the exogenous sequence in a mammalian cell. The first nucleic acid sequence may further comprise at least one sequence encoding an insulator. The first nucleic acid sequence may further comprise a polyadenosine (polyA) sequence. The sequence encoding a promoter capable of expressing the exogenous sequence in a mammalian cell may express the exogenous sequence in a human cell. The promoter may be a constitutive promoter or an inducible promoter.

[0010] The at least one exogenous sequence may comprise a sequence encoding a non-naturally occurring antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof. In a preferred embodiment, the non-naturally occurring antigen receptor comprises a chimeric antigen receptor (CAR). The CAR may comprise (a) an ectodomain comprising an antigen recognition region, (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain. The antigen recognition region may comprise at least one single chain variable fragment (scFv), a single domain antibody, centyrin, or a combination thereof. The single domain antibody may be a VHH or a VH.

[0011] The antigen recognition region may comprise at least one anti-BCMA centilin. Preferably, the anti-BCMA centilin comprises the amino acid sequence of SEQ ID NO: 29. The antigen recognition region may comprise at least one anti-BCMA VH. Preferably, the anti-BCMA VH comprises the amino acid sequence of SEQ ID NO: 97. The antigen recognition region may comprise at least one anti-PSMA centilin. Preferably, the anti-PSMA centilin comprises the amino acid sequence of SEQ ID NO: 94.

[0012] The ectodomain may further comprise a signal peptide. The CAR may further comprise a hinge region between the antigen recognition region and the transmembrane domain. The transmembrane domain may comprise a sequence encoding a CD8 transmembrane domain. The at least one costimulatory domain may comprise a CD3 zeta costimulatory domain, a 4-1BB costimulatory domain, or a combination thereof. The at least one costimulatory domain may comprise a CD3 zeta costimulatory domain and a 4-1BB costimulatory domain, the 4-1BB costimulatory domain being located between the transmembrane domain and the CD3 zeta costimulatory domain. The at least one exogenous sequence may comprise a sequence encoding an inducible pro-apoptotic polypeptide, a sequence encoding a second selection marker, a sequence encoding a chimeric stimulatory receptor (CSR), a sequence encoding a transferase, a sequence encoding a self-cleaving peptide, or a combination thereof. The second selection marker may comprise a sequence encoding a mutant protein enzyme of dihydrofolate reductase (DHFR).

[0013] The present disclosure also provides a polynucleotide comprising a nucleic acid sequence encoding a composition (e.g., a transposon or nanotransposon) disclosed herein, and / or a polynucleotide comprising a nucleic acid sequence encoding a CAR disclosed herein.

[0014] The present disclosure also provides a cell comprising a composition (e.g., a transposon or nanotransposon) disclosed herein. The present disclosure also provides a population of cells, where the plurality of populations is modified to express a CAR or a composition (e.g., a transposon or nanotransposon) disclosed herein. In one embodiment, the plurality of modified cells is a plurality of modified immune cells. In one embodiment, the plurality of modified cells is a plurality of modified T cells. In one embodiment, at least 50% of the plurality of modified T cells express one or more cell surface markers, including CD45RA and CD62L, and do not express one or more cell surface markers, including CD45RO.

[0015] The present disclosure also provides a pharmaceutical composition comprising a CAR or a composition disclosed herein (e.g., a transposon or nanotransposon), and further comprising a pharma- ceutically acceptable carrier.

[0016] The present disclosure also provides a method of administering a therapeutically effective amount of any of the compositions (e.g., transposons or nanotransposons), CARs, cells, cell populations, or pharmaceutical compositions disclosed herein to treat a proliferative disease in a subject in need thereof. In one embodiment, the proliferative disease is cancer. The cancer may be a BCMA-positive cancer or a PSMA-positive cancer. The cancer may be a primary tumor, a metastatic cancer, a multidrug-resistant cancer, an advanced tumor, or a recurrent cancer. The cancer may be a solid tumor or a hematological cancer. The cancer may be lung cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, skin cancer, esophageal cancer, lymphoma, leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, acute myeloid leukemia (AML), acute myeloid leukemia, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), Hodgkin's disease, non-Hodgkin's lymphoma, or multiple myeloma.

[0017] This patent or application file contains one or more color drawings. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a pair of schematic diagrams comparing maps of the piggyBac complete plasmid and the piggyBac nanotransposon (NT). [Diagram 2] FIG. 2 is a graph showing improved transfer by piggyBac NT in human pan-T cells. [Diagram 3]FIG. 3 is a pair of schematic diagrams comparing the maps of piggyBac NT and piggyBac short NT. [Figure 4] FIG. 4 is a graph showing that piggyBac transposition in human pan T cells is enhanced by shortening the inter-ITR sequence (eg, shortening the distance adjacent to the ITRs). [Diagram 5] FIG. 5 is a pair of graphs showing increased metastasis by anti-BCMA chimeric antigen receptor (CAR) NT and anti-PSMA CAR NT in human pan-T cells. [Figure 6] FIG. 6 is a series of graphs showing that human CAR-T cells generated with anti-BCMA CAR NT or anti-PSMA CAR NT were able to kill target tumor cells. [Figure 7] FIG. 7 is a series of graphs showing that human CAR-T cells generated with anti-BCMA CAR NT or anti-PSMA CAR NT had comparable phenotypic composition. [Figure 8] FIG. 8 is a series of graphs showing that human CAR-T cells generated with anti-BCMA CAR NT or anti-PSMA CAR NT have similar integration copy numbers. [Figure 9] FIG. 9 is a photograph of a gel electrophoresis analysis showing that purity of monomeric NT correlates with transfer efficiency in human pan-T cells. [Figure 10] FIG. 10 is a pair of graphs showing that purity of monomeric NT correlates with transfer efficiency in human pan-T cells. [Figure 11] FIG. 11 is a schematic showing preclinical evaluation of the P-PSMA-101 transposon when delivered with full-length plasmid (FLP) versus NT at a stress dose using a mouse xenograft model. [Figure 12] FIG. 12 is a series of graphs showing tumor volume assessment of mice treated with P-PSMA-101 transposon when delivered with FLP versus NT. [Figure 13]Figure 13 is a schematic diagram showing the P-BCMA-101 piggyBac NT encoding BCMA CARTyrin. The nanotransposon encodes ITR#1, insulator#1, EF1α promoter, BMCA CARTyrin, SV40 PA, insulator#2, and ITR#2. This sequence also encodes the nanotransposon elements RNA-OUT and R6K origin. [Figure 14] Figure 14 is a schematic diagram showing the P-PSMA-101 piggyBac NT encoding PSMA CARTyrin. The nanotransposon encodes ITR#1, insulator#1, EF1α promoter, PSMA CARTyrin, SV40 PA, insulator#2, and ITR#2. This sequence also encodes the nanotransposon elements RNA-OUT and R6K origin. [Figure 15] 15 is a schematic diagram showing the P-BCMA-ALLO1 piggyBac nanotransposon encoding the BCMA VH CAR (VCAR). The nanotransposon encodes ITR#1, insulator#1, EF1α promoter, BMCA VCAR, SV40 PA, insulator#2, and ITR#2. This sequence also encodes the nanotransposon elements RNA-OUT and R6K origin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] All documents cited herein, including any cross-referenced patents or related patents or applications, are incorporated herein by reference in their entirety for all purposes unless expressly excluded or otherwise limited. The citation of any document is not an admission that the document is prior art with respect to any invention disclosed or claimed herein, or that it alone, or in any combination with other references, teaches, suggests, or discloses that invention. Furthermore, in the event that a meaning or definition of a term in this document conflicts with a meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall control.

[0020] Detailed Description of the Invention The present disclosure provides nanotransposons, compositions and cells comprising the nanotransposons, methods of making the nanotransposons, and methods of using the nanotransposons, compositions, and cells disclosed herein.

[0021] The nanotransposon of the present disclosure is designed to minimize the inter-ITR sequence of the nanotransposon and to bring the first and second ITR sequences as close as possible, thereby improving the efficacy and efficiency of transposition.The nanotransposon of the present disclosure and the composition comprising the nanotransposon are effective in all cell types, but they are particularly effective for use in human cells.As described herein, the nanotransposon of the present disclosure can be used to increase transposition, and thus increase gene transfer into human cells to a sufficiently high cell percentage in a plurality of cells.

[0022] Without wishing to be bound by theory, by minimizing the inter-ITR sequence or distance, the corresponding transposase can bring both ITR sequences together, leading to increased excision of the sequence within the ITR from the nanotransposon and / or increased integration of the sequence within the ITR into the target site.Furthermore, in a preferred embodiment of the present disclosure, the nanotransposon, its backbone, and / or the inter-ITR sequence do not contain foreign DNA sequences.The absence of foreign DNA further improves the efficacy and efficiency of transposition, especially when compared with non-nanotransposons.

[0023] Disclosed Compositions

[0024] The present disclosure provides a composition comprising: a first nucleic acid sequence, comprising (a) a first inverted terminal repeat (ITR) or a sequence encoding the first ITR, (b) a second ITR or a sequence encoding the second ITR, and (c) an intra-ITR sequence or a sequence encoding the intra-ITR sequence, the intra-ITR sequence comprising a transposon sequence or a sequence encoding a transposon sequence; and a second nucleic acid sequence comprising an inter-ITR sequence or a sequence encoding an inter-ITR sequence, the inter-ITR sequence being 700 nucleotides or less in length. The second nucleic acid sequence is also referred to herein as a backbone region or a non-integration region. In one embodiment, the composition is a circular or linear DNA. In one embodiment, the composition is a plasmid or vector. In one embodiment, the composition is a transposon. In a preferred embodiment, the composition is a nanotransposon.

[0025] In some embodiments, the length of the inter-ITR sequence is 650 nucleotides or less, 600 nucleotides or less, 550 nucleotides or less, 500 nucleotides or less, 450 nucleotides or less, 400 nucleotides or less, 350 nucleotides or less, 300 nucleotides or less, 250 nucleotides or less, 200 nucleotides or less, 150 nucleotides or less, 100 nucleotides or less, 50 nucleotides or less, 25 nucleotides or less, or 10 nucleotides or less. In some embodiments, the length of the second nucleic acid sequence is 700 nucleotides or less, 650 nucleotides or less, 600 nucleotides or less, 550 nucleotides or less, 500 nucleotides or less, 450 nucleotides or less, 400 nucleotides or less, 350 nucleotides or less, 300 nucleotides or less, 250 nucleotides or less, 200 nucleotides or less, 150 nucleotides or less, 100 nucleotides or less, 50 nucleotides or less, 25 nucleotides or less, or 10 nucleotides or less.

[0026] The present disclosure provides a composition comprising: a first nucleic acid sequence, which comprises: (a) a first inverted terminal repeat (ITR) or a sequence encoding a first ITR; (b) a second ITR or a sequence encoding a second ITR; and (c) an intra-ITR sequence or a sequence encoding an intra-ITR sequence, said intra-ITR sequence comprising a transposon sequence or a sequence encoding a transposon sequence; and a second nucleic acid sequence comprising an inter-ITR sequence or a sequence encoding an inter-ITR sequence, the inter-ITR sequence being between 1 and 700 nucleotides in length. The second nucleic acid sequence is also referred to herein as a backbone region or a non-integration region. In one embodiment, the composition is a circular or linear DNA. In one embodiment, the composition is a plasmid or vector. In one embodiment, the composition is a transposon. In a preferred embodiment, the composition is a nanotransposon.

[0027] In some embodiments, the length of the inter-ITR sequence is 1 to 650 nucleotides, 1 to 600 nucleotides, 1 to 550 nucleotides, 1 to 500 nucleotides, 1 to 450 nucleotides, 1 to 400 nucleotides, 1 to 350 nucleotides, 1 to 300 nucleotides, 1 to 250 nucleotides, 1 to 200 nucleotides, 1 to 150 nucleotides, 1 to 100 nucleotides, 1 to 50 nucleotides, 1 to 25 nucleotides, or 1 to 10 nucleotides, inclusive of both ends of each range. In some embodiments, the length of the second nucleic acid sequence is 1 to 650 nucleotides, 1 to 600 nucleotides, 1 to 550 nucleotides, 1 to 500 nucleotides, 1 to 450 nucleotides, 1 to 400 nucleotides, 1 to 350 nucleotides, 1 to 300 nucleotides, 1 to 250 nucleotides, 1 to 200 nucleotides, 1 to 150 nucleotides, 1 to 100 nucleotides, 1 to 50 nucleotides, 1 to 25 nucleotides, or 1 to 10 nucleotides, inclusive of both ends of each range.

[0028] In some embodiments, the length of the inter-ITR sequence is 1 to 25 nucleotides, 1 to 50 nucleotides, 25 to 50 nucleotides, 50 to 100 nucleotides, 100 to 150 nucleotides, 150 to 200 nucleotides, 200 to 250 nucleotides, 250 to 300 nucleotides, 300 to 350 nucleotides, 350 to 400 nucleotides, 400 to 450 nucleotides, 450 to 500 nucleotides, 500 to 550 nucleotides, 550 to 600 nucleotides, 600 to 650 nucleotides, and 650 to 700 nucleotides, inclusive of each end of the range. In some embodiments, the length of the second nucleic acid sequence is 1 to 25 nucleotides, 1 to 50 nucleotides, 25 to 50 nucleotides, 50 to 100 nucleotides, 100 to 150 nucleotides, 150 to 200 nucleotides, 200 to 250 nucleotides, 250 to 300 nucleotides, 300 to 350 nucleotides, 350 to 400 nucleotides, 400 to 450 nucleotides, 450 to 500 nucleotides, 500 to 550 nucleotides, 550 to 600 nucleotides, 600 to 650 nucleotides, and 650 to 700 nucleotides, inclusive of each end of the range.

[0029] In some embodiments involving a short nanotransposon (NTS) of the present disclosure, the length of the inter-ITR sequence is 1-10 nucleotides, 10-20 nucleotides, 20-30 nucleotides, 30-40 nucleotides, 40-50 nucleotides, 50-60 nucleotides, 60-70 nucleotides, 70-80 nucleotides, 80-90 nucleotides, or 90-100 nucleotides, inclusive of the ends of each range. In some embodiments involving a short nanotransposon (NTS) of the present disclosure, the length of the nucleic acid sequence is 1-10 nucleotides, 10-20 nucleotides, 20-30 nucleotides, 30-40 nucleotides, 40-50 nucleotides, 50-60 nucleotides, 60-70 nucleotides, 70-80 nucleotides, 80-90 nucleotides, or 90-100 nucleotides, inclusive of the ends of each range.

[0030] In some embodiments, the length of the sequence within the ITR is 100 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 1500 nucleotides or more, 2000 nucleotides or more, 2500 nucleotides or more, 3000 nucleotides or more, 3500 nucleotides or more, 4000 nucleotides or more, 4500 nucleotides or more, 5000 nucleotides or more, 5500 nucleotides or more, 6000 nucleotides or more, 6500 nucleotides or more, 7000 nucleotides or more, 7500 nucleotides or more, 8000 nucleotides or more, 8500 nucleotides or more, 9000 nucleotides or more, 9500 nucleotides or more, 10000 nucleotides (10 kilobases (kb)), 50000 nucleotides (50 kb), 100000 nucleotides (100 kb), 150000 nucleotides (150 kb), 200000 nucleotides (200 kb), 250000 nucleotides (250 kb), 300000 nucleotides (300 kb), 350000 nucleotides (350 kb), 400000 nucleotides (400 kb), 450000 nucleotides (400 kb), 500000 nucleotides (5 ... The nucleic acid sequence may be 300,000 nucleotides (300 kb) or more, 350,000 nucleotides (350 kb) or more, 400,000 nucleotides (400 kb) or more, 450,000 nucleotides (450 kb) or more, 500,000 nucleotides (500 kb) or more, or any number of nucleotides in between.In some embodiments, the length of the second nucleotide sequence is greater than or equal to 100 nucleotides, greater than or equal to 500 nucleotides, greater than or equal to 1000 nucleotides, greater than or equal to 1500 nucleotides, greater than or equal to 2000 nucleotides, greater than or equal to 2500 nucleotides, greater than or equal to 3000 nucleotides, greater than or equal to 3500 nucleotides, greater than or equal to 4000 nucleotides, greater than or equal to 4500 nucleotides, greater than or equal to 5000 nucleotides, greater than or equal to 5500 nucleotides, greater than or equal to 6000 nucleotides, greater than or equal to 6500 nucleotides, greater than or equal to 7000 nucleotides, greater than or equal to 7500 nucleotides, greater than or equal to 8000 nucleotides, greater than or equal to 8500 nucleotides, greater than or equal to 9000 nucleotides, greater than or equal to 9500 nucleotides, greater than or equal to 10000 nucleotides (10 kilobases (kb)), greater than or equal to 50000 nucleotides (50 kb), greater than or equal to 100000 nucleotides (100 kb), greater than or equal to 150000 nucleotides (150 kb), greater than or equal to 200000 nucleotides (200 kb), greater than or equal to 250000 nucleotides (250 The nucleic acid sequence may be 300,000 nucleotides (300 kb) or more, 350,000 nucleotides (350 kb) or more, 400,000 nucleotides (400 kb) or more, 450,000 nucleotides (450 kb) or more, 500,000 nucleotides (500 kb) or more, or any number of nucleotides in between.

[0031] The composition may further comprise a sequence encoding an origin of replication or a replication sequence. The first nucleic acid sequence or the second nucleic acid sequence may further comprise a sequence encoding an origin of replication or a replication sequence. Preferably, the first nucleic acid sequence comprises a sequence encoding an origin of replication or a replication sequence.

[0032] In some embodiments, the length of the origin of replication sequence is 450 nucleotides or less, 400 nucleotides or less, 350 nucleotides or less, 300 nucleotides or less, 250 nucleotides or less, 200 nucleotides or less, 150 nucleotides or less, 100 nucleotides or less, 50 nucleotides or less, 25 nucleotides or less, or 10 nucleotides or less. In some embodiments, the length of the origin of replication sequence is 1 to 450 nucleotides, 1 to 400 nucleotides, 1 to 350 nucleotides, 1 to 300 nucleotides, 1 to 250 nucleotides, 1 to 200 nucleotides, 1 to 150 nucleotides, 1 to 100 nucleotides, 1 to 50 nucleotides, 1 to 25 nucleotides, or 1 to 10 nucleotides, inclusive of each end of the range.

[0033] The origin of replication sequence may comprise an R6K origin of replication. The R6K origin of replication may comprise an R6Kγ origin of replication. The origin of replication sequence may comprise a mini origin of replication. The mini origin of replication may comprise an R6K mini origin of replication. The R6K mini origin of replication may comprise an R6Kγ mini origin of replication. The R6Kγ mini origin of replication is 281 nucleotides (281 base pairs) in length and comprises, consists essentially of, or consists of the nucleic acid sequence of SEQ ID NO:15.

[0034] The composition may further comprise a first selection marker or a sequence encoding the first selection marker. The first nucleic acid sequence or the second nucleic acid sequence may further comprise a first selection marker or a sequence encoding the first selection marker. Preferably, the first nucleic acid sequence comprises a first selection marker or a sequence encoding the first selection marker.

[0035] In some embodiments, the length of the first selection marker is 450 nucleotides or less, 200 nucleotides or less, 150 nucleotides or less, 100 nucleotides or less, 50 nucleotides or less, 25 nucleotides or less, or 10 nucleotides or less. In some embodiments, the length of the first selection marker is 1-200 nucleotides, 1-150 nucleotides, 1-100 nucleotides, 1-50 nucleotides, 1-25 nucleotides, or 1-10 nucleotides, inclusive of each end of the range.

[0036] The first selection marker may comprise a sucrose selection marker, a fluorescent marker, a cell surface marker, or a combination thereof. In a preferred embodiment, the first selection marker comprises, consists essentially of, or consists of a sucrose selection marker. In a preferred embodiment, the sucrose selection marker comprises an RNA-OUT selection marker. The RNA-OUT selection marker is 139 nucleotides (139 base pairs) in length and comprises, consists essentially of, or consists of the nucleic acid sequence of SEQ ID NO:16.

[0037] The first ITR-encoding sequence or the second ITR-encoding sequence may comprise a recognition sequence for TTAA, TTAT, or TTAX. The first ITR-encoding sequence or the second ITR-encoding sequence may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleotides.

[0038] The first ITR-encoding sequence or the second ITR-encoding sequence may comprise, consist essentially of, or consist of a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identity to SEQ ID NO: 24. The first ITR-encoding sequence or the second ITR-encoding sequence may comprise, consist essentially of, or consist of a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identity to SEQ ID NO: 25. The first ITR-encoding sequence or the second ITR-encoding sequence may comprise, consist essentially of, or consist of a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identity to SEQ ID NO: 26. The first ITR-encoding sequence or the second ITR-encoding sequence may comprise, consist essentially of, or consist of a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identity to SEQ ID NO: 27. The first ITR-encoding sequence or the second ITR-encoding sequence may comprise, consist essentially of, or consist of a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identity to SEQ ID NO:2. The first ITR-encoding sequence or the second ITR-encoding sequence may comprise, consist essentially of, or consist of a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identity to SEQ ID NO:14.

[0039] In one embodiment, the sequence encoding the first ITR comprises the nucleic acid sequence of SEQ ID NO: 24 and the second sequence encoding the second ITR comprises the nucleic acid sequence of SEQ ID NO: 25. In one embodiment, the sequence encoding the first ITR comprises the nucleic acid sequence of SEQ ID NO: 24 and the second sequence encoding the second ITR comprises the nucleic acid sequence of SEQ ID NO: 26. In one embodiment, the sequence encoding the first ITR comprises the nucleic acid sequence of SEQ ID NO: 24 and the second sequence encoding the second ITR comprises the nucleic acid sequence of SEQ ID NO: 27.

[0040] The first nucleic acid sequence may further comprise at least one exogenous sequence and at least one promoter capable of expressing the exogenous sequence in mammalian cells.In a preferred embodiment, the promoter is capable of expressing the exogenous sequence in human cells.In a preferred embodiment, the transposon sequence of the composition comprises at least one exogenous sequence and at least one promoter capable of expressing the exogenous sequence in mammalian cells.

[0041] The promoter may be a constitutive promoter. The promoter may be an inducible promoter. The promoter may be a cell type or tissue type specific promoter. The promoter may be an EF1a promoter (SEQ ID NO: 4), a CMV promoter, an MND promoter, an SV40 promoter, a PGK1 promoter, a Ubc promoter, a CAG promoter, an H1 promoter, or a U6 promoter. In a preferred embodiment, the promoter is an EF1a promoter. In one embodiment, the first nucleic acid sequence comprises a first sequence encoding a first promoter capable of expressing a first exogenous sequence in a mammalian cell, and a second sequence encoding a second promoter capable of expressing a second exogenous sequence in a mammalian cell, the first promoter being a constitutive promoter and the second promoter being an inducible promoter. In one embodiment, the first sequence encoding the first promoter and the second sequence encoding the second promoter are inverted.

[0042] At least one exogenous sequence comprises, consists essentially of, or consists of a sequence encoding a non-natural antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof. The non-natural antigen receptor may comprise a chimeric antigen receptor (CAR), a T cell receptor (TCR), a chimeric stimulating receptor (CSR), an HLA class I histocompatibility antigen, an alpha chain E recombinant polypeptide (HLA-E), a beta-2-microglobulin (B2M) recombinant polypeptide, or a combination thereof. TCR, CSR, HLA-E, and B2M are described in detail herein. In a preferred embodiment, the non-natural antigen receptor comprises a CAR.

[0043] The at least one exogenous sequence may further comprise, consist essentially of, or consist of sequences encoding an inducible pro-apoptotic polypeptide, which are described in detail herein.

[0044] The at least one exogenous sequence may further comprise, consist essentially of, or consist of a sequence encoding a second selection marker. The second selection marker may encode a gene product essential for cell viability and survival. The second selection marker may encode a gene product essential for cell viability and survival when stimulated by selective cell culture conditions. The selective cell culture conditions may include a compound detrimental to cell viability and survival, and the gene product confers resistance to the compound. Non-limiting examples of selection genes include neo (which confers resistance to neomycin), DHFR (which encodes dihydrofolate reductase and confers resistance to methotrexate), TYMS (which encodes thymidylate synthase), MGMT (which encodes O(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (which encodes aldehyde dehydrogenase 1 family, member A1), FRANCF, RAD51C (which encodes RAD51 paralog C), GCS (which encodes glucosylceramide synthase), NKX2.2 (which encodes NK2 homeobox 2), or any combination thereof.

[0045] The second selection marker may be a detectable marker. The detectable marker may be a fluorescent marker, a cell surface marker, or a metabolic marker. In a preferred embodiment, the second selection marker comprises a sequence encoding a dihydrofolate reductase (DHFR) mutein enzyme. The DHFR mutein enzyme comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:52. The DHFR mutein enzyme is encoded by a polynucleotide comprising, consists essentially of, or consists of the nucleic acid sequence of SEQ ID NO:53 or SEQ ID NO:11. The amino acid sequence of the DHFR mutein enzyme may further comprise a mutation at one or more of positions 80, 113, or 153. The amino acid sequence of the DHFR mutein enzyme may comprise one or more of a phenylalanine (F) or leucine (L) substitution at position 80, a leucine (L) or valine (V) substitution at position 113, and a valine (V) or aspartic acid (D) substitution at position 153.

[0046] The at least one exogenous sequence may further comprise, consist essentially of, or consist of a sequence encoding at least one self-cleaving peptide. For example, the self-cleaving peptide may be located between the CAR and the inducible pro-apoptotic polypeptide, or the self-cleaving peptide may be located between the CAR and the second selection marker.

[0047] At least one exogenous sequence may further comprise, essentially consist of, or consist of a sequence encoding at least two self-cleaving peptides. For example, the first self-cleaving peptide is located upstream or immediately upstream of the CAR, and the second self-cleaving peptide is located downstream or immediately downstream of the CAR, or the first self-cleaving peptide and the second self-cleaving peptide are adjacent to the CAR. For example, the first self-cleaving peptide is located upstream or immediately upstream of the inducible pro-apoptotic polypeptide, and the second self-cleaving peptide is located downstream or immediately downstream of the inducible pro-apoptotic polypeptide, or the first self-cleaving peptide and the second self-cleaving peptide are adjacent to the inducible pro-apoptotic polypeptide. For example, the first self-cleaving peptide is located upstream or immediately upstream of the second selection marker, and the second self-cleaving peptide is located downstream or immediately downstream of the second selection marker, or the first self-cleaving peptide and the second self-cleaving peptide are adjacent to the second selection marker.

[0048] Non-limiting examples of self-cleaving peptides include T2A peptide, GSG-T2A peptide, E2A peptide, GSG-E2A peptide, F2A peptide, GSG-F2A peptide, P2A peptide, or GSG-P2A peptide. A T2A peptide comprises, consists essentially of, or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO: 54. A GSG-T2A peptide comprises, consists essentially of, or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO: 55. GSG-T2A polypeptides are encoded by polypeptides comprising, consisting essentially of, or consisting of a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:56. E2A peptides comprise, consist essentially of, or consist of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO:57. The GSG-E2A peptide comprises, consists essentially of, or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identity to SEQ ID NO: 58. The F2A peptide comprises, consists essentially of, or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identity to SEQ ID NO: 59.The GSG-F2A peptide comprises, consists essentially of, or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO: 60. The P2A peptide comprises, consists essentially of, or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO: 61. A GSG-P2A peptide comprises, consists essentially of, or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO:62.

[0049] The first nucleic acid sequence comprising at least one exogenous sequence and at least one promoter capable of expressing the exogenous sequence in a mammalian cell may further comprise at least one sequence encoding an insulator. In one aspect, the first nucleic acid sequence may comprise a first sequence encoding a first insulator and a second sequence encoding a second insulator. In some embodiments, the sequence encoding the first or second insulator comprises, consists essentially of, or consists of a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO:3 or SEQ ID NO:13.

[0050] The first nucleic acid sequence comprising at least one exogenous sequence and at least one promoter capable of expressing the exogenous sequence in a mammalian cell may further comprise a polyadenosine (polyA) sequence. The first nucleic acid sequence comprising at least one exogenous sequence, at least one promoter capable of expressing the exogenous sequence in a mammalian cell, and at least one sequence encoding an insulator may further comprise a polyadenosine (polyA) sequence. The polyA sequence may be isolated or derived from a viral polyA sequence. The polyA sequence may be isolated or derived from a (SV40) polyA sequence. In some embodiments, the sequence encoding the first or second insulator comprises, consists essentially of, or consists of a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO: 12.

[0051] In one embodiment, the composition does not include a sequence encoding foreign DNA. In one embodiment, the first nucleic acid sequence does not include a sequence encoding foreign DNA. In one embodiment, the second nucleic acid sequence does not include a sequence encoding foreign DNA. In one embodiment, the composition includes a sequence encoding foreign DNA. In one embodiment, the first nucleic acid sequence includes a sequence encoding foreign DNA. In one embodiment, the second nucleic acid sequence includes a sequence encoding foreign DNA. The foreign DNA is a DNA sequence that is not derived from or obtained from the same organism as the mammalian cell in which the exogenous sequence is expressed. For example, the foreign DNA can be DNA from a virus rather than a mammal, or the foreign DNA can be DNA from a reptile rather than a mammal. In another embodiment, the foreign DNA can be derived from one mammal, but the mammal is different from the mammal in which the exogenous sequence is expressed. For example, the foreign DNA is derived from a rat rather than a human.

[0052] In one aspect, the composition does not include recombination sites, excision sites, ligation sites, or combinations thereof. In one aspect, the composition does not include the products of recombination events, excision events, ligation events, or combinations thereof. In one aspect, the composition is not derived from recombination events, excision events, ligation events, or combinations thereof.

[0053] In one embodiment, the first nucleic acid sequence does not include a recombination site, an excision site, a ligation site, or a combination thereof. In one embodiment, the first nucleic acid sequence does not include the product of a recombination event, an excision event, a ligation event, or a combination thereof. In one embodiment, the first nucleic acid sequence does not originate from a recombination event, an excision event, a ligation event, or a combination thereof.

[0054] In one embodiment, the second nucleic acid sequence does not include a recombination site, an excision site, a ligation site, or a combination thereof. In one embodiment, the second nucleic acid sequence does not include the product of a recombination event, an excision event, a ligation event, or a combination thereof. In one embodiment, the second nucleic acid sequence does not result from a recombination event, an excision event, a ligation event, or a combination thereof.

[0055] Recombination sites may comprise sequences that result from a recombination event, may comprise sequences that are the product of a recombination event, or may comprise the activity of a recombinase (eg, a recombinase site).

[0056] Chimeric antigen receptors (CARs)

[0057] The present disclosure also provides a composition (e.g., a nanotransposon) comprising a CAR, the CAR comprising an external domain comprising an antigen recognition region, a transmembrane domain, and an internal domain comprising at least one costimulatory domain. The CAR may further comprise a hinge region between the antigen recognition domain and the transmembrane domain.

[0058] The antigen recognition region may comprise at least one single chain variable fragment (scFv), centrin, single domain antibody, or a combination thereof. In one embodiment, at least one single domain antibody is a VHH. In one embodiment, at least one single domain antibody is a VH.

[0059] scFv

[0060] The composition (e.g., transposon or nanotransposon) of the present disclosure may comprise a CAR, and in some embodiments, the antigen recognition region of the CAR may comprise one or more scFv compositions to recognize and bind to a specific target protein / antigen. The antigen recognition region may comprise at least two scFvs. The antigen recognition region may comprise at least three scFvs. In one embodiment, the CAR of the present disclosure is a bispecific CAR that comprises at least two scFvs that specifically bind to two distinct antigens.

[0061] The scFv composition comprises the heavy and light chain variable regions of an antibody. An scFv is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin, in which the VH and VL domains are connected by a short peptide linker. An scFv can retain the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker. In some embodiments, the linker polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:33. The linker polypeptide may be encoded by a polynucleotide comprising, consists essentially of, or consists of the nucleic acid sequence of SEQ ID NO:34.

[0062] Sentinel

[0063] The compositions (e.g., transposons or nanotransposons) of the present disclosure may comprise a CAR, and in some embodiments, the antigen recognition region of the CAR may comprise one or more centrilin compositions for recognizing and binding to a specific target protein / antigen. Centilin, which specifically binds to an antigen, can be used to direct the specificity of cells (e.g., cytotoxic immune cells) toward a specific antigen. A CAR comprising centrilin is referred to herein as a CARTyrin.

[0064] The centrin of the present disclosure may include a protein scaffold, which can specifically bind to an antigen. The centrin of the present disclosure may include a protein scaffold including a consensus sequence of at least one fibronectin type III (FN3) domain, which can specifically bind to an antigen. The at least one fibronectin type III (FN3) domain may be derived from a human protein. The human protein may be tenascin-C. The consensus sequence comprises, consists essentially of, or consists of an amino acid sequence having at least 74%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identity to SEQ ID NO:84, or the consensus sequence comprises, consists essentially of, or consists of an amino acid sequence having at least 74%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identity to SEQ ID NO:85. The consensus sequence comprises, consists essentially of, or is encoded by a polynucleotide that consists of a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity (or any percentage therebetween) to SEQ ID NO:86.

[0065] The consensus sequence may be modified at one or more of: (a) an AB loop comprising, or consisting of, the amino acid residues TEDS (SEQ ID NO: 87) at positions 13-16 of the consensus sequence; b) a BC loop comprising, or consisting of, the amino acid residues TAPDAAF (SEQ ID NO: 88) at positions 22-28 of the consensus sequence; (c) a CD loop comprising, or consisting of, the amino acid residues SEKVGE (SEQ ID NO: 89) at positions 38-43 of the consensus sequence; (d) a DE loop comprising, or consisting of the amino acid residues GSER (SEQ ID NO: 90) at positions 51-54 of the consensus sequence; (e) an EF loop comprising, or consisting of the amino acid residues GLKPG (SEQ ID NO: 91) at positions 60-64 of the consensus sequence; (f) an FG loop comprising, or consisting of the amino acid residues KGGHRSN (SEQ ID NO: 92) at positions 75-81 of the consensus sequence; or (g) any combination of (a)-(f). A centirin of the present disclosure may comprise a consensus sequence of at least five fibronectin type III (FN3) domains, at least ten fibronectin type III (FN3) domains, or at least fifteen fibronectin type III (FN3) domains.

[0066] The term "antibody mimic" is intended to describe an organic compound that specifically binds to a target sequence and has a structure different from that of naturally occurring antibodies. Antibody mimics may include proteins, nucleic acids, or small molecules. The target sequence to which the antibody mimics of the present disclosure specifically bind may be an antigen. Antibody mimics can offer superior properties over antibodies, including, but not limited to, superior solubility, tissue penetration, thermal and enzymatic stability (e.g., resistance to enzymatic degradation), and lower manufacturing costs. Examples of antibody mimics include, but are not limited to, affibodies, affrylins, affimers, affitins, alphabodies, anticalins, and avimers (also known as avidity multimers), DARPins (Designed Ankyrin Repeat Proteins), finomers, Kunitz domain peptides, and monobodies.

[0067] The affibody molecule of the present disclosure comprises a protein scaffold that comprises or consists of one or more α-helices without disulfide bridges. Preferably, the affibody molecule of the present disclosure comprises or consists of three α-helices. For example, the antibody molecule of the present disclosure may comprise an immunoglobulin binding domain. The affibody molecule of the present disclosure may comprise the Z domain of Protein A.

[0068] Affilin molecules of the present disclosure include, for example, protein scaffolds generated by modification of exposed amino acids of either gamma-B crystallin or ubiquitin. Affilin molecules functionally mimic the affinity of antibodies for antigens, but do not structurally mimic antibodies. In the protein scaffolds used to generate affilins, amino acids accessible to solvent or potential binding partners in a properly folded protein molecule are considered exposed amino acids. Any one or more of these exposed amino acids may be modified to specifically bind to a target sequence or antigen.

[0069] The Affimer molecules of the present disclosure include protein scaffolds that include highly stable proteins engineered to display peptide loops that provide high affinity binding sites for specific target sequences. Exemplary Affimer molecules of the present disclosure include protein scaffolds based on cystatin proteins or their tertiary structures. Exemplary Affimer molecules of the present disclosure may share a common tertiary structure that includes alpha helices positioned at the apexes of antiparallel beta sheets.

[0070] The affitin molecules of the present disclosure include an artificial protein scaffold, the structure of which may be derived from, for example, a DNA-binding protein (e.g., the DNA-binding protein Sac7d). The affitins of the present disclosure selectively bind to a target sequence, which may be the whole or part of an antigen. Exemplary affitins of the present disclosure are produced by randomizing one or more amino acid sequences at the binding surface of a DNA-binding protein and subjecting the resulting protein to ribosome display and selection. The target sequence of the affitins of the present disclosure may be found, for example, in a genome or on the surface of a peptide, protein, virus, or bacteria. In some embodiments, the affitin molecules may be used as specific inhibitors of enzymes. The affitin molecules of the present disclosure may include a heat-resistant protein or a derivative thereof.

[0071] The Alphabody molecules of the present disclosure may also be referred to as cell-penetrating Alphabody (CPAB). The Alphabody molecules of the present disclosure comprise small proteins (typically less than 10 kDa) that bind to a variety of target sequences (including antigens). The Alphabody molecules are capable of reaching and binding to intracellular target sequences. Structurally, the Alphabody molecules of the present disclosure comprise artificial sequences that form single-stranded alpha helices (similar to naturally occurring coiled coil structures). The Alphabody molecules of the present disclosure may comprise a protein scaffold that comprises one or more amino acids modified to specifically bind to a target protein. Regardless of the binding specificity of the molecule, the Alphabody molecules of the present disclosure maintain correct folding and thermal stability.

[0072] The anticalin molecules of the present disclosure include artificial proteins that bind to target sequences or target sites within proteins or small molecules. The anticalin molecules of the present disclosure may include artificial proteins derived from human lipocalins. The anticalin molecules of the present disclosure may be used, for example, in place of monoclonal antibodies or fragments thereof. The anticalin molecules may exhibit better tissue penetration and thermal stability than monoclonal antibodies or fragments thereof. An example of an anticalin molecule of the present disclosure may include about 180 amino acids with a mass of about 20 kDa. Structurally, the anticalin molecules of the present disclosure include a barrel structure that includes antiparallel β-strands and attached α-helices connected in pairs by loops. In some embodiments, the anticalin molecules of the present disclosure include a barrel structure that includes eight antiparallel β-strands and attached α-helices connected in pairs by loops.

[0073] The avimer molecules of the present disclosure include artificial proteins that specifically bind to a target sequence (which may be an antigen). The avimers of the present disclosure can recognize multiple binding sites within the same target or within different targets. When the avimers of the present disclosure recognize multiple targets, the avimers mimic the function of bispecific antibodies. The artificial protein avimers may include two or more peptide sequences of about 30-35 amino acids each. These peptides may be linked via one or more linker peptides. The amino acid sequence of one or more peptides of the avimer may be derived from the A domain of a membrane receptor. Avimers have a rigid structure that may optionally include disulfide bonds and / or calcium. The avimers of the present disclosure may exhibit higher thermal stability compared to antibodies.

[0074] The DARPins (Designed Ankyrin Repeat Proteins) of the present disclosure comprise engineered recombinant or chimeric proteins with high specificity and high affinity for target sequences. In some embodiments, the DARPins of the present disclosure are derived from ankyrin proteins, and in some cases comprise at least three repeat motifs (also called repeat structural units) of ankyrin proteins. Ankyrin proteins mediate high affinity protein-protein interactions. The DARPins of the present disclosure comprise large target interaction surfaces.

[0075] Fynomers of the present disclosure include small molecule binding proteins (approximately 7 kDa) derived from the human Fyn SH3 domain and engineered to bind target sequences and molecules with affinity and specificity comparable to antibodies.

[0076] The Kunitz domain peptides of the present disclosure include protein scaffolds that include a Kunitz domain. The Kunitz domain includes an active site for inhibiting protease activity. Structurally, the Kunitz domain of the present disclosure includes a disulfide-rich α+β type fold. This structure is exemplified by bovine pancreatic trypsin inhibitor. The Kunitz domain peptides recognize specific protein structures and function as competitive protease inhibitors. The Kunitz domain of the present disclosure may include ecallantide (derived from human lipoprotein-associated coagulation inhibitor (LACI)).

[0077] The monobodies of the present disclosure are small proteins (containing about 94 amino acids and having a mass of about 10 kDa) of a size comparable to a single chain antibody. These engineered proteins specifically bind to target sequences, including antigens. The monobodies of the present disclosure can specifically target one or more separate proteins or target sequences. In some embodiments, the monobodies of the present disclosure comprise a protein scaffold that mimics the structure of human fibronectin, more preferably mimics the structure of the 10th extracellular type III domain of fibronectin. The 10th extracellular type III domain of fibronectin and its monobody mimics comprise seven beta sheets that form a barrel and three exposed loops on either side, corresponding to the three complementarity determining regions (CDRs) of an antibody. In contrast to the variable domain structure of an antibody, the monobody does not contain any binding sites for metal ions as well as a central disulfide bond. Multispecific monobodies may be optimized by modifying the loops BC and FG. The monobodies of the present disclosure may comprise adnectins.

[0078] VHH

[0079] The composition (e.g., transposon or nanotransposon) of the present disclosure may comprise a CAR, and in some embodiments, the antigen recognition region of the CAR may comprise at least one single domain antibody (SdAb) to recognize and bind to a specific target protein / antigen. In one embodiment, the single domain antibody is a VHH. VHH is a heavy chain antibody found in camelids. A VHH that specifically binds to an antigen can be used to direct the specificity of a cell (e.g., a cytotoxic immune cell) toward a specific antigen. The antigen recognition region may comprise at least two VHHs. The antigen recognition region may comprise at least three VHHs. In one embodiment, the CAR of the present disclosure is a bispecific CAR that comprises at least two VHHs that specifically bind to two distinct antigens. A CAR that comprises a VHH is referred to herein as a VCAR.

[0080] At least one VHH protein or VCAR of the present disclosure can optionally be produced by a cell line, a mixed cell line, an immortalized cell, or a clonal population of immortalized cells, as is well known in the art. See, e.g., Ausubel, et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, NY (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); and Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001).

[0081] As is known in the art, amino acids can be altered, added and / or deleted from a VHH protein to reduce immunogenicity or to reduce, enhance or modify binding, affinity, association constant, dissociation constant, avidity, specificity, half-life, stability, solubility, or other suitable properties.

[0082] In some cases, VHH proteins may be engineered to retain high affinity for the antigen and other favorable biological properties. To achieve this goal, VHH proteins may be prepared using three-dimensional models of the parental and engineered sequences, possibly through a process of analysis of the parental sequences and various conceptually engineered products. Three-dimensional models are commonly available and familiar to those skilled in the art. Computer programs are available (e.g., the Immunofilter program from Xencor, Inc., Monrovia, Calif) that illustrate and display possible three-dimensional structural structures of selected candidate sequences and can measure possible immunogenicity. Inspection of these displays allows analysis of the likely role of functional residues in the candidate sequences, i.e., residues that affect the ability of the candidate VHH protein to bind its antigen. In this way, residues from the parental and reference sequences can be selected and combined to achieve the desired properties, such as affinity for the target antigen. Alternatively, or in addition to the above procedures, other suitable engineering methods may be used. Screening of VHHs for specific binding to similar proteins or fragments can be conveniently achieved using nucleotide (DNA or RNA display) or peptide display libraries, e.g., in vitro display. Competition analyses may be performed on the VHHs or VCARs of the disclosure to determine which proteins, antibodies, and other competitors compete for binding to a target protein with the VHHs or VCARs of the disclosure and / or share epitopic regions. These analyses, which are well known to those of skill in the art, assess competition between competitors or ligands for a limited number of binding sites on the surface of a protein.

[0083] VH

[0084] The composition (e.g., transposon or nanotransposon) of the present disclosure may comprise a CAR, and in some embodiments, the antigen recognition region of the CAR may comprise at least one single domain antibody (SdAb) to recognize and bind a specific target protein / antigen. In one embodiment, the single domain antibody is a VH. The VH is a single domain binder derived from a common IgG. The VH that specifically binds to an antigen may be used to direct the specificity of a cell (e.g., a cytotoxic immune cell) toward a specific antigen. The antigen recognition region may comprise at least two VHs. The antigen recognition region may comprise at least three VHs. In one embodiment, the CAR of the present disclosure is a bispecific CAR that comprises at least two VHs that specifically bind two distinct antigens.

[0085] The VH can be isolated or derived from human sequences. The VH may comprise human CDR sequences and / or human framework sequences, as well as non-human or humanized sequences (e.g., rat Fc domain). In some embodiments, the VH is a fully humanized VH. In some embodiments, the VH is not a naturally occurring antibody or a naturally occurring antibody fragment. In some embodiments, the VH is not a fragment of a monoclonal antibody. In some embodiments, the VH is a UniDab antibody (TeneoBio). In some embodiments, the VH is modified to remove the Fc domain or a portion thereof. In some embodiments, the framework sequences of the VH are modified, e.g., to improve expression, reduce immunogenicity, or improve function.

[0086] VHs can be fully engineered using the UniRat (TeneoBio) system and "NGS-based Discovery" to generate VHs. With this method, specific VHs are not of natural origin, but are generated using a fully engineered system. VHs are not derived from naturally occurring monoclonal antibodies (mAbs) isolated directly from a host (e.g., mouse, rat, or human) or from a single clone (hybrid cell) of a cell or cell line. These VHs were not subsequently cloned from said cell line. Instead, VH sequences are fully engineered using the UniRat system as transgenes containing human variable regions (VH domains) with rat Fc domains, and thus are human / rat chimeras with no light chains, which differs from standard mAb formats. The natural rat genes have been knocked out, and only antibodies expressed in rats are derived from transgenes with VH domains that bind to rat Fc (UniAbs). These are the exclusive Abs expressed in UniRat. Next generation sequencing (NGS) and bioinformatics are used to identify the complete antigen-specific population of heavy chain antibodies generated by UniRat after immunization. A proprietary gene assembly method is then used to convert the sequence information of the antibody population into a large collection of fully human heavy chain antibodies that can be screened in vitro for various functions. In some embodiments, fully humanized VHs are generated in vitro by fusing human VH domains with human Fc (to generate recombinant VH antibodies of non-natural origin). In some embodiments, VHs are fully humanized, but they are expressed in vivo as human / rat chimeras without light chains (human VH, rat Fc). Fully humanized VHs are expressed in vivo, as the human / rat chimeras without light chains (human VH, rat Fc) are approximately 80 kDa (vs. 150 kDa).

[0087] The CAR of this disclosure is 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -13 M or less, 10 -14 M or less, and 10-15 K below M D The K D can be measured by any means, including but not limited to, surface plasmon resonance.

[0088] In one embodiment, the antigen recognition region of the disclosed CAR comprises at least one anti-BCMA centilin. The anti-BCMA centilin comprises, consists essentially of, or consists of an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO: 29. The anti-BCMA centilin is encoded by a polynucleotide comprising, consists essentially of, or consists of a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO: 28.

[0089] A CAR comprising an anti-BCMA centirin is referred to herein as a BCMA CARTyrin. In a preferred embodiment, the BCMA CARTyrin comprises, consists essentially of, or consists of an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO: 30. The BCMA CARTyrin is encoded by a polynucleotide comprising, consists essentially of, or consists of a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO: 9.

[0090] Compositions of the disclosure that include BCMA CARTyrin (e.g., nanotransposons) comprise, consist essentially of, or consist of an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO: 17. Compositions of the disclosure that include BCMA CARTyrin (e.g., nanotransposons) are encoded by a polynucleotide that comprises, consists essentially of, or consists of a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO: 1. Compositions of the disclosure that include BCMA CARTyrin (e.g., nanotransposons) are referred to herein as P-BCMA-101-transposons (as shown in FIG. 13).

[0091] In one embodiment, the antigen recognition region of the disclosed CAR comprises at least one anti-PSMA centilin. The anti-PSMA centilin comprises, consists essentially of, or consists of an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO: 94. The anti-PSMA centilin is encoded by a polynucleotide comprising, consists essentially of, or consists of a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO: 93.

[0092] A CAR comprising an anti-PSMA centrin is referred to herein as a PSMA CARTyrin. In a preferred embodiment, the PSMA CARTyrin comprises, consists essentially of, or consists of an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO: 95. The PSMA CARTyrin is encoded by a polynucleotide comprising, consists essentially of, or consists of a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO: 19.

[0093] A composition of the present disclosure comprising a PSMA CARTyrin (e.g., a nanotransposon) comprises, consists essentially of, or consists of an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO: 20. A composition of the present disclosure comprising a PSMA CARTyrin (e.g., a nanotransposon) is encoded by a polynucleotide comprising, consists essentially of, or consists of a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO: 18. A composition of the present disclosure comprising a PSMA CARTyrin (e.g., a nanotransposon) is referred to herein as a P-PSMA-101 transposon (as shown in FIG. 14).

[0094] In one embodiment, the antigen recognition region of the disclosed CAR comprises at least one anti-BCMA VH. The anti-BCMA VH comprises, consists essentially of, or consists of an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO: 97. The anti-BCMA VH is encoded by a polynucleotide that comprises, consists essentially of, or consists of a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO: 96.

[0095] A CAR comprising an anti-BCMA VH is referred to herein as a BCMA VCAR. In a preferred embodiment, the BCMA VCAR comprises, consists essentially of, or consists of an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO: 98. The BCMA VCAR is encoded by a polynucleotide comprising, consists essentially of, or consists of a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO: 22.

[0096] Compositions of the disclosure comprising BCMA VCAR (e.g., nanotransposons) comprise, consist essentially of, or consist of an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO: 23. Compositions of the disclosure comprising BCMA VCAR (e.g., nanotransposons) are encoded by a polynucleotide comprising, consist essentially of, or consist of a nucleic acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO: 21. Compositions of the disclosure comprising BCMA VCAR (e.g., nanotransposons) are referred to herein as P-BCMA-ALLO1-transposons (as shown in FIG. 15).

[0097] The ectodomain may comprise a signal peptide. The signal peptide may comprise a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB, or GM-CSFR signal peptide. In a preferred embodiment, the signal peptide comprises, consists essentially of, or consists of a human CD8α signal peptide (SP) or a portion thereof. Human CD8α SP comprises, consists essentially of, or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO:31. Preferably, human CD8α SP comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:31.

[0098] Human CD8α SP comprises, consists essentially of, or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO: 32. Preferably, human CD8α SP is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:32.

[0099] The hinge domain or region may comprise the sequence of human CD8α, IgG4, CD4, or a combination thereof. In a preferred embodiment, the hinge may comprise, consist essentially of, or consist of a human CD8α hinge or a portion thereof. The human CD8α hinge comprises, consists essentially of, or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO: 35. Preferably, the human CD8α hinge domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 35.

[0100] The human CD8α hinge is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO: 36. Preferably, the human CD8α hinge domain is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO:36.

[0101] The transmembrane domain may comprise, consist essentially of, or consist of sequences encoding the transmembrane domains of human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB, or GM-CSFR. Preferably, the transmembrane domain may comprise, consist essentially of, or consist of the human CD8α transmembrane domain, or a portion thereof. The CD8α transmembrane domain comprises, consists essentially of, or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO:37. Preferably, the human CD8α transmembrane domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:37.

[0102] The CD8 alpha transmembrane domain is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO: 38. Preferably, the CD8 alpha transmembrane domain is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO:38.

[0103] The at least one costimulatory domain may comprise, consist essentially of, or consist of the intracellular domains of human 4-1BB, CD28, CD3zeta, CD40, ICOS, MyD88, OX-40, or any combination thereof. Preferably, the at least one costimulatory domain comprises the costimulatory domain of CD3zeta, 4-1BB, or a combination thereof.

[0104] The 4-1BB intracellular domain comprises, consists essentially of, or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO: 39. Preferably, the 4-1BB intracellular domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 39.

[0105] The 4-1BB intracellular domain is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO: 40. Preferably, the 4-1BB intracellular domain is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 40.

[0106] The CD3 zeta intracellular domain comprises, consists essentially of, or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO: 41. Preferably, the CD3 zeta intracellular domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 41.

[0107] The CD3 zeta intracellular domain is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO: 42. Preferably, the CD3 zeta intracellular domain is encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 42.

[0108] Transposon and vector compositions

[0109] Transference System

[0110] The present disclosure provides a transposon or nanotransposon comprising: a first nucleic acid sequence comprising: (a) a first inverted terminal repeat (ITR) or a sequence encoding a first ITR, (b) a second ITR or a sequence encoding a second ITR, and (c) an intra-ITR sequence or a sequence encoding an intra-ITR sequence, wherein the intra-ITR sequence comprises a transposon sequence or a sequence encoding a transposon sequence; and a second nucleic acid sequence comprising an inter-ITR sequence or a sequence encoding an inter-ITR sequence, wherein the length of the inter-ITR sequence is 700 nucleotides or less.

[0111] The transposon or nanotransposon of the present disclosure comprises a protein scaffold (e.g., a CAR comprising at least one scFv, single domain antibody, or centilin). The transposon or nanotransposon may be a plasmid DNA transposon comprising a sequence encoding a protein scaffold (e.g., a CAR comprising at least one scFv, single domain antibody, or centilin) ​​flanked by two cis-regulatory insulator elements. The transposon or nanotransposon may further comprise a plasmid comprising a sequence encoding a transferase. The sequence encoding the transferase may be a DNA sequence or an RNA sequence. Preferably, the sequence encoding the transferase is an mRNA sequence.

[0112] The transposon or nanotransposon of the present disclosure may be a piggyBac™ (PB) transposon. In some embodiments where the transposon is a PB transposon, the transposase is a piggyBac™ (PB) transposase, a piggyBac-like (PBL) transposase, or a Super piggyBac™ (SPB) transposase. Preferably, the sequence encoding the SPB transposase is an mRNA sequence.

[0113] 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 International Patent Publication WO 2010 / 099296.

[0114] PB, PBL and SPB transposases recognize transposon-specific inverted terminal repeats (ITRs) on the terminal surface of the transposon and insert their contents between the ITRs at a 5'-TTAT-3' sequence within the chromosomal site (TTAT target sequence) or a 5'-TTAA-3' sequence within the chromosomal site (TTAA target sequence). The target sequences of PB or PBL transposons are 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'-T TAC-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 does not limit the amount of loading of the gene of interest that may be included between the ITRs.

[0115] Exemplary amino acid sequences of one or more of PB, PBL, and SPB transferases 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. In a preferred embodiment, the PB transferase comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO:63.

[0116] The PB or PBL transferase may comprise or consist of an amino acid sequence having an amino acid substitution at two or more, three or more, or each of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 63. The transferase may be an SPB transferase comprising or consisting of the amino acid sequence of SEQ ID NO: 63, where the amino acid substitution at position 30 may be a substitution of isoleucine (I) with valine (V), the amino acid substitution at position 165 may be a substitution of glycine (G) with serine (S), the amino acid substitution at position 282 may be a substitution of methionine (M) with valine (V), and the amino acid substitution at position 538 may be a substitution of asparagine (N) with lysine (K). In a preferred embodiment, the SPB transferase comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO:64.

[0117] In certain embodiments in which the transferase comprises a mutation as described above at positions 30, 165, 282, and / or 538, the PB, PBL, and SPB transferases 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:63 or SEQ ID NO:64, which are described in International Patent Publication WO 2006 / 023636. This is described in further detail in International Patent Application No. PCT / US2019 / 173636 and International Patent Application No. PCT / US2019 / 049816.

[0118] The PB, PBL, or SPB transferase may be isolated or derived from an insect, vertebrate, crustacean, or tunicate, as described in further detail in International Patent Publication No. WO 2019 / 173636 and International Patent Application No. PCT / US2019 / 049816. In a preferred embodiment, the PB, PBL, or SPB transferase is isolated or derived from the insect Trichoplusia ni (GenBank Accession No. AAA87375) or the silkworm Bombyx mori (GenBank Accession No. BAD11135).

[0119] A hyperactive PB or PBL transferase is a transferase that is more active than the naturally occurring mutant from which it is derived. In a preferred embodiment, the hyperactive PB or PBL transferase is isolated or derived from the silkworm or Xenopus tropicalis. Examples of hyperactive PB or PBL transferase are disclosed in U.S. Pat. No. 6,218,185, U.S. Pat. No. 6,962,810, U.S. Pat. No. 8,399,643, and International Patent Publication WO 2019 / 173636. A list of hyperactive amino acid substitutions is disclosed in U.S. Pat. No. 10,041,077.

[0120] In some embodiments, PB or PBL transposase is integration-defective. An integration-defective PB or PBL transposase is a transposase that can excise its corresponding transposon, but integrates the excised transposon at a lower frequency than its corresponding wild-type transposase. Examples of integration-defective PB or PBL transposase 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 International Patent Publication WO 2019 / 173636. A list of integration-defective amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[0121] In some embodiments, PB or PBL transferase is fused to a nuclear localization signal. Examples of PB or PBL transferase 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 International Patent Publication WO 2019 / 173636.

[0122] The transposon or nanotransposon of the present disclosure may be a Sleeping Beauty transposon. In some embodiments, when the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase (e.g., as disclosed in U.S. Pat. No. 9,228,180), or a hyperactive Sleeping Beauty (SB100X) transposase. In preferred embodiments, the Sleeping Beauty transposase comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO:65. In preferred embodiments, the hyperactive Sleeping Beauty (SB100X) transferase comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO:66.

[0123] The transposon or nanotransposon of the present disclosure may be a Helraiser transposon. An example of a Helraiser transposon is Helibat1, which comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO: 67. In some embodiments, when the transposon is a Helraiser transposon, the transposase is a Helitron transposase (e.g., as disclosed in International Patent Publication WO 2019 / 173636). In preferred embodiments, the Helitron transferase comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO:68.

[0124] The transposon or nanotransposon of the present disclosure may be a Tol2 transposon. An exemplary Tol2 transposon, including an inverted repeat sequence, a subterminal sequence, and a Tol2 transposase, comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO: 69. In some embodiments, when the transposon is a Tol2 transposon, the transposase is a Tol2 transposase (e.g., as disclosed in International Patent Publication WO 2019 / 173636). In a preferred embodiment, the Tol2 transferase comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO:70.

[0125] The transposon or nanotransposon of the present disclosure may be a TcBuster transposon. In some embodiments, when the transposon is a TcBuster transposon, the transposase is a TcBuster transposase or a hyperactive TcBuster transposase (e.g., as disclosed in International Patent Publication WO 2019 / 173636). The TcBuster transposase may comprise or consist of a naturally occurring or non-naturally occurring amino acid sequence. In preferred embodiments, the TcBuster transposase comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity (or any percentage therebetween) to SEQ ID NO: 71. The polynucleotide encoding the TcBuster transposase may comprise or consist of a naturally occurring or non-naturally occurring nucleic acid sequence. In preferred embodiments, the TcBuster transferase is encoded by a polynucleotide comprising or consisting of a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity (or any percentage therebetween) to SEQ ID NO:72.

[0126] In some embodiments, the mutant TcBuster transferase comprises one or more sequence mutations when compared to wild-type TcBuster transferase, as described in more detail in International Patent Publication WO 2019 / 173636 and International Patent Application PCT / US2019 / 049816.

[0127] The cell delivery compositions (e.g., transposons) disclosed herein may include nucleic acids encoding therapeutic proteins or therapeutic agents. Examples of therapeutic proteins include those disclosed in International Patent Publication WO 2019 / 173636 and International Patent Application PCT / US2019 / 049816.

[0128] Vector System

[0129] In some embodiments, the compositions of the present disclosure (e.g., nanotransposons) may be utilized in combination with another transposon or nanotransposon, or with a vector. The vectors of the present disclosure may be viral or recombinant vectors. The viral vectors may include sequences isolated or derived from retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or any combination thereof. The viral vectors may include sequences isolated or derived from adeno-associated viruses (AAV). The viral vectors may include recombinant AAV (rAAV). Examples of adeno-associated viruses and recombinant adeno-associated viruses include two or more inverted terminal repeat (ITR) sequences located in cis next to the sequences encoding the scFv or CAR of the present disclosure. Examples of 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). Examples of adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids that contain the genome of one serotype and the capsid of another serotype (e.g., AAV2 / 5, AAV-DJ, and AAV-DJ8). Examples of adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, rAAV-LK03.

[0130] The vector of the present disclosure may 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.Nanoparticle vectors may pass through cell membranes passively or actively.

[0131] The cell delivery compositions (e.g., transposons, vectors) disclosed herein may include nucleic acids encoding therapeutic proteins or therapeutic agents. Examples of therapeutic proteins include those disclosed in International Patent Publication WO 2019 / 173636 and International Patent Application PCT / US2019 / 049816.

[0132] Disclosed Cells and Modified Cells

[0133] The cells and modified cells of the present disclosure may be mammalian cells. Preferably, the cells and modified cells are human cells. The cells and modified cells of the present disclosure may be immune cells. The immune cells of the present disclosure include lymphoid progenitor cells, natural killer (NK) cells, T lymphocytes (T cells), stem memory T cells (T SCM cells), central memory T cells (T CM ), 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.

[0134] Immune progenitor cells may include any cell capable of differentiating into one or more immune cell types. Immune progenitor cells may include pluripotent stem cells capable of self-renewal and maturing into immune cells. Immune progenitor cells may include hematopoietic stem cells (HSCs) or their progeny. Immune progenitor cells may include progenitor cells capable of maturing into immune cells. Immune progenitor cells may include hematopoietic progenitor cells (HPCs).

[0135] Hematopoietic stem cells (HSCs) are multipotent self-renewing cells. All differentiated blood cells from 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.

[0136] HSCs may be isolated or derived from primary or cultured stem cells. HSCs may be isolated or derived from embryonic stem cells, pluripotent stem cells, multipotent stem cells, adult stem cells, or induced pluripotent stem cells (iPSCs).

[0137] Immune progenitor cells may include HSCs or HSC progeny. Non-limiting examples of HSC progeny include pluripotent 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.

[0138] HSCs produced by the disclosed methods can retain the characteristics of "primitive" stem cells that share the characteristics of embryonic stem cells when isolated or derived from adult stem cells and committed to a single lineage. For example, "primitive" HSCs produced by the disclosed methods retain their "stemness" after division and do not differentiate. As a result, as adoptive cell therapy, "primitive" HSCs produced by the disclosed methods not only replenish their numbers but also expand in vivo. "Primitive" HSCs produced by the disclosed methods are therapeutically effective when administered as a single dose.

[0139] The primitive HSCs may be CD34+. The primitive HSCs may be CD34+ and CD38-. The primitive HSCs may be CD34+, CD38-, and CD90+. The primitive HSCs may be CD34+, CD38-, CD90+, and CD45RA-. The primitive HSCs may be CD34+, CD38-, CD90+, CD45RA-, and CD49f+. The primitive HSCs may be CD34+, CD38-, CD90+, CD45RA-, and CD49f+.

[0140] Primitive HSCs, HSCs, and / or HSC progeny cells can be modified according to the disclosed methods to express exogenous sequences (e.g., chimeric antigen receptors or therapeutic proteins). The modified primitive HSCs, HSCs, and / or HSC progeny cells can then be differentiated to produce modified immune cells, such as, but not limited to, modified T cells, modified natural killer cells, and / or modified B cells.

[0141] The modified immune cells or immune precursor cells may be NK cells. NK cells may be cytotoxic lymphocytes differentiated from lymphoid progenitor cells. Modified NK cells may be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs. In some embodiments, non-activated NK cells are derived by leukapheresis depleted of CD3 (including CD14 / CD19 / CD56+ cells).

[0142] The modified immune cells or immune precursor cells may be B cells. B cells are a type of lymphocyte that express a B cell receptor on the cell surface. The B cell receptor binds to a specific antigen. The modified B cells may be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

[0143] The modified T cells of the present disclosure may be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs. Unlike conventional biologics and chemotherapy, the modified T cells of the disclosure have the ability to rapidly regenerate upon antigen recognition, potentially eliminating the need for repeated treatment. To achieve this, in some embodiments, the modified T cells not only drive the initial response, but also persist in the patient as a stable population of viable memory T cells to prevent potential relapse. Alternatively, in some embodiments, the modified T cells do not persist in the patient if this is not desired.

[0144] Development of antigen receptor molecules that do not cause T cell exhaustion through antigen-independent (tonic) signaling, as well as the ability to inhibit early memory T cells, especially stem memory cells (T SCMIntensive efforts have been made to develop modified T cell products that contain stem cell-like T cells (T cells). The stem cell-like modified T cells of the present disclosure have the greatest capacity for self-renewal and the ability to differentiate into central memory T cells (T CM ) or T CM -like cells, effector memory cells (T EM ) and effector T cells (T E ), resulting in better tumor eradication potential and long-term engraftment of modified T cells. The lineage pathway of differentiation is N )>T SCM >T CM >T EM >T E >T TE may be involved in the generation of these cells, as shown in N is T SCM directly produces T CM The composition of T cells of the present disclosure may include a fraction of one or more of each of the parent T cells, SCM Cells are the most abundant (e.g., T SCM >T CM >T EM >T E >T TE ).

[0145] Immune cell precursors include early memory T cells, stem cell-like T cells, and naive T cells (T N ), T SCM , T CM , T EM , T E , or T TE The immune cell precursor may be a primitive HSC, HSC, or HSC progeny cell of the present disclosure. The immune cell may be an early memory T cell, a stem cell-like T cell, a naive T cell (T N ), T SCM , T CM , T EM , T E , or T TE may be also possible.

[0146] The disclosed methods can modify and / or produce a population of modified T cells, in which 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, a plurality of modified T cells express one or more cell surface markers of early memory T cells. The population of modified early memory T cells includes a plurality of modified stem cell-like T cells. The population of modified early memory T cells can include a plurality of modified T cells. SCM The population of modified early memory T cells includes multiple modified T CM Contains cells.

[0147] The disclosed methods can modify and / or produce a population of modified T cells, where 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, a plurality of modified T cells express one or more cell surface markers of stem cell-like T cells. A population of modified stem cell-like T cells can be a plurality of modified T cells. SCM The population of modified stem cell-like T cells comprises a plurality of modified T CM Contains cells.

[0148] In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, or any percentage in between, of the plurality of modified T cells in the population are stem memory T cells (T SCM ) or T SCMThe cells express one or more cell surface markers of CD45-like cells, the one or more cell surface markers including CD45RA and CD62L. The cell surface markers may include one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95, and IL-2Rβ. The cell surface markers may include one or more of CD45RA, CD95, IL-2Rβ, CCR7, and CD62L.

[0149] In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the plurality of modified T cells in the population are central memory T cells (T CM ) or T CM The one or more cell surface markers of CD45-like cells may include one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L.

[0150] The disclosed methods can modify and / or produce a population of modified T cells, where 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, a plurality of modified T cells are naive T cells (T N ) The cell surface markers may include one or more of CD45RA, CCR7, and CD62L.

[0151] The disclosed methods can modify and / or produce a population of modified T cells, where 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, a plurality of modified T cells within the population are effector T cells (modified T EFF ) The cell surface markers may include one or more of CD45RA, CD95, and IL-2Rβ.

[0152] The disclosed methods can modify and / or produce a population of modified T cells, where 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 a plurality of modified T cells are stem cell-like T cells, stem memory cells (T SCM ), or central memory T cells (T CM ) express one or more cell surface markers.

[0153] A population of multiple modified cells comprises a transgene or a sequence encoding a transgene (e.g., a CAR), and 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 multiple cells in the population comprise a transgene or a sequence encoding a transgene, and at least 70%, at least 75%, at least 80 ...0%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least 90%, at least %, 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 modified cells express one or more cell surface markers including CD34, or alternatively at least about 70% to about 99%, about 75% to about 95%, or about 85% to about 95% of the modified cells in the population express one or more cell surface markers including CD34 (e.g., comprising CD34+ as a cell surface marker phenotype).

[0154] A population of multiple modified cells comprises a transgene or a sequence encoding a transgene (e.g., a CAR), and 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 multiple cells in the population comprise a transgene or a sequence encoding a transgene, and 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 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 modified cells express one or more cell surface markers including CD34 and do not express one or more cell surface markers including CD38, or at least about 45% to about 90%, about 50% to about 80%, or about 65% to about 75% of the modified cells in the population express one or more cell surface markers including CD34 and do not express one or more cell surface markers including CD38 (e.g., cell surface marker phenotypes include CD34+ and CD38-).

[0155] A population of multiple modified cells comprises a transgene or a sequence encoding a transgene (e.g., a CAR), and 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 multiple cells in the population comprise a transgene or a sequence encoding a transgene, and at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 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 modified cells contain CD34 and CD90. or at least about 0.2% to about 40%, about 0.2% to about 30%, about 0.2% to about 2%, or about 0.5% to about 1.5% of the modified cells in the population express one or more cell surface markers, including CD34 and CD90, and do not express one or more cell surface markers, including CD38 (e.g., cell surface marker phenotypes include CD34+, CD38-, and CD90+).

[0156] A population of multiple modified cells comprises a transgene or a sequence encoding a transgene (e.g., a CAR), and 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 multiple cells in the population comprise a transgene or a sequence encoding a transgene, and at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% of the modified cells express one or more cell surface markers including CD34 and CD90. and do not express one or more cell surface markers, including CD38 and CD45RA, or at least about 0.2% to about 40%, about 0.2% to about 30%, about 0.2% to about 2%, or about 0.5% to about 1.5% of the modified cells within the population express one or more cell surface markers, including CD34 and CD90, and do not express one or more cell surface markers, including CD38 and CD45RA (e.g., cell surface marker phenotypes include CD34+, CD38-, CD90+, and CD45RA-).

[0157] A population of multiple modified cells comprises a transgene or a sequence encoding a transgene (e.g., a CAR), and 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 multiple cells in the population comprise a transgene or a sequence encoding a transgene, and at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 1.6%, at least 1.8%, at least 1.9%, at least 2.0%, at least 2.5 ... %, 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 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 modified cells express one or more cell surface markers including CD34, CD90, and CD49f and do not express one or more cell surface markers including CD38 and CD45RA, or at least about 0.02% to about 30%, about 0.02% to about 2%, about 0.04% to about 2%, or about 0.Between 0.4% and approximately 1% of the modified cells express one or more cell surface markers including CD34, CD90, and CD49f, and do not express one or more cell surface markers including CD38 and CD45RA (e.g., cell surface marker phenotypes include CD34+, CD38-, CD90+, CD45RA-, and CD49f+).

[0158] A population of multiple modified cells comprises a transgene or a sequence encoding a transgene (e.g., a CAR), and 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 multiple cells in the population comprise a transgene or a sequence encoding a transgene, and at least 0.01%, at least 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 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 9 9.9%, or 100% of the modified cells express one or more cell surface markers including CD34 and CD90 and do not express one or more cell surface markers including CD45RA, or at least about 0.2% to about 5%, about 0.2% to about 3%, or about 0.4% to about 3% of the modified cells within the population express one or more cell surface markers including CD34 and CD90 and do not express one or more cell surface markers including CD45RA (e.g., cell surface marker phenotypes include CD34+, CD90+, and CD45RA-).

[0159] Compositions and methods for producing and / or expanding immune cells or immune progenitor cells (e.g., the disclosed modified T cells), as well as buffers for maintaining or enhancing cell viability levels and / or stem-like phenotype of immune cells or immune progenitor cells (e.g., the disclosed modified T cells), are described elsewhere herein and are disclosed in more detail in U.S. Pat. No. 10,329,543 and International Patent Publication WO 2019 / 173636.

[0160] The cells and modified cells of the present disclosure may be somatic cells. The cells and modified cells of the present disclosure may be differentiated cells. The cells and modified cells of the present disclosure may be autologous or allogeneic cells. Allogeneic cells are engineered to prevent adverse reactions to engraftment after administration to a subject. Allogeneic cells may be any cell type. Allogeneic cells may be stem cells or may be derived from stem cells. Allogeneic cells may be differentiated somatic cells.

[0161] Methods for expressing chimeric antigen receptors

[0162] The present disclosure provides a method of expressing a CAR on a cell surface, the method comprising: (a) harvesting a cell population; (b) contacting the cell population with a composition comprising a CAR or a sequence encoding a CAR under conditions sufficient to translocate the CAR across the cell membrane of at least one cell in the cell population, thereby generating a modified cell population; (c) culturing the modified cell population under conditions suitable for incorporation of the sequence encoding the CAR; and (d) expanding and / or selecting at least one cell from the modified cell population that expresses the CAR on its cell surface.

[0163] In some embodiments, the cell population may include leukocytes and / or CD4+ and CD8+ leukocytes. The cell population may include CD4+ and CD8+ leukocytes in an optimal ratio. The optimal ratio of CD4+ to CD8+ leukocytes does not occur naturally in vivo. The cell population may include tumor cells.

[0164] In some embodiments, the conditions sufficient to transport the CAR, or the sequence encoding the CAR, transposon, or vector across the cell membrane of at least one cell in the cell population include at least one of the following: application of one or more electrical pulses at a specific voltage, a buffer, and one or more supplementary factors. In some embodiments, the conditions suitable for integration of the sequence encoding the CAR include at least one of a buffer and one or more supplementary factors.

[0165] The buffer may include PBS, HBSS, OpitMEM, BTXpres, Amaxa Nucleofocter, human T cell nucleofection buffer, or any combination thereof. The one or more supplemental factors may include (a) recombinant human cytokines, chemokines, interleukins, or any combination thereof; (b) salts, minerals, metabolites, or any combination thereof; (c) cell culture medium; (d) inhibitors of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathways, or combinations thereof; (e) one or more nucleic acid modifying or stabilizing reagents. The recombinant human cytokine, chemokine, interleukin, or any combination thereof may be selected from the group consisting of 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-γ, IL-1α / IL-1F1, IL-1β / IL-1F2, IL-12p70, IL-12 / IL-35p35, IL-1 3, IL-17 / IL-17A, IL-17A / F heterodimer, IL-17F, IL-18 / IL-1F4, IL-23, IL-24, IL-32, IL-32β, IL-32γ, IL-33, LAP (TGF-β1), lymphotoxin-α / TNF-β, TGF-β, TNF-α, TRANCE / TNFSF11 / RANK L, or any combination thereof.The salts, minerals, metabolites, or any combination thereof may include HEPES, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solution, ascorbic acid, nucleosides, FBS / FCS, human serum, serum replacements, antibiotics, pH adjusters, Earle's salts, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, Nucleofector PLUS adduct, KCl, MgCl2, Na2HPO4, NaH2PO4, sodium lactobionate, mannitol, sodium succinate, sodium chloride, CINa, glucose, Ca(NO3)2, Tris / HCl, K2HPO4, KH2PO4, polyethyleneimine, polyethylene glycol, Poloxamer 188, Poloxamer 181, Poloxamer 407, polyvinylpyrrolidone, Pop313, Crown-5, or any combination thereof. The cell culture medium may include 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. Inhibitors of cellular DNA sensing, metabolism, differentiation, signal transduction, one or more apoptotic pathways, or combinations thereof include inhibitors of TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-KB, type 1 interferon, proinflammatory 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-3β, e.g., TWS119), or any combination thereof. Examples of such inhibitors may include bafilomycin, chloroquine, quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK, or any combination thereof.Reagents that modify or stabilize one or more nucleic acids include pH adjusters, DNA binding proteins, lipids, phospholipids, CaPO4, net neutral charge DNA binding peptides with or without NLS sequences, TREX1 enzyme, or any combination thereof.

[0166] The growth and selection steps may be performed simultaneously or sequentially. Growth may be performed prior to selection. Growth may be performed after selection, and in some cases growth may be followed by a further (i.e. second) round of selection. Growth and selection may be performed simultaneously. The growth and / or selection steps may take between 10 and 14 days inclusive.

[0167] The expansion may include contacting at least one cell of the cell population to be modified with an antigen to stimulate at least one cell via the CAR, thereby producing an expanded cell population. The antigen may be presented on a substrate surface. The substrate may be in any form, including but not limited to a surface, a well, a bead or beads, and a matrix. The substrate may further include a paramagnetic or magnetic member. The antigen may be presented on a substrate surface, the substrate being a magnetic bead, and the magnetic bead can be removed or separated from the modified and expanded cell population using a magnet. The antigen may be presented on a cell surface or an artificial antigen-presenting cell surface. The artificial antigen-presenting cell may include but is not limited to tumor cells and stem cells.

[0168] In some embodiments in which the transposon or vector comprises a selection gene, the selection step comprises contacting at least one cell of the modified cell population with a compound to which the selection gene confers resistance, whereby cells that survive the selection step are identified as cells that express the selection gene and cells that do not survive the selection step are identified as cells that do not express the selection gene.

[0169] The present disclosure provides compositions comprising cell populations that are modified, expanded, and selected by the methods described herein.

[0170] A more detailed description of methods for expressing CAR on the cell surface is disclosed in International Patent Publication WO 2019 / 049816 and International Patent Publication PCT / US2019 / 049816.

[0171] The present disclosure provides a cell or population of cells, the cells comprising a composition comprising (a) an inducible transgene construct comprising a sequence encoding an inducible promoter and a sequence encoding a transgene, and (b) a receptor construct comprising a sequence encoding a constitutive promoter and a sequence encoding an exogenous receptor, such as a CAR, wherein the (a) construct and the (b) construct are integrated into the genomic sequence of the cell to express the exogenous receptor, which upon binding to a ligand or antigen transmits an intracellular signal that directly or indirectly targets the inducible promoter that regulates expression of the inducible transgene (a) to modify gene expression.

[0172] The composition can modify gene expression by reducing gene expression. The composition can modify gene expression by temporarily modifying gene expression (e.g., while a ligand is bound to an exogenous receptor). The composition can modify gene expression acutely (e.g., a ligand is reversibly bound to an exogenous receptor). The composition can modify gene expression chronically (e.g., a ligand is irreversibly bound to an exogenous receptor).

[0173] Exogenous receptors may include endogenous receptors with respect to the genomic sequence of the cell. Examples of receptors include, but are not limited to, intracellular receptors, cell surface receptors, transmembrane receptors, ligand-gated ion channels, and G protein-coupled receptors.

[0174] The exogenous receptor may include a non-naturally occurring receptor. The non-naturally occurring receptor may be a synthetic, modified, recombinant, mutated, or chimeric receptor. The non-naturally occurring receptor may include one or more sequences isolated or derived from a T cell receptor (TCR). The non-naturally occurring receptor may include one or more sequences isolated or derived from a scaffold protein. In some embodiments, such as those in which the non-naturally occurring receptor does not include a transmembrane domain, the non-naturally occurring receptor interacts with a second transmembrane, membrane-bound, and / or intracellular receptor that transmits an intracellular signal following contact with the non-naturally occurring receptor. The non-naturally occurring receptor may include a transmembrane domain. The non-naturally occurring receptor may interact with an intracellular receptor that transmits an intracellular signal. The non-naturally occurring receptor may include an intracellular signaling domain. The non-naturally occurring receptor may be a chimeric ligand receptor (CLR). The CLR may be a chimeric antigen receptor (CAR).

[0175] The sequence encoding the inducible promoter includes a sequence encoding an NFκB promoter, a sequence encoding an interferon (IFN) promoter, or a sequence encoding an interleukin-2 promoter. In some embodiments, the IFN promoter is an IFNγ promoter. The inducible promoter may be isolated or derived from a cytokine or chemokine promoter. The cytokine or chemokine may 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), CC-C motif chemokine ligand 5 (CCL5), CC-C motif chemokine ligand 4 (Ccl4), CC-C motif chemokine ligand 3 (Ccl3), XC motif chemokine ligand 1 (Xcl1), or LIF interleukin 6 family cytokine (Lif).

[0176] The inducible promoter may be isolated or derived from the promoter of a gene, including a surface protein involved in cell differentiation, activation, depletion, and function. In some embodiments, the gene includes CD69, CD71, CTLA4, PD-1, TIGIT, LAG3, TIM-3, GITR, MHCII, COX-2, FASL, or 4-1BB.

[0177] The inducible promoter may be isolated or derived from the promoters of genes involved in CD40 metabolism and differentiation. The inducible promoter may be isolated or derived from the promoters of Nr4a1, Nr4a3, Tnfrsf9(4-1BB), Sema7a, Zfp36l2, Gadd45b, Dusp5, Dusp6, and Neto2.

[0178] In some embodiments, the inducible transgene construct comprises or drives expression of inhibitory checkpoint signals, transcription factors, cytokines or cytokine receptors, chemokines or chemokine receptors, cell death or apoptosis receptors / ligands, metabolic sensing molecules, proteins that confer sensitivity to cancer treatments, and downstream signaling components of oncogenes or tumor suppressor genes, non-limiting examples of which are disclosed in International Patent Publication WO 2019 / 173636 and International Patent Application PCT / US2019 / 049816.

[0179] Armored Cells

[0180] The modified cells (e.g., CAR T cells) of the present disclosure may be further modified to enhance their therapeutic capabilities. Alternatively, or in addition, the modified cells may be further modified to reduce their sensitivity to immunological and / or metabolic checkpoints. Following modification, cells with this type of "armored" modification may be referred to herein as "armored" cells (e.g., armed T cells). Armed cells may be produced, for example, in the tumor immunosuppressive microenvironment, naturally blocking and / or diluting certain checkpoint signals delivered to the cells (e.g., checkpoint inhibition).

[0181] The armed cells of the present disclosure may be derived from any cell, such as, for example, T cells, NK cells, hematopoietic progenitor cells, T cells from peripheral blood (PB) (such as T cells isolated or derived from G-CSF mobilized peripheral blood), or T cells from umbilical cord blood (UCB). The armed cells (e.g., armed T cells) may comprise one or more of chimeric ligand receptors (CLRs comprising protein scaffolds, antibodies, ScFvs, or antibody mimetics) / chimeric antigen receptors (CARs comprising protein scaffolds, antibodies, ScFvs, or antibody mimetics), CARTyrin (CARs comprising centrin), and / or VCARs (CARs comprising camelid VHHs or single domain VHs). The armed cells (e.g., armed T cells) may comprise an inducible pro-apoptotic polypeptide as disclosed herein. The armed cells (e.g., armed T cells) may comprise an exogenous sequence. The exogenous sequence may comprise a sequence encoding a therapeutic protein. Exemplary therapeutic proteins may be nuclear, cytoplasmic, intracellular, transmembrane, cell surface-associated, or secreted proteins. Exemplary therapeutic proteins expressed by armed cells (e.g., armed T cells) may modify the activity of the armed cell or may modify the activity of a second cell. Armed cells (e.g., armed T cells) may include a selection gene or selection marker. Armed cells (e.g., armed T cells) may include a synthetic gene expression cassette (also referred to herein as an "inducible transgene construct").

[0182] The modified cells (e.g., CAR T cells) of the present disclosure may be further modified to silence or reduce the expression of one or more genes encoding a receptor of an inhibitory checkpoint signal to produce an armed cell (e.g., armed CAR T cell). The receptor of the inhibitory checkpoint signal is expressed on the cell surface or in the cytoplasm of the cell. Silencing or reducing the expression of the gene encoding the receptor of the inhibitory checkpoint signal leads to a loss of expression of the inhibitory checkpoint receptor protein on the surface or in the cytoplasm of the armed cell. Thus, armed cells with silenced or reduced expression of one or more genes encoding an inhibitory checkpoint receptor are resistant, non-receptive, or insensitive to checkpoint signals. The reduced resistance or sensitivity of armed cells to inhibitory checkpoint signals improves the therapeutic ability of the armed cells in the presence of these inhibitory checkpoint signals. Non-limiting examples of inhibitory checkpoint signals (and proteins that induce immune suppression) are disclosed in International Patent Publication WO 2019 / 173636. Preferred examples of inhibitory checkpoint signals that may be silenced include, but are not limited to, PD-1 and TGFRII.

[0183] The modified cells (e.g., CAR T cells) of the present disclosure may be further modified to silence or reduce expression of one or more genes encoding intracellular proteins involved in checkpoint signaling to produce armed cells (e.g., armed CAR T cells). The activity of the modified cells may be enhanced by targeting any intracellular signaling protein involved in a checkpoint signaling pathway, thereby achieving checkpoint inhibition or interference with one or more checkpoint pathways. Non-limiting examples of intracellular signaling proteins involved in checkpoint signaling are disclosed in International Patent Publication WO 2019 / 173636.

[0184] The modified cells (e.g., CAR T cells) of the present disclosure may be further modified to silence or reduce the expression of one or more genes encoding transcription factors that interfere with the efficacy of the treatment to produce armed cells (e.g., armed CAR T cells). The activity of the modified cells may be enhanced or regulated by silence or reduce the expression (or inhibit the function) of a transcription factor that interferes with the efficacy of the treatment. Non-limiting examples of transcription factors that may be modified to silence or reduce the expression or inhibit the function include, but are not limited to, the exemplary transcription factors disclosed in International Patent Publication WO 2019 / 173636.

[0185] The modified cells (e.g., CAR T cells) of the present disclosure may be further modified to silence or reduce the expression of one or more genes encoding cell death receptors or cell apoptosis receptors to produce armed cells (e.g., armed CAR T cells). The interaction of a cell death receptor with its endogenous ligand initiates apoptosis. Disruption of the expression, activity, or interaction of a cell death receptor and / or apoptosis receptor and / or ligand makes the modified cell less sensitive to death signals, and thus makes the armed cell more effective in the tumor environment. Non-limiting examples of cell death receptors and / or cell apoptosis receptors and ligands are disclosed in International Patent Publication WO 2019 / 173636. A preferred example of a cell death receptor that may be modified is Fas (CD95).

[0186] The modified cells (e.g., CAR T cells) of the present disclosure may be further modified to silence or reduce expression of one or more genes encoding metabolic sensing proteins to produce armed cells (e.g., armed CAR T cells). Disruption of metabolic sensing of the immunosuppressive tumor microenvironment (characterized by low levels of oxygen, pH, glucose, and other molecules) by the modified cells may preserve T cell function longer, resulting in more tumor cells dying per cell. Non-limiting examples of metabolic sensing genes and proteins are disclosed in International Patent Publication WO 2019 / 173636. Preferred examples, HIF1a and VHL, play a role in T cell function in a hypoxic environment. Armed T cells may have silenced or reduced expression of one or more genes encoding HIF1a or VHL.

[0187] The modified cells (e.g., CAR T cells) of the present disclosure may be further modified to silence or reduce expression of one or more genes encoding proteins that confer sensitivity to cancer treatments, including monoclonal antibodies, to produce armed cells (e.g., armed CAR T cells). Thus, the armed cells can operate and exhibit superior function or efficacy in the presence of a cancer treatment (e.g., chemotherapy, monoclonal antibody therapy, or another anti-tumor treatment). Non-limiting examples of proteins involved in conferring sensitivity to cancer treatments are disclosed in International Patent Publication WO 2019 / 173636.

[0188] The modified cells (e.g., CAR T cells) of the present disclosure may be further modified to silence or reduce expression of one or more genes encoding growth effectors to produce armed cells (e.g., armed CAR T cells). Silencing or reducing expression of an oncogene can confer growth effectivity to the cells. For example, silence or reduction (e.g., disruption of expression) of the TET2 gene during the CAR T cell production process can result in the production of armed CAR T cells with significant capacity for proliferation and subsequent eradication of tumors compared to unarmed CAR T cells that lack the capacity for proliferation. This approach may be combined with a safety switch (e.g., the iC9 safety switch described herein), which allows for targeted destruction of armed CAR T cells in the event of adverse reactions from the subject or uncontrolled proliferation of the armed CAR T cells. Non-limiting examples of growth effectors are disclosed in International Patent Publication WO 2019 / 173636.

[0189] The modified cells of the present disclosure (e.g., CAR T cells) may be further modified to express modified / chimeric checkpoint receptors to produce armed T cells of the present disclosure.

[0190] The modified / chimeric checkpoint receptor may include a null receptor, a decoy receptor, or a dominant negative receptor. The null receptor, the decoy receptor, or the dominant negative receptor may be a modified / chimeric receptor / protein. The null receptor, the decoy receptor, or the dominant negative receptor may be truncated for expression of the intracellular signaling domain. Alternatively, or in addition, the null receptor, the decoy receptor, or the dominant negative receptor may be mutated at one or more defined amino acid positions in the intracellular signaling domain or at one or more amino acid positions required for effective signaling. The truncation or mutation of the null receptor, the decoy receptor, or the dominant negative receptor may result in the loss of the receptor's ability to transport or transmit checkpoint signals to or within the cell.

[0191] For example, dilution or blocking of immune suppressive checkpoint signals from PD-L1 receptors expressed on the surface of tumor cells may be achieved by expressing modified / chimeric PD-1 null receptors on the surface of armed cells (e.g., armed CAR T cells), which effectively compete with endogenous (unmodified) PD-1 receptors also expressed on the surface of the armed cells, reducing or inhibiting the transmission of immune suppressive checkpoint signals through the endogenous PD-1 receptors of the armed cells. In this non-limiting example, this competition between two different receptors for binding to PD-L1 expressed on the surface of tumor cells reduces or decreases the level of effective checkpoint signaling, thereby enhancing the therapeutic capacity of the armed cells expressing the PD-1 null receptors.

[0192] Modified / chimeric checkpoint receptors may include null receptors, decoy receptors, or dominant-negative receptors that are transmembrane receptors, membrane-associated or membrane-bound receptors / proteins, or intracellular receptors / proteins. Examples of null, decoy, or dominant-negative intracellular receptors / proteins include, but are not limited to, downstream signaling components of inhibitory checkpoint signals, transcription factors, cytokines or cytokine receptors, chemokines or chemokine receptors, cell death or apoptosis receptors / ligands, metabolic sensing molecules, proteins that confer sensitivity to cancer treatment, and oncogenes or tumor suppressor genes. Non-limiting examples of cytokines, cytokine receptors, chemokines, and chemokine receptors are disclosed in International Patent Publication WO 2019 / 173636.

[0193] Modified / chimeric checkpoint receptors may include switch receptors. Examples of switch receptors include modified / chimeric receptors / proteins in which a native or wild-type intracellular signaling domain is switched or replaced with a different intracellular signaling domain that is non-native to the protein and / or is not a wild-type domain. For example, an inhibitory signaling domain is replaced with a stimulatory signaling domain, switching the immunosuppressive signal to an immunostimulatory signal. Alternatively, the inhibitory signaling domain can be replaced with a different inhibitory domain to reduce or increase the level of inhibitory signaling. Expression or overexpression of a switch receptor can result in dilution and / or blocking of the cognate checkpoint signal through competition with endogenous wild-type checkpoint receptors (not the switch receptor) for binding to the cognate checkpoint receptor expressed in the immunosuppressive tumor microenvironment. Armed cells (e.g., armed CAR T cells) may include sequences encoding switch receptors, which lead to the expression of one or more switch receptors, thereby altering the activity of the armed cells. Armed cells (e.g., armed CAR T cells) can express switch receptors that target checkpoint receptors, transcription factors, cytokine receptors, death receptors, metabolic sensing molecules, cancer therapeutics, oncogenes, and / or tumor suppressor proteins or intracellularly expressed proteins downstream of genes.

[0194] Examples of switch receptors may include or be derived from, but are not limited to, inhibitory checkpoint signals, transcription factors, cytokines or cytokine receptors, chemokines or chemokine receptors, cell death or apoptosis receptors / ligands, metabolic sensing molecules, proteins that confer sensitivity to cancer therapies, and proteins that contain signaling components downstream of oncogenes or tumor suppressor genes.

[0195] The modified cells of the present disclosure (e.g., CAR T cells) may be further modified to express a CLR / CAR that mediates conditional gene expression to produce armed T cells. The combination of the CLR / CAR and the conditional gene expression system in the nucleus of the armed T cell constitutes a synthetic gene expression system that is conditionally activated upon binding of the cognate ligand to the CLR or the cognate antigen to the CAR. This system may help to improve the "arming" or therapeutic capacity of the modified T cell, for example, by reducing or limiting the expression of the synthetic gene at the binding site of the ligand or antigen in or within the tumor environment.

[0196] Gene editing compositions and methods

[0197] The modified cells are produced by introducing a transgene into the cells. The introducing step may include delivery of a nucleic acid sequence, a transgene, and / or a genome editing construct via a non-transfer delivery system.

[0198] Introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells in vitro, in vivo, in a laboratory tool, or in situ may include one or more of local delivery, adsorption, absorption, electroporation, roll-transduction, co-culture, transduction, mechanical delivery, sonic delivery, vibration delivery, magnetic delivery, or nanoparticle-mediated delivery. Introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells in vitro, in vivo, in a laboratory tool, or in situ may include liposome transduction, calcium phosphate transduction, fugene transduction, and dendrimer-mediated transduction. Introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells in vitro, in vivo, in a laboratory tool, or in situ by mechanical transduction may include cell squeezing, cell bombardment, or gene gun techniques. Introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells by ex vivo, in vivo, in labware, or in situ nanoparticle-mediated transduction may include liposome delivery, micelle delivery, and polymerase-mediated delivery.

[0199] The introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells in vitro, in vivo, in laboratory equipment, or in situ may include non-viral vectors. Non-viral vectors may include nucleic acids. Non-viral vectors may include plasmid DNA, linear double-stranded DNA (dsDNA), linear single-stranded DNA (ssDNA), DoggyBone™ DNA, nanoplasmid, minicircle DNA, single-stranded oligodeoxynucleotides (ssODN), DDNA oligonucleotides, single-stranded mRNA (ssRNA), and double-stranded mRNA (dsRNA). Non-viral vectors may include transposons as described herein.

[0200] The introduction of the nucleic acid sequence, transgene, and / or genome editing construct into cells in vitro, in vivo, in laboratory equipment, or in situ may include a viral vector. The viral vector may be a non-integrated non-chromosomal vector. Non-limiting examples of non-integrated non-chromosomal vectors include adeno-associated virus (AAV), adenovirus, and herpes virus. The viral vector may be an integrated chromosomal vector. Non-limiting examples of integrated chromosomal vectors include adeno-associated vector (AAV), lentivirus, and gamma retrovirus.

[0201] The introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells in vitro, in vivo, in a laboratory device, or in situ may include a combination of vectors.Non-limiting examples of vector combinations include a viral vector and a non-viral vector, multiple non-viral vectors, or multiple viral vectors.Non-limiting examples of vector combinations include a DNA-derived vector and an RNA-derived vector, a RNA and a reverse transcriptase, a transposon and a transferase, a non-viral vector and an endonuclease, and a viral vector and an endonuclease.

[0202] Genome modification may include introducing a nucleic acid sequence, a transgene, and / or a genome editing construct into a cell in vivo, in vivo, in a laboratory device, or in situ to stably integrate the nucleic acid sequence, to transiently integrate the nucleic acid sequence, to generate site-specific integration of the nucleic acid sequence, or to generate biased integration of the nucleic acid sequence. The nucleic acid sequence may be a transgene.

[0203] Genome modification may include introducing nucleic acid sequences, transgenes, and / or genome editing constructs into cells in vitro, in vivo, in a laboratory device, or in situ to stably integrate the nucleic acid sequences. Stable chromosomal integration may be random, site-specific, or biased. Site-specific integration may be unassisted or assisted. Assisted site-specific integration is delivered simultaneously with a site-specific nuclease. The site-specific nuclease includes a transgene with 5' and 3' nucleotide sequence extensions that include a percentage of homology with the upstream and downstream regions of the genome integration site. The transgene with the nucleotide extensions of homology allows genome integration by homologous recombination, microhomology-mediated end joining, or non-homologous end joining. Site-specific integration may occur at a safe harbor site. Genomic safe harbor sites can accommodate integration of novel genetic material to ensure that the newly inserted genetic element is functional (e.g., expressed at therapeutically effective expression levels) and do not cause deleterious changes to the host genome that pose a risk to the host organism. Non-limiting examples of potential genomic safe harbors include intronic sequences in the human albumin gene, adeno-associated virus site 1 (AAVS1), the naturally occurring integration site of the AAV virus on the surface of chromosome 19, the chemokine (CC motif) receptor 5 (CCR5) gene site, and the human ortholog of the mouse Rosa26 locus.

[0204] Site-specific transgene integration can occur at the site that disrupts the expression of target gene.Disruption of target gene expression can occur by site-specific integration at intron, exon, promoter, genetic element, enhancer, suppressor, start codon, stop codon, and response element.Non-limiting examples of target genes that site-specific integration targets include TRAC, TRAB, PDI, any immunosuppressive gene, and genes involved in allogeneic rejection.

[0205] Site-specific transgene integration may occur at sites that enhance expression of the target gene. Enhancement of target gene expression may occur by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.

[0206] Enzymes can be used to form strand breaks in the host genome to facilitate the delivery or integration of transgenes. Enzymes can form single-strand breaks or double-strand breaks. Non-limiting examples of break-inducing enzymes include transferases, integrases, endonucleases, CRISPR-Cas9, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), Cas-CLOVER™, and CPF1. Break-inducing enzymes can be delivered to cells as DNA-encoded and mRNA-encoded as proteins or as nucleoprotein complexes with guide RNA (gRNA).

[0207] Site-specific transgene integration can be controlled by vector-mediated integration site bias. Vector-mediated integration site bias can be controlled by the selected lentiviral vector or the selected gamma retroviral vector.

[0208] The site-specific transgene integration site may be a non-stable chromosomal insertion. The integrated transgene may be in a stopped, removed, excised, or further modified form. The genome modification may be a non-stable integration of the transgene. The non-stable integration may be a transient non-chromosomal integration, a semi-stable non-chromosomal integration, a semi-persistent non-chromosomal insertion, or a non-stable chromosomal insertion. The transient non-chromosomal insertion may be epichromosomal or cytoplasmic. In one aspect, the transient non-chromosomal insertion of the transgene is not integrated into the chromosome, and the modified genetic material is not replicated during cell division.

[0209] Genomic modification may be semistable or persistent non-chromosomal integration of the transgene. The DNA vector encodes a scaffold / matrix-associated region (S-MAR) module that binds to nuclear matrix proteins for episomal retention of non-viral vectors, allowing autonomous replication in the nucleus of dividing cells.

[0210] The genomic modification may be a non-stable chromosomal integration of the transgene. The integrated transgene may be terminated, removed, excised, or further modified.

[0211] Modification of genome by inserting transgene may occur through homologous recombination (HR), microhomology-mediated end joining (MMEJ), non-homologous end joining (NHEJ), transferase-mediated modification, integrase-mediated modification, endonuclease-mediated modification, or host cell-directed double-strand break repair (homology-directed repair) by recombinase-mediated modification. Modification of genome by inserting transgene may occur through CRISPR-Cas9, TALEN, ZFN, Cas-CLOVER™, and cpf1.

[0212] In gene editing systems involving the insertion of new or existing nucleotides / nucleic acids, an insertion tool (e.g., DNA template vector, transposable element (transposon or retrotransposon)) needs to be delivered to the cell in addition to a cutting enzyme (e.g., nuclease, recombinase, integrase, or transferase). An example of this insertion tool for a recombinase may include a DNA vector. In other gene editing systems, an integration enzyme needs to be delivered with an insertion vector, a transferase with a transposon / retrotransposon, and the like. An example of a recombinase that may be used as a cutting enzyme is CRE recombinase. Non-limiting examples of integration enzymes that may be used in the insertion tool include viral enzymes taken from any of several viruses, such as AAV, gamma retrovirus, and lentivirus. Examples of transposons / retrotransposons that may be used in the insertion tool are described in more detail herein.

[0213] The cell with the ex vivo, in vivo, in labware, or in situ genomic modification may be a germline cell or a somatic cell. The modified cell may be a human, non-human, mammalian, rat, mouse, or canine cell. The modified cell may be a differentiated, undifferentiated, or immortalized cell. The undifferentiated modified cell may be a stem cell. The undifferentiated modified cell may be an induced pluripotent stem cell. The modified cell may be an immune cell. The modified cell may be a T cell, a hematopoietic stem cell, a natural killer cell, a macrophage, a dendritic cell, a monocyte, a megakaryocyte, or an osteoclast. The modified cell may be modified while the cell is in quiescence, activation, quiescence, interphase, prophase, metaphase, anaphase, or teleophase. The modified cell may be a fresh and cryopreserved population, separated into subpopulations from whole blood, from leukapheresis, or from an immortalized cell line. A detailed description of leukapheresis products or isolation of cells from blood is disclosed in International Patent Publication No. WO 2019 / 173636 and International Patent Application No. PCT / US2019 / 049816.

[0214] The present disclosure provides a gene editing composition and / or a cell comprising the gene editing composition. The gene editing composition may comprise a sequence encoding a DNA binding domain and a sequence encoding a nuclease protein or its nuclease domain. The sequence encoding a nuclease protein or its nuclease domain may comprise a DNA sequence, an RNA sequence, or a combination thereof. The nuclease or its nuclease domain may comprise one or more CRISPR / Cas proteins, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and endonucleases.

[0215] The nuclease or its nuclease domain may comprise a nuclease-inactivated Cas (dCas) protein and an endonuclease. The endonuclease may comprise a Clo051 nuclease or its nuclease domain. The gene editing composition may comprise a fusion protein. The fusion protein may comprise a nuclease-inactivated Cas9 (dCas9) protein and a Clo051 nuclease or a Clo051 nuclease domain. The gene editing composition may further comprise a guide sequence. The guide sequence comprises an RNA sequence.

[0216] The present disclosure provides a composition comprising a small molecule Cas9 operably linked to an effector. The present disclosure provides a fusion protein comprising, consisting essentially of, or consisting of a DNA localization component and an effector molecule, the effector comprising a small molecule Cas9. The small molecule Cas9 construct of the present disclosure may comprise an effector comprising a type IIS endonuclease. Staphylococcus aureus Cas9 with an active catalytic site comprises the amino acid sequence of SEQ ID NO: 43.

[0217] The present disclosure provides a composition comprising an inactivated small molecule Cas9 (dSaCas9) operably linked to an effector. The present disclosure provides a fusion protein comprising, consisting essentially of, or consisting of a DNA localization component and an effector molecule, wherein the effector comprises an inactivated small molecule Cas9 (dSaCas9). The inactivated small molecule Cas9 (dSaCas9) construct of the present disclosure may comprise an effector comprising a type IIS endonuclease. The dSaCas9 comprises the amino acid sequence of SEQ ID NO: 44 and comprises the D10A and N580A mutations that inactivate the catalytic site.

[0218] The present disclosure provides a composition comprising an inactivated Cas9 (dCas9) operably linked to an effector. The present disclosure provides a fusion protein comprising, consisting essentially of, or consisting of a DNA localization component and an effector molecule, wherein the effector comprises an inactivated Cas9 (dCas9). The inactivated Cas9 (dCas9) construct of the present disclosure may comprise an effector comprising a type IIS endonuclease.

[0219] The dCas9 may be isolated or derived from Streptococcus pyogenes. The dCas9 may include dCas9 with substitutions at amino acid positions 10 and 840 that inactivate the catalytic site. In some embodiments, these substitutions are D10A and H840A. The dCas9 may include the amino acid sequence of SEQ ID NO:45 or SEQ ID NO:46.

[0220] An example of a Clo051 nuclease domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:47.

[0221] The dCas9-Clo051 (Cas-CLOVER) fusion protein may, for example, comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 48. The dCas9-Clo051 fusion protein may, for example, be encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 49. The nucleic acid encoding the dCas9-Clo051 fusion protein may be DNA or RNA.

[0222] An exemplary dCas9-Clo051 (Cas-CLOVER) fusion protein may comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 50. An exemplary dCas9-Clo051 fusion protein may be encoded by a polynucleotide that comprises, consists essentially of, or consists of the nucleic acid sequence of SEQ ID NO: 51. The nucleic acid encoding the dCas9-Clo051 fusion protein may be DNA or RNA.

[0223] The cell comprising the gene editing composition can express the gene editing composition stably or transiently.Preferably, the gene editing composition is expressed transiently.The guide RNA can comprise a sequence that is complementary to the target sequence in the genomic DNA sequence.The target sequence in the genomic DNA sequence can be the target sequence in the safe harbor site of the genomic DNA sequence.

[0224] 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.

[0225] Gene editing tools may also be delivered to cells using one or more polyhistidine-based micelles. Polyhistidine (e.g., poly(L-histidine)) is a pH-sensitive polymer due to the imidazole ring that provides a lone pair of electrons on the unsaturated nitrogen surface. That is, polyhistidine has amphoteric properties due to protonation-deprotonation. In particular, at a certain pH, polyhistidine-containing triblock copolymers assemble into micelles with positively charged polyhistidine units on the surface, thereby allowing complexation with negatively charged gene editing molecules. These nanoparticles can be used to bind and release proteins and / or nucleic acids in a pH-dependent manner, providing an efficient and selective mechanism to perform the desired genetic modifications. In particular, this micelle-based delivery system offers substantial flexibility for charged materials, as well as large loading capacity and targeted release of nanoparticle loading capacity. In one example, site-specific cleavage of double-stranded DNA is possible by delivery of nucleases using polyhistidine-based micelles. Without wishing to be bound by any particular theory, it is believed that in micelles formed by various triblock copolymers, the hydrophobic blocks aggregate to form a nucleus, leaving hydrophilic blocks and polyhistidine blocks at the termini to form one or more coating layers.

[0226] In one aspect, the present disclosure provides a triblock copolymer consisting of a hydrophilic block, a hydrophobic block, and a charged block. In some aspects, the hydrophilic block can be polyethylene oxide (PEO) and the charged block can be poly(L-histidine). An example of a usable triblock copolymer is PEO-b-PLA-b-PHIS, where the number of repeat units in each block varies by design.

[0227] Diblock copolymers that can be used as intermediates to prepare triblock copolymers may have hydrophilic biocompatible polyethylene oxide (PEO), chemically synonymous with PEG, conjugated to a variety of hydrophobic aliphatic polyanhydrides, polynucleic acids, polyesters, polyorthoesters, polypeptides, polyphosphazenes, and polysaccharides, including, but not limited to, polylactide (PLA), polyglycolide (PLGA), poly(lactic-co-glycolic acid) (PLGA), poly(ε-caprolactone) (PCL), and polytrimethylene carbonate (PTMC). Polymeric micelles composed of 100% PEGylated surfaces exhibit improved chemical stability in vitro, increased bioavailability in vivo, and extended half-life in the blood circulation.

[0228] Polymeric vesicles, e.g., micelles comprising triblock copolymers, polymersomes, and polyhistidine-based micelles, and methods of making them, are described in further detail in U.S. Pat. Nos. 7,217,427; 7,868,512; 6,835,394; 8,808,748; 10,456,452; U.S. Patent Publication Nos. 2014 / 0363496; 2017 / 0000743; 2019 / 0255191; and International Patent Publication No. WO 2019 / 126589.

[0229] Inducible Pro-Apoptotic Polypeptides

[0230] The inducible pro-apoptotic polypeptides disclosed herein are superior to existing inducible polypeptides because the inducible pro-apoptotic polypeptides disclosed herein are much less immunogenic. The inducible pro-apoptotic polypeptides are recombinant polypeptides and therefore of non-natural origin. Furthermore, the sequences that are recombined to produce an inducible pro-apoptotic polypeptide that does not contain non-human sequences that can be recognized as "non-self" by the host human immune system result in the induction of an immune response in a subject receiving the inducible pro-apoptotic polypeptide, a cell comprising the inducible pro-apoptotic polypeptide, or a composition comprising the inducible pro-apoptotic polypeptide, or a cell comprising the inducible pro-apoptotic polypeptide.

[0231] The present disclosure provides an inducible pro-apoptotic polypeptide comprising a ligand-binding region, a linker, and a pro-apoptotic peptide, the inducible pro-apoptotic polypeptide being free of non-human sequences. In certain embodiments, the non-human sequences comprise a restriction site. In certain embodiments, the ligand-binding region may be a multimeric ligand-binding region. In certain embodiments, the pro-apoptotic peptide is a caspase polypeptide. Non-limiting examples of caspase polypeptides include caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, and caspase 14. Preferably, the caspase polypeptide is a caspase 9 polypeptide. The caspase 9 polypeptide may be a truncated caspase 9 polypeptide. The inducible pro-apoptotic polypeptide may be of non-natural origin. When the caspase is caspase 9 or truncated caspase 9, the inducible pro-apoptotic polypeptide is sometimes referred to as the "iC9 safety switch."

[0232] The inducible caspase polypeptide may comprise (a) a ligand binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise non-human sequences. In certain embodiments, the inducible caspase polypeptide comprises (a) a ligand binding region, (b) a linker, and (c) a truncated caspase 9 polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise non-human sequences.

[0233] The ligand binding region may comprise an FK506 binding protein 12 (FKBP12) polypeptide. The amino acid sequence of the ligand binding region comprising an FK506 binding protein 12 (FKBP12) polypeptide may comprise a modification at position 36 of the sequence. The modification may be a substitution of phenylalanine (F) at position 36 with valine (V) (F36V). The FKBP12 polypeptide may comprise, consist essentially of, or consist of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity (or any percentage therebetween) to SEQ ID NO:73. The FKBP12 polypeptide may be encoded by a polynucleotide comprising or consisting of a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO:74.

[0234] The linker region may comprise, consist essentially of, or consist of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO: 75, or the linker region may be encoded by a polynucleotide that comprises or consists of a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identity to SEQ ID NO: 76. In some embodiments, the nucleic acid sequence encoding the linker does not contain a restriction site.

[0235] The truncated caspase 9 polypeptide may comprise an amino acid sequence that does not include an arginine (R) at position 87 of the sequence. Alternatively, or in addition, the truncated caspase 9 polypeptide may comprise an amino acid sequence that does not include an alanine (A) at position 282 of the sequence. The truncated caspase 9 polypeptide may comprise, consist essentially of, or consist of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identity to SEQ ID NO: 77, or the truncated caspase 9 polypeptide may be encoded by a polynucleotide that comprises or consists of a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identity to SEQ ID NO: 78.

[0236] In certain embodiments in which the polypeptide comprises a truncated caspase-9 polypeptide, the inducible pro-apoptotic polypeptide comprises, consists essentially of, or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identity to SEQ ID NO:79, or the inducible pro-apoptotic polypeptide may be encoded by a polynucleotide that comprises or consists of a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identity to SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:80.

[0237] The inducible pro-apoptotic polypeptide may be expressed in a cell under the transcriptional control of any promoter known in the art that is capable of initiating and / or regulating expression of the inducible pro-apoptotic polypeptide in a cell.

[0238] Activation of inducible pro-apoptotic polypeptides can be achieved, for example, through chemically induced dimerization (CID) mediated by an inducing agent to produce a conditionally regulated protein or polypeptide. Not only are the pro-apoptotic polypeptides inducible, but induction of these polypeptides is also reversible by degradation of the labile dimerizing agent or administration of a monomeric competitive inhibitor.

[0239] In certain embodiments, in which the ligand binding region comprises an FKBP12 polypeptide having a substitution of phenylalanine (F) with valine (V) at position 36 (F36V), the inducer is the synthetic drug AP1903 (CAS Index Name: 2-piperidinecarboxylic acid, 1-[(2S)-1-oxo-2-(3,4,5-trimethoxyphenyl)butyl]-, 1,2-ethanediylbis[imino(2-oxo-2,1-ethanediyl)oxy-3,1-phenylene[(1R )-3-(3,4-dimethoxyphenyl)propylidene]] ester, [2S-[1(R*),2R*[S*[S*[1(R*),2R*]]]]]-(9Cl), CAS Registry Number: 195514-63-7; molecular formula: C78H98N4O20; molecular weight: 1411.65)), AP20187 (CAS Registry Number: 195514-80-8 and molecular formula: C82H107N5O20), or an AP20187 analog such as AP1510. As used herein, the inducers AP20187, AP1903 and AP1510 may be used interchangeably.

[0240] Inducible pro-apoptotic peptides and methods for inducing these peptides are described in detail in International Patent Publications WO 2019 / 0225667 and WO 2018 / 068022.

[0241] Chimeric stimulating receptors and recombinant HLA-E polypeptides

[0242] An adoptive cell composition that is "universally" safe for administration to any patient requires a significant reduction or elimination of alloreactivity. To this end, the cells (e.g., allogeneic cells) of the present disclosure may be modified to disrupt the expression or function of T cell receptor (TCR) and / or a type of major histocompatibility complex (MHC). TCR mediates graft-versus-host (GvH) responses, while MHC mediates host-versus-graft (HvG) responses. In preferred embodiments, either TCR expression and / or function is eliminated to prevent T cell-mediated GvH, which may result in death of the subject. Thus, in preferred embodiments, the present disclosure provides a pure TCR-negative allogeneic T cell composition (e.g., each cell of the composition expresses at such low levels that it is either undetectable or absent).

[0243] The expression and / or function of MHC class I (MHC-I, specifically HLA-A, HLA-B, and HLA-C) is reduced or eliminated to prevent HvG, thereby improving the engraftment of cells in the subject. Improved engraftment results in longer persistence of the cells, thereby providing a larger therapeutic window for the subject. Specifically, the expression and / or function of beta-2-microglobulin (B2M), a structural component of MHC-I, is reduced or eliminated.

[0244] The above mentioned approach poses additional challenges. Knockout (KO) of the T cell receptor (TCR) in T cells results in loss of expression of CD3ζ, which is part of the TCR complex. Loss of CD3ζ in TCR-KO T cells dramatically reduces the ability of these cells to be optimally activated and expanded using standard stimulating / activating reagents, such as, but not limited to, agonist anti-CD3 mAbs. Blocking the expression or function of any one component of the TCR complex results in loss of all components of the complex, including TCRα, TCRβ, CD3γ, CD3ε, CD3δ, and CD3ζ. Both CD3ε and CD3ζ are required for T cell activation and expansion. Agonist anti-CD3 mAbs usually recognize CD3ε and possibly another protein in the complex that in turn signals to CD3ζ. CD3ζ provides the primary stimulus for T cell activation (along with a secondary costimulatory signal) for optimal activation and expansion. Under normal conditions, full T cell activation depends on engagement of the TCR in concert with a second signal mediated by one or more costimulatory receptors (e.g., CD28, CD2, 4-1BBL) that promotes the immune response. However, in the absence of the TCR, stimulation with standard activating / stimulating reagents, including agonist anti-CD3 mAbs, significantly reduces T cell proliferation. Indeed, T cell proliferation is reduced to only 20-40% of normal proliferation levels when stimulated with standard activating / stimulating reagents, including agonist anti-CD3 mAbs.

[0245] Thus, the present disclosure provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an ectodomain comprising an activating component isolated or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain comprising at least one signaling domain isolated or derived from a second protein, where the first protein and the second protein are not identical.

[0246] The activating component includes a portion of one or more of a T cell receptor (TCR) component, a TCR complex component, a TCR co-receptor component, a TCR co-stimulatory protein component, a TCR inhibitory protein component, a cytokine receptor, and a chemokine receptor to which the activating component's agent binds. The activating component may include the CD2 extracellular domain or a portion thereof to which the agent binds.

[0247] The signaling domain may comprise one or more of a human signaling domain component, a T cell receptor (TCR), a TCR complex component, a TCR co-receptor component, a TCR co-stimulatory protein component, a TCR inhibitory protein component, a cytokine receptor, and a chemokine receptor. The signaling domain may comprise a CD3 protein or a portion thereof. The CD3 protein may comprise a CD3 zeta protein or a portion thereof.

[0248] The endodomain may further comprise a cytoplasmic domain. The cytoplasmic domain may be isolated from or derived from a third protein. The first protein and the third protein may be identical. The ectodomain may further comprise a signal peptide. The signal peptide may be derived from a fourth protein. The first protein and the fourth protein may be identical. The transmembrane domain may be isolated from or derived from a fifth protein. The first protein and the fifth protein may be identical.

[0249] In some embodiments, the activating component does not bind to a naturally occurring molecule. In some embodiments, the activating component binds to a naturally occurring molecule, but CSR does not transmit a signal when the activating component binds to a naturally occurring molecule. In some embodiments, the activating component binds to a non-naturally occurring molecule. In some embodiments, the activating component does not bind to a naturally occurring molecule, but binds to a non-naturally occurring molecule. CSR can selectively transmit a signal when the activating component binds to a non-naturally occurring molecule.

[0250] In a preferred embodiment, the present disclosure provides a non-naturally occurring chimeric stimulating receptor (CSR) comprising: (a) an ectodomain comprising a signal peptide comprising a CD2 signal peptide or a portion thereof, and an activating moiety comprising a CD2 extracellular domain or a portion thereof to which an agent binds; (b) a transmembrane domain comprising a CD2 transmembrane domain or a portion thereof; and (c) an endodomain comprising a cytoplasmic domain comprising a CD2 cytoplasmic domain or a portion thereof, and at least one signaling domain comprising a CD3 zeta protein or a portion thereof. In some embodiments, the non-naturally occurring CSR comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO:81. In a preferred embodiment, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO:81.

[0251] The present disclosure also provides a non-naturally occurring chimeric stimulating receptor (CSR) in which the ectodomain comprises a modification. The modification may comprise a mutation or truncation of the amino acid sequence of the activating component or the first protein when compared to the wild-type sequence of the activating component or the first protein. The mutation or truncation of the amino acid sequence of the activating component may comprise a mutation or truncation of the CD2 extracellular domain or a portion thereof to which the agent binds. The mutation or truncation of the CD2 extracellular domain can reduce or eliminate binding to naturally occurring CD58. In some embodiments, the CD2 extracellular domain comprising the mutation or truncation comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO:82. In a preferred embodiment, the CD2 extracellular domain comprising the mutation or truncation comprises the amino acid sequence of SEQ ID NO:82.

[0252] In a preferred embodiment, the present disclosure provides a non-naturally occurring chimeric stimulating receptor (CSR) comprising: (a) an ectodomain comprising a signal peptide comprising a CD2 signal peptide or a portion thereof, and an activating component comprising a CD2 extracellular domain or a portion thereof, comprising a mutation or truncation, to which an agent binds; (b) a transmembrane domain comprising a CD2 transmembrane domain or a portion thereof; and (c) an endodomain comprising a cytoplasmic domain comprising a CD2 cytoplasmic domain or a portion thereof, and at least one signaling domain comprising a CD3 zeta protein or a portion thereof. In some embodiments, the non-naturally occurring CSR comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identity to SEQ ID NO:83. In a preferred embodiment, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO:83.

[0253] The present disclosure provides a nucleic acid sequence encoding any of the CSRs disclosed herein.The present disclosure provides a transposon or vector comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.

[0254] The present disclosure provides a cell comprising any of the CSRs disclosed herein.The present disclosure provides a cell comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.The present disclosure provides a cell comprising a vector comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.The present disclosure provides a cell comprising a transposon comprising a nucleic acid sequence encoding any of the CSRs disclosed herein.

[0255] The modified cell disclosed herein can be an allogeneic cell or an autologous cell. In some preferred embodiments, the modified cell is an allogeneic cell. In some preferred embodiments, the modified cell is an autologous T cell or a modified autologous CAR T cell. In some preferred embodiments, the modified cell is an allogeneic T cell or a modified allogeneic CAR T cell.

[0256] The present disclosure provides a composition comprising any of the CSRs disclosed herein. The present disclosure provides a composition comprising a nucleic acid sequence encoding any of the CSRs disclosed herein. The present disclosure provides a composition comprising a vector comprising a nucleic acid sequence encoding any of the CSRs disclosed herein. The present disclosure provides a composition comprising a transposon comprising a nucleic acid sequence encoding any of the CSRs disclosed herein. The present disclosure provides a composition comprising a modified cell disclosed herein, or a composition comprising a plurality of modified cells disclosed herein.

[0257] The present disclosure provides modified T lymphocytes (T cells) comprising: (a) a modification of an endogenous sequence encoding a T cell receptor (TCR) that reduces or eliminates the level of expression or activity of the TCR; and (b) a chimeric stimulating receptor (CSR) comprising (i) an ectodomain comprising an activating component isolated from or derived from a first protein, (ii) a transmembrane domain, and (iii) an endodomain comprising at least one signaling domain isolated from or derived from a second protein, where the first protein and the second protein are not identical.

[0258] The modified T cells may further comprise an inducible pro-apoptotic polypeptide. The modified T cells may further comprise a modification of the endogenous sequence encoding beta-2-microglobulin (B2M) that reduces or eliminates the level of major histocompatibility complex (MHC) class I (MHC-I) expression or activity.

[0259] The modified T cell may further comprise a non-naturally occurring polypeptide comprising an HLA class I histocompatibility antigen, alpha chain E (HLA-E) polypeptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide may further comprise a B2M signal peptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide may further comprise a B2M polypeptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide may further comprise a linker, the linker being located between the B2M polypeptide and the HLA-E polypeptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide may further comprise a peptide and a B2M polypeptide. The non-naturally occurring polypeptide comprising an HLA-E may further comprise a first linker located between the B2M signal peptide and the peptide, and a second linker located between the B2M polypeptide and the peptide encoding HLA-E.

[0260] The modified T cells may comprise a non-naturally occurring antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof. The non-naturally occurring antigen receptor may comprise a chimeric antigen receptor (CAR).

[0261] The CSR can be transiently expressed in the modified T cells. The CSR can be stably expressed in the modified T cells. A polypeptide comprising an HLA-E polypeptide can be transiently expressed in the modified T cells. A polypeptide comprising an HLA-E polypeptide can be stably expressed in the modified T cells. An inducible pro-apoptotic polypeptide can be transiently expressed in the modified T cells. An inducible pro-apoptotic polypeptide can be stably expressed in the modified T cells. A sequence encoding a non-naturally occurring antigen receptor or therapeutic protein can be transiently expressed in the modified T cells. A sequence encoding a non-naturally occurring antigen receptor or therapeutic protein can be stably expressed in the modified T cells.

[0262] As described in detail herein, gene editing compositions including, but not limited to, RNA-guided fusion proteins including dCas9-Clo051 can be used to target and reduce or eliminate the expression of endogenous T cell receptors. In a preferred embodiment, the gene editing composition targets and eliminates the gene, part of the gene, or regulatory element of the gene (such as the promoter) that encodes the endogenous T cell receptor. Non-limiting examples of primers (such as T7 promoters, genomic target sequences, and gRNA scaffolds) for generating guide RNA (gRNA) templates for targeting and eliminating TCR-α, targeting and eliminating TCR-β, and targeting and eliminating β-2-microglobulin (β2M) are disclosed in International Patent Application PCT / US2019 / 049816.

[0263] Gene editing compositions including, but not limited to, RNA guide fusion proteins including dCas9-Clo051 can be used to target and reduce or eliminate the expression of endogenous MHCI, MHCII, or MHC activators. In a preferred embodiment, the gene editing composition targets and eliminates the gene, part of a gene, or regulatory element of a gene (such as a promoter) that encodes one or more components of endogenous MHCI, MHCII, or MHC activators. Non-limiting examples of guide RNAs (gRNAs) for targeting and eliminating MHC activators are disclosed in International Patent Application PCT / US2019 / 049816.

[0264] A detailed description of the genetic modifications of endogenous sequences encoding chimeric stimulating receptors of non-natural origin, TCR-α, TCR-β, and / or β-2-microglobulin (β2M), and non-natural polypeptides, including HLA class I histocompatibility antigens, alpha chain E (HLA-E) polypeptides, is disclosed in International Patent Application PCT / US2019 / 049816.

[0265] Formulations, Dosages and Modes of Administration

[0266] The present disclosure provides formulations, dosages, and methods for administering the compositions described herein.

[0267] The disclosed compositions and pharmaceutical compositions may further comprise at least one of any suitable auxiliary agent, such as, but not limited to, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, excipients, etc. Pharmaceutically acceptable auxiliary agents are preferred. Non-limiting examples of such sterile solutions and methods for their preparation are well known in the art and are described, for example, but not limited to, in Gennaro, ed., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co. (Easton, Pa.) 1990, and "Physician's Desk Reference", 52nd ed., Medical Economics (Montvale, NJ) 1998. As is well known in the art or described herein, suitable pharma-ceutically acceptable carriers can be routinely selected in terms of the mode of administration, solubility, and / or stability of the protein scaffold, its fragment, or variant composition.

[0268] Non-limiting examples of pharmaceutical excipients and additives suitable for use include proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars; and polysaccharides or sugar polymers), which may be present alone or in combination at 1-99.99% by weight or volume. Non-limiting examples of protein excipients include serum albumins such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acids / protein components that can contribute to buffering performance include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is glycine.

[0269] Non-limiting examples of carbohydrate excipients suitable for use include monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), myo-inositol, etc. Preferably, the carbohydrate excipient is mannitol, trehalose, and / or raffinose.

[0270] The composition may also include a buffer or a pH adjuster, and typically the buffer is a salt prepared from an organic acid or an organic base.Representative buffers include organic acid salts such as citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid salts; Tris, tromethamine hydrochloride, or phosphate buffers.Preferred buffers are organic acid salts such as citrate salts.

[0271] Additionally, the disclosed compositions may include polymeric excipients / additives such as polyvinylpyrrolidone, Ficoll (polymeric sugars), dextrates (e.g., cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycol, flavorings, antimicrobials, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "TWEEN® 20" and "TWEEN® 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

[0272] Many known and developed methods may be used to administer the therapeutically effective amount of the compositions or pharmaceutical compositions disclosed herein.Non-limiting examples of administration methods include bolus injection, oral, infusion, intra-articular, intrabronchial, intra-abdominal, intra-capsular, intra-cartilaginous, intra-cavitary, intraperitoneal, intra-cerebellar, intraventricular, intra-colonic, intracervical, intra-gastric, intra-hepatic, intra-lesional, intra-muscular, intra-myocardial, intra-nasal, intra-ocular, intra-osseous, intra-skeletal, intra-pelvic, intra-pericardial, intra-peritoneal, intra-pleural, intra-prostatic, intra-pulmonary, intra-rectal, intra-renal, intra-retinal, intra-spinal, intra-synovial, intra-thoracic, intra-uterine, intra-tumor, intravenous, intravesical, oral, parenteral, rectal, sublingual, subcutaneous, transdermal, or intra-vaginal administration means.

[0273] The compositions of the present disclosure may be prepared for use in parenteral (subcutaneous, intramuscular, or intravenous) or any other administration, particularly in the form of liquid solutions or suspensions; for use in vaginal or rectal administration, particularly in semi-solid forms such as, but not limited to, creams and suppositories; for use in buccal or sublingual administration, such as, but not limited to, in the form of tablets or capsules; for use in intranasal administration, such as, but not limited to, powders, nose drops, aerosols, or in the form of certain medications; or for use in transdermal administration, such as, but not limited to, gels, ointments, lotions, suspensions, or patch delivery systems. Here, the patch delivery systems include chemical enhancers such as dimethylsulfoxide to modify skin structure or increase drug concentration in transdermal patches (Junginger, et al. "Drug Permeation Enhancement"; Hsieh, DS, Eds., pp. 59-90, Marcel Dekker, Inc. New York 1994), or oxidizing agents to allow application of formulations containing proteins and peptides to the skin (International Patent Application WO 98 / 53847), or application of electric fields to create temporary transport pathways such as electroporation and to enhance the mobility of charged drugs through the skin such as iontophoresis, or application of ultrasound such as phonophoresis (U.S. Patent Nos. 4,309,989 and 4,767,402) (the above publications and patents are incorporated herein by reference in their entirety).

[0274] For parenteral administration, any composition disclosed herein may be formulated as a solution, suspension, emulsion, granules, powder, or lyophilized powder, either in combination with a pharma- ceutically acceptable parenteral excipient or provided separately. Preparations for parenteral administration may contain, as common excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like. Aqueous or oily suspensions for injection may be prepared according to known methods using suitable emulsifiers or wetting agents and suspending agents. Injectable agents may be non-toxic parenterally administrable diluents, such as aqueous solutions, sterile injectable solutions, or suspensions in solvents. Excipients or solvents that can be used include water, Ringer's solution, isotonic saline, and the like, and sterile fixed oils may be used as normal solvents or suspension solvents. For these purposes, any kind of fixed oils and fatty acids, such as natural or synthetic or semi-synthetic fatty oils or fatty acids, and natural or synthetic or semi-synthetic mono- or di- or triglycerides, may be used. Parenteral administration is known in the art and includes, but is not limited to, conventional injection means, the gas pressurized needleless injection device described in U.S. Pat. No. 5,851,198, and the laser perforation device described in U.S. Pat. No. 5,839,446.

[0275] Formulations for oral administration rely on the co-administration of adjuvants (e.g., resorcinol and non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether) to artificially increase the permeability of the intestinal wall, as well as enzyme inhibitors (e.g., pancreatic trypsin inhibitor, diisopropylfluorophosphate (DFF), and trasylol) to inhibit enzymatic degradation. Formulations for delivering hydrophilic drugs, including proteins and protein scaffolds, and combinations of at least two surfactants intended for oral, buccal, mucosal, nasal, pulmonary, and vaginal transmembrane, or rectal administration are described in U.S. Pat. No. 6,309,663. The active ingredient compound in the solid dosage form for oral administration may be mixed with at least one additive such as sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginic acid, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, and glycerides. These dosage forms may also contain other additive types, such as inert diluents, lubricants such as magnesium stearate, preservatives such as parabens, sorbic acid, antioxidants such as ascorbic acid, α-tocopherol, cysteine, disintegrants, binders, thickeners, buffers, sweeteners, flavors, fragrances, etc.

[0276] Tablets and pills may also be prepared into enteric formulations. Liquid formulations for oral administration include emulsions, syrups, elixirs, suspensions, and solution formulations that are acceptable for medical use. These formulations may contain inert diluents that are commonly used in the field, such as water. Liposomes have also been described as drug delivery systems for insulin and heparin (U.S. Pat. No. 4,239,754). More recently, microspheres of artificial polymers of mixed amino acids (proteinoids) have been used to deliver pharmaceuticals (U.S. Pat. No. 4,925,673). In addition, carrier compounds described in U.S. Pat. Nos. 5,879,681 and 5,871,753 and used to deliver biologically active agents orally are known in the art.

[0277] For pulmonary administration, the compositions or pharmaceutical compositions described herein are preferably delivered in a particle size effective to reach the lungs or the lower respiratory tract of the paranasal sinuses. The compositions or pharmaceutical compositions may be delivered by any of a variety of inhalation or nasal devices known in the art for administration of therapeutic agents by inhalation. These devices capable of depositing an aerosolized formulation in the paranasal sinuses or alveoli of a patient include metered dose inhalers, nebulizer inhalers (e.g., jet nebulizer inhalers, ultrasonic nebulizer inhalers), dry powder generators, nebulizers, and the like. All such devices may employ formulations suitable for administration of the compositions or pharmaceutical compositions described herein in an aerosol-dispensed manner. The aerosol may be comprised of either a solution (both aqueous and non-aqueous solutions) or solid particles. Additionally, a spray comprising the compositions or pharmaceutical compositions described herein may be generated by forcing a suspension or solution of at least one protein scaffold through a nozzle under pressure. In a metered dose inhaler (MDI), the propellant, the compositions or pharmaceutical compositions described herein, and any excipients or other additives are packaged in a container as a mixture with a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol containing particles in the size range of less than about 10 μm, preferably about 1 μm to about 5 μm, and most preferably about 2 μm to about 3 μm. A more detailed description of pulmonary administration, formulations, and associated devices is disclosed in International Patent Publication WO 2019 / 049816.

[0278] In the case of absorption through a mucosal surface, the composition comprises an emulsion comprising a plurality of submicron particles, a mucoadhesive polymer, a bioactive peptide, and an aqueous continuous phase, which achieves mucoadhesion of the emulsion particles to facilitate absorption through the mucosal surface (US Pat. No. 5,514,670). Mucosal surfaces suitable for use of the emulsions of the present disclosure may include corneal, conjunctival, oral, sublingual, nasal, vaginal, pulmonary, gastric, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, such as suppositories, may include excipients such as polyalkylene glycols, petrolatum, cocoa butter, and the like. Formulations for intranasal administration may be solid and may include excipients such as lactose, or may be aqueous or oily solutions of nasal drops. In the case of intrabuccal administration, excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch, and the like (US Pat. No. 5,849,695). A more detailed description of mucosal administration and formulations thereof is disclosed in International Patent Publication WO 2019 / 049816.

[0279] For transdermal administration, the compositions or pharmaceutical compositions disclosed herein are encapsulated in delivery devices such as liposomes, or polymeric nanoparticles, microparticles, microcapsules, or microspheres (collectively referred to as microparticles unless otherwise specified). Many suitable devices are known, including microparticles made of synthetic polymers such as polyhydroxy acids, such as polylactic acid, polyglycolic acid, and their copolymers, polyorthoesters, polyanhydrides, and polyphosphazenes, and natural polymers such as collagen, polyamino acids, albumin and other proteins, alginates and other polysaccharides, and combinations thereof (U.S. Patent No. 5,814,599). A more detailed description of transdermal administration, formulations, and suitable devices is disclosed in International Patent Publication WO 2019 / 049816.

[0280] It may be desirable to deliver the disclosed compounds to a subject over an extended period of time, for example, from one week to one year from a single administration. A variety of sustained release, depot, or transfer dosage forms may be utilized. For example, the dosage form may include a pharma- ceutically acceptable non-toxic salt of a compound having a low level of solubility in bodily fluids, such as (a) an acid addition salt with a polybasic acid, such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene monosulfonic acid or disulfonic acid, polygalacturonic acid, (b) a salt with a polyvalent metal cation, such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, or an organic cation, such as formed from N,N'-dibenzyl-ethylenediamine or ethylenediamine; or (c) a combination of (a) and (b), such as zinc tannate salt. In addition, the disclosed compounds, or preferably relatively insoluble salts such as the salts just described, may be formulated into gels suitable for injection, such as aluminum monostearate gels with sesame oil. Particularly preferred salts are zinc salts, zinc tannate salts, pamoate salts, and the like. Another type of sustained release depot formulation for injection includes the compound or salt dispersed for encapsulation in slowly degrading non-toxic, non-antigenic polymers, such as polylactic acid / polyglycolic acid polymers, as described, for example, in U.S. Pat. No. 3,773,919. The compounds, or preferably relatively insoluble salts such as the salts just described, may also be formulated into cholesterol-based silicone pellets, particularly for use in animals. Additional sustained release formulations, depot formulations, or transfer formulations, such as gaseous or liquid liposomes, are known in the literature (U.S. Pat. No. 5,770,222 and “Sustained and Controlled Release Drug Delivery Systems”, J.R. Robinson ed., Marcel Dekker, Inc., NY, 1978).

[0281] Appropriate dosages are well known in the art.See, for example, Wells et al., eds., Pharmacotherapy Handbook, 2nd ed., Appleton and Lange, Stamford, Conn. (2000);PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Special Edition, Tarascon Publishing, Loma Linda, Calif. (2000);Nursing 2001 Handbook of Drugs, 21st ed., Springhouse Corp., Springhouse, Pa., 2001;and Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, NJ. Preferred doses may include, as the case may be, about 0.1-99 and / or 100-500 mg / kg per administration, or any range, value, or fraction thereof, or to achieve a serum concentration of about 0.1-5000 μg / ml per single or multiple administrations, or any range, value, or fraction thereof. Preferred dose ranges for the compositions or pharmaceutical compositions disclosed herein are from about 1 mg / kg up to about 3, about 6, or about 12 mg / kg of subject body weight.

[0282] Alternatively, the dosage may vary depending on known factors such as the pharmacodynamic properties of the particular agent and its mode and route of administration; the age, health, and weight of the patient; the nature and extent of the symptoms, the type of disease being treated concomitantly, the frequency of treatment, and the desired effect. Typically, the dosage of the active ingredient may be about 0.1 to 100 mg per kg of body weight. Typically, in a single dose or in sustained release form, 0.1 to 50 mg per kg, preferably 0.1 to 10 mg, is effective to obtain the desired results.

[0283] As a non-limiting example, for treatment of humans or animals, a single or periodic dose of the compositions or pharmaceutical compositions disclosed herein may be provided using a single infusion or multiple administrations, from about 0.1 to 100 mg / kg, or any range, value, or fraction thereof, daily, for at least one day out of 1 day to 40 days, or alternatively or additionally, for at least one week out of 1 week to 52 weeks, or alternatively or additionally, for at least one year out of 1 year to 20 years, or any combination thereof.

[0284] Dosage forms suitable for internal administration typically contain from about 0.001 mg to 500 mg of active ingredient per unit or packet. In these pharmaceutical compositions, the active ingredient is typically present in an amount of from about 0.5 to 99.999% by weight based on the total weight of the composition.

[0285] An effective amount may include an amount of about 0.001 to about 500 mg / kg, in a single dose (e.g., a bolus dose), multiple doses, or continuous administration, or an amount that will achieve a serum concentration of about 0.01 to 5000 μg / ml, or any effective range, value, or fraction thereof, in a single dose, multiple dose, or continuous administration, which may be performed or determined using known methods described herein or known in the relevant art.

[0286] In embodiments where the composition administered to a subject in need thereof is a modified cell disclosed herein, the cells may be present in an amount of about 1×10 3 ~1x10 15 cells; approx. 1x10 4 ~1x10 12 cells; approximately 1x10 5 ~1x10 10 cells; approximately 1x10 6 ~1x10 9 cells; approximately 1x10 6 ~x10 8 cells; approximately 1x10 6 ~1x10 7 cells; or approximately 1x10 6 ~25x10 6In one embodiment, the cells are administered in an amount of about 5x10 6 ~25x10 6 It is administered in units of cells.

[0287] A more detailed description of the pharma- ceutically acceptable excipients, formulations, dosages, and methods of administration of the disclosed compositions and pharmaceutical compositions is disclosed in International Patent Publication WO 2019 / 049816.

[0288] Methods of Using the Compositions of the Present Disclosure

[0289] The present disclosure provides a use of the disclosed composition for improving transposition efficiency. Specifically, the present disclosure includes contacting a cell or a plurality of cells with the following composition, the composition includes a first nucleic acid sequence including (a) a first inverted terminal repeat (ITR) or a sequence encoding the first ITR, (b) a second ITR or a sequence encoding the second ITR, and (c) an intra-ITR sequence or a sequence encoding the intra-ITR sequence including a transposon sequence or a sequence encoding a transposon, and a second nucleic acid sequence including an inter-ITR sequence or a sequence encoding an inter-ITR sequence, wherein the length of the inter-ITR sequence is 700 nucleotides or less, and the transposition efficiency in the cell or a plurality of cells is improved compared to the same composition including a second nucleic acid sequence or an inter-ITR sequence that is more than 700 nucleotides. In one aspect, the transfer efficiency is improved by 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 90%.

[0290] The present disclosure provides for the use of compositions and pharmaceutical compositions of the present disclosure for the treatment of a disease or disorder in a cell, tissue, organ, animal, or subject, as known in the art or as described herein, for example, by administering or contacting a therapeutically effective amount of the composition or pharmaceutical composition to the cell, tissue, organ, animal, or subject. In one aspect, the subject is a mammal. Preferably, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0291] The present disclosure provides a method for modulating or treating at least one malignant disease or disorder in a cell, tissue, organ, animal, or subject. Preferably, the malignant disease is cancer. Non-limiting examples of malignant diseases or disorders include leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, B-cell, T-cell, or FAB ALL, acute myeloid leukemia (AML), acute myeloid leukemia, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's lymphoma, and the like. These include sarcoma, colorectal cancer, pancreatic cancer, nasopharyngeal cancer, malignant histiocytosis, paraneoplastic syndrome / hypercalcemia associated with malignancy, solid tumors, bladder cancer, breast cancer, colon cancer, endometrial cancer, head cancer, neck cancer, hereditary nonpolyposis carcinoma, Hodgkin lymphoma, liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, testicular cancer, adenocarcinoma, sarcoma, melanoma, hemangioma, metastatic disease, cancer-related bone resorption, and cancer-related bone pain.

[0292] In a preferred embodiment, the treatment of malignant disease or disorder includes adoptive cell therapy. For example, in one embodiment, the present disclosure provides modified cells expressing at least one disclosed protein scaffold and / or CAR comprising the protein scaffold (e.g., scFv, single domain antibody, centilin delivered to cells with the composition of the present disclosure) selected and / or expanded for administration to a subject in need thereof. The modified cells may be formulated for storage at any temperature, such as room temperature and body temperature. The modified cells may be formulated for cryopreservation and then thawed. The modified cells may be formulated in a pharmaceutically acceptable carrier for direct administration to a subject from a sterile package. The modified cells may be formulated in a pharmaceutically acceptable carrier with an indicator of cell viability and / or CAR expression level to ensure a minimum level of cell function and CAR expression. The modified cells may be formulated in a pharmaceutically acceptable carrier at a predetermined density with one or more reagents to inhibit further proliferation and / or prevent cell death.

[0293] Any of the methods may include administering an effective amount of any of the compositions or pharmaceutical compositions disclosed herein to a cell, tissue, organ, animal, or subject in need of modulation, treatment, or therapy. The methods may optionally further include co-administration or combination therapy to treat the disease or disorder, and the administration of any of the compositions or pharmaceutical compositions disclosed herein further includes administration prior to, concurrently with, and / or subsequent to at least one chemotherapeutic agent (e.g., alkylating agents, mitotic inhibitors, radiopharmaceuticals).

[0294] In some embodiments, the subject does not develop graft versus host (GvH) and / or host versus graft (HvG) after administration. In one embodiment, administration is systemic. Systemic administration may be by any method known in the art and described in detail herein. Preferably, systemic administration is by intravenous injection or intravenous infusion. In one embodiment, administration is local. Local administration may be by any method known in the art and described in detail herein. Preferably, local administration is by intratumoral, intraspinal, intraventricular, intraocular, or intraosseous injection or infusion.

[0295] In some embodiments, the therapeutically effective dose is a single dose.In some embodiments, 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 therebetween that are simultaneously produced.In some embodiments, where the composition is autologous or allogeneic cells, the dose is sufficient for cells to engraft and / or last for a sufficient time to treat disease or disorder.

[0296] 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 protein scaffold or a composition comprising a CAR (e.g., scFv, single domain antibody, centilin) ​​comprising a protein scaffold. The antibody or CAR specifically binds to an antigen on the surface of a tumor cell. In an embodiment in which the composition comprises a modified cell or a modified cell population, the cell or cell population may be autologous or allogeneic.

[0297] In some embodiments of the therapeutic methods described herein, the treatment may be altered or terminated. Specifically, in embodiments in which the composition used in the treatment comprises an inducible pro-apoptotic polypeptide, apoptosis may be selectively induced in the cells by contacting the cells with an inducer. For example, the treatment may be altered or terminated in response to signs of recovery or signs of a decrease in disease severity / progression, signs of disease remission / cessation, and / or the occurrence of an adverse event. In some embodiments, the method includes administering an inhibitor of the inducer to prevent the alteration of the cell therapy, thereby restoring the function and / or efficacy of the cell therapy (e.g., if signs or symptoms of the disease recur, or if increased severity and / or adverse events are resolved).

[0298] Protein scaffold production, selection and purification

[0299] At least one protein scaffold of the present disclosure (e.g., monoclonal antibody, chimeric antibody, single domain antibody, VHH, VH, single chain variable fragment (scFv), centrin, antigen binding fragment (Fab) or Fab fragment) may optionally be produced by a cell line, a mixed cell line, an immortalized cell, or a clonal population of immortalized cells, as is well known in the art. See, e.g., Ausubel, et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, NY (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); and Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001).

[0300] Amino acids from the protein scaffold may be altered, added and / or deleted to reduce immunogenicity or to reduce, enhance or alter binding, affinity, on-rate, off-rate, avidity, specificity, half-life, stability, solubility, or other suitable properties known in the art.

[0301] In some cases, protein scaffolds can be engineered with high affinity for antigens and other favorable biological properties. To achieve this goal, scaffold proteins can be prepared by a process of analysis of parent sequences and various conceptual engineered products using three-dimensional models of the parent and engineered sequences. Three-dimensional models are commonly available and familiar to those skilled in the art. Computer programs are available that can illustrate and display the possible three-dimensional conformational structures of selected candidate sequences and measure their possible immunogenicity (e.g., the Immunofilter program from Xencor, Inc., Monrovia, Calif.). Inspection of these displays allows analysis of the possible role of residues in the functionalization of the candidate sequence, i.e., analysis of residues that affect the ability of the candidate protein scaffold to bind to its antigen. In this way, residues from parent and reference sequences can be selected and combined to achieve the desired properties, such as affinity for the target antigen. Alternatively or in addition to the above procedures, other suitable engineering methods can be used.

[0302] Screening of protein scaffolds for specific binding to similar proteins or fragments can be conveniently accomplished using nucleotide (DNA or RNA display) or peptide display libraries, e.g., in vitro display. This method involves screening a large population of peptides for individual members having a desired function or structure. The display nucleotide or peptide sequences may be 3-5000 or more nucleotides or amino acids long, frequently 5-100 amino acids long, and more often about 8-25 amino acids long. In addition to direct chemical synthesis methods for generating peptide libraries, several recombinant DNA methods have been described. One method involves displaying peptide sequences on the surface of a bacteriophage or cell. Each bacteriophage or cell contains a nucleotide sequence that codes for a particular display peptide sequence. This method is described in International Patent Publications WO 91 / 17271, WO 91 / 18980, WO 91 / 19818, and WO 93 / 08278.

[0303] Other systems for generating peptide libraries include aspects of both in vitro chemical synthesis and recombinant methods. See International Patent Publications WO 92 / 05258, WO 92 / 14843, and WO 96 / 19256. See U.S. Patents 5,658,754 and 5,643,768. Peptide display libraries, vectors, and selection kits are commercially available from companies such as Invitrogen (Carlsbad, Calif.), and Cambridge Antibody Technologies (Cambridgeshire, UK). See, e.g., U.S. Patent 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, and 5,856,456, all assigned to Enzon Corporation; 5,223,409, 5,403,484, 5,571,698, and 5,837,500, all assigned to Dyax Corporation; 5,427,908 and 5,580,717, all assigned to Affymax Corporation; Cambridge Antibody See No. 5,885,793, assigned to Genentech, Inc.; No. 5,750,373, assigned to Genentech, Inc.; Nos. 5,618,920, 5,595,898, 5,576,195, 5,698,435, 5,693,493, and 5,698,417, assigned to Xoma, Inc.; Colligan, supra; Ausubel, supra; or Sambrook, supra.

[0304] The protein scaffolds of the present disclosure can bind to human or other mammalian proteins with a wide range of affinities (KD). In a preferred embodiment, at least one protein scaffold of the present disclosure has a KD of about 10, for example, as measured by surface plasmon resonance or KinExa, as may be practiced by those skilled in the art. -7 The antibody can bind to the target protein with high affinity, having a K value of less than or equal to M. For example, the K value can be, but is not limited to, 0.1 to 9.9 (or any range or value therebetween) x 10-8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , 10 -15 , or any range or value therebetween.

[0305] The affinity or avidity of a protein scaffold for an antigen can be determined experimentally using any suitable method (see, e.g., Berzofsky, et al., "Antibody-Antigen Interactions," In Fundamental Immunology, Paul, WE, Ed., Raven Press: New York, NY (1984); Kuby, Janis Immunology, WH Freeman and Company: New York, NY (1992); and methods described herein). The measured affinity of a particular protein scaffold-antigen interaction may vary when 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 performed using standardized solutions of the protein scaffold and antigen, as well as standardized buffers, such as those described herein.

[0306] Competitive measurements can be performed with protein scaffolds to determine which proteins, antibodies, and other competitors compete with the protein scaffold for binding to the target protein and / or share epitope regions. These measurements, well known to those skilled in the art, evaluate the competition between competitors or ligands for a limited number of binding sites on the protein surface. Proteins and / or antibodies are immobilized or insolubilized before or after competition, and the target protein-bound sample is separated from the unbound sample, for example, by decanting (if the protein / antibody is pre-insolubilized) or centrifugation (if the protein / antibody is precipitated after the competitive reaction). Competitive binding can also be determined by whether function is altered by the binding or lack of binding of the protein scaffold to the target protein, for example, whether the protein scaffold inhibits or enhances an enzymatic activity such as a label. ELISA and other functional measurements can be used, as is well known in the art.

[0307] nucleic acid molecule

[0308] The nucleic acid molecule of the present disclosure encoding a protein scaffold may 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 combination thereof. The DNA may be triple-stranded, double-stranded, or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA may be the coding strand, also known as the sense strand, or the non-coding strand, also known as the antisense strand.

[0309] The isolated nucleic acid molecule of the present disclosure may optionally include a nucleic acid molecule that includes one or more introns, such as, but not limited to, an open reading frame (ORF) with at least one specific portion of at least one protein scaffold; a nucleic acid molecule that includes a coding sequence of a protein scaffold or loop region that binds to a target protein; and a nucleic acid molecule that includes a substantially different nucleotide sequence from the above sequences, but still codes for a protein scaffold as described herein and / or known in the art due to the degeneracy of the genetic code. It is understood that the genetic code is well known in the art. Therefore, it is routine for a person skilled in the art to generate such degenerate nucleic acid variants that code for a specific protein scaffold of the present disclosure. For example, see Ausubel et al., supra, and it will be understood that such nucleic acid variants are included in the present disclosure.

[0310] As provided herein, the nucleic acid molecules of the present disclosure, including nucleic acids encoding protein scaffolds, may include, but are not limited to, nucleic acid molecules that themselves encode the amino acid sequence of a protein scaffold fragment; coding sequences for the entire protein scaffold or a portion thereof; coding sequences for the protein scaffold, fragments, or portions, as well as additional sequences, such as coding sequences for 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 transcribed non-translated sequences that play a role in transcription or mRNA processing, including transcription, splicing, and polyadenylation signals (e.g., ribosome binding and mRNA stability); and additional coding sequences that encode additional amino acids, such as amino acids that provide additional functions. Thus, the protein scaffold-encoding sequence can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fusion protein scaffold comprising the protein scaffold fragment or portion thereof.

[0311] Polynucleotides that selectively hybridize to the polynucleotides described herein

[0312] The present disclosure provides an isolated nucleic acid that hybridizes to the polynucleotide disclosed herein under selective hybridization conditions. The polynucleotide can therefore be used to isolate, detect, and / or quantify the nucleic acid that contains the polynucleotide. For example, the polynucleotide of the present disclosure can be used to identify, isolate, or amplify partial or full-length clones in a deposited library. The polynucleotide can be a genomic or cDNA sequence isolated from a human or mammalian nucleic acid library, or a genomic or cDNA sequence complementary to the cDNA.

[0313] Preferably, the cDNA library comprises at least 80% full-length sequences, preferably at least 85% or 90% full-length sequences, more preferably at least 95% full-length sequences. The cDNA library may be normalized to increase the representation of rare sequences. Low or moderate stringency hybridization conditions are typically, but not exclusively, used with sequences that have reduced sequence identity compared to complementary sequences. Moderate and high stringency conditions may be used for sequences with higher identity in some cases. Low stringency conditions allow selective hybridization of sequences with about 70% sequence identity, and can be used to distinguish orthologous or paralogous sequences.

[0314] In some cases, the polynucleotide encodes at least a portion of the protein scaffold encoded by the polynucleotide described herein. The polynucleotide comprises a nucleic acid sequence that can be used to selectively hybridize to the polynucleotide encoding the protein scaffold of the present disclosure.See, for example, Ausubel, supra, and Colligan, supra, each of which is incorporated herein by reference in its entirety.

[0315] Nucleic acid construction

[0316] Isolated nucleic acids of the disclosure can be produced using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and / or (d) combinations thereof, as known in the art.

[0317] The nucleic acid may conveniently contain sequences in addition to the polynucleotide of the disclosure. For example, a multicloning site containing one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in the isolation of the polynucleotide. Also, a translatable sequence may be inserted to aid in the isolation of the translated polynucleotide of the disclosure. For example, a hexahistidine marker sequence provides a convenient means for purifying the protein of the disclosure. The nucleic acid of the disclosure, excluding the coding sequence, is optionally a vector, adapter, or linker for the cloning and / or expression of the polynucleotide of the disclosure.

[0318] Additional sequences can be added to the cloning and / or expression sequences to optimize their function in cloning and / or expression, to aid in isolation of the polynucleotide, or to improve introduction of the polynucleotide into cells. The use of cloning vectors, expression vectors, adapters, and linkers are well known in the art (see, e.g., Ausubel, supra, and Sambrook, supra).

[0319] Recombinant methods for constructing nucleic acids

[0320] The isolated nucleic acid composition of the present disclosure, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using several cloning methods known to those skilled in the art.In some embodiments, the oligonucleotide probe that selectively hybridizes to the polynucleotide of the present disclosure under stringent conditions is used to identify the desired sequence in a cDNA or genomic DNA library.The isolation of RNA and the construction of cDNA and genomic libraries are well known to those skilled in the art (see, for example, Ausubel, supra, and Sambrook, supra).

[0321] Methods for selecting and isolating nucleic acids

[0322] A cDNA or genomic library can be screened using a probe based on the sequence of the polynucleotide of the present disclosure. The probe can be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms. Those skilled in the art will appreciate that hybridization of various degrees of stringency can be used for the measurement, and either the hybridization or the washing medium can be stringent. The more stringent the hybridization conditions, the higher the degree of complementarity between the probe and the target is required 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 altering the polarity of the reactant solution, for example, by manipulating the concentration of formamide in the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding is altered according to the stringency of the hybridization medium and / or the washing medium. The degree of complementarity will optimally be 100%, or between 70-100%, or any range or value therein, although, of course, minor sequence variations in the probes and primers can be compensated for by reducing the stringency of the hybridization and / or washing medium.

[0323] Methods for amplifying RNA or DNA are well known in the art and can be used in accordance with the present disclosure without undue experimentation, based on the teachings and guidance presented herein.

[0324] Known methods of DNA or RNA amplification include, but are not limited to, the polymerase chain reaction (PCR) and its related amplification processes (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, and 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.; 5,122,464 to Innis and 5,122,512 to Wilson et al.; 5,122,512 ... Nos. 5,091,310 to Gyllensten et al., 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, and 4,656,134 to Ringold), and RNA-mediated amplification (U.S. Patent No. 5,130,238 to Malek et al., under the trade name NASBA), which uses antisense RNA to a target sequence as a template for double-stranded DNA synthesis. The entire contents of these reference documents are incorporated herein by reference (see, e.g., Ausubel, supra, and Sambrook, supra).

[0325] For example, the sequences of the polynucleotides and related genes of the present disclosure can be directly amplified from genomic DNA or cDNA libraries using polymerase chain reaction (PCR) technology. PCR and other in vitro amplification methods are also useful techniques, for example, to clone nucleic acid sequences that code for expressed proteins and to generate nucleic acids to be used as probes for detecting the presence of desired mRNA in a sample, for diffusion sequencing, or for other purposes. Examples of techniques sufficient to guide the skilled artisan through in vitro amplification methods can be found in Berger, Sambrook, and Ausubel, supra, as well as in 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, for example, Advantage-GC Genomic PCR Kit (Clontech). Additionally, the yield of long PCR products can be improved using, for example, T4 gene 32 protein (Boehringer Mannheim).

[0326] Synthetic methods for constructing nucleic acids

[0327] The isolated nucleic acid of the present disclosure may also be prepared by direct chemical synthesis according to known methods (see, for example, 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. Chemical synthesis of DNA is limited to sequences of about 100 bases or more, but those skilled in the art will recognize that longer sequences can be obtained by linking shorter sequences.

[0328] Recombinant Expression Cassettes

[0329] The present disclosure further provides a recombinant expression cassette comprising the nucleic acid of the present disclosure. The nucleic acid sequence of the present disclosure, for example, the cDNA or genomic sequence encoding the protein scaffold of the present disclosure, can be used to construct a recombinant expression cassette that can be introduced into at least one desired host cell. The recombinant expression cassette typically comprises the polynucleotide of the present disclosure operably linked to a transcription initiation regulatory sequence that induces the transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to induce the expression of the nucleic acid of the present disclosure.

[0330] In some embodiments, isolated nucleic acids functioning as promoters, enhancers, or other elements may be introduced into a suitable location (upstream, downstream, or in an intron) of a non-heterologous form of a polynucleotide of the disclosure to upregulate or downregulate expression of the polynucleotide of the disclosure. For example, endogenous promoters may be altered in vivo or in vitro by mutation, deletion, and / or substitution.

[0331] Expression vectors and host cells

[0332] The present disclosure also relates to vectors comprising the isolated nucleic acid molecules of the present disclosure, host cells genetically engineered with recombinant vectors, and the production of at least one protein scaffold by recombinant techniques, as known in the art.See, for example, Sambrook et al., supra, and Ausubel et al., supra, each of which is incorporated herein by reference in its entirety.

[0333] Polynucleotide may be optionally linked to a vector that contains a selection marker for propagation in a host.Generally, plasmid vector is introduced into precipitate such as calcium phosphate precipitate or into a complex with charged lipid.When vector is a virus, it can be packaged in vitro using suitable packaging cell line and transduced into host cell.

[0334] The DNA insert should be operably linked to a suitable promoter. The expression construct further comprises a transcription initiation site, a termination site, and in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct preferably comprises translation initiation 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 being preferred for mammalian or eukaryotic cell expression.

[0335] Expression vectors optionally but preferably include at least one selectable marker, such as, but 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, see U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739), blasticidin (bsd gene), or a combination of ... p Resistance genes for eukaryotic cell culture such as 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 culture in E. coli and other bacteria or prokaryotes (the above mentioned patents are incorporated herein by reference in their entirety). Appropriate culture media and conditions for the above mentioned host cells are known in the art. Appropriate vectors will be readily apparent to those skilled in the art. Introduction of the vector construct into the host cell can be achieved by calcium phosphate transduction, DEAE-dextran mediated transduction, cationic lipid mediated transduction, electroporation, transduction, infection or other known methods. These methods are explained in the art, such as in Sambrook et al., supra, Chapters 1-4 and 16-18; Ausubel et al., supra, Chapters 1, 9, 13, 15 and 16.

[0336] The expression vector may, but preferably, include at least one selection cell surface marker for the isolation of cells modified by the compositions and methods of the present disclosure. The selection cell surface markers of the present disclosure include surface proteins, glycoproteins, or groups of proteins that distinguish a cell or a fraction of cells from another defined fraction of cells. Preferably, the selection cell surface marker distinguishes cells that have been modified by the compositions or methods of the present disclosure from cells that have not been modified by the compositions or methods of the present disclosure. Such cell surface markers include, for example, "designated cluster" or "classification determinant" proteins (often abbreviated as "CD"), such as, but not limited to, truncated or full-length versions of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof. Cell surface markers further include the suicide gene marker RQR8 (see Philip B et al. Blood. 2014 Aug 21; 124(8):1277-87).

[0337] The expression vector may, but preferably, include at least one selective drug resistance marker for the isolation of cells modified by the compositions and methods of the present disclosure. The selective drug resistance markers of the present disclosure may include wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

[0338] At least one protein scaffold of the present disclosure may be expressed in modified form, such as a fusion protein, and may include additional heterologous functional regions, as well as secretion signals. For example, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the protein scaffold to improve stability and persistence in the host cell during purification or subsequent manipulation and storage. Peptide moieties may also be added to the protein scaffold of the present disclosure to facilitate purification. Such regions may be removed prior to final preparation of the protein scaffold or at least one fragment thereof. This method is described in many standard laboratory manuals, such as Chapters 17.29-17.42 and 18.1-18.74 of Sambrook, supra; Chapters 16, 17, and 18 of Ausubel, supra.

[0339] Those skilled in the art are familiar with the many expression systems available for expressing the nucleic acid encoding the protein of the present disclosure.Alternatively, the nucleic acid of the present disclosure can be expressed in a host cell by starting (by engineering) in a host cell that contains endogenous DNA encoding the protein scaffold of the present disclosure.Such methods are well known in the art, for example, as described in U.S. Patent Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, the entirety of which is incorporated herein by reference.

[0340] Examples of cell cultures useful for the production of protein scaffolds, specific portions or variants thereof are bacteria, yeast, and mammalian cells known in the art. Mammalian cell systems are often in the form of a monolayer of cells, although suspensions of mammalian cells or biochemical reactants may also be used. A number of suitable host cell lines capable of expressing intact glycosylated proteins have been developed in the art, including 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, for example, from the 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 No. CRL-1580) and SP2 / 0-Ag14 cells (ATCC Accession No. CRL-1851). In a preferred embodiment, the recombinant cell is a P3X63Ab8.653 or SP2 / 0-Ag14 cell.

[0341] Expression vectors for these cells may include one or more of the following expression control sequences, including, but not limited to, an origin of replication; a promoter (e.g., a late or early SV40 promoter, a CMV promoter (U.S. Pat. Nos. 5,168,062; 5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an EF-1α 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., the SV40 large T Ag polyA addition site), and transcription terminator sequences. See, e.g., Ausubel et al., supra, and Sambrook et al., supra. Other cells useful for producing the nucleic acids or proteins of the disclosure are known and / or available, for example, from the American Cell Line Organization's catalog of cell lines and fusion cells (www.atcc.org) or other known commercial sources.

[0342] When eukaryotic host cells are used, polyadenylation sequences or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for precise splicing of the transcript may also be included. An example of a splicing sequence is the VP1 intron from SV40 (see Sprague, et al., J. Virol. 45:773-781 (1983)). In addition, gene sequences for controlling replication in host cells may be incorporated into the vector, as known in the art.

[0343] Purification of protein scaffolds

[0344] Protein scaffolds can be recovered and purified from recombinant cell cultures by well-known methods, including, but not limited to, Protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography.High performance liquid chromatography ("HPLC") may also be used for purification.See, for example, Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), for example, chapters 1, 4, 6, 8, 9, and 10, each of which is incorporated herein by reference in its entirety.

[0345] The protein scaffolds of the present disclosure include purified products, products of chemical synthesis procedures, and products produced by recombinant technology from prokaryotic or eukaryotic hosts, including, for example, E. coli, yeast, higher plants, insects, and mammalian cells. Depending on the host used in the recombinant production procedure, the protein scaffolds of the present disclosure may be glycosylated or non-glycosylated. This method is described in many standard laboratory manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18, and 20; Colligan, Protein Science, supra, Chapters 12-14, each of which is incorporated herein by reference in its entirety.

[0346] Amino acid code

[0347] The amino acids constituting the protein scaffolds of the present disclosure are often abbreviated. The designation of an amino acid can be indicated by designating the amino acid by its one-letter code, its three-letter code, name, or three-nucleotide codon, as is well known in the art (see Alberts, B., et al., Molecular Biology of The Cell, 3rd ed., Garland Publishing, Inc., New York, 1994). The protein scaffolds of the present disclosure may include one or more amino acid substitutions, deletions, or additions from spontaneous mutations and / or human manipulation, as specified herein. The amino acids in the protein scaffolds of the present disclosure that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (see, e.g., Ausubel, supra, Chapter 8, 15; Cunningham and Wells, Science 244:1081-1085 (1989)). In the latter procedure, single alanine mutations are introduced at all residues 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 important for protein scaffold binding can also be identified by structural analysis, such as crystallization, nuclear magnetic resonance, or photoaffinity labeling (see Smith, et al., J. Mol. Biol. 224:899-904 (1992) and de Vos, et al., Science 255:306-312 (1992)).

[0348] It will be understood by those skilled in the art that the present disclosure includes at least one biologically active protein scaffold of the disclosure. A biologically active protein scaffold has at least 20%, 30%, or 40%, preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95%-99% or more of the specific activity of a natural (non-synthetic), endogenous or related known protein scaffold. Methods for measuring and quantifying enzyme activity and substrate specificity measurements are well known to those skilled in the art.

[0349] In another aspect, the present disclosure relates to protein scaffolds and fragments thereof described herein that are modified by the covalent attachment of organic moieties. Such modifications can generate protein scaffold fragments with improved pharmacokinetic properties (e.g., increased serum half-life in vivo). The organic moieties can be linear or branched hydrophilic polymeric groups, fatty acid groups, or fatty acid ester groups. In certain aspects, the hydrophilic polymeric groups can have a molecular weight of about 800 to about 120,000 Da and can be polyalkane glycols (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymers, amino acid polymers, or polyvinylpyrrolidone, and the fatty acid or fatty acid ester groups can contain about 8 to about 40 carbon atoms.

[0350] The modified protein scaffolds and fragments of the present disclosure may include one or more organic moieties that are directly or indirectly covalently attached to an antibody. Each organic moiety that is attached to a protein scaffold or fragment of the present disclosure may be independently a hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. As used herein, the term "fatty acid" includes monocarboxylic and dicarboxylic acids. As used herein, the term "hydrophilic polymer group" 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, the protein scaffold modified by the covalent attachment of polylysine is encompassed by the present disclosure. Hydrophilic polymers suitable for modifying the protein scaffolds of the present disclosure may be linear or branched and include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG, etc.), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides, etc.), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartic acid, etc.), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide, etc.), and polyvinylpyrrolidone. Preferably, the hydrophilic polymers modifying the protein scaffolds of the present disclosure have a molecular weight of about 800 to about 150,000 Da as separate molecular entities. For example, PEG 5000 and PEG20,000 may be used, where the subscript is the average molecular weight of the polymer in Daltons (Da). The hydrophilic polymeric group may be substituted with 1 to about 6 alkyl fatty acid or fatty acid ester groups. Hydrophilic polymers substituted with fatty acid or fatty acid ester groups can be prepared using suitable methods. For example, a polymer containing an amine group may be attached to a carboxylate of a fatty acid or fatty acid ester, and an activated carboxylate (e.g., activated with N,N-carbonyldiimidazole) of the fatty acid or fatty acid ester surface can be attached to hydroxyl groups on the polymer surface.

[0351] The fatty acids and fatty acid esters suitable for modifying the protein scaffold of the present disclosure may be saturated or may contain one or more units of unsaturation.The fatty acids suitable for modifying the protein scaffold of the present 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, octadecanedioic acid, docosanedioic acid, etc. Suitable fatty acid esters include monoesters of dicarboxylic acids containing a straight or branched chain lower alkyl group. The lower alkyl group may contain from 1 to about 12, preferably from 1 to about 6, carbon atoms.

[0352] Modified protein scaffolds and fragments can be prepared using suitable methods, such as by reaction with one or more modifying agents. The term "modifying agent" as used herein refers to a suitable organic group (e.g., hydrophilic polymer, fatty acid, fatty acid ester) that includes an activating group. An "activating group" is a chemical moiety or functional group that can react with a second chemical group under suitable conditions, 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 (chloride, bromide, fluoride, iodide), N-hydroxysuccinimidyl ester (NHS), etc. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acryloyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), etc. Aldehyde functional groups can be attached to amine- or hydrazide-containing molecules, and azide groups can react with trivalent phosphorus groups to form phosphoramidate or phosphoimide bonds. Suitable methods for introducing activating groups into molecules are known in the art (see, for example, Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)). Activating groups can be attached directly to organic groups (e.g., hydrophilic polymers, fatty acids, fatty acid esters) or can be attached via a linker moiety, e.g., a divalent C1-C12 group in which one or more carbon atoms can be replaced with 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-. A modifying agent containing a linker moiety may be, for example, a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) reacted 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 may be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine which can be coupled to another carboxylate as described, or may be reacted with maleic anhydride and the resulting cyclized product to produce an activated maleimide derivative of a fatty acid (see, e.g., Thompson et al., International Patent Publication WO 92 / 16221, the teachings of which are incorporated herein by reference in their entirety).

[0353] The modified protein scaffolds of the present disclosure can be generated by reacting a protein scaffold or fragment with a modifying agent. For example, organic moieties can be attached to the protein scaffold in a non-site-specific manner using amine-reactive modifiers such as NHS esters of PEG. Modified protein scaffolds and fragments containing organic moieties that are attached to specific sites of the protein scaffolds of the present 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 methods described in Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996).

[0354] definition

[0355] As used throughout this 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 "dosage" includes reference to one or more dosages and equivalents thereof known to those of skill in the art.

[0356] The term "about" or "approximately" means within an acceptable error range for a particular numerical value as determined by one of ordinary skill in the art, which depends in part on how the numerical value is measured or determined, e.g., on the limitations of the measurement system. For example, "about" means within one or more standard deviations. Alternatively, "about" may mean within a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given numerical value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of the numerical value. When a specific numerical value is described in an application and claims, the term "about" should be assumed to mean within an acceptable error range of the specific numerical value, unless otherwise indicated.

[0357] The present 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 natural environment of origin. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular materials or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. Optimally, an "isolated" polynucleotide does not include 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 embodiments, an isolated polynucleotide may include less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. Proteins that are substantially free of cellular material include preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating proteins. When the proteins of the present disclosure or biologically active portions thereof are recombinantly produced, optimal media refers to less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or chemicals other than the protein of interest.

[0358] The present disclosure provides fragments and variants of the disclosed DNA sequences and proteins encoded by these DNA sequences. The term "fragment" used throughout this disclosure refers to a portion of a DNA sequence or a portion of an amino acid sequence and thus the protein they encode. A fragment of a DNA sequence, including a coding sequence, may encode a protein fragment that retains the biological activity of the native protein, thus DNA recognition or binding activity to a target DNA sequence as described herein. Alternatively, a fragment of a DNA sequence useful as a hybridization probe generally does not encode a protein that retains biological activity or does not retain promoter activity. Thus, a fragment of a DNA sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, to the full length of the polynucleotide of the present disclosure.

[0359] The nucleic acids or proteins of the disclosure can be constructed by a modular approach that involves preassembling monomeric and / or repeating units into a target vector that can then be assembled into a final destination vector. The polypeptides of the disclosure can be constructed by a modular approach by preassembling repeating units into a target vector that may include repeating monomers of the disclosure and can then be assembled into a final destination vector. The disclosure provides polypeptides produced by this method, as well as nucleic acid sequences encoding these polypeptides. The disclosure provides host organisms and cells that contain nucleic acid sequences encoding the polypeptides produced by this modular approach.

[0360] The term "antibody" is used in the broadest sense, specifically including single monoclonal antibodies (including agonist and antagonist antibodies), and antibody compositions with polyepitopic specificity. It is also within the scope of the present specification to use natural or synthetic analogs, mutants, variants, alleles, homologs, and orthologs (collectively referred to herein as "analogs") of the antibodies of the present specification as defined herein. Thus, according to one aspect of the present specification, the term "antibody of the present specification" in its broadest sense also includes such analogs. In general, such analogs may have one or more amino acid residues substituted, deleted, and / or added compared to the antibodies of the present specification as defined herein.

[0361] As used herein, an "antibody fragment," and all literal variants thereof, is defined as a portion of an intact antibody that contains the antigen-binding site or variable region of the intact antibody, which does not include the constant heavy chain domains of the Fc region of the intact antibody (i.e., CH2, CH3, and CH4, depending on the antibody isotype). Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one continuous sequence of adjacent amino acid residues, including, but not limited to, (1) a single-chain Fv (scFv) molecule, (2) a single-chain polypeptide containing only one light chain variable domain, or a fragment thereof containing the three CDRs of a light chain variable domain without the associated heavy chain portion, and (3) a single-chain polypeptide containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of a heavy chain variable region without the associated light chain portion (referred to herein as a "single-chain antibody fragment" or "single-chain polypeptide"); and multispecific or multivalent structures formed from antibody fragments. In antibody fragments comprising one or more heavy chains, the heavy chain may comprise any of the constant domain sequences found in the non-Fc region in intact antibodies (e.g., CHI in IgG isotypes), and / or any of the hinge region sequences found in intact antibodies, and / or a leucine zipper sequence fused to or located within the hinge region sequence or constant domain sequence of the heavy chain. The term further includes single domain antibodies ("sdAB"), which generally refer to antibody fragments having a single monomeric variable antibody domain (e.g., from camelids). Such antibody fragment species are readily apparent to those skilled in the art.

[0362] "Binding" refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific.

[0363] The term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements of any essential importance in the combination when used for the intended purpose. Thus, a composition consisting essentially of the elements defined herein does not exclude trace amounts of contaminants or inert agents. "Consisting of" is intended to mean excluding more than trace elements of other components or substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0364] The term "epitope" refers to an antigenic determinant of a polypeptide. An epitope may contain three amino acids that are unique to the epitope in a spatial conformation. In general, an epitope consists of at least 4, 5, 6, or 7 of these amino acids, and more generally, at least 8, 9, or 10 amino acids. Methods for determining the spatial conformation of amino acids are known in the art, and include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0365] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0366] "Gene expression" refers to the conversion of information contained within a gene into a gene product. A gene product may be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, microRNA, structural RNA, or other types of RNA) or a protein produced by translation of an mRNA. Gene products also include RNA that is modified by processes such as capping, polyadenylation, methylation, editing, and proteins that are modified by methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0367] "Modulation" or "regulation" of gene expression refers to a change in the activity of a gene. Modulation of expression includes, but is not limited to, gene activation and gene repression.

[0368] The term "operatively linked" or its equivalents (e.g., linked operatively) means that two or more molecules are positioned relative to one another such that they are capable of an interaction that affects a function attributable to one or both of the molecules, or a combination of the molecules.

[0369] Non-covalently linked components and methods of producing 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. operably linked) proteins are used to allow for 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 allows for functional association only or primarily under circumstances where this association is required for the desired activity. The association may persist for a sufficient time to allow for the desired effect.

[0370] A method for targeting a protein to a specific locus in the genome of an organism is disclosed. The method may include providing a DNA localization component and providing an effector molecule, wherein the DNA localization component and the effector molecule may be operably linked via a non-covalent bond.

[0371] The term "scFv" refers to a single chain variable fragment. An scFv is a fusion protein of the variable regions of an immunoglobulin heavy (VH) and light (VL) chains, linked to a linker peptide. The linker peptide may be about 5-40 amino acids in length, or about 10-30 amino acids in length, or about 5, 10, 15, 20, 25, 30, 35, or 40 amino acids in length. Single chain variable fragments do not contain the constant Fc region found in intact antibody molecules, and therefore do not contain common binding sites (e.g., protein G) used in antibody purification. The term further includes scFvs that are intracellular antibodies, antibodies that are stable in the cytoplasm of a cell and can bind to intracellular proteins.

[0372] The term "single domain antibody" refers to an antibody fragment having a single monomeric variable antibody domain capable of selectively binding to a specific antigen. Single domain antibodies are peptide chains, typically about 110 amino acids long, containing one variable domain (VH) of a heavy chain antibody or common IgG, which generally have similar affinity for antigen as whole antibodies, but are more heat-resistant and stable to detergents and high concentrations of urea. Examples are antibodies derived from camel or fish antibodies. Alternatively, single domain antibodies can be generated from conventional mouse IgG or human IgG, which have four chains.

[0373] As used herein, the terms "specifically bind" and "specific binding" refer to the ability of an antibody, antibody fragment, or nanobody to preferentially bind to a particular antigen present in a homogenous mixture of different antigens. In some embodiments, the specific binding interaction distinguishes between desired and undesired antigens in a sample. In some embodiments, the distinction is greater than about 10-fold to 100-fold or greater (e.g., greater than about 1000-fold or 10,000-fold). "Specificity" refers to the ability of an immunoglobulin or immunoglobulin fragment, such as a nanobody, to preferentially bind to one antigen target versus a different antigen target, and does not necessarily imply high affinity.

[0374] A "target site" or "target sequence" is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, assuming sufficient conditions for binding exist.

[0375] The term "nucleic acid" or "oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked to each other. The representation of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid may also encompass the complementary strand of the single strand represented. The nucleic acids of the present disclosure also encompass substantially identical nucleic acids and their complements that retain the same structure or code for the same protein.

[0376] A probe of the present disclosure may comprise a single-stranded nucleic acid capable of hybridizing to a target sequence under stringent hybridization conditions. Thus, a nucleic acid of the present disclosure may refer to a probe that hybridizes under stringent hybridization conditions.

[0377] The nucleic acids of the present disclosure may be single-stranded or double-stranded. The nucleic acids of the present disclosure may be predominantly single-stranded or may contain double-stranded sequences. The nucleic acids of the present disclosure may be predominantly double-stranded or may contain single-stranded sequences. The nucleic acids of the present disclosure may include genomic DNA, cDNA, RNA, or hybrids thereof. The nucleic acids of the present disclosure may include combinations of deoxyribonucleotides and ribonucleotides. The nucleic acids of the present disclosure may include combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. The nucleic acids of the present disclosure may be synthesized to include non-natural amino acid modifications. The nucleic acids of the present disclosure may be produced by chemical synthesis or recombinant methods.

[0378] The nucleic acids of the present disclosure, i.e., either their entire sequence or any part thereof, may be of non-natural origin. The nucleic acids of the present disclosure may contain one or more mutations, substitutions, deletions, or insertions that are not naturally occurring, making the entire nucleic acid sequence of non-natural origin. The nucleic acids of the present disclosure may contain one or more duplicated sequences, inverted sequences, or repeated sequences, the resulting sequences of which are not naturally occurring, making the entire nucleic acid sequence of non-natural origin. The nucleic acids of the present disclosure may contain modified nucleotides, artificial nucleotides, or synthetic nucleotides that are not naturally occurring, making the entire nucleic acid sequence of non-natural origin.

[0379] Given the redundancy in the genetic code, multiple nucleotide sequences may encode any particular protein, all of which are contemplated herein.

[0380] The term "operably linked" as used throughout this disclosure refers to the expression of a gene under the control of a promoter to which it is spatially linked. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which it is derived. Variation in the distance between the promoter and the gene may be accommodated without loss of promoter function.

[0381] The term "promoter" as used throughout this disclosure refers to a synthetic or naturally derived molecule that can confer, activate, or enhance expression of a nucleic acid in a cell. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. A promoter may also contain distal enhancer or repressor elements that can be located as far as several thousand base pairs from the start site of transcription. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters can constitutively or specifically regulate expression of genetic components with respect to the cell, tissue or organ in which expression occurs, or with respect to the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include a bacteriophage T7 promoter, a bacteriophage T3 promoter, an SP6 promoter, a lac operator promoter, a tac promoter, an SV40 late promoter, an SV40 early promoter, an RSV-LTR promoter, a CMV IE promoter, an EF-1α promoter, a CAG promoter, an SV40 early promoter or an SV40 late promoter, and a CMV IE promoter.

[0382] The term "substantially complementary" as used throughout this disclosure refers to a first sequence that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids, or where the two sequences hybridize under stringent hybridization conditions.

[0383] The term "substantially identical" as used throughout this disclosure refers to a first and second sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity 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 to nucleic acids, where the first sequence is substantially complementary to the complement of the second sequence.

[0384] The term "variant" as used to describe nucleic acids throughout this disclosure refers to: (i) a portion or fragment of a reference nucleotide sequence; (ii) a complement of a reference nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to a reference nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions to a reference nucleic acid, its complement, or a sequence substantially identical thereto.

[0385] The term "vector" used throughout this disclosure refers to a nucleic acid sequence that includes an origin of replication. A vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector may be a DNA vector or an RNA vector. A vector may be a self-replicating extrachromosomal vector, preferably a DNA plasmid. A vector may include a combination of amino acids and DNA sequences, RNA sequences, or both DNA and RNA sequences.

[0386] The term "variant" as used throughout this disclosure to describe a peptide or polypeptide refers to a peptide or polypeptide that differs in amino acid sequence by amino acid insertion, deletion, or conservative substitution, but retains at least one biological activity. Variant may also refer to a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity.

[0387] Conservative substitutions of amino acids, i.e., replacement of an amino acid with a different amino acid with similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are recognized in the art as typically involving minor changes. These minor changes can be identified in part by considering the hydrophobicity / hydrophilicity index of the amino acid, as is known in the art. See Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydrophobicity / hydrophilicity index of an amino acid is based on considering its hydrophobicity and charge. Amino acids with similar hydrophobicity / hydrophilicity indexes can be substituted and still retain protein function. In one embodiment, amino acids with hydrophobicity / hydrophilicity indexes of ±2 are substituted. The hydrophilicity of an amino acid can be used to clearly indicate substitutions that result in a protein that retains biological function. Considering the hydrophilicity of an amino acid in the context of a peptide allows for calculation of the maximum local average hydrophilicity of the peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. See U.S. Patent No. 4,554,101, which is incorporated herein by reference in its entirety.

[0388] Substitution of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, such as immunogenicity. Substitutions can be made with amino acids with hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with this observation, it has been found that amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids, and in particular on the side chains of those amino acids, as indicated by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0389] "Conservative" amino acid substitutions as used herein may be defined as shown in Tables A, B, or C below. In some embodiments, the fusion polypeptide and / or the nucleic acid encoding the fusion polypeptide comprises conservative substitutions introduced by modification of the polynucleotide encoding the polypeptide of the present disclosure. Amino acids may be classified according to their physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is the replacement of one amino acid with another amino acid with similar properties. Examples of conservative substitutions are shown in Table A.

[0390] [Table 1]

[0391] Alternatively, conservative amino acids can be grouped into the groups described by Lehninger (Biochemistry, 2nd ed.; Worth Publishers, Inc. NY, NY (1975), pp. 71-77), which are set forth in Table B.

[0392] [Table 2]

[0393] Alternatively, examples of conservative substitutions are shown in Table C.

[0394] [Table 3]

[0395] Of course, the polypeptides of the present disclosure are intended to include polypeptides having one or more insertions, deletions, or substitutions of amino acid residues, or any combination thereof, as well as modifications of amino acid residues other than insertions, deletions, or substitutions. The polypeptides or nucleic acids of the present disclosure may also include one or more conservative substitutions.

[0396] As used throughout this disclosure, the term "two or more" 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, 20, or more of the listed amino acid substitutions. The term "two or more" may also refer to 2, 3, 4, or 5 of the listed amino acid substitutions.

[0397] The polypeptides and proteins of the present disclosure may be of non-natural origin, either in their entirety or in any part thereof. The polypeptides and proteins of the present disclosure may contain one or more mutations, substitutions, deletions, or insertions that are not naturally occurring, making the entire amino acid sequence of non-natural origin. The polypeptides and proteins of the present disclosure may contain one or more duplicated, inverted, or repeated sequences, resulting in a sequence that is not naturally occurring, making the entire amino acid sequence of non-natural origin. The polypeptides and proteins of the present disclosure may contain modified, artificial, or synthetic amino acids that are not naturally occurring, making the entire amino acid sequence of non-natural origin.

[0398] "Sequence identity" as used throughout this disclosure may be determined using a self-executable BLAST engine program (bl2seq) for resolving two sequences, which can be retrieved from the National Center for Biotechnology Information (NCBI) FTP site using default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; incorporated herein by reference in its entirety). The term "identical" or "identity" when used in the context of two or more nucleic acid or polypeptide sequences refers to a certain percentage of residues that are identical over a particular region of each sequence. This percentage is determined by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions at which identical residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to calculate the percentage of sequence identity. If the two sequences are of different lengths, or the alignment produces one or more staggered ends, and only a single sequence is included in the designated comparison region, the residues of the single sequence are included in the numerator but not the denominator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity determinations may be performed manually or using computer sequence algorithms such as BLAST or BLAST 2.0.

[0399] The term "endogenous" as used throughout this disclosure refers to a nucleic acid or protein sequence that is naturally associated with the target gene or host cell into which it is introduced.

[0400] The term "exogenous" as used throughout this disclosure refers to a nucleic acid or protein sequence that is not naturally associated with a target gene or host cell into which it is introduced, and includes naturally occurring nucleic acids, e.g., non-naturally occurring multiple copies of a DNA sequence, or a naturally occurring nucleic acid sequence located in a non-naturally occurring genomic location.

[0401] The present disclosure provides a method for introducing a polynucleotide construct comprising a DNA sequence into a host cell. By "introducing" it is intended to present the polynucleotide construct to the cell so that the construct can gain access to the interior of the host cell. The method of the present disclosure does not depend on a particular method for introducing a polynucleotide construct into a host cell, only on the method by which the polynucleotide construct gains access to the interior of one of the host's cells. Methods for introducing polynucleotide constructs into bacteria, plants, fungi, and animals are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. EXAMPLES

[0402] Example 1: Improved transposition with PiggyBac nanotransposon

[0403] The piggyBac transposon plasmid backbone was truncated to evaluate the effect of shortening the distance between the ITRs on the transposition efficiency into the genome of human pan-T cells. A full-length piggyBac plasmid (FP) (Figure 1), piggyBac nanotransposon (NT) (Figure 1), and piggyBac short nanotransposon (NTS) (Figure 3) were constructed, each containing a transposon encoding GFP. The FP backbone encoded a bacterial pUC origin of replication as well as Kan / Neo resistance genes. The NT backbone encoded an antibiotic-free, sucrose-selectable nanoplasmid backbone containing an RNA-OUT element as well as an R6K mini origin of replication. The NTS differed from the NT in that the RNA-OUT element and the R6K mini origin of replication of the backbone were placed inside the transposon element, thereby further shortening the distance between the ITRs. Figure 1 shows the differences between the full-length plasmid and the NT nanotransposon. The size of the transposon remained constant (3,606 bp), but the distance between adjacent ITRs was effectively truncated four-fold from 2,034 bp to 493 bp, making it shorter in the NT scaffold, as detailed in Table 1. Figure 3 shows the differences between piggyBac NT and piggyBac NTS: the size of the transposon increased from 3,614 bp to 4,069 bp (to incorporate the RNA-OUT and R6K sequences), but the distance between adjacent ITRs was effectively truncated ten-fold from 485 bp to 48 bp, making it shorter in the NTS scaffold, as detailed in Table 2.

[0404] [Table 4]

[0405] [Table 5]

[0406] piggyBac FP or piggyBac NT was delivered by electroporation (EP) into human pan-T cells either with or without mRNA encoding Super piggyBac transposase (SPB) (Figure 2). Furthermore, FP and NT were delivered to cells in equimolar or equal mass amounts. Following EP, cells were stimulated with standard TCR activation reagents and GFP expression was assessed by FACS 15 days later. These data show that truncating the distance between adjacent ITRs by a factor of four (from 2,034 bp to 493 bp) resulted in higher levels of GFP transposition at both equimolar and equal mass amounts compared to the FP GFP transposon plasmid. Furthermore, GFP expression was not detected in T cells electroporated in the absence of SPB, suggesting that GFP expression is the result of stable integration of the transposon.

[0407] Figures 1 and 2 show that truncating the piggyBac transposon plasmid backbone increased the efficiency of transposition into human pan T cells. However, because the total size of the plasmid is not identical between FP and NT, it remains unclear whether the improved transposition efficiency with NT is due to a smaller plasmid (equimolar; less DNA delivered to the cell), a greater total amount of plasmid delivered (equivalent mass), or a shorter distance between adjacent piggyBac ITRs. Because DNA delivered to human pan T cells can induce immunomodulatory effects and exhibit toxicity, the improved transposition efficiency with NT could be the result of either a smaller total amount of DNA delivered (equimolar) or a greater amount of plasmid delivered (equivalent mass). To clarify this, an NTS was constructed such that the nanoplasmid backbone was relocated within the transposon and placed between the insulator and the ITR (Figure 3). The sizes of NT and NTS remained constant (4,099 bp and 4,117 bp), but the distance between adjacent ITRs in the NTS was shortened by 10-fold (from 485 bp to 48 bp) (Table 2). Equimolar / mass amounts of NT or NTS were delivered by electroporation (EP) together with mRNA encoding SPB into human pan-T cells. Following EP, cells were stimulated with standard TCR activation reagents and GFP expression was assessed by FACS 15 days later. These data indicate that truncating the distance between adjacent ITRs by 10-fold (from 485 bp to 48 bp) while keeping the total plasmid size constant resulted in higher levels of GFP transposition by the NTS compared to the GFP nanotransposon in the NT (Figure 4).

[0408] Example 2: Transposition of BCMA CAR and PSMA CAR nanotransposons

[0409] Anti-BCMA CAR and anti-PSMA CAR encoding full-length piggyBac plasmid (FP) or piggyBac nanotransposon (NT) were delivered to human pan T cells at equal masses by electroporation (EP) together with mRNA encoding super piggyBac transferase (Figure 5). Following EP, cells were stimulated with standard TCR activation reagents in the absence of selection reagents and CAR expression was assessed by FACS 5 days later. These data show that compared to FP transposon plasmid, NT in both conditions resulted in higher levels of transfer at equal masses. This result was the same when CAR-T cells were generated from human pan T cells from two different normal donors. Anti-BCMA CAR cells and anti-PSMA CAR cells generated with either full-length piggyBac plasmid (FP) or piggyBac nanotransposon (NT) were produced as described herein. Killing rates of K562 cells engineered to express either BCMA (K562.BCMA) or PSMA (K562.PSMA) by CAR-T cells at the indicated effector-to-target ratios are shown (Figure 6). These data indicate that all CAR-T cells, whether generated with FP or NT, were able to kill target tumor cells in an antigen-dependent manner. This result was the same for CAR-T cells generated from human pan-T cells from two different normal donors. Figure 7 is a series of graphs showing that human CAR-T cells generated with anti-BCMA CAR or anti-PSMA CAR nanotransposon (NT) had comparable phenotypic composition. Anti-BCMA CAR and anti-PSMA CAR T cells generated with either full-length piggyBac plasmid (FP) or piggyBac nanotransposon (NT) were generated as described herein. Phenotypic analysis of memory T cell markers and activation / exhaustion markers was performed (data not shown).These data show that all CAR-T cells, whether generated with FP or NT, displayed identical phenotypic composition of CD45RA+CD62L+(Tscm), CD45RA-CD62L+(Tcm), CD45RA-CD62L-(Tem), and CD45RA+CD62L-(Teff) cells. Furthermore, comparable levels of expression of CCR7 (CD197), CD127, CD27, LAG3, TIM3, CXCR3, PD-1, and CD25 were observed (data not shown). This result was the same for CAR-T cells generated from human pan-T cells from two different normal donors. The average copy number of integrated transposon was measured by quantitative PCR. These data show that all CAR-T cells, whether generated with FP or NT, from two different donors, displayed similar integrated copy numbers of transposon (Figure 8).

[0410] Anti-BCMA CAR piggyBac nanotransposons of different monomeric purity were generated by mixing a high concentration multimeric lot (7% monomer) with a high concentration monomeric lot (87% monomer) in different ratios. Both lots were confirmed by gene sequencing to be identical at the primary level in monomeric or multimeric structure and differ only at the tertiary level. Each new lot of mixed anti-BCMA CAR NT was run on an agarose gel in the absence of restriction digestion to obtain each ratio of monomeric to multimeric nanotransposons, i.e. lots of different monomeric purities (7%, 32%, 45%, 59%, 65%, 72%, and 87%) were generated. On the gel surface, multimeric NTs migrated slower than monomeric NTs (Figure 9). The gel bands shown in Figure 9 are enclosed in rectangles to indicate multimeric (top) and monomeric (bottom) nanotransposons, and the numbering of the rectangles, going from top to bottom and left to right, is 1 (multimeric) and 2 (monomeric) [7% monomer purity], 3 and 4 [32% monomer purity], 5 and 6 [45% monomer purity], 7 and 8 [59% monomer purity], 9 and 10 [65% monomer purity], 11 and 12 [72% monomer purity], 13 and 14 [no lot], and 15 and 16 [87% monomer purity].

[0411] Anti-BCMA CAR piggyBac NT of different monomeric purity was delivered into human pan-T cells by electroporation (EP) together with mRNA encoding Super piggyBac transferase (Figure 10). As a control, full-length anti-BCMA CAR plasmid (FP) of 94% monomeric purity was also delivered in equimolar amounts. Following EP, cells were stimulated with standard TCR activation reagents in the absence of selection reagent, and CAR expression was assessed by FACS 5 days later. These data show that in two separate donors (donor #3 and donor #2), monomeric purity positively impacts transposition efficiency. Furthermore, these data show that NT resulted in higher levels of transposition when compared to equimolar amounts of FP transposon plasmid.

[0412] Example 3: Preclinical evaluation of P-PSMA-101 nanotransposon

[0413] A mouse xenograft model was used to evaluate the efficacy of the P-PSMA-101 transposon when delivered with a "stress" dose of full-length plasmid (FLP) versus nanotransposon (NT) in a preclinical setting. Utilizing a mouse xenograft model using a luciferase-expressing LNCaP cell line (LNCaP.luc) injected subcutaneously (SC) into NSG mice, the in vivo antitumor efficacy of the P-PSMA-101 transposon was evaluated in vivo when delivered by full-length plasmid (FLP) or nanotransposon (NT) in a "stress" dose (2.5x10) of two different total CAR-T cells from two different normal donors. 6 or 4x10 6 ) (Figure 11). All CAR-T cells were generated using piggyBac (PB) delivery of the P-PSMA-101 transposon using either FLP or NT delivery. Mice were injected with LNCaP under the armpits and tumors formed (100-200 mm by caliper measurement). 3 ) Mice were treated with two different "stress" doses (2.5x10) to increase resolution in detecting functional differences in efficacy between FLP and NT transposon delivery. 6 or 4x10 6 ) P-PSMA-101 CAR-T by intravenous injection. Tumor volume assessed by caliper measurements in control mice (black), donor #1 FLP mice (red), donor #1 NT mice (blue), donor #2 FLP mice (orange), and donor #2 NT mice (green) are shown as means with error bars for the group (top) and individual mice (bottom) (Figure 12). The y-axis shows tumor volume (mm) assessed by caliper measurements. 3 ) are shown. The x-axis indicates days after treatment with T cells. NT, or "stress" doses of P-PSMA-101 transposon delivered demonstrated improved antitumor efficacy against established SC LNCaP.luc solid tumors by caliper measurements versus FLP and control mice.

Claims

1. below: a first nucleic acid sequence comprising (a) a first inverted terminal repeat (ITR), (b) a second ITR, and (c) an inter-ITR sequence, the inter-ITR sequence comprising a transposon sequence; and a second nucleic acid sequence comprising an inter-ITR sequence, wherein the length of said inter-ITR sequence is 1-600 nucleotides; A composition comprising a polynucleotide having the following structure:

2. The composition of claim 1, wherein the inter-ITR sequence is 1 to 100 nucleotides in length.

3. The composition of claim 1 or 2, wherein the first nucleic acid sequence or the second nucleic acid sequence further comprises an origin of replication sequence.

4. The composition of claim 3, wherein the length of the origin of replication sequence is between 1 and 450 nucleotides.

5. 5. The composition of claim 3, wherein the origin of replication sequence comprises an R6K origin of replication.

6. The composition of any one of claims 1 to 5, wherein the first nucleic acid or the second nucleic acid sequence further comprises a sequence encoding a first selectable marker.

7. The composition of claim 6, wherein the first selection marker is 1 to 200 nucleotides in length.

8. 8. The composition of claim 6 or 7, optionally wherein the first selection marker is a sucrose selection marker.

9. The composition of claim 8, wherein the sucrose selection marker is an RNA-OUT selection marker.

10. 10. The composition of any one of claims 1 to 9, wherein the first nucleic acid sequence or the second nucleic acid sequence does not contain any recombination sites, excision sites, ligation sites, or combinations thereof, or the first nucleic acid sequence or the second nucleic acid sequence does not contain any sequence encoding foreign DNA.

11. The composition of any one of claims 1 to 10, wherein the first nucleic acid sequence further comprises at least one exogenous sequence and a sequence encoding a promoter capable of expressing the exogenous sequence in a mammalian cell.

12. The composition of claim 11 , wherein the first nucleic acid sequence further comprises at least one sequence encoding an insulator and / or the first nucleic acid sequence further comprises a polyadenosine (polyA) sequence.

13. The composition of claim 11, wherein the sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell is capable of expressing an exogenous sequence in a human cell.

14. The composition of claim 13 , wherein the promoter is a constitutive promoter or an inducible promoter.

15. The composition of claim 11 , wherein the at least one exogenous sequence comprises a sequence encoding a non-naturally occurring antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof.

16. The composition of claim 15, wherein the non-naturally occurring antigen receptor comprises a chimeric antigen receptor (CAR).

17. The composition according to any one of claims 1 to 16, wherein the composition is a transposon.

18. 18. The composition of claim 17, wherein the transposon is a piggyBac transposon.

19. A polynucleotide comprising a nucleic acid sequence encoding the composition of any one of claims 1 to 18.

20. A cell comprising the composition according to any one of claims 1 to 18.

21. 17. A population of cells, wherein a plurality of said population of cells are engineered to express the composition of claim 16, and at least 50% of the plurality of engineered T cells express a CAR and express one or more cell surface markers comprising CD45RA and CD62L, and do not express one or more cell surface markers comprising CD45RO.

22. A pharmaceutical composition comprising the composition of any one of claims 1 to 18 and a pharma- ceutically acceptable carrier.

23. A composition according to any one of claims 1 to 18, or a pharmaceutical composition according to claim 22, for use in the treatment of cancer in a subject in need thereof.

24. 24. The composition of claim 23, wherein the cancer is a BCMA positive cancer or a PSMA positive cancer, or the cancer is a primary tumor, a metastatic cancer, a multidrug resistant cancer, an aggressive tumor, or a recurrent cancer, or the cancer is lung cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, liver cancer, pancreatic cancer, gastric cancer, colon cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, skin cancer, esophageal cancer, lymphoma, leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, acute myeloid leukemia (AML), acute myeloid leukemia, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), Hodgkin's disease, non-Hodgkin's lymphoma, or multiple myeloma.

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