Anti-Muc1 Compositions and Methods of Use

Abstract

Disclosed are antibodies to MUC1, MUC1-CAR compositions, and methods of using these antibodies and compositions to target the MUC1 protein, where cells expressing the MUC1 protein can be targeted and killed, for example, by cytotoxic T cells.

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 951,257, filed December 20, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION The present disclosure is directed to molecular biology, and more particularly to antibodies and chimeric antigen receptors that specifically bind to target proteins with high affinity and avidity.

[0003] Incorporation by Reference of Sequence Listing "POTH-040" was created on December 15, 2020 and is 412KB in size. 001WO Sequence Listing The contents of the file "ST25" are incorporated herein by reference in their entirety. [Background technology]

[0004] The discovery of substances that can recognize and bind to specific target proteins with high affinity and avidity has been a focus of the biopharmaceutical industry. There remains a need for more effective drugs that are smaller, more soluble, and more stable than available options. Summary of the Invention

[0005] The present disclosure provides an isolated antibody comprising a heavy chain variable region comprising the following amino acid sequence:

[0006] [ka]

[0007] wherein X1 of SEQ ID NO:1 is V or I, X2 of SEQ ID NO:1 is R or K, X3 of SEQ ID NO:1 is A or R, X4 of SEQ ID NO:1 is G or A, X5 of SEQ ID NO:1 is V or A, X6 of SEQ ID NO:1 is T or S, and X7 of SEQ ID NO:1 is D or A; and the light chain variable region comprises the following amino acid sequence:

[0008] [ka]

[0009] Here, X1 of SEQ ID NO: 2 is T or S, X2 of SEQ ID NO: 2 is L or V, and X3 of SEQ ID NO: 2 is T or D.

[0010] The isolated antibody can be humanized. The isolated antibody can be an IgG. The isolated antibody can bind to human MUC1-C, and the isolated antibody can be a monoclonal antibody, a chimeric antibody, a single domain antibody, a VHH, a VH, a single-chain variable fragment (scFv), a Fab, or a Fab fragment. Preferably, the isolated antibody is an scFv.

[0011] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. The light chain variable region comprises the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0012] The scFv may comprise a linker between the heavy chain variable region and the light chain variable region. In one embodiment, the linker comprises the amino acid sequence of SEQ ID NO:59.

[0013] The scFv can comprise SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140 or SEQ ID NO: 141. In one embodiment, the scFv comprises the amino acid sequence of SEQ ID NO: 125.

[0014] The present disclosure also provides a chimeric antigen receptor (CAR) comprising the antibody disclosed herein. Preferably, the CAR comprises an scFv disclosed herein. The CAR can comprise: (a) an ectodomain comprising an antigen-recognition region, the antigen-recognition region comprising at least one anti-MUC1 single-chain variable fragment (scFv); (b) a transmembrane domain; and (c) an endodomain comprising at least one costimulatory domain, wherein the scFv comprises a heavy chain variable region comprising the amino acid sequence:

[0015] [ka]

[0016] wherein X1 of SEQ ID NO: 1 is V or I, X2 of SEQ ID NO: 1 is R or K, X3 of SEQ ID NO: 1 is A or R, X4 of SEQ ID NO: 1 is G or A, X5 of SEQ ID NO: 1 is V or A, X6 of SEQ ID NO: 1 is T or S, and X7 of SEQ ID NO: 1 is D or A; and a light chain variable region comprising the following amino acid sequence:

[0017] [ka]

[0018] Here, X1 of SEQ ID NO: 2 is T or S, X2 of SEQ ID NO: 2 is L or V, and X3 of SEQ ID NO: 2 is T or D.

[0019] The heavy chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. The light chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0020] The heavy chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:3, and the light chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:9. The heavy chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:4, and the light chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:9. The heavy chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:5, and the light chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:9. The heavy chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:6, and the light chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:9. The heavy chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:7, and the light chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:9. The heavy chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:8, and the light chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:9. The heavy chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:3, and the light chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:10. The heavy chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:4, and the light chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:10. The heavy chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:5, and the light chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:10. The heavy chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:6, and the light chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:10. The heavy chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:7, and the light chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:10. The heavy chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:8, and the light chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:10. The heavy chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:3, and the light chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:11. The heavy chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:4, and the light chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:11. The heavy chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:5, and the light chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO:11.The heavy chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO: 6, and the light chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO: 11. The heavy chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO: 7, and the light chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO: 11. The heavy chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO: 8, and the light chain variable region of the CAR can comprise the amino acid sequence of SEQ ID NO: 11.

[0021] The scFv can comprise a linker between the heavy chain variable region and the light chain variable region. Preferably, the linker comprises the amino acid sequence of SEQ ID NO:59. The scFv can comprise SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, or SEQ ID NO:141. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO:125. The ectodomain can further comprise a signal peptide. Preferably, the signal peptide comprises the amino acid sequence of SEQ ID NO:57. The CAR can further comprise a hinge region between the antigen recognition region and the transmembrane domain. Preferably, the hinge region comprises the amino acid sequence of SEQ ID NO:61. The transmembrane domain can comprise a sequence encoding a CD8 transmembrane domain. Preferably, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO:63. At least one costimulatory domain can comprise a CD3ζ costimulatory domain, a 4-1BB costimulatory domain, or a combination thereof. In one embodiment, the at least one costimulatory domain comprises a CD3ζ costimulatory domain and a 4-1BB costimulatory domain, wherein the 4-1BB costimulatory domain is located between the transmembrane domain and the CD3ζ costimulatory domain. Preferably, the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 65. Preferably, the CD3ζ costimulatory domain comprises the amino acid sequence of SEQ ID NO: 67.

[0022] The present disclosure also provides a chimeric antigen receptor (CAR) comprising: (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one anti-MUC1 single-chain variable fragment (scFv); (b) a transmembrane domain; and (c) an endodomain comprising at least one costimulatory domain, wherein the scFv comprises a heavy chain variable region comprising the following amino acid sequence:

[0023] [ka]

[0024] wherein X1 of SEQ ID NO: 1 is V or I, X2 of SEQ ID NO: 1 is R or K, X3 of SEQ ID NO: 1 is A or R, X4 of SEQ ID NO: 1 is G or A, X5 of SEQ ID NO: 1 is V or A, X6 of SEQ ID NO: 1 is T or S, and X7 of SEQ ID NO: 1 is D or A; and a light chain variable region comprising the following amino acid sequence:

[0025] [ka]

[0026] wherein X1 in SEQ ID NO: 2 is T or S, X2 in SEQ ID NO: 2 is L or V, and X3 in SEQ ID NO: 2 is T or D; the scFv comprises a linker between the heavy chain variable region and the light chain variable region; the ectodomain comprises a signal peptide; the CAR further comprises a hinge region between the antigen recognition region and the transmembrane domain; the transmembrane domain comprises a sequence comprising a CD8 transmembrane domain; and the at least one costimulatory domain comprises a CD3ζ costimulatory domain and a 4-1BB costimulatory domain, and the 4-1BB costimulatory domain is located between the transmembrane domain and the CD3ζ costimulatory domain.

[0027] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 125, the signal peptide comprises SEQ ID NO: 57, the hinge region comprises SEQ ID NO: 61, the CD8 transmembrane domain comprises SEQ ID NO: 63, the 4-1BB costimulatory domain comprises SEQ ID NO: 65, and the CD3ζ costimulatory domain comprises SEQ ID NO: 67.

[0028] In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the amino acid sequence of the CAR is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 167. In some embodiments, the amino acid sequence of the CAR is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 167.

[0029] The present disclosure also provides polynucleotides comprising a polynucleotide comprising a nucleic acid sequence encoding an antibody disclosed herein; a polynucleotide comprising a nucleic acid sequence encoding an scFv disclosed herein; and / or a polynucleotide comprising a nucleic acid sequence encoding a CAR disclosed herein.

[0030] The present disclosure also provides a transposon comprising a nucleic acid encoding a CAR disclosed herein. In some embodiments, the nucleic acid sequence comprises a CAR comprising the amino acid sequence of SEQ ID NO: 13. The transposon can further comprise a nucleic acid encoding an inducible caspase polypeptide, a nucleic acid encoding a chimeric stimulatory receptor, a nucleic acid encoding a selection gene, a nucleic acid encoding a therapeutic agent, or a combination thereof. The selection gene can comprise a DHFR resistance gene. Preferably, the transposon is a piggyBac transposon. In some embodiments, the transposon comprises the nucleic acid sequence of SEQ ID NO: 172. The present disclosure also provides a plasmid or vector comprising any of the polynucleotides disclosed herein or any of the transposons disclosed herein.

[0031] The present disclosure also provides cells comprising any of the antibodies, scFvs, CARs, or transposons disclosed herein. The present disclosure also provides cell populations, wherein a plurality of the populations are modified to express any of the antibodies, scFvs, CARs, or transposons as 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 some embodiments, the plurality of cell populations comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of cells expressing the CAR. In one embodiment, the CAR comprises the amino acid sequence of SEQ ID NO: 13. 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.

[0032] The present disclosure also provides a composition comprising any of the antibodies, scFvs, CARs, transposons, cells, or cell populations disclosed herein. In one aspect, the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier.

[0033] The present disclosure also provides methods of treating a proliferative disorder in a subject in need thereof by administering a therapeutically effective amount of any of the antibodies, scFvs, CARs, transposons, cells, cell populations, compositions, or pharmaceutical compositions as disclosed herein. In one embodiment, the proliferative disorder is cancer. In certain embodiments, the cancer can be a MUC1-positive cancer. In one embodiment, the cancer is a MUC1-C-positive cancer. The cancer can be a primary tumor, a metastatic cancer, a multidrug-resistant cancer, an advanced tumor, or a recurrent cancer. The cancer can be a solid tumor. The cancer can be 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, or esophageal cancer.

[0034] The patent or application file will contain at least one drawing executed in color. Copies of this patent or patent application publication and any color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0035] [Figure 1A] Figures 1A-1B are a pair of schematic diagrams showing the structure of the MUC1 heterodimer. Figure 1A shows MUC1 undergoing autoproteolysis at the SEA domain (sea urchin sperm protein, enterokinase, and agrin domains) to generate two subunits that form a stable noncovalent heterodimer. The nomenclature MUC1-N and MUC1-C is used to designate the position of the subunits after cleavage and to distinguish them from genetic isoforms, which are subclassified by the Greek phenotype. [Figure 1B]Figure 1B shows details of the MUC1-C subunit. The 58-amino acid extracellular domain of MUC1-C is glycosylated at asparagine 36, the N36LT site. The amino acid sequence of the C-terminal extracellular domain of MUC1-C (MUC1-C / ECD) is shown (SEQ ID NO: 77). The 72-amino acid cytoplasmic domain of MUC1-C is sufficient to interact with multiple effectors and induce oncogenic transformation. Figures 1A-1B are reproduced from Kufe DW, Oncogene, 32(9):1073. [Figure 2A] FIG. 2A is a pair of schematic diagrams showing ribbon structures of either full-length MUC1 (PDB: 2ACM) or the predicted structure of the MUC1-C domain. [Figure 2B] FIG. 2B is a series of graphs showing MUC1 expression in different cell types. [Figure 3] FIG. 3 is a series of flow cytometry graphs showing Muc1 expression in different cancer cell lines. [Figure 4] Figure 4 is a schematic diagram showing an exemplary construction of a humanized MUC1-C chimeric antigen receptor (CAR). Heavy chain (4 + 2 variants (H1B and H2B)) and light chain (3) sequences were assembled in different light chain combinations to construct 14 novel candidate humanized MUC1-C CARs. The following humanized MUC1-C CAR structure was used: signal peptide (CD8α)-light chain-linker-heavy chain-hinge (CD8α)-transmembrane (CD8α)-intracellular signaling (4-1BB)-intracellular signaling (CD3ζ). [Figure 5A] FIG. 5A shows an amino acid sequence alignment of the heavy and light chain variable regions of humanized anti-MUC-1C antibodies of the present disclosure. [Figure 5B] 5B shows an amino acid sequence alignment of the heavy and light chain variable regions of humanized anti-MUC-1C antibodies of the disclosure. [Figure 6]Figure 6 is a schematic diagram of the disclosed piggyBac nanotransposon containing the MUC1-C CAR. The MUC1-C CAR was subcloned into a tricistronic piggyBac transposon. The transposon contains the EF1α promoter-iC9 safety switch-T2A-MUC1-C CAR (L1-linker-H1B scFv)-T2A-DHFR selection gene construct. [Figure 7] Figure 7 is a series of flow cytometry graphs showing that candidate MUC1-C CARs are expressed on the surface of T cells. All candidate MUC1-C CARs were expressed and detected on the surface of piggyBac-producing CAR-T cells. [Figure 8] Figure 8 is a graph showing specific killing of MUC1+ target cells by candidate MUC1-C CAR T cells. Killing of MDA-MB-468 is shown for each CAR in a line graph. All CAR+ T cells exhibited specific killing of MUC1+ MDA-MB-468 cells. Mock T cells did not specifically kill target cells (blue series). [Figure 9] Figure 9 is a graph showing the specific killing of MUC1+ target cells by candidate MUC1-C CAR T cells. Killing of each cell line is shown for each CAR in a bar graph, and error bars represent the standard deviation of samples performed in triplicate. All CAR+ T cells expressed specific killing of the MUC1+ engineered Raji cell line, but not MUC1-Raji cells. Mock transposon T cells did not kill either the Raji or engineered Raji target cell line. [Figure 10] Figure 10 is a schematic diagram of the study design for preclinical evaluation of candidate humanized MUC1-C CAR-T cells at a "stress" dose using a mouse xenograft model. DETAILED DESCRIPTION OF THE INVENTION

[0036] Unless expressly excluded or otherwise limited, all documents cited herein are incorporated by reference for all purposes. The citation of a document is not an admission that it is prior art with respect to any invention disclosed or claimed herein, or that it alone, or in any combination with other references or references, teaches, suggests, or discloses such invention. Further, to the extent 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.

[0037] Detailed Description of the Invention The present disclosure provides compositions and methods for using these compositions to recognize and bind with high affinity and high affinity to a specific target protein, mucin 1, cell surface-associated (MUC1) protein.

[0038] MUC1 MUC1 is an extensively O-glycosylated mucin protein expressed primarily by epithelial cells. Secreted and membrane-bound, MUC1 forms a physical barrier that protects the apical border of epithelial cells from damage induced by toxins, microorganisms, and other stresses that occur at the interface with the external environment. Aberrant overexpression of MUC1, as observed in most human carcinomas, confers anchorage-independent growth and tumorigenicity. Overexpression of MUC1 confers resistance to apoptosis induced by oxidative stress and genotoxic anticancer drugs.

[0039] Human MUC1 is a heterodimeric glycoprotein that is translated as a single polypeptide and cleaved in the endoplasmic reticulum into N- and C-terminal subunits (MUC1-N and MUC1-C). This cleavage may be mediated by an autocatalytic process. The >250 kDa MUC1 N-terminal (MUC1 N-ter or MUC1-N) subunit is incomplete with highly conserved variations and contains a variable number of tandem repeats of 20 amino acids that are modified by O-linked glycans. MUC1-N is bound to the cell surface by dimerization with the approximately 23 kDa C-terminal subunit (MUC1 C-ter or MUC1-C), which contains a 58-amino acid extracellular region, a 28-amino acid transmembrane domain, and a 72-amino acid cytoplasmic domain (Figure 1B). MUC1-C contains the amino acid sequence of (SEQ ID NO: 76).

[0040] [ka]

[0041] The compositions of the present disclosure can bind to the underlined 58 amino acid portion of MUC1-C / ECD. The bolded sequence represents the CD, and the italicized portion represents the oligomer-inhibiting peptide. When normal epithelium transforms into cancer, MUC1 is abnormally overexpressed throughout the cytoplasm and plasma membrane. Plasma membrane-bound MUC1 is targeted to endosomes via clathrin-mediated endocytosis. Furthermore, MUC1-C targets the nucleus and mitochondria, whereas MUC1-N does not.

[0042] The compositions of the present disclosure can selectively bind to one or more amino acids of the "epitope" MUC1-C / extracellular domain (MUC1-C / ECD). The epitope can be linear or conformational. As used herein, the term "epitope" is meant to refer to one or more amino acids to which the disclosed compositions specifically bind. One or more amino acids of the epitope of the present disclosure can be arranged in a linear, non-linear, contiguous, or discontinuous manner. The epitope of the present disclosure can be "conformational," meaning that it binds with higher affinity or higher selectivity to one or more amino acids of the epitope when the protein backbone is presented in the conformation of a properly folded peptide, protein, or protein complex. In some embodiments, a composition that binds to a conformational epitope may not bind to a linear epitope.

[0043] The compositions of the present disclosure can selectively bind to one or more amino acids of the MUC1-C / extracellular domain (MUC1-C / ECD) defined by the amino acid sequence SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAG (SEQ ID NO: 77) (see FIG. 1B). Alternatively, or in addition, the compositions selectively bind to one or more amino acids of a mutant MUC1-C / extracellular domain (MUC1-C / ECD). Mutant MUC1-C / ECD peptides of the present disclosure include, but are not limited to, MUC1-C / ECD-L6A, MUC1-C / ECD-L8A, MUC1-C / ECD-L6,8A, MUC1-C / ECD-Q23V, MUC1-C / ECD-Q26V, and MUC1-C / ECD-N36A, numbered according to SEQ ID NO: 76 or SEQ ID NO: 77.

[0044] The compositions of the disclosure can selectively bind to one or more amino acids of the following peptides derived from the MUC1-C / extracellular domain (MUC1-C / ECD): SVVVQLTLAFREGTINVHDVET ("Peptide 1," SEQ ID NO: 78), VETQFNQYKTEAASRYNLTISD ("Peptide 2," SEQ ID NO: 79), or TISDVSVSDVPFPFSAQSGAG ("Peptide 3," SEQ ID NO: 80).

[0045] The compositions of the disclosure can selectively bind to the alpha 3 (α3) helix or the alpha 4 (α4) helix in MUC1-C / ECD. In some embodiments, the MUC1-C / ECD comprises the following amino acid sequence:

[0046] [ka]

[0047] The α3 helix is ​​in bold, the α4 helix is ​​in bold and italics. The MUC1-C / ED α3 helix can comprise, essentially consist of, or consist of the following amino acid sequence:

[0048] [ka]

[0049] The MUC1-C / ED α4 helix can comprise, consist essentially of, or consist of the following amino acid sequence:

[0050] [ka]

[0051] The epitope of the compositions of the present disclosure can comprise, consist of, or consist of the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:82. The epitope can be linear or conformational. In some embodiments, the epitope is discontinuous, optionally consisting essentially of, or consisting of, two or more discontinuous amino acids of the amino acid sequence of SEQ ID NO:81 or SEQ ID NO:82.

[0052] Disclosure Structure The present disclosure provides antibodies comprising a heavy chain variable region that consists essentially of or consists of at least 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) of the amino acid sequence.

[0053] [ka]

[0054] wherein X1 of SEQ ID NO:1 is V or I, X2 of SEQ ID NO:1 is R or K, X3 of SEQ ID NO:1 is A or R, X4 of SEQ ID NO:1 is G or X5 of SEQ ID NO:1 is V or A, X6 of SEQ ID NO:1 is T or S, and X7 of SEQ ID NO:1 is D or A; the light chain variable region comprises, consists essentially of, or consists of an amino acid sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical (or any percentage therebetween) to the following sequence:

[0055] [ka]

[0056] Here, X1 of SEQ ID NO: 2 is T or S, X2 of SEQ ID NO: 2 is L or V, and X3 of SEQ ID NO: 2 is T or D.

[0057] Preferably, the heavy variable chain variable region comprises the amino acid sequence of SEQ ID NO: 1, wherein X2 is R or K, X3 is A or R, X4 is G or A, and X5 is V or A. Preferably, the light variable chain variable region comprises the amino acid sequence of SEQ ID NO: 2, wherein X1 is T or S, X2 is L or V, and X3 is T or D.

[0058] In some embodiments, the antibody binds to a human MUC1 polypeptide (UniProt Accession No. P15941-1) consisting of, or consisting of, SEQ ID NO: 160. In one embodiment, the antibody binds to a human MUC1-C polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO: 76. In another embodiment, the antibody binds to a human MUC1-N polypeptide (a subunit of the MUC1 receptor, also known as the alpha chain, mature chain) consisting of, consisting essentially of, or consisting of SEQ ID NO: 161.

[0059] In some embodiments, the antibody binds to a human MUC1-C extracellular domain (ED) consisting essentially of, or consisting of, SEQ ID NO: 77. In some embodiments, the antibody binds to the MUC1-C ED (SEQ ID NO: 77) with greater affinity than to the human full-length MUC1 polypeptide (SEQ ID NO: 160). In some embodiments, the antibody binds to the human MUC1-C ED (SEQ ID NO: 77) with greater affinity than to the MUC1-N polypeptide (SEQ ID NO: 161). In some embodiments, the antibody binds to the MUC1-C ED (SEQ ID NO: 77) but does not bind to the human full-length MUC1 polypeptide (SEQ ID NO: 160). In some embodiments, the antibody binds to the human MUC1-C ED (SEQ ID NO: 77) but does not bind to the MUC1-N polypeptide (SEQ ID NO: 161).

[0060] In some embodiments, the human variable heavy chain framework acceptor comprises, consists essentially of, or consists of the polypeptide of IGHV1-69 08 of SEQ ID NO: 162. In some aspects, the human variable light chain framework acceptor comprises, consists essentially of, or consists of the polypeptide of IGKV6-21 02 of SEQ ID NO: 163.

[0061] The heavy chain variable region comprises a complementarity determining region 1 (CDRH1) comprising SEQ ID NO: 69. The heavy chain variable region comprises a CDRH2 comprising SEQ ID NO: 70 or SEQ ID NO: 75. The heavy chain variable region comprises a CDRH3 comprising SEQ ID NO: 71.

[0062] The light chain variable region comprises complementarity determining region 1 (CDRL1) comprising SEQ ID NO: 72. The light chain variable region comprises CDRL2 comprising SEQ ID NO: 73. The light chain variable region comprises CDRL3 comprising SEQ ID NO: 74.

[0063] The heavy chain variable region comprises, consists essentially of, or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. In a preferred embodiment, the heavy chain variable region comprises or consists essentially of the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0064] The heavy chain variable region is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:164. In a preferred embodiment, the heavy chain variable region is encoded by a polynucleotide that consists essentially of or consists of the nucleic acid sequence of SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:164.

[0065] The light chain variable region comprises, consists essentially of, or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11. In a preferred embodiment, the light chain variable region comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11.

[0066] The light chain variable region is encoded by a polynucleotide that consists essentially of or consists of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 or SEQ ID NO:165. In a preferred embodiment, the light chain variable region is encoded by a polynucleotide that consists essentially of or consists of the nucleic acid sequence of SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 or SEQ ID NO:165.

[0067] The antibody heavy chain variable region can comprise the amino acid sequence of SEQ ID NO:3, and the antibody light chain variable region can comprise the amino acid sequence of SEQ ID NO:9. The antibody heavy chain variable region can comprise the amino acid sequence of SEQ ID NO:4, and the antibody light chain variable region can comprise the amino acid sequence of SEQ ID NO:9. The antibody heavy chain variable region can comprise the amino acid sequence of SEQ ID NO:5, and the antibody light chain variable region can comprise the amino acid sequence of SEQ ID NO:9. The antibody heavy chain variable region can comprise the amino acid sequence of SEQ ID NO:6, and the antibody light chain variable region can comprise the amino acid sequence of SEQ ID NO:9. The antibody heavy chain variable region can comprise the amino acid sequence of SEQ ID NO:7, and the antibody light chain variable region can comprise the amino acid sequence of SEQ ID NO:9. The antibody heavy chain variable region can comprise the amino acid sequence of SEQ ID NO:8, and the antibody light chain variable region can comprise the amino acid sequence of SEQ ID NO:9.

[0068] The heavy chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:3, and the light chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:10. The heavy chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:4, and the light chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:10. The heavy chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:5, and the light chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:10. The heavy chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:6, and the light chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:10. The heavy chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:7, and the light chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:10. The heavy chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:8, and the light chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:10.

[0069] The heavy chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:3, and the light chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:11. The heavy chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:4, and the light chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:11. The heavy chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:5, and the light chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:11. The heavy chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:6, and the light chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:11. The heavy chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:7, and the light chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:11. The heavy chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:8, and the light chain variable region of the antibody can comprise the amino acid sequence of SEQ ID NO:11.

[0070] In some embodiments, the antibody comprises human or humanized sequences. In some embodiments, the CDRs comprise humanized sequences. In some embodiments, the variable region comprises humanized sequences. In some embodiments, the framework region comprises humanized sequences. The framework region can further comprise one or more sequence mutations to reduce immunogenicity or improve production of the antibody. In some embodiments, the antibody is an IgG.

[0071] The antibody may be a monoclonal antibody, a chimeric antibody, a single domain antibody, a VHH, a VH, a single chain variable fragment (scFv), an antigen-binding fragment (Fab) or a Fab fragment. In a preferred embodiment, the antibody is an scFv.

[0072] The present disclosure provides scFv compositions and methods for using these compositions to recognize and bind to specific target proteins (e.g., MUC1) with high affinity and avidity. The scFv compositions can include the heavy chain variable region and the light chain variable region of an anti-MUC1 antibody.

[0073] The scFv can comprise a linker polypeptide between the heavy chain variable region and the light chain variable region. In certain embodiments, the linker polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 59. The linker polypeptide can be encoded by a polynucleotide consisting essentially of, or consisting of, the nucleic acid sequence of SEQ ID NO: 60.

[0074] The scFv comprises, consists essentially of, or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29. In a preferred embodiment, the scFv comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29.

[0075] The scFv comprises, consists essentially of, or consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 or SEQ ID NO: 166. In a preferred embodiment, the scFv comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 or SEQ ID NO:166.

[0076] The present disclosure also provides a chimeric antigen receptor (CAR) comprising an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one anti-MUC1 single-chain variable fragment (scFv) of the present disclosure; a transmembrane domain; and an endodomain comprising at least one costimulatory domain. The CAR may further comprise a hinge region between the antigen recognition domain and the transmembrane domain. The antigen recognition region may comprise at least two anti-MUC1 scFvs. The antigen recognition region may comprise at least three anti-MUC1 scFvs. In one embodiment, the CAR of the present disclosure is a bispecific CAR comprising at least two scFvs that specifically bind to two different antigens.

[0077] The ectodomain can comprise a signal peptide. The signal peptide can 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 CD8 alpha (CD8α) signal peptide (SP) or a portion thereof. The human CD8α SP comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO:57. Preferably, the human CD8α SP comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:57.

[0078] Human CD8α SP is encoded by a polynucleotide that consists essentially of, or consists of, a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 58. Preferably, human CD8α SP is encoded by a polynucleotide that consists essentially of, or consists of, the amino acid sequence of SEQ ID NO: 58.

[0079] The hinge domain or hinge region can comprise a human CD8α, IgG4, CD4 sequence, or a combination thereof. In a preferred embodiment, the hinge can comprise, essentially comprise, or consist of a human CD8α (CD8α) hinge or a portion thereof. The human CD8a hinge comprises, essentially comprises, or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO: 61. Preferably, the human CD8α hinge domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 61.

[0080] The human CD8 alpha hinge is encoded by a polynucleotide that essentially consists of or consists of a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 62 or SEQ ID NO: 168. Preferably, the human CD8 alpha hinge domain is encoded by a polynucleotide that consists essentially of or consists of the nucleic acid sequence of SEQ ID NO: 62 or SEQ ID NO: 168.

[0081] The transmembrane domain can comprise, consist essentially of, or consist of a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB, or GM-CSFR transmembrane domain. Preferably, the transmembrane domain can comprise, essentially comprise, or consist of a human CD8α (CD8α) transmembrane domain, or a portion thereof. The CD8a transmembrane domain comprises, consists of, or consists essentially of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO: 63. Preferably, the human CD8α transmembrane domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 63.

[0082] The CD8α transmembrane domain is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 64 or SEQ ID NO: 169. Preferably, the CD8α transmembrane domain is encoded by a polynucleotide that consists essentially of or consists of the nucleic acid sequence of SEQ ID NO: 64 or SEQ ID NO: 169.

[0083] At least one costimulatory domain can comprise or consist essentially of human 4-1BB, CD28, CD3 zeta (CD3ζ), CD40, ICOS, MyD88, OX-40 intracellular domain, or any combination thereof. Preferably, at least one costimulatory domain comprises a CD3ζ, 4-1BB costimulatory domain, or a combination thereof.

[0084] The 4-1BB intracellular domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 65. Preferably, the 4-1BB intracellular domain consists of, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 65.

[0085] The 4-1BB intracellular domain is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 66 or SEQ ID NO: 170. 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: 66 or SEQ ID NO: 170.

[0086] The CD3 zeta intracellular domain comprises, consists essentially of, or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any percentage therebetween) to SEQ ID NO: 67. Preferably, the CD3 zeta intracellular domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 67.

[0087] The CD3 zeta intracellular domain is encoded by a polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 68 or SEQ ID NO: 171. Preferably, the CD3 zeta intracellular domain is encoded by a polynucleotide that comprises, consists essentially of, or comprises the nucleic acid sequence of SEQ ID NO: 68 or SEQ ID NO: 171.

[0088] The compositions of the disclosure (e.g., anti-MUC1 scFv, CARs comprising anti-MUC1 scFv) can be administered in a dose of 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 can bind to human MUC1 with at least one affinity selected from K D may be determined by any means, including but not limited to, surface plasmon resonance.

[0089] A composition comprising an anti-MUC1 scFv or a CAR comprising an anti-MUC1 scFv can be incorporated into a cell delivery composition (e.g., a transposon or vector) and, optionally, into a cell, as described in detail herein.

[0090] Cells (e.g., immune cells and cytotoxic immune cells) modified by contact with and / or uptake of the disclosed compositions can specifically target MUC1-expressing cells. A preferred embodiment of the disclosed methods uses MUC1-C scFv binding agents to redirect cytotoxic cell types that mediate the destruction of cells expressing MUC1-C (MUC1-C+). For example, for treating proliferative disorders such as cancer, modified cells expressing an anti-MUC1 scFv or a CAR comprising an anti-MUC1 scFv of the present disclosure demonstrate improved in vivo persistence and anti-tumor efficacy. In some embodiments, modified cells expressing an anti-MUC1 scFv or a CAR comprising an anti-MUC1 scFv of the present disclosure exhibit improved potency and reduced immunogenicity compared to cells expressing a murine anti-MUC1 scFv or a CAR comprising a murine anti-MUC1 scFv. In some embodiments, modified cells expressing an anti-MUC1 scFv or a CAR comprising an anti-MUC1 scFv of the present disclosure exhibit improved potency and reduced immunogenicity compared to cells expressing an scFv or a CAR comprising an scFv comprising the heavy variable region of SEQ ID NO: 176 and the light variable region of SEQ ID NO: 177. In some embodiments, modified cells expressing an anti-MUC1 scFv or a CAR comprising an anti-MUC1 scFv of the present disclosure exhibit improved potency and reduced immunogenicity compared to cells expressing an scFv, or a CAR comprising an scFv, comprising the heavy variable region of SEQ ID NO: 178 and the light variable region of SEQ ID NO: 179. In some embodiments, modified cells expressing an anti-MUC1 scFv or a CAR comprising an anti-MUC1 scFv of the present disclosure exhibit improved potency and reduced immunogenicity compared to cells expressing, or a CAR comprising, an scFv comprising the heavy variable region of SEQ ID NO: 180 and the light variable region of SEQ ID NO: 181.

[0091] The "L1H1" scFv comprises a light chain variable region "L1" having the amino acid sequence of SEQ ID NO:9 encoded by the nucleic acid sequence of SEQ ID NO:54, and a heavy chain variable region "H1" having the amino acid sequence of SEQ ID NO:3 encoded by the nucleic acid sequence of SEQ ID NO:48. The "L1H1" scFv comprises the amino acid sequence of SEQ ID NO:124 encoded by the nucleic acid sequence of SEQ ID NO:142. The "L1H1" CAR comprises the amino acid sequence of SEQ ID NO:12 encoded by the nucleic acid sequence of SEQ ID NO:30.

[0092] The "L1H1B" scFv comprises a light chain variable region "L1" having the amino acid sequence of SEQ ID NO:9 encoded by the nucleic acid sequence of SEQ ID NO:54 or SEQ ID NO:165, and a heavy chain variable region "H1B" having the amino acid sequence of SEQ ID NO:4 encoded by the nucleic acid sequence of SEQ ID NO:49 or SEQ ID NO:164. The "L1H1B" scFv comprises the amino acid sequence of SEQ ID NO:125 encoded by the nucleic acid sequence of SEQ ID NO:143 or SEQ ID NO:166. The "L1H1B" CAR comprises the amino acid sequence of SEQ ID NO:13 encoded by the nucleic acid sequence of SEQ ID NO:31 or SEQ ID NO:167.

[0093] The "L1H2" scFv comprises a light chain variable region "L1" having the amino acid sequence of SEQ ID NO:9 encoded by the nucleic acid sequence of SEQ ID NO:54, and a heavy chain variable region "H2" having the amino acid sequence of SEQ ID NO:5 encoded by the nucleic acid sequence of SEQ ID NO:50. The "L1H2" scFv comprises the amino acid sequence of SEQ ID NO:126 encoded by the nucleic acid sequence of SEQ ID NO:144. The "L1H2" CAR comprises the amino acid sequence of SEQ ID NO:14 encoded by the nucleic acid sequence of SEQ ID NO:32.

[0094] The "L1H2B" scFv comprises a light chain variable region "L1" having the amino acid sequence of SEQ ID NO:9 encoded by the nucleic acid sequence of SEQ ID NO:54, and a heavy chain variable region "H2B" having the amino acid sequence of SEQ ID NO:6 encoded by the nucleic acid sequence of SEQ ID NO:51. The "L1H2B" scFv comprises the amino acid sequence of SEQ ID NO:127 encoded by the nucleic acid sequence of SEQ ID NO:145. The "L1H2B" CAR comprises the amino acid sequence of SEQ ID NO:15 encoded by the nucleic acid sequence of SEQ ID NO:33.

[0095] The "L1H3" scFv comprises a light chain variable region "L1" having the amino acid sequence of SEQ ID NO:9 encoded by the nucleic acid sequence of SEQ ID NO:54, and a heavy chain variable region "H3" having the amino acid sequence of SEQ ID NO:7 encoded by the nucleic acid sequence of SEQ ID NO:52. The "L1H3" scFv comprises the amino acid sequence of SEQ ID NO:128 encoded by the nucleic acid sequence of SEQ ID NO:146. The "L1H3" CAR comprises the amino acid sequence of SEQ ID NO:16 encoded by the nucleic acid sequence of SEQ ID NO:34.

[0096] The "L1H4" scFv comprises a light chain variable region "L1" having the amino acid sequence of SEQ ID NO:9 encoded by the nucleic acid sequence of SEQ ID NO:54, and a heavy chain variable region "H4" having the amino acid sequence of SEQ ID NO:8 encoded by the nucleic acid sequence of SEQ ID NO:53. The "L1H4" scFv comprises the amino acid sequence of SEQ ID NO:129 encoded by the nucleic acid sequence of SEQ ID NO:147. The "L1H4" CAR comprises the amino acid sequence of SEQ ID NO:17 encoded by the nucleic acid sequence of SEQ ID NO:35.

[0097] The "L2H1" scFv comprises a light chain variable region "L2" having the amino acid sequence of SEQ ID NO: 10, encoded by the nucleic acid sequence of SEQ ID NO: 55, and a heavy chain variable region "H1" having the amino acid sequence of SEQ ID NO: 3, encoded by the nucleic acid sequence of SEQ ID NO: 48. The "L2H1" scFv comprises the amino acid sequence of SEQ ID NO: 130, encoded by the nucleic acid sequence of SEQ ID NO: 148. The "L2H1" CAR comprises the amino acid sequence of SEQ ID NO: 18, encoded by the nucleic acid sequence of SEQ ID NO: 36.

[0098] The "L2H1B" scFv comprises a light chain variable region "L2" having the amino acid sequence of SEQ ID NO: 10, encoded by the nucleic acid sequence of SEQ ID NO: 55, and a heavy chain variable region "H1B" having the amino acid sequence of SEQ ID NO: 4, encoded by the nucleic acid sequence of SEQ ID NO: 49. The "L2H1B" scFv comprises the amino acid sequence of SEQ ID NO: 131, encoded by the nucleic acid sequence of SEQ ID NO: 149. The "L2H1B" CAR comprises the amino acid sequence of SEQ ID NO: 19, encoded by the nucleic acid sequence of SEQ ID NO: 37.

[0099] The "L2H2" scFv comprises a light chain variable region "L2" having the amino acid sequence of SEQ ID NO: 10, encoded by the nucleic acid sequence of SEQ ID NO: 55, and a heavy chain variable region "H2" having the amino acid sequence of SEQ ID NO: 5, encoded by the nucleic acid sequence of SEQ ID NO: 50. The "L2H2" scFv comprises the amino acid sequence of SEQ ID NO: 132, encoded by the nucleic acid sequence of SEQ ID NO: 150. The "L2H2" CAR comprises the amino acid sequence of SEQ ID NO: 20, encoded by the nucleic acid sequence of SEQ ID NO: 38.

[0100] The "L2H2B" scFv comprises a light chain variable region "L2" having the amino acid sequence of SEQ ID NO: 10, encoded by the nucleic acid sequence of SEQ ID NO: 55, and a heavy chain variable region "H2B" having the amino acid sequence of SEQ ID NO: 6, encoded by the nucleic acid sequence of SEQ ID NO: 51. The "L2H2B" scFv comprises the amino acid sequence of SEQ ID NO: 133, encoded by the nucleic acid sequence of SEQ ID NO: 151. The "L2H2B" CAR comprises the amino acid sequence of SEQ ID NO: 21, encoded by the nucleic acid sequence of SEQ ID NO: 39.

[0101] The "L2H3" scFv comprises a light chain variable region "L2" having the amino acid sequence of SEQ ID NO: 10, encoded by the nucleic acid sequence of SEQ ID NO: 55, and a heavy chain variable region "H3" having the amino acid sequence of SEQ ID NO: 7, encoded by the nucleic acid sequence of SEQ ID NO: 52. The "L2H3" scFv comprises the amino acid sequence of SEQ ID NO: 134, encoded by the nucleic acid sequence of SEQ ID NO: 152. The "L2H3" CAR comprises the amino acid sequence of SEQ ID NO: 22, encoded by the nucleic acid sequence of SEQ ID NO: 40.

[0102] The "L2H4" scFv comprises a light chain variable region "L2" having the amino acid sequence of SEQ ID NO: 10, encoded by the nucleic acid sequence of SEQ ID NO: 55, and a heavy chain variable region "H4" having the amino acid sequence of SEQ ID NO: 8, encoded by the nucleic acid sequence of SEQ ID NO: 53. The "L2H4" scFv comprises the amino acid sequence of SEQ ID NO: 135, encoded by the nucleic acid sequence of SEQ ID NO: 153. The "L2H4" CAR comprises the amino acid sequence of SEQ ID NO: 23, encoded by the nucleic acid sequence of SEQ ID NO: 41.

[0103] The "L3H1" scFv comprises a light chain variable region "L3" having the amino acid sequence of SEQ ID NO: 11 encoded by the nucleic acid sequence of SEQ ID NO: 56, and a heavy chain variable region "H1" having the amino acid sequence of SEQ ID NO: 3 encoded by the nucleic acid sequence of SEQ ID NO: 48. The "L3H1" scFv comprises the amino acid sequence of SEQ ID NO: 136 encoded by the nucleic acid sequence of SEQ ID NO: 154. The "L3H1" CAR comprises the amino acid sequence of SEQ ID NO: 24 encoded by the nucleic acid sequence of SEQ ID NO: 42.

[0104] The "L3H1B" scFv comprises a light chain variable region "L3" having the amino acid sequence of SEQ ID NO: 11 encoded by the nucleic acid sequence of SEQ ID NO: 56, and a heavy chain variable region "H1B" having the amino acid sequence of SEQ ID NO: 4 encoded by the nucleic acid sequence of SEQ ID NO: 49. The "L3H1B" scFv comprises the amino acid sequence of SEQ ID NO: 137 encoded by the nucleic acid sequence of SEQ ID NO: 155. The "L3H1B" CAR comprises the amino acid sequence of SEQ ID NO: 25 encoded by the nucleic acid sequence of SEQ ID NO: 43.

[0105] The "L3H2" scFv comprises a light chain variable region "L3" having the amino acid sequence of SEQ ID NO: 11 encoded by the nucleic acid sequence of SEQ ID NO: 56, and a heavy chain variable region "H2" having the amino acid sequence of SEQ ID NO: 5 encoded by the nucleic acid sequence of SEQ ID NO: 50. The "L3H2" scFv comprises the amino acid sequence of SEQ ID NO: 138 encoded by the nucleic acid sequence of SEQ ID NO: 156. The "L3H2" CAR comprises the amino acid sequence of SEQ ID NO: 26 encoded by the nucleic acid sequence of SEQ ID NO: 44.

[0106] The "L3H2B" scFv comprises a light chain variable region "L3" having the amino acid sequence of SEQ ID NO: 11 encoded by the nucleic acid sequence of SEQ ID NO: 56, and a heavy chain variable region "H2B" having the amino acid sequence of SEQ ID NO: 6 encoded by the nucleic acid sequence of SEQ ID NO: 51. The "L3H2B" scFv comprises the amino acid sequence of SEQ ID NO: 139 encoded by the nucleic acid sequence of SEQ ID NO: 157. The "L3H2B" CAR comprises the amino acid sequence of SEQ ID NO: 27 encoded by the nucleic acid sequence of SEQ ID NO: 45.

[0107] The "L3H3" scFv comprises a light chain variable region "L3" having the amino acid sequence of SEQ ID NO: 11, encoded by the nucleic acid sequence of SEQ ID NO: 56, and a heavy chain variable region "H3" having the amino acid sequence of SEQ ID NO: 7, encoded by the nucleic acid sequence of SEQ ID NO: 52. The "L3H3" scFv comprises the amino acid sequence of SEQ ID NO: 140, encoded by the nucleic acid sequence of SEQ ID NO: 158. The "L3H3" CAR comprises the amino acid sequence of SEQ ID NO: 28, encoded by the nucleic acid sequence of SEQ ID NO: 46.

[0108] The "L3H4" scFv comprises a light chain variable region "L3" having the amino acid sequence of SEQ ID NO: 11 encoded by the nucleic acid sequence of SEQ ID NO: 56, and a heavy chain variable region "H4" having the amino acid sequence of SEQ ID NO: 8 encoded by the nucleic acid sequence of SEQ ID NO: 53. The "L3H4" scFv comprises the amino acid sequence of SEQ ID NO: 141 encoded by the nucleic acid sequence of SEQ ID NO: 159. The "L3H4" CAR comprises the amino acid sequence of SEQ ID NO: 29 encoded by the nucleic acid sequence of SEQ ID NO: 47.

[0109] Tables 1A and 1B show exemplary MUC-1C CAR components and sequences of the present disclosure.

[0110] [Table 1-1]

[0111] [Table 1-2]

[0112] [Table 1-3]

[0113] [Table 1-4]

[0114] [Table 1-5]

[0115] [Table 1-6]

[0116] [Table 1-7]

[0117] [Table 1-8]

[0118] [Table 2-1]

[0119] [Table 2-2]

[0120] Cells and modified cells of the present disclosure The disclosed cells and modified cells can be mammalian cells. Preferably, the cells and modified cells are human cells. The disclosed cells and modified cells can be immune cells. The immune cells of the present disclosure can include lymphoid progenitor cells, natural killer (NK) cells, T lymphocytes (T cells), stem memory T cells (TSCM cells), central memory T cells (TCM), stem cell-like T cells, B lymphocytes (B cells), antigen-presenting cells (APCs), cytokine-induced killer (CIK) cells, myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, macrophages, platelets, erythrocytes, red blood cells (RBCs), megakaryocytes, or osteoclasts.

[0121] Immune progenitor cells can include any cells that can differentiate into one or more types of immune cells. Immune progenitor cells can include pluripotent stem cells that are self-renewing and capable of developing into immune cells. Immune progenitor cells can include hematopoietic stem cells (HSCs) or their progeny. Immune progenitor cells can include progenitor cells that can develop into immune cells. Immune progenitor cells can include hematopoietic progenitor cells (HPCs).

[0122] Hematopoietic stem cells (HSCs) are multipotent self-renewing cells. All differentiated blood cells from the lymphoid and myeloid lineages arise from HSCs. HSCs can be found in adult bone marrow, peripheral blood, mobilized peripheral blood, peritoneal dialysis effluent, and umbilical cord blood.

[0123] HSCs can be isolated or derived from primary or cultured stem cells. HSCs can be isolated or derived from embryonic stem cells, pluripotent stem cells, multipotent stem cells, adult stem cells, or induced pluripotent stem cells (iPSCs).

[0124] Immune progenitor cells can include HSCs or HSC progeny, including, but not limited to, pluripotent stem cells, lymphoid progenitor cells, natural killer cells, T lymphocytes, B lymphocytes, myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, and macrophages.

[0125] HSCs produced by the disclosed methods can be isolated or derived from adult stem cells and, while committed to a single lineage, retain the characteristics of "primitive" stem cells that share the characteristics of embryonic stem cells. For example, "primitive" HSCs generated by the disclosed methods retain "stemness" after division and do not differentiate. Thus, 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 can be therapeutically effective when administered as a single dose.

[0126] Primitive HSCs can be CD34+. Primitive HSCs can be CD34+ and CD38-. Primitive HSCs can be CD34+, CD38- and CD90+. Primitive HSCs can be CD34+, CD38-, CD90+ and CD45RA-. Primitive HSCs can be CD34+, CD38-, CD90+, CD45RA- and CD49f+. Primitive HSCs can be CD34+, CD38-, CD90+, CD45RA- and CD49f+.

[0127] Primitive HSCs, HSCs, and / or progeny of HSCs can be modified according to the disclosed methods to express exogenous sequences (e.g., chimeric antigen receptors or therapeutic proteins). The modified primitive HSCs, modified HSCs, and / or progeny of the modified HSCs can be forward differentiated to produce modified immune cells, including, but not limited to, modified T cells, modified natural killer cells, and / or modified B cells.

[0128] The modified immune or immune progenitor cells can be NK cells. NK cells can be cytotoxic lymphocytes differentiated from lymphoid progenitor cells. The modified NK cells can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs. In some embodiments, the non-activated NK cells are derived from CD3-depleted leukapheresis (containing CD14 / CD19 / CD56+ cells).

[0129] The modified immune or immune precursor cells can 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 can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs.

[0130] The modified T cells of the present disclosure can be derived from modified hematopoietic stem and progenitor cells (HSPCs) or modified HSCs. Unlike conventional biologics and chemotherapeutics, the disclosed modified T cells have the ability to rapidly regenerate upon antigen recognition, thereby potentially avoiding the need for retreatment. 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, preventing potential relapse. Alternatively, in some aspects, the modified T cells do not persist in the patient if this is undesirable.

[0131] The development of antigen receptor molecules that do not cause T cell exhaustion through antigen-independent (tonic) signaling, and the development of early memory T cells, especially stem cell memory (T SCM Intensive efforts are being made to develop modified T cell products that include central memory (T) or stem cell-like T cells. The stem cell-like modified T cells of the present disclosure are CM ) T cells or T CM Cells, effector memory (T EM ) and effector T cells (T E ) exhibit the greatest self-renewal and pluripotency potential to induce T cells such as T cells, thereby producing successful tumor eradication and long-term modified T cell engraftment. A linear pathway of differentiation may be involved in the generation of these cells: naive T cells (T N )>T SCM >T CM >T EM >T E >T TE , thereby T N is directly T SCM These are the parent progenitor cells that give rise to T CMThe T cell compositions of the present disclosure produce the most abundant T SCM Cells (e.g., T SCM >T CM >T EM >TO E >T TE ) can include one or more of each parental T cell subset having

[0132] Immune cell precursors include early memory T cells, stem cell-like T cells, and naïve T cells (T N ), T SCM , T CM , T EM , T E , or T TE The immune cell precursors can be primitive HSCs, HSCs, or HSC progeny cells of the present disclosure. The immune cells can be early memory T cells, stem cell-like T cells, naive T cells (T N ), T SCM , T CM , T EM , T E , or T TE It could be.

[0133] The methods of the present disclosure can modify and / or produce a population of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween, of a plurality of modified T cells in the population express one or more cell surface markers of early memory T cells. The population of modified early memory T cells comprises a plurality of modified stem cell-like T cells. The population of modified early memory T cells can comprise a plurality of modified T cells. SCM The population of modified early memory T cells includes multiple modified T CM Contains cells.

[0134] The disclosed methods can modify and / or produce a population of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween, of a plurality of modified T cells in the population express one or more cell surface markers of stem cell-like T cells. A population of modified stem cell-like T cells can include a plurality of modified T cells. SCM The population of modified stem cell-like T cells comprises multiple modified T CM Contains cells.

[0135] 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 therebetween, of the plurality of modified T cells in the population are stem memory T cells (T SCM ) or T SCM The one or more cell surface markers may include one or more of 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.

[0136] 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 include CD45RO and CD62L. The cell surface markers can include one or more of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L.

[0137] The disclosed methods can modify and / or produce a population of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween, of the plurality of modified T cells in the population are naive T cells (T N ) The cell surface markers can include one or more of CD45RA, CCR7, and CD62L.

[0138] The disclosed methods can modify and / or produce a population of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween, of the plurality of modified T cells in the population are effector T cells (modified T EFF ) The cell surface markers can include one or more of CD45RA, CD95, and IL-2Rβ.

[0139] The disclosed methods can modify and / or produce a population of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or any percentage therebetween, of a plurality of modified T cells in the population are stem cell-like T cells, stem memory T cells (T SCM ) or central memory T cells (T CM ) express one or more cell surface markers.

[0140] A plurality of modified cells of the population comprises a transgene or a sequence encoding a transgene (e.g., a CAR), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% of the plurality of cells of the population comprise a transgene or a sequence encoding a transgene, and 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 therebetween of the plurality of modified T cells of the population are stem cell-like T cells, stem memory T cells (T SCM ) or central memory T cells (T CM In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 13 encoded by the nucleic acid sequence of SEQ ID NO: 31 or SEQ ID NO: 167.

[0141] A plurality of the modified cells of the population comprises a transgene or a sequence encoding a transgene (e.g., a CAR), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% of the plurality of cells of the population comprise a transgene or a sequence encoding a transgene, wherein at least 70%, at least 75%, at least 80%, at least 99.9%, at least 99.9%, or at least 100% of the plurality of cells of the population comprise a transgene or a sequence encoding a transgene, 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% express one or more cell surface markers including CD34, or at least about 70% to about 99%, about 75% to about 95%, or about 85% to about 95% of the population of modified cells express one or more cell surface markers including CD34 (e.g., comprising the cell surface marker phenotype CD34+).

[0142] A plurality of the modified cells of the population comprise a transgene or a sequence encoding a transgene (e.g., a CAR), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% of the plurality of cells of the population comprise a transgene or a sequence encoding a transgene, wherein at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% express one or more cell surface markers including CD34 but 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 population of modified cells express one or more cell surface markers including CD34 but do not express one or more cell surface markers including CD38 (e.g., comprising the cell surface marker phenotypes CD34+ and CD38-).

[0143] A plurality of the modified cells of the population 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 plurality of cells of 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 4 ... 0%, 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% are CD34 and CD or at least about 0.2% to about 40%, about 0.2% to about 30%, about 0.2% to about 2%, or 0.5% to about 1.5% of the population of modified cells express one or more cell surface markers including CD34 and CD90 but do not express one or more cell surface markers including CD38 (e.g., including cell surface marker phenotypes CD34+, CD38- and CD90+).

[0144] A plurality of the modified cells of the population comprises a transgene or a sequence encoding a transgene (e.g., a CAR), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% of the plurality of cells of the population comprise a transgene or a sequence encoding a transgene, wherein at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 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 cells express one or more 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 0.5% to about 1.5% of the population of modified cells express one or more cell surface markers including CD34 and CD90 but do not express one or more cell surface markers including CD38 and CD45RA (phenotypes including CD34+, CD38-, CD90+ and CD45RA-).

[0145] A plurality of the modified cells of the population comprises a transgene or a sequence encoding a transgene (e.g., a CAR), wherein at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% of the plurality of cells of the population comprise a transgene or a sequence encoding a transgene, and wherein at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.10%, at least 0.11%, at least 0.12%, at least 0.13%, at least 0.14%, at least 0.15%, at least 0.16%, at least 0.17%, at least 0.18%, at least 0.19 ... %, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20 %, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or 100% are CD34, express one or more cell surface markers including CD90 and CD49f but 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 0.04% to about 1% of the population of modified cells express one or more cell surface markers including CD34, CD90, and CD49f but do not express one or more cell surface markers including CD38 and CD45RA (including phenotypes CD34+, CD38-, CD90+, CD45RA-, and CD49+).

[0146] A plurality of the modified cells of the population 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 plurality of cells of the population comprise a transgene or a sequence encoding a transgene, and at least 0% of the plurality of cells of the population comprise a transgene or a sequence encoding a transgene. 01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5% , at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% express one or more cell surface markers including CD34 and CD90 but 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 population of modified cells express one or more cell surface markers including CD34 and CD90 but do not express one or more cell surface markers including CD45RA (phenotypes including CD34+, CD90+, and CD45RA-).

[0147] Compositions and methods for producing and / or expanding immune cells or immune progenitor cells (e.g., the disclosed modified T cells), and buffers for maintaining or enhancing cell viability and / or stem-like phenotype of immune cells or immune progenitor cells (e.g., the disclosed modified T cells), are disclosed elsewhere herein and in further detail in U.S. Pat. No. 10,329,543 and PCT Publication WO 2019 / 173636.

[0148] The disclosed cells and modified cells can be somatic cells. The disclosed cells and modified cells can be differentiated cells. The disclosed cells and modified cells can be autologous or allogeneic cells. Allogeneic cells are engineered to prevent adverse reactions to engraftment after administration to a subject. Allogeneic cells can be any type of cell. Allogeneic cells can be stem cells or derived from stem cells. Allogeneic cells can be differentiated somatic cells.

[0149] Method for expressing chimeric antigen receptors The present disclosure provides a method for expressing a CAR on the surface of a cell, the method including: (a) obtaining a cell population; (b) contacting the cell population with a composition comprising a CAR or a sequence encoding a CAR under conditions sufficient to transduce the CAR across the cell membrane of at least one cell in the cell population, thereby producing a modified cell population; (c) culturing the modified cell population under conditions suitable for integration of the sequence encoding the CAR; and (d) expanding and / or selecting at least one cell from the modified cell population that expresses the CAR on its cell surface.

[0150] In some embodiments, the cell population can comprise leukocytes and / or CD4+ and CD8+ leukocytes. The cell population can comprise CD4+ and CD8+ leukocytes in an optimized ratio. The optimized ratio of CD4+ to CD8+ leukocytes does not naturally occur in vivo. The cell population can comprise tumor cells.

[0151] In some embodiments, the conditions sufficient to transfer the CAR or sequence encoding the CAR, transposon, or vector across the cell membrane of at least one cell in the cell population comprise at least one of application of one or more electrical pulses at a particular voltage, a buffer, and one or more cofactors. In some embodiments, the conditions suitable for integration of the sequence encoding the CAR comprise at least one of a buffer and one or more cofactors.

[0152] The buffer can include PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, human T cell nucleofection buffer, or any combination thereof. The one or more cofactors can 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; and (e) one or more nucleic acid modifying or stabilizing reagents. Recombinant human cytokines, chemokines, interleukins, or any combination thereof, include IL2, IL7, IL12, IL15, IL21, IL1, IL3, IL4, IL5, IL6, IL8, CXCL8, IL9, IL10, IL11, IL13, IL14, IL16, IL17, IL18, IL19, IL20, IL22, IL23, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL35, IL36, GM-CSF, IFN-gamma, IL-1 alpha / IL-1F1, IL-1 beta / IL-1F2, IL-12 p70, IL-12 / IL-35 The proteins may include p35, IL-13, IL-17 / IL-17A, IL-17A / F heterodimer, IL-17F, IL-18 / IL-1F4, IL-23, IL-24, IL-32, IL-32beta, IL-32gamma, IL-33, LAP (TGF-beta 1), lymphotoxin-alpha / TNF-beta, TGF-beta, TNF-alpha, TRANCE / TNFSF11 / RANK L, or any combination thereof.The salts, minerals, metabolites, or any combination thereof can include HEPES, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solution, ascorbic acid, nucleosides, FBS / FCS, human serum, serum substitutes, antibiotics, pH adjusters, Earle's salts, 2-mercaptoethanol, human transferrin, recombinant human insulin, human serum albumin, Nucleofector PLUS supplement, KCL, MgCl2, Na2HPO4, NAH2PO4, sodium lactobionate, mannitol, sodium succinate, sodium chloride, CINa, glucose, Ca(NO3)2, Tris / HCl, K2HPO4, KH2PO4, polyethyleneimine, polyethylene glycol, poloxamer 188, poloxamer 181, poloxamer 407, polyvinylpyrrolidone, Pop313, Crown-5, or any combination thereof. The cell culture medium can 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 apoptosis pathways, or combinations thereof, include inhibitors of TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-κB, type 1 interferon, proinflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNA pol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, Caspase 1, Pro-IL1B, PI3K, Akt, Wnt3A, glycogen synthase kinase-3β (GSK-3β) (e.g., TWS119), or any combination thereof. Examples of such inhibitors 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 modifiers, DNA binding proteins, lipids, phospholipids, CaPO4, net neutral charge DNA binding peptides with or without NLS sequences, TREX1 enzyme, or any combination thereof.

[0153] The expansion and selection steps can be performed simultaneously or sequentially. The expansion can occur before selection. The expansion can occur after selection, and optionally, a further (i.e., second) selection can occur after expansion. Simultaneous expansion and selection are possible. The expansion and / or selection steps can proceed for 10 to 14 days (inclusive).

[0154] This expansion can include contacting at least one cell of the modified cell population with an antigen to stimulate the at least one cell via the CAR, thereby generating an expanded cell population. The antigen can be presented on the surface of a substrate. The substrate can have any form, including, but not limited to, a surface, a well, beads, or multiple beads, and a matrix. The substrate can further include a paramagnetic or magnetic component. The antigen can be presented on the surface of a substrate, where the substrate is a magnetic bead, where the magnetic bead can be used to remove or separate from the modified and expanded cell population. The antigen can be presented on the surface of a cell or an artificial antigen-presenting cell. Artificial antigen-presenting cells can include, but are not limited to, tumor cells and stem cells.

[0155] 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, thereby identifying cells that express the selection gene as surviving the selection and cells that do not express the selection gene as being unable to survive the selection step.

[0156] The present disclosure provides compositions comprising the modified, expanded and selected cell populations described herein.

[0157] A more detailed description of methods for expressing a CAR on the surface of a cell is disclosed in PCT Publications WO2019 / 049816 and PCT / US2019 / 049816.

[0158] The present disclosure provides a cell or cell population, wherein the cell comprises a composition comprising (a) an inducible transgene construct comprising a sequence encoding an inducible promoter and a sequence encoding a transgene, and (b) a receptor construct comprising a sequence encoding a constitutive promoter and a sequence encoding an exogenous receptor, such as a CAR, wherein, upon integration of the construct of (a) and the construct of (b) into the genomic sequence of the cell, the exogenous receptor is expressed, and upon binding to a ligand or antigen, the exogenous receptor transduces an intracellular signal that directly or indirectly targets the inducible promoter that regulates expression of the inducible transgene (a), thereby modifying gene expression.

[0159] The composition can modify gene expression by decreasing gene expression. The composition can modify gene expression by temporarily modifying gene expression (e.g., the duration of binding of a ligand to an exogenous receptor). The composition can acutely modify gene expression (e.g., a ligand reversibly binds to an exogenous receptor). The composition can chronically modify gene expression (e.g., a ligand irreversibly binds to an exogenous receptor).

[0160] Exogenous receptors can include endogenous receptors in relation to the genomic sequence of a 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.

[0161] The exogenous receptor can include a non-naturally occurring receptor. The non-natural receptor can be synthetic, modified, recombinant, mutated, or chimeric. The non-naturally occurring receptor can include one or more sequences isolated or derived from a T cell receptor (TCR). The non-naturally occurring receptor can include one or more sequences isolated or derived from a scaffold protein. In some embodiments, including those in which the non-natural receptor does not include a transmembrane domain, the non-natural receptor interacts with a second transmembrane, membrane-bound, and / or intracellular receptor that transduces an intracellular signal after contact with the non-natural receptor. The non-natural receptor can include a transmembrane domain. The non-natural receptor can interact with an intracellular receptor that transduces an intracellular signal. The non-natural receptor can include an intracellular signaling domain. The non-naturally occurring receptor can be a chimeric ligand receptor (CLR). The CLR can be a chimeric antigen receptor (CAR).

[0162] The sequence encoding the inducible promoter includes a sequence encoding an NFκB promoter, a sequence encoding an interferon promoter, or a sequence encoding an interleukin-2 promoter. In some embodiments, the IFN promoter is an IFNγ promoter. The inducible promoter can be isolated or derived from a cytokine or chemokine promoter. The cytokine or chemokine can include 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).

[0163] Inducible promoters can be isolated from or derived from promoters of genes including surface proteins involved in cell differentiation, activation, exhaustion, and function. In some embodiments, the genes include CD69, CD71, CTLA4, PD-1, TIGIT, LAG3, TIM-3, GITR, MHCII, COX-2, FASL, or 4-1BB.

[0164] The inducible promoter can be isolated from or derived from the promoters of genes involved in CD4+ metabolism and differentiation, such as Nr4a1, Nr4a3, Tnfrsf9(4-1BB), Sema7a, Zfp3612, Gadd45b, Dusp5, Dusp6, and Neto2.

[0165] 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 therapy, and downstream signaling components of oncogenes or tumor suppressor genes, non-limiting examples of which are disclosed in PCT Publication WO2019 / 173636 and PCT Application No. PCT / US2019 / 049816.

[0166] The present disclosure provides methods for producing a modified T cell population, comprising, or consisting of, introducing a composition comprising a CAR of the present disclosure into a plurality of primary human T cells, the composition comprising a CAR of the present disclosure or a sequence encoding the same to produce a plurality of modified T cells. The present disclosure provides compositions comprising a population of modified T cells produced by the method. 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 population expresses a CAR of the present disclosure.

[0167] armed cells The disclosed modified cells (e.g., CAR T cells) can be further modified to enhance their therapeutic potential. Alternatively, or in addition, the modified cells can be further modified to make them less susceptible to immunological and / or metabolic checkpoints. This type of "coated" cell modification, following modification, may be referred to herein as "coated" cells (e.g., coated T cells). Coated cells can be generated within the tumor immunosuppressive microenvironment, for example, by blocking and / or diluting certain checkpoint signals that are naturally delivered to the cells (e.g., checkpoint inhibition).

[0168] The armed cells of the present disclosure can be derived from any cell, such as T cells, NK cells, hematopoietic progenitor cells, peripheral blood-derived T cells (including T cells derived from G-CSF-mobilized peripheral blood), or umbilical cord blood-derived T cells. Armed cells (e.g., armed T cells) can comprise one or more of a chimeric ligand receptor (CLR comprising a protein scaffold, antibody, Scan Fv, or antibody mimic) / chimeric antigen receptor (CAR comprising a protein scaffold, antibody, Scan Fv, or antibody mimic), a CARTyrin (CAR comprising centyrin), and / or a VCAR (CAR comprising a camelid VHH or single-domain VH). Armed cells (e.g., armed T cells) can comprise an inducible pro-apoptotic polypeptide as disclosed herein. Armed cells (e.g., armed T cells) can comprise an exogenous sequence, which can include a sequence encoding a therapeutic protein. Exemplary therapeutic proteins can be nuclear proteins, cytoplasmic proteins, intracellular proteins, transmembrane proteins, cell surface-associated proteins, or secreted proteins. Exemplary therapeutic proteins expressed by an armed cell (e.g., an armed T cell) can modify the activity of the armed cell or can modify the activity of a second cell. An armed cell (e.g., an armed T cell) can include a selection gene or selection marker. An armed cell (e.g., an armed T cell) can include a synthetic gene expression cassette (also referred to herein as an inducible transgene construct).

[0169] The disclosed modified cells (e.g., CAR T cells) can be further modified to silence or reduce expression of one or more gene(s) encoding a receptor for an inhibitory checkpoint signal to generate an armed cell (e.g., an armed CAR T cell). Receptors for inhibitory checkpoint signals are expressed on the cell surface or in the cytoplasm of the cell. Silencing or reducing expression of a gene encoding a receptor for an inhibitory checkpoint signal results in loss of protein expression of the inhibitory checkpoint receptor on the surface or in the cytoplasm of the coated cell. Thus, armed cells with silenced or reduced expression of one or more genes encoding inhibitory checkpoint receptors are resistant, non-receptive, or insensitive to checkpoint signals. The reduced resistance or sensitivity of armed cells to inhibitory checkpoint signals enhances the therapeutic potential of the armed cells in the presence of these inhibitory checkpoint signals. Non-limiting examples of inhibitory checkpoint signals (and proteins that induce immunosuppression) are disclosed in PCT Publication WO2019 / 173636. Preferred examples of inhibitory checkpoint signals that can be inhibited include, but are not limited to, PD-1 and TGFβRII.

[0170] The disclosed modified cells (e.g., CAR T cells) can be further modified to silence or reduce expression of one or more genes encoding intracellular proteins involved in checkpoint signaling (e.g., armed CAR T cells) to generate armed cells (e.g., armed CAR T cells). The activity of the modified cells can 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 PCT Publication WO2019 / 173636.

[0171] The disclosed modified cells (e.g., CAR T cells) can be further modified to inactivate or reduce expression of one or more gene(s) encoding transcription factors that interfere with the efficacy of a therapy to produce armed cells (e.g., armed CAR T cells). The activity of the modified cells can be enhanced or modulated by silencing or reducing the expression (or inhibiting the function) of a transcription factor that interferes with the efficacy of a therapy. Non-limiting examples of transcription factors that can be modified to inhibit expression, reduce expression, or inhibit the function include, but are not limited to, exemplary transcription factors disclosed in PCT Publication WO2019 / 173636.

[0172] The disclosed modified cells (e.g., CAR T cells) can be further modified to suppress or reduce the expression of one or more genes encoding cell death or cell apoptosis receptors to generate armed cells (e.g., armed CAR T cells). The interaction of a death receptor with its endogenous ligand results in the initiation of apoptosis. Disruption of the expression, activity, or interaction of a cell death and / or cell apoptosis receptor and / or ligand makes the modified cell less susceptible to death signals, thereby making the coated cells more effective in the tumor environment. Non-limiting examples of cell death and / or cell apoptosis receptors and ligands are disclosed in PCT Publication WO2019 / 173636. A preferred example of a cell death receptor that can be modified is Fas (CD95).

[0173] The disclosed modified cells (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more gene(s) encoding metabolic sensing proteins to generate 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 results in prolonged retention of T cell function, resulting in the killing of more tumor cells per cell. Non-limiting examples of metabolic sensing genes and proteins are disclosed in PCT Publication WO2019 / 173636. As a preferred example, HIF1a and VHL play a role in T cell function in hypoxic environments. Armed T cells may have silenced or reduced expression of one or more genes encoding HIF1a or VHL.

[0174] The disclosed modified cells (e.g., CAR T cells) can be further modified to silence or reduce expression of one or more genes (e.g., coated CAR T cells) that encode proteins that confer sensitivity to cancer treatments, including monoclonal antibodies, to produce coated cells (e.g., coated CAR T cells). The armed cells can thus function and exhibit superior function or efficacy in the presence of cancer treatments (e.g., chemotherapy, monoclonal antibody therapy, or other anti-tumor treatments). Non-limiting examples of proteins involved in conferring sensitivity to cancer treatments are disclosed in PCT Publication WO2019 / 173636.

[0175] The disclosed modified cells (e.g., CAR T cells) can be further modified to suppress or reduce the expression of one or more gene(s) encoding a growth advantage to generate armed cells (e.g., armed CAR T cells). Silencing or reducing the expression of an oncogene can favor cell proliferation. For example, silencing or reducing (e.g., disrupting) the expression of the TET2 gene during the CAR T cell manufacturing process results in the generation of armed CAR T cells with significant capacity for tumor expansion and subsequent eradication when compared to disarmed CAR T cells that lack this expansion capacity. This strategy can be coupled to a safety switch (e.g., the iC9 safety switch described herein) that allows for targeted destruction of armed CAR T cells in the event of an adverse reaction from the subject or uncontrolled proliferation of the armed CAR T cells. Non-limiting examples of growth advantage factors are disclosed in PCT Publication WO2019 / 173636.

[0176] The modified cells (e.g., CAR T cells) of the disclosure can be further modified to express modified / chimeric checkpoint receptors to generate armed T cells of the disclosure.

[0177] The modified / chimeric checkpoint receptor can include a null receptor, a decoy receptor, or a dominant-negative receptor. The null receptor, decoy receptor, or dominant-negative receptor can be a modified / chimeric receptor / protein. The null receptor, decoy receptor, or dominant-negative receptor can be truncated to express the intracellular signaling domain. Alternatively, or in addition, the null receptor, decoy receptor, or dominant-negative receptor can be mutated within the intracellular signaling domain at one or more amino acid positions that are critical or required for effective signaling. Truncation or mutation of the null receptor, decoy receptor, or dominant-negative receptor can result in the loss of the receptor's ability to transmit or transduce a checkpoint signal into or within a cell.

[0178] For example, dilution or blocking of immunosuppressive checkpoint signals from PD-L1 receptors expressed on the surface of tumor cells can be achieved by expressing a modified / chimeric PD-1 null receptor on the surface of armed cells (e.g., armed CAR T cells), which also effectively competes with the endogenous (unmodified) PD-1 receptor expressed on the surface of the armed cells, reducing or inhibiting the transduction of immunosuppressive checkpoint signals through the armed cells' endogenous PD-1 receptor. In this non-limiting example, competition between two different receptors for binding to PD-L1 expressed on tumor cells reduces or diminishes the level of effective checkpoint signaling, thereby enhancing the therapeutic potential of armed cells expressing PD-1 null receptors.

[0179] Modified / chimeric checkpoint receptors may include null, decoy, or dominant-negative receptors that are transmembrane receptors, membrane-bound or membrane-associated receptors / proteins, or intracellular receptors / proteins. Exemplary 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 therapy, and oncogenes or tumor suppressor genes. Non-limiting examples of cytokines, cytokine receptors, chemokines, and chemokine receptors are disclosed in PCT Publication WO2019 / 173636.

[0180] Modified / chimeric checkpoint receptors can include switch receptors. Exemplary 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 the wild-type domain. For example, replacing an inhibitory signaling domain with a stimulatory signaling domain will switch an immunoinhibitory signal to an immunostimulatory signal. Alternatively, replacing an inhibitory signaling domain with a different inhibitory domain can reduce or enhance the level of inhibitory signaling. Expression or overexpression of a switch receptor can result in dilution and / or blockage of a cognate checkpoint signal through competition with endogenous wild-type checkpoint receptors (not the switch receptor) for binding to the cognate checkpoint receptor expressed within the immunosuppressive tumor microenvironment. Armed cells (e.g., armed CAR T cells) can include a sequence encoding a switch receptor that directs the expression of one or more switch receptors, thereby altering the activity of the armed cell. 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 proteins expressed in cells downstream of tumor suppressor proteins or genes.

[0181] Exemplary switch receptors may include or be derived from proteins, including, but not limited to, signaling components downstream 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 therapy, and oncogenes or tumor suppressor genes.

[0182] The disclosed modified cells (e.g., CAR T cells) can be further modified to express a CLR / CAR that mediates conditional gene expression to generate armed T cells. The combination of a CLR / CAR and a 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 CLR or cognate antigen to the cognate ligand of the CAR. This system can aid in "arming" or enhance the therapeutic potential of the modified T cell by, for example, reducing or limiting synthetic gene expression at the ligand or antigen binding site in or within the tumor environment.

[0183] Gene editing compositions and methods The modified cells are generated by introducing a transgene into the cells. The introducing step can include delivery of a nucleic acid sequence, a transgene, and / or a genome editing construct via a non-transfer delivery system.

[0184] Introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ can be by local delivery, adsorption, absorption, electroporation, spinfection, co-culture, transfection, mechanical delivery, sonic delivery, vibration delivery, magnetofection, or nanoparticle-mediated delivery. Introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ can include liposome transfection, calcium phosphate transfection, gene transfection, and dendrimer-mediated transfection. Introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ by mechanical transfection can include cell squeezing, cell bombardment, or gene gun techniques. Introducing nucleic acid sequences, transgenes and / or genome editing constructs into cells ex vivo, in vivo, in vitro or in situ by nanoparticle-mediated transfection can include liposome delivery, micelle delivery, and polymerosome delivery.

[0185] Introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ can include non-viral vectors. Non-viral vectors can include nucleic acids. Non-viral vectors can 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 can include transposons as described herein.

[0186] Introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ can involve viral vectors. The viral vector can be a non-integrating, non-chromosomal vector. Non-limiting examples of non-integrating, non-chromosomal vectors include adeno-associated virus (AAV), adenovirus, and herpes virus. The viral vector can be an integrating chromosomal vector. Non-limiting examples of integrating chromosomal vectors include adeno-associated vector (AAV), lentivirus, and gamma-retrovirus.

[0187] Introducing nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ can involve 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-based vector and an RNA-based vector, an RNA-based vector and a reverse transcriptase, a transposon-based vector and a transposase, a non-viral vector and an endonuclease, and a viral vector and an endonuclease.

[0188] Genome modification can include introducing a nucleic acid sequence, transgene, and / or genome editing construct into a cell ex vivo, in vivo, in vitro, or in situ to stably integrate a nucleic acid sequence, to transiently integrate a nucleic acid sequence, to generate site-specific integration of a nucleic acid sequence, to generate site-specific integration of a nucleic acid sequence, or to generate biased integration of a nucleic acid sequence. The nucleic acid sequence can be a transgene.

[0189] Genome modification can include introducing nucleic acid sequences, transgenes, and / or genome editing constructs into cells ex vivo, in vivo, in vitro, or in situ to stably integrate the nucleic acid sequences. Stable chromosomal integration can be random, site-specific, or biased integration. Site-specific integration can be unassisted or assisted. Assisted site-specific integration is delivered simultaneously with site-specific nucleases. The site-specific nucleases include transgenes with 5' and 3' nucleotide sequence extensions that contain percentage homology to regions upstream and downstream of the genomic integration site. Transgenes with homologous nucleotide extensions allow for genome integration by homologous recombination, microhomology-mediated end joining, or non-homologous end joining. Site-specific integration can occur at safe harbor sites. Genomic safe harbor sites can accommodate the integration of new genetic material in a way that ensures that the newly inserted genetic element is functional (e.g., expressed at therapeutically effective expression levels), does not cause deleterious changes to the host genome, and poses no risk to the host organism. Non-limiting examples of potential genomic safe harbors include intron sequences of the human albumin gene, the adeno-associated virus site 1 (AAVS1), the naturally occurring site of AAV viral integration on chromosome 19, the site of the chemokine (CC motif) receptor 5 (CCR5) gene, and the site of the human ortholog of the mouse Rosa26 locus.

[0190] Site-specific transgene integration can occur at a site that disrupts expression of a target gene. Disruption of target gene expression can occur by site-specific integration at an intron, exon, promoter, genetic element, enhancer, suppressor, start codon, stop codon, and response element. Non-limiting examples of target genes targeted by site-specific integration include TRAC, TRAB, PDI, any immunosuppressive gene, and genes involved in allogeneic rejection.

[0191] Site-specific transgene integration can occur at sites that enhance expression of the target gene. Enhancement of target gene expression can occur by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.

[0192] Enzymes can be used to create strand breaks in the host genome to facilitate transgene delivery or integration. Enzymes can create single-strand or double-strand breaks. Non-limiting examples of cleavage-inducing enzymes include transposases, integrases, endonucleases, CRISPR-Cas9, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), Cas-CLOVER™, and CPF1. Cleavage-inducing enzymes can be delivered to cells DNA-encoded, mRNA-encoded, as proteins, or as nucleoprotein complexes with guide RNAs (gRNAs).

[0193] Site-specific transgene integration can be controlled by vector-mediated integration site bias, which can be controlled by the selected lentiviral or gamma-retroviral vector.

[0194] The site-specific transgene integration site may be a non-stable chromosomal insertion. The integrated transgene may be silenced, removed, excised, or further modified. 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 embodiment, the transient non-chromosomal insertion of the transgene is not integrated into a chromosome, and the modified genetic material is not replicated during cell division.

[0195] The genome modification can be semistable or persistent non-chromosomal integration of the transgene. The DNA vector encodes a scaffold / matrix attachment region (S-MAR) module that binds to nuclear matrix proteins for episomal retention of the non-viral vector, allowing autonomous replication within the nuclei of dividing cells.

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

[0197] Modification of the genome by inserting a transgene can occur through homologous recombination (HR), microhomology-mediated end joining (MMEJ), non-homologous end joining (NHEJ), transposase enzyme-mediated modification, integrase enzyme-mediated modification, endonuclease enzyme-mediated modification, or host cell-directed double-strand break repair (homology-directed repair) by recombinase enzyme-mediated modification. Modification of the genome by inserting a transgene can occur through CRISPR-Cas9, TALEN, ZFN, Cas-CLOVER™, and cpf1.

[0198] In gene editing systems involving the insertion of new or existing nucleotides / nucleic acids, an insertion tool (e.g., a DNA template vector, a transposable element (transposon or retrotransposon)) must be delivered to a cell in addition to a cleavage enzyme (e.g., a nuclease, a recombinase, an integrase, or a transposase). Examples of such insertion tools for recombination enzymes may include a DNA vector. Other gene editing systems require the delivery of an integrase along with the insertion vector, or a transposase along with the transposon / retrotransposon. An example of a recombination enzyme that can be used as a cleavage enzyme is CRE recombinase. Non-limiting examples of integrases that can be used in insertion tools include viral-based enzymes taken from any of a number of viruses, including AAV, gamma retrovirus, and lentivirus. Examples of transposons / retrotransposons that can be used in insertion tools are described in more detail herein.

[0199] Cells with ex vivo, in vivo, in vitro, or in situ genomic modifications can be germline or somatic cells. The modified cells can be human, non-human, mammalian, rat, mouse, or canine cells. The modified cells can be differentiated, undifferentiated, or immortalized. The modified undifferentiated cells can be stem cells. The modified undifferentiated cells can be induced pluripotent stem cells. The modified cells can be immune cells. The modified cells can be T cells, hematopoietic stem cells, natural killer cells, macrophages, dendritic cells, monocytes, megakaryocytes, or osteoclasts. The modified cells can be modified while the cells are in a resting, activated, quiescent, interphase, prophase, metaphase, anaphase, or telophase state. The modified cells can be fresh, cryopreserved, bulk, sorted into subpopulations, selected from whole blood, leukapheresis, or immortalized cell lines. Detailed descriptions for isolating cells from leukapheresis products or blood are disclosed in PCT Publications WO2019 / 173636 and PCT / US2019 / 049816.

[0200] The present disclosure provides a gene editing composition and / or a cell comprising the gene editing composition. The gene editing composition may include a sequence encoding a DNA binding domain and a sequence encoding a nuclease protein or its nuclease domain. The sequence encoding the nuclease protein or its nuclease domain may include a DNA sequence, an RNA sequence, or a combination thereof. The nuclease or its nuclease domain may include one or more of a CRISPR / Cas protein, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), and an endonuclease.

[0201] The nuclease or nuclease domain thereof can comprise a nuclease-inactivated Cas protein and an endonuclease. The endonuclease can comprise Clo051 nuclease or a nuclease domain thereof. The gene editing composition can comprise a fusion protein. The fusion protein can comprise a nuclease-inactivated Cas9 (dCas9) protein and Clo051 nuclease or a Clo051 nuclease domain. The gene editing composition can further comprise a guide sequence. The guide sequence comprises an RNA sequence.

[0202] The present disclosure provides compositions comprising a small Cas9 (Cas9) operatively linked to an effector. The present disclosure provides fusion proteins consisting essentially of or consisting of a DNA localization component and an effector molecule, where the effector comprises a small Cas9 (Cas9). The small Cas9 construct of the present disclosure can include an effector comprising a type IIS endonuclease. A Staphylococcus aureus Cas9 having an active catalytic site comprises the amino acid sequence of SEQ ID NO:83.

[0203] The present disclosure provides compositions comprising an inactivated small Cas9 (dSaCas9) operably linked to an effector. The present disclosure provides fusion proteins consisting essentially of or consisting of a DNA localization component and an effector molecule, wherein the effector comprises a small inactivated Cas9 (dSaCas9). The small inactivated Cas9 (dSaCas9) construct of the present disclosure can include an effector comprising a type IIS endonuclease. The SadA Cas9 comprises the amino acid sequence of SEQ ID NO: 84, including D10A and N580A mutations to inactivate the catalytic site.

[0204] The present disclosure provides compositions comprising an inactivated Cas9 (dCas9) operably linked to an effector. The present disclosure provides fusion proteins consisting essentially of or consisting of a DNA localization component and an effector molecule, where the effector comprises an inactivated Cas9 (dCas9). The inactivated Cas9 (dCas9) constructs of the present disclosure can include an effector comprising a Type IIS endonuclease.

[0205] dCas9 can be isolated or derived from Streptococcus pyogenes. dCas9 can comprise substitutions at amino acid positions 10 and 840 that inactivate the catalytic site. In some embodiments, these substitutions are D10A and H840A. dCas9 can comprise the amino acid sequence of SEQ ID NO:85 or SEQ ID NO:86.

[0206] An exemplary Clo051 nuclease domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:87.

[0207] An exemplary dCas9-Clo051 (Cas-CLOVER) fusion protein can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 88. An exemplary dCas9-Clo051 fusion protein can be encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 89. The nucleic acid encoding the dCas9-Clo051 fusion protein can be DNA or RNA.

[0208] An exemplary dCas9-Clo051 (Cas-CLOVER) fusion protein can comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 90. An exemplary dCas9-Clo051 fusion protein can be encoded by a polynucleotide comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO: 91. The nucleic acid encoding the dCas9-Clo051 fusion protein can be DNA or RNA.

[0209] The cell containing the gene editing composition can stably or transiently express the gene editing composition. Preferably, the gene editing composition is transiently expressed. The guide RNA can comprise a sequence complementary to the target sequence in the genomic DNA sequence. The target sequence in the genomic DNA sequence can be a target sequence in a safe harbor site of the genomic DNA sequence.

[0210] 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.

[0211] Gene editing tools can also be delivered to cells using one or more poly(histidine)-based micelles. Poly(histidine) (e.g., poly(L-histidine)) is a pH-sensitive polymer with imidazole rings that provide electron lone pairs on the unsaturated nitrogen. That is, poly(histidine) has amphoteric properties through protonation-deprotonation. In particular, at a specific pH, poly(histidine)-containing triblock copolymers can assemble into micelles with positively charged poly(histidine) units on the surface, thereby enabling complexation with negatively charged gene editing molecules. Using these nanoparticles to bind and release proteins and / or nucleic acids in a pH-dependent manner can provide an efficient and selective mechanism for achieving desired genetic modifications. In particular, this micelle-based delivery system offers substantial flexibility in terms of charged materials, large payload capacity, and targeted release of nanoparticle payloads. In one example, site-specific cleavage of double-stranded DNA is enabled by the delivery of nucleases using poly(histidine)-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 core, leaving the hydrophilic and polyblocks at the ends to form one or more surrounding layers.

[0212] In one aspect, the present disclosure provides triblock copolymers consisting of a hydrophilic block, a hydrophobic block, and a charged block. In some embodiments, the hydrophilic block can be poly(ethylene oxide) (PEO) and the charged block can be poly(L-histidine). An exemplary triblock copolymer that can be used is PEO-b-PLA-b-PHIS, where the number of repeating units in each block varies by design.

[0213] Diblock copolymers that can be used as intermediates for preparing triblock copolymers can include hydrophilic, biocompatible poly(ethylene oxide) (PEO), which is chemically equivalent to PEG, and are conjugated to various hydrophobic aliphatic poly(anhydrides), poly(nucleic acids), poly(esters), poly(orthoesters), poly(peptides), poly(phosphazenes), and poly(saccharides), including, but not limited to, poly(lactide) (PLA), poly(glycolide) (PLGA), poly(lactic-co-glycolic acid) (PLGA), poly(ε-caprolactone) (PCL), and poly(trimethylene carbonate) (PTMC). Polymeric micelles composed of 100% PEGylated surfaces have improved in vitro chemical stability, increased in vivo bioavailability, and extended blood circulation half-lives.

[0214] Polymeric vesicles, polymersomes, and poly(histidine)-based micelles (including those containing triblock copolymers), and methods for their production, 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. Application Publication Nos. 2014 / 0363496; 2017 / 0000743; and 2019 / 0255191; and PCT Publication No. WO2019 / 126589.

[0215] Transposon and Vector Compositions The present disclosure provides compositions and methods for delivering an antibody (e.g., scFv) or a CAR (e.g., comprising an scFv) to a cell or cell population. Non-limiting examples of compositions for delivery of the compositions of the present disclosure to a cell or cell population include a transposon or a vector. Thus, the present disclosure provides a transposon comprising an antibody (e.g., scFv) or a CAR (e.g., comprising an scFv), or a vector comprising an antibody (e.g., scFv) or a CAR (e.g., comprising an scFv).

[0216] A transposon comprising a CAR of the present disclosure, or a vector comprising a CAR of the present disclosure, can further comprise a sequence encoding an inducible pro-apoptotic polypeptide. Alternatively or additionally, one transposon or one vector can comprise a CAR of the present disclosure, and a second transposon or second vector can comprise a sequence encoding an inducible pro-apoptotic polypeptide of the present disclosure. Inducible apoptotic polypeptides are described in more detail herein.

[0217] A transposon comprising a CAR of the present disclosure, or a vector comprising a CAR of the present disclosure, can further comprise a sequence encoding a chimeric stimulating receptor (CSR). Alternatively, or in addition, one transposon or one vector can comprise a CAR of the present disclosure, and a second transposon or second vector can comprise a sequence encoding a CSR of the present disclosure. Chimeric stimulating receptors are described in more detail herein.

[0218] A transposon comprising a CAR of the present disclosure or a vector comprising a CAR of the present disclosure can further comprise a sequence encoding a recombinant HLA-E polypeptide. Alternatively, or in addition, one transposon or one vector can comprise a CAR of the present disclosure, and a second transposon or second vector can comprise a sequence encoding a recombinant HLA-E polypeptide. Recombinant HLA-E polypeptides are described in more detail herein.

[0219] A transposon comprising a CAR of the present disclosure, or a vector comprising a CAR of the present disclosure, can further comprise a selection gene. The selection gene can encode a gene product essential for cell survival and viability. The selection gene can encode a gene product essential for cell survival and viability when challenged under selective cell culture conditions. The selective cell culture conditions can include a compound deleterious to cell survival or viability, where the gene product confers resistance to the compound. Non-limiting examples of selection genes include neo (confers resistance to neomycin), DHFR (encodes dihydrofolate reductase, conferring resistance to methotrexate), TYMS (encodes thymidylate synthetase), MGMT (encodes O(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encodes aldehyde dehydrogenase 1 family, member A1), FRANCF, RAD51C (encodes RAD51 paralog C), GCS (encodes glucosylceramide synthase), NKX2.2 (encodes NK2 homeobox 2), or any combination thereof.

[0220] In a preferred embodiment, the selection gene encodes a DHFR mutein enzyme. The DHFR mutein enzyme comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:92. The DHFR mutein enzyme consists of, or is encoded by, a polynucleotide consisting of, the nucleic acid sequence of SEQ ID NO:93 or SEQ ID NO:174. The amino acid sequence of the DHFR mutein enzyme can further comprise a mutation at one or more of positions 80, 113, or 153. The amino acid sequence of the DHFR mutein enzyme can include 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.

[0221] A transposon comprising a CAR of the present disclosure or a vector comprising a CAR of the present disclosure can further comprise at least one self-cleaving peptide. For example, the self-cleaving peptide can be located between the CAR (e.g., comprising an scFv) and the inducible pro-apoptotic polypeptide; or the self-cleaving peptide can be located between the CAR (e.g., comprising an scFv) and the protein encoded by the selection gene.

[0222] A transposon comprising a CAR of the present disclosure or a vector comprising a CAR of the present disclosure can further comprise 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 apoptosis-inducing polypeptide, and the second self-cleaving peptide is located downstream or immediately downstream of the inducible apoptosis-inducing polypeptide; or the first self-cleaving peptide and the second self-cleaving peptide are adjacent to the inducible apoptosis-inducing polypeptide. For example, the first self-cleaving peptide is located upstream or immediately upstream of the protein encoded by the selection gene, and the second self-cleaving peptide is located downstream or immediately downstream of the protein encoded by the selection gene; or the first self-cleaving peptide and the second self-cleaving peptide are adjacent to the protein encoded by the selection gene.

[0223] 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 that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 94. A GSG-T2A peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 95. A GSG-T2A polypeptide is encoded by a polynucleotide comprising or consisting of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 96. An E2A peptide comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 97. A GSG-E2A peptide comprises, consists essentially of, or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 98. The F2A peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 99. The GSG-F2A peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 100.A P2A peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 101. A GSG-P2A peptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 102.

[0224] In some embodiments, a transposon of the present disclosure comprises a nucleic acid encoding a CAR of SEQ ID NO: 13. In some embodiments, the nucleic acid encoding the CAR is adjacent to a nucleic acid encoding a T2A peptide of SEQ ID NO: 95. In some embodiments, the transposon further comprises a nucleic acid encoding a DHFR enzyme of SEQ ID NO: 92. In some embodiments, the transposon further comprises a nucleic acid encoding an ic9 safety switch peptide of SEQ ID NO: 172.

[0225] In some embodiments, a transposon of the present disclosure comprises, consists essentially of, or consists of a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 175.

[0226] Dislocation system The present disclosure provides a transposon comprising a protein scaffold disclosed herein, or the present disclosure provides a transposon comprising an antibody (e.g., scFv) or CAR (e.g., comprising an scFv) disclosed herein. In a preferred embodiment, the transposon is a plasmid DNA transposon flanked by a nucleotide sequence (e.g., comprising an scFv) encoding an scFv or CAR disclosed herein and two cis-regulatory insulator elements. The present disclosure also provides a composition comprising the transposon. In a preferred embodiment, the composition comprising the transposon further comprises a plasmid comprising a nucleotide sequence encoding a transposase. The nucleotide sequence encoding the transposase can be a DNA sequence or an RNA sequence. Preferably, the sequence encoding the transposase is an mRNA sequence.

[0227] The transposon of the present disclosure can 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. The sequence encoding the SPB transposase is an mRNA sequence.

[0228] Non-limiting examples of PB transposons and PB, PBL, and SPB transposases are described in detail in U.S. Patent No. 6,218,182; U.S. Patent No. 6,962,810; U.S. Patent No. 8,399,643, and PCT Publication WO2010 / 099296.

[0229] PB, PBL, and SPB transposases recognize transposon-specific inverted terminal repeats (ITRs) at the ends of transposons and insert the contents between the ITRs into a 5'-TTAT-3' sequence (TTAT target sequence) or a 5'-TTAA-3' sequence (TTAA target sequence) within the chromosomal site. The target sequences of the PB or PBL transposon 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'- TTAC-3', 5'-ACTA-3', 5'-AGGG-3', 5'-CTAG-3', 5'-TGAA-3', 5'-AGGT-3', 5'-ATCA-3', 5'-CTCC-3', 5'-TAAA-3', 5'-TCTC-3', 5'TGAA-3', 5'-AA The PB or PBL transposon system can comprise or consist of AT-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 have payload restrictions on the gene of interest that can be contained between the ITRs.

[0230] Exemplary amino acid sequences of one or more PB, PBL, and SPB transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810; and U.S. Patent No. 8,399,643. In preferred embodiments, the PB transposase comprises or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO:103.

[0231] The PB or PBL transposase may comprise or consist of an amino acid sequence having two or more, three or more, or more amino acid substitutions at each of positions 30, 165, 282, or 538 of the sequence of SEQ ID NO: 103. The transposase may be an SPB transposase comprising or consisting of the amino acid sequence of the sequence of SEQ ID NO: 103, wherein the amino acid substitution at position 30 may be a substitution of valine (V) for isoleucine (I), the amino acid substitution at position 165 may be a substitution of serine (S) for glycine (G), the amino acid substitution at position 282 may be a substitution of valine (V) for methionine (M), and the amino acid substitution at position 538 may be a substitution of lysine (K) for asparagine (N). In preferred embodiments, the SPB transposase comprises or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 104.

[0232] In certain embodiments where the transposase comprises the above mutations at positions 30, 165, 282, and / or 538, the PB, PBL, and SPB transposases can further comprise amino acid substitutions 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:103 or SEQ ID NO:104, as described in more detail in PCT Publications WO2019 / 173636 and PCT / US2019 / 049816.

[0233] The PB, PBL, or SPB transposase can be isolated or derived from an insect, vertebrate, crustacean, or chordate, as described in detail in PCT Publications WO2019 / 173636 and PCT / US2019 / 049816. In a preferred embodiment, the PB, PBL, or SPB transposase is isolated or derived from the insect Trichoplusia ni (GenBank Accession No. AAA87375) or Bombyx mori (GenBank Accession No. BAD11135).

[0234] A hyperactive PB or PBL transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In a preferred embodiment, the hyperactive PB or PBL transposase is isolated or derived from Bombyx mori or Xenopus tropicalis. Examples of hyperactive PB or PBL transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810; U.S. Patent No. 8,399,643; and WO2019 / 173636. A list of hyperactive amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[0235] In some embodiments, the PB or PBL transposase is integration-deficient. An integration-deficient PB or PBL transposase is a transposase that can excise the corresponding transposon but integrates the excised transposon at a lower frequency than the corresponding wild-type transposase. Examples of integration-deficient PB or PBL transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810; U.S. Patent No. 8,399,643 and WO2019 / 173636. A list of integration-deficient amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[0236] In some embodiments, the PB or PBL transposase is fused to a nuclear localization signal. Examples of PB or PBL transposase 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 WO2019 / 173636.

[0237] The transposon of the present disclosure can be a Sleeping Beauty transposon. When the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase (e.g., 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 that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO: 105. In preferred embodiments, the hyperactive Sleeping Beauty (SB100X) transposase comprises or consists of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 106.

[0238] The transposon of the present disclosure may be a Helraiser transposon. Exemplary Helraiser transposons include Helibat1, which comprises or consists of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 107. In one embodiment, when the transposon is a Helraiser transposon, the transposase is a Helitron transposase (e.g., as disclosed in WO2019 / 173636). In a preferred embodiment, the Helitron transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 108.

[0239] The transposon of the present disclosure may be a Tol2 transposon. An exemplary Tol2 transposon, including an inverted repeat, a subterminal sequence, and a Tol2 transposase, comprises or consists of a nucleic acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 109. In some embodiments, when the transposon is a Tol2 transposon, the transposase is a Tol2 transposase (e.g., as disclosed in WO2019 / 173636). In a preferred embodiment, the Tol2 transposase comprises or consists of an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 110.

[0240] The transposon of the present disclosure can 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 WO2019 / 173636). The TcBuster transposase can 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 that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO: 111. The polynucleotide encoding the TcBuster transposase can comprise or consist of a naturally occurring or non-naturally occurring nucleic acid sequence. In preferred embodiments, the TcBuster transposase is encoded by a polynucleotide comprising or consisting of a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any percentage therebetween) to SEQ ID NO:112.

[0241] In some embodiments, the mutant TcBuster transposase comprises one or more sequence mutations when compared to the wild-type TcBuster transposase, as described in detail in PCT Publications WO2019 / 173636 and PCT / US2019 / 049816.

[0242] The transposon is a nanotransposon. The nanotransposon comprises, consists essentially of, or consists of: (a) a transposon insert-encoding sequence, including a sequence encoding a first inverted terminal repeat (ITR), a sequence encoding a second inverted terminal repeat (ITR), and an inter-ITR sequence; (b) a backbone-encoding sequence, the backbone-encoding sequence including a sequence encoding an origin of replication having 1 to 450 nucleotides, inclusive, and a sequence encoding a selectable marker having 1 to 200 nucleotides, inclusive, and (c) an inter-ITR sequence. In some embodiments, the inter-ITR sequence of (c) comprises the sequence of (b). In some embodiments, the inter-ITR sequence of (a) comprises the sequence of (b).

[0243] The sequence encoding the backbone can comprise 1 to 600 nucleotides, including the endpoints. In some embodiments, the sequence encoding the backbone consists of 1 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, or 550 to 600 nucleotides, each range including the endpoints.

[0244] The inter-ITR sequence can comprise 1 to 1,000 nucleotides, inclusive of the endpoints. In some embodiments, the inter-ITR sequence consists of 1 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, 650 to 700 nucleotides, 700 to 750 nucleotides, 750 to 800 nucleotides, 800 to 850 nucleotides, 850 to 900 nucleotides, 900 to 950 nucleotides, or 950 to 1,000 nucleotides, each range including the endpoints.

[0245] The nanotransposon can be a short nanotransposon (SNT), where the inter-ITR sequence comprises 1 to 200 nucleotides, including the endpoints, and the inter-ITR sequence is 1 to 10 nucleotides, 10 to 20 nucleotides, 20 to 30 nucleotides, 30 to 40 nucleotides, 40 to 50 nucleotides, 50 to 60 nucleotides, 60 to 70 nucleotides, 70 to 80 nucleotides, 80 to 90 nucleotides, or 90 to 100 nucleotides, each including the endpoints.

[0246] A selectable marker having 1 to 200 nucleotides including an end point can comprise a sequence encoding a sucrose selectable marker. The sequence encoding a sucrose selectable marker can comprise a sequence encoding an RNA-OUT sequence. The sequence encoding the RNA-OUT sequence can comprise or consist of 137 base pairs (bp). A selectable marker having 1 to 200 nucleotides including an end point can comprise a sequence encoding a fluorescent marker. A selectable marker having 1 to 200 nucleotides including an end point can comprise a sequence encoding a cell surface marker.

[0247] The sequence encoding an origin of replication having 1 to 450 nucleotides including the end points can comprise a sequence encoding a miniorigin of replication. In some embodiments, the sequence encoding an origin of replication having 1 to 450 nucleotides including the end points comprises a sequence encoding an R6K origin of replication. The R6K origin of replication can comprise an R6K gamma origin of replication. The R6K origin of replication can comprise an R6K miniorigin of replication. The R6K origin of replication can comprise an R6K gamma miniorigin of replication. The R6K gamma miniorigin of replication can comprise or consist of 281 base pairs (bp).

[0248] In some embodiments of the nanotransposon, the backbone-encoding sequence does not comprise a recombination site, an excision site, a ligation site, or a combination thereof. In some embodiments, neither the nanotransposon nor the backbone-encoding sequence comprises the product of a recombination site, an excision site, a ligation site, or a combination thereof. In some embodiments, neither the nanotransposon nor the backbone-encoding sequence is derived from a recombination site, an excision site, a ligation site, or a combination thereof.

[0249] In some embodiments of the nanotransposon, the recombination sites comprise sequences that result from a recombination event. In certain embodiments, the recombination sites comprise sequences that are the product of a recombination event. In some embodiments, the recombination event comprises the activity of a recombinase enzyme (e.g., a recombinase site).

[0250] In some embodiments of the nanotransposon, the backbone-encoding sequence does not further comprise a sequence encoding foreign DNA.

[0251] In some embodiments of the nanotransposon, the inter-ITR sequence does not comprise a recombination site, an excision site, a ligation site, or a combination thereof. In some embodiments, the inter-ITR sequence does not comprise the product of a recombination event, an excision event, a ligation event, or a combination thereof. In some embodiments, the inter-ITR sequence does not result from a recombination event, an excision event, a ligation event, or a combination thereof. In some embodiments, the inter-ITR sequence comprises a sequence encoding foreign DNA. In some embodiments, the inter-ITR sequence comprises at least one sequence encoding an insulator and a sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell. The mammalian cell can be a human cell. In some embodiments, the inter-ITR sequence comprises a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell, and a second sequence encoding an insulator. In some embodiments, the intra-ITR sequence comprises a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing an exogenous sequence in a mammalian cell, a polyadenosine (polyA) sequence, and a second sequence encoding an insulator. In some embodiments, the sequence within the ITR comprises a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing the exogenous sequence in a mammalian cell, at least one exogenous sequence, a polyadenosine (polyA) sequence, and a second sequence encoding an insulator.

[0252] Nanotransposons are described in detail in PCT / US2019 / 067758.

[0253] Vector System The vectors of the present disclosure may be viral vectors or recombinant vectors. Viral vectors may contain sequences isolated or derived from retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or any combination thereof. Viral vectors may contain sequences isolated or derived from adeno-associated viruses (AAV). Viral vectors may include recombinant AAV (rAAV). Exemplary adeno-associated viruses and recombinant adeno-associated viruses contain two or more inverted terminal repeat (ITR) sequences located in cis next to the scFv or CAR encoding sequence of the present disclosure. Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, all serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids that contain the genome of one serotype and the capsid of another serotype (e.g., AAV2 / 5, AAV-DJ, and AAV-DJ8). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, rAAV-LK03.

[0254] 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 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 can be passively or actively transported across cell membranes.

[0255] The cell delivery compositions (e.g., transposons, vectors) disclosed herein can include nucleic acids encoding therapeutic proteins or therapeutic agents. Examples of therapeutic proteins include those disclosed in PCT Publications WO2019 / 173636 and PCT / US2019 / 049816.

[0256] Inducible Pro-Apoptotic Polypeptides The inducible pro-apoptotic polypeptides disclosed herein are superior to existing inducible polypeptides because the inducible pro-apoptotic polypeptides of the present disclosure are much less immunogenic. The inducible pro-apoptotic polypeptides are recombinant polypeptides and therefore do not occur in nature. Furthermore, they have been engineered to produce inducible pro-apoptotic polypeptides that do not contain non-human sequences that could be recognized as "non-self" by the host human immune system, thereby inducing an immune response in a subject receiving the inducible pro-apoptotic polypeptide, in cells containing the inducible pro-apoptotic polypeptide, or in cells containing a composition comprising the inducible pro-apoptotic polypeptide.

[0257] The present disclosure provides an inducible pro-apoptotic polypeptide comprising a ligand-binding region, a linker, and a pro-apoptotic peptide, wherein the inducible pro-apoptotic polypeptide does not comprise a non-human sequence. In certain embodiments, the non-human sequence comprises a restriction site. In some embodiments, the ligand-binding region can 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 can be a cleaved caspase 9 polypeptide. The inducible apoptotic polypeptide can be non-naturally occurring. When the caspase is caspase 9 or cleaved caspase 9, the inducible pro-apoptotic polypeptide can also be referred to as an "iC9 safety switch."

[0258] The inducible caspase polypeptide can 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 some embodiments, the inducible caspase polypeptide comprises (a) a ligand-binding region, (b) a linker, and (c) a cleaved caspase 9 polypeptide, wherein the inducible pro-apoptotic polypeptide does not comprise non-human sequences.

[0259] The ligand-binding region can 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 can comprise a modification at position 36 of the sequence. This modification can be a substitution of valine (V) for phenylalanine (F) at position 36 (F36V). The FKBP12 polypeptide can comprise, consist essentially of, or consist of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 113. The FKBP12 polypeptide can be encoded by a polynucleotide comprising or consisting of a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:114.

[0260] The linker region can comprise, consist essentially of, or consist of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 115, or the linker can be encoded by a polynucleotide that comprises or consists of a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 116. In some embodiments, the nucleic acid sequence encoding the linker does not contain any restriction sites.

[0261] 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 that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:117, or the truncated caspase-9 polypeptide may be encoded by a polynucleotide that comprises or consists of a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:118.

[0262] In particular embodiments where the polypeptide comprises a cleaved caspase-9 polypeptide, the inducible pro-apoptotic polypeptide can comprise, consist essentially of, or consist of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:119, or the inducible pro-apoptotic polypeptide can be encoded by a polynucleotide that comprises or consists of a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:120.

[0263] In particular embodiments where the polypeptide comprises a cleaved caspase-9 polypeptide, the inducible pro-apoptotic polypeptide can comprise, consist essentially of, or consist of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 172, or the inducible pro-apoptotic polypeptide can be encoded by a polynucleotide that comprises or consists of a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 173.

[0264] The inducible apoptosis-inducing 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 apoptosis-inducing polypeptide in that cell.

[0265] Activation of inducible pro-apoptotic polypeptides can be achieved, for example, by generating conditionally controlled proteins or polypeptides through chemically induced dimerization (CID) mediated by an inducer. Not only are the pro-apoptotic polypeptides inducible, but the induction of these polypeptides is also reversible due to degradation of the labile dimerizer or administration of a monomeric competitive inhibitor.

[0266] In certain embodiments, when the ligand binding region comprises an FKBP12 polypeptide with a substitution of valine (V) for phenylalanine (F) at position 36 (F36V), the inducer is AP1903, a synthetic drug (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 can be used interchangeably.

[0267] Inducible apoptosis-inducing peptides and methods for inducing these peptides are described in detail in U.S. Patent Publication No. WO2019 / 0225667 and PCT Publication No. WO2018 / 068022.

[0268] Chimeric stimulatory receptors and recombinant HLA-E polypeptides 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 disclosed cells (e.g., allogeneic cells) can be modified to disrupt expression or function of T cell receptors (TCRs) and / or classes of major histocompatibility complexes (MHCs). TCRs mediate graft-versus-host (GvH) reactions, while MHCs mediate host-versus-graft (HvG) reactions. In preferred embodiments, any expression and / or function of TCRs is removed to prevent T cell-mediated GvH, which can cause death in the subject. Thus, in preferred embodiments, the present disclosure provides pure TCR-negative allogeneic T cell compositions (e.g., each cell of the composition expresses TCRs at such low levels that they are undetectable or nonexistent).

[0269] 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 and consequently improve cell engraftment in a subject. Improved engraftment results in longer cell persistence and, therefore, a larger therapeutic window for the subject. Specifically, the expression and / or function of a structural component of MHC-I, beta-2-microglobulin (B2M), is reduced or eliminated.

[0270] The above strategies pose additional challenges. T cell receptor (TCR) knockout (KO) in T cells results in the loss of expression of CD3-zeta (CD3z or CD3ζ), a part of the TCR complex. Loss of CD3ζ in TCR-KO T cells dramatically reduces the ability to optimally activate and expand these cells using standard stimulating / activating reagents, including, but not limited to, agonistic anti-CD3 mAbs. Disruption of expression or function of any component of the TCR complex results in the loss of all components of the complex, including TCR-alpha (TCRα), TCR-beta (TCRβ), CD3-gamma (CD3γ), CD3-epsilon (CD3ε), CD3-delta (CD3δ), and CD3-zeta (CD3ζ). CD3ε and CD3ζ are required for T cell activation and proliferation. Agonistic anti-CD3 mAbs typically recognize CD3ε and possibly other proteins within the complex, which then signal to CD3ζ. CD3ζ serves as the primary stimulus (together with a secondary costimulatory signal) for optimal T cell activation and proliferation. Under normal conditions, full T cell activation depends on engagement of the TCR 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 activation / stimulation reagents, including agonist anti-CD3 mAbs, significantly reduces T cell proliferation. In fact, T cell proliferation is reduced to only 20–40% of normal expansion levels when stimulated with standard activation / stimulation reagents, including agonist anti-CD3 mAbs.

[0271] Accordingly, the present disclosure provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an ectodomain comprising an activating component, wherein the activating component is isolated or derived from a first protein; (b) a transmembrane domain; and (c) an endodomain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein, wherein the first protein and the second protein are not identical.

[0272] The activating component can include one or more portions of a T cell receptor (TCR), a TCR complex, a TCR co-receptor, a TCR co-stimulatory protein, a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor to which an agonist of the activating component binds. The activating component can include the CD2 extracellular domain or a portion thereof to which an agonist binds.

[0273] The signaling domain can include one or more of a human signaling domain, a T cell receptor (TCR), a component of a TCR complex, a component of a TCR coreceptor, a component of a TCR costimulatory protein, a component of a TCR inhibitory protein, a cytokine receptor, and a chemokine receptor. The signaling domain can include a CD3 protein or a portion thereof. The CD3 protein can include a CD3 zeta protein or a portion thereof.

[0274] The endodomain may further comprise a cytoplasmic domain. The cytoplasmic domain may be isolated or derived from a third protein. The first protein and the third protein are the same. 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 are the same. The transmembrane domain may be isolated or derived from a fifth protein. The first protein and the fifth protein are the same.

[0275] 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 transduce 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 transduce a signal when the activating component binds to a non-naturally occurring molecule.

[0276] In a preferred embodiment, the present disclosure provides a non-natural chimeric stimulating receptor (CSR) comprising: (a) an ectodomain comprising a signal peptide and an activation component, wherein the ectodomain comprises the signal peptide or a portion thereof, and the activation component comprises the CD2 extracellular domain or a portion thereof to which an agonist binds; (b) a transmembrane domain comprising the CD2 transmembrane domain or a portion thereof; and (c) an endodomain comprising a cytoplasmic domain and at least one signaling domain, wherein the cytoplasmic domain comprises the CD2 cytoplasmic domain or a portion thereof, and the at least one signaling domain comprises the CD2 cytoplasmic domain or a portion thereof, and the at least one signaling domain comprises the CD3 zeta protein or a portion thereof. In some embodiments, the non-natural CSR comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 121. In a preferred embodiment, the non-natural CSR comprises the amino acid sequence of SEQ ID NO: 121.

[0277] The present disclosure also provides non-natural chimeric stimulating receptors (CSRs) whose ectodomains comprise modifications. The modifications can include mutations or truncations of the amino acid sequence of the activating component or first protein compared to the wild-type sequence of the activating component or first protein. The mutations or truncations of the amino acid sequence of the activating component can include mutations or truncations of the CD2 extracellular domain or a portion thereof to which an agonist binds. The mutations or truncations of the CD2 extracellular domain can reduce or eliminate binding to naturally occurring CD58. In some embodiments, the CD2 extracellular domain comprising the mutations or truncations comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 122. In a preferred embodiment, the CD2 extracellular domain comprising the mutations or truncations comprises the amino acid sequence of SEQ ID NO: 122.

[0278] In a preferred aspect, the present disclosure provides a non-natural chimeric stimulating receptor (CSR) comprising: (a) an ectodomain comprising a signal peptide and an activation component, wherein the ectodomain comprises the signal peptide or a portion thereof, and wherein the activation component comprises the CD2 extracellular domain or a portion thereof to which an agonist binds, and the ectodomain comprises the CD2 extracellular domain or a portion thereof to which an agonist binds; (b) a transmembrane domain comprising the CD2 transmembrane domain or a portion thereof, and wherein the transmembrane domain comprises the CD2 transmembrane domain or a portion thereof; and (c) a cytoplasmic domain and at least one signaling domain, wherein the cytoplasmic domain comprises the CD2 cytoplasmic domain or a portion thereof, and at least one signaling domain comprises the CD2 cytoplasmic domain or a portion thereof, and the at least one signaling domain comprises a CD3ζ protein or a portion thereof. In some embodiments, the non-natural CSR comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO: 123. In preferred embodiments, the non-natural CSR comprises the amino acid sequence of SEQ ID NO: 123.

[0279] 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.

[0280] 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.

[0281] The modified cells disclosed herein can be allogeneic or autologous cells. In some preferred embodiments, the modified cells are allogeneic cells. In some embodiments, the modified cells are autologous T cells or modified autologous CAR T cells. In some preferred embodiments, the modified cells are allogeneic T cells or modified allogeneic CAR T cells.

[0282] The present disclosure provides compositions comprising any of the CSRs disclosed herein. The present disclosure provides compositions comprising a nucleic acid sequence encoding any of the CSRs disclosed herein. The present disclosure provides compositions comprising a vector comprising a nucleic acid sequence encoding any of the CSRs disclosed herein. The present disclosure provides compositions comprising a transposon comprising a nucleic acid sequence encoding any of the CSRs disclosed herein. The present disclosure provides compositions comprising a modified cell disclosed herein, or a composition comprising a plurality of modified cells disclosed herein.

[0283] The present disclosure provides (a) modified T cell receptors (TCRs), wherein the modification is in an endogenous sequence encoding the T cell receptor (TCR), which reduces or eliminates the expression or activity level of the TCR, and (b) modified T lymphocytes (T cells), comprising a chimeric stimulating receptor (CSR), wherein the CSR comprises: (i) an ectodomain comprising an activating component, wherein the activating component is isolated or derived from a first protein; (ii) a transmembrane domain; and (iii) an endodomain comprising at least one signaling domain, wherein the at least one signaling domain is isolated or derived from a second protein, wherein the first protein and the second protein are not identical.

[0284] The modified T cells can further comprise an inducible pro-apoptotic polypeptide. The modified T cells can further comprise a modification of the endogenous sequence encoding beta-2-microglobulin (B2M), which reduces or eliminates major histocompatibility complex (MHC) class I (MHC-I) expression or activity levels.

[0285] The modified T cells can 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 can further comprise a B2M signal peptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide can further comprise a B2M polypeptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide can further comprise a linker, wherein the linker is positioned between the B2M polypeptide and the HLA-E polypeptide. The non-naturally occurring polypeptide comprising an HLA-E polypeptide can further comprise a peptide and a B2M polypeptide. The non-naturally occurring polypeptide comprising HLA-E can further comprise a first linker positioned between the B2M signal peptide and the peptide, and a second linker positioned between the B2M polypeptide and the peptide encoding HLA-E.

[0286] The modified T cells can further comprise a non-native antigen receptor, a sequence encoding a therapeutic polypeptide, or a combination thereof. The non-native antigen receptor can include a chimeric antigen receptor (CAR).

[0287] 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-natural antigen receptor or a therapeutic protein can be transiently expressed in the modified T cells. A sequence encoding a non-natural antigen receptor or a therapeutic protein can be stably expressed in the modified T cells.

[0288] As described in detail herein, gene editing compositions, including but not limited to RNA-guided fusion proteins comprising dCas9-Clo051, can be used to target, reduce, or eliminate the expression of endogenous T cell receptors. In a preferred embodiment, the gene editing composition targets and deletes a gene, a portion of a gene, or a regulatory element (e.g., a promoter) of a gene encoding an endogenous T cell receptor. Non-limiting examples of primers (including T7 promoters, genomic target sequences, and gRNA scaffolds) for generating guide RNA (gRNA) templates for targeting and deleting TCR-alpha (TCR-α), TCR-beta (TCR-β), and beta-2-microglobulin (β2M) are disclosed in PCT application PCT / US2019 / 049816.

[0289] Gene editing compositions, including, but not limited to, RNA-guided fusion proteins, including dCas9-Clo051, can be used to target, reduce, or eliminate the expression of endogenous MHC1, MHCII, or MHC activators. In a preferred embodiment, the gene editing composition targets and deletes a gene, a portion of a gene, or a regulatory element (e.g., a promoter) of a gene encoding one or more components of endogenous MHC1, MHCII, or MHC activators. Non-limiting examples of guide RNAs (gRNAs) for targeting and deleting MHC activators are disclosed in PCT application PCT / US2019 / 049816.

[0290] A detailed description of genetic modifications of endogenous sequences encoding non-natural chimeric stimulating receptors, TCR-alpha (TCR-α), TCR-beta (TCR-β), and / or beta-2-microglobulin (β2M), and non-natural polypeptides, including HLA class I histocompatibility antigen, alpha chain E (HLA-E) polypeptides, is disclosed in PCT application PCT / US2019 / 049816.

[0291] Formulation, dosage and administration method The present disclosure provides formulations, dosages and methods for administration of the compositions described herein.

[0292] The disclosed compositions and pharmaceutical compositions can further comprise at least one of any suitable auxiliary agent, including, but not limited to, a diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant, 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, in Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990, and "Physician's Desk Reference," 25th Edition, Medical Economics (Montvale, NJ) 1998. Pharmaceutically acceptable carriers are well known in the art or can be routinely selected as appropriate for the mode of administration, solubility, and / or stability of the protein scaffold, fragment, or variant compositions as described herein.

[0293] Non-limiting examples of pharmaceutical excipients and additives suitable for use include proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; derivatized sugars, e.g., alditols, aldonic acids, esterified sugars, and the like; and polysaccharides or sugar polymers), which may be present alone or in combination and may comprise 1 to 99.99% by weight or volume, alone or in combination. Non-limiting examples of protein excipients include serum albumins, such as human serum albumin, recombinant human albumin (rHA), gelatin, and casein. Representative amino acids / protein components that also function in buffering capacity 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.

[0294] 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; alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), myo-inositol, etc. Preferably, the carbohydrate excipient is mannitol, trehalose, and / or raffinose.

[0295] The composition can also contain a buffer or pH adjuster, and typically, the buffer is a salt prepared from an organic acid or 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; Tris, tromethamine hydrochloride, or phosphate buffer. Preferred buffers are organic acid salts such as citrate.

[0296] Additionally, the disclosed compositions can include polymeric excipients / additives such as polyvinylpyrrolidone, Ficoll (polymeric sugars), dextrates (e.g., cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycol, flavoring agents, antimicrobial agents, 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).

[0297] Many known and developed modes can be used to administer a therapeutically effective amount of the compositions or pharmaceutical compositions disclosed herein. Non-limiting examples of modes of administration include bolus, buccal, infusion, intra-articular, intrabronchial, intraperitoneal, intravesical, intrachondral, intracavity, intracellular, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intralesional, intramuscular, intramyocardial, intranasal, intraocular, intraosseous, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intratumoral, intravenous, intravesical, oral, parenteral, rectal, sublingual, subcutaneous, transdermal, or vaginal means.

[0298] The compositions of the present disclosure may be formulated for parenteral administration (subcutaneous, intramuscular, intravenous) or any other administration, particularly in the form of a solution or suspension; for vaginal or rectal administration, particularly in semi-solid form (non-limiting examples: creams, suppositories, etc.); for buccal or sublingual administration, particularly in the form of tablets or capsules; for intranasal administration, particularly in the form of powders, nasal drops, aerosols, or some other pharmaceutical form; for transdermal administration, particularly in the form of gels, ointments, lotions, suspensions, or patch delivery systems, which may contain chemical enhancers (such as dimethyl sulfoxide) to alter the skin structure or to increase the drug concentration in the transdermal patch (Junginger et al., in Drug Permeation Enhancement, edited by Hsieh, DS, pp. 59-90 (Marcel Dekker, Inc., New York, 1994; which are incorporated herein by reference in their entireties), using oxidizing agents that allow formulations containing proteins and peptides to be applied to the skin (WO 98 / 53847), applying electric fields (e.g., iontophoresis) or ultrasound (e.g., sonophoresis) to create transient transport pathways (e.g., electroporation) or to increase the mobility of charged drugs through the skin (U.S. Patent Nos. 4,309,989 and 4,767,402) (the above publications and patents are incorporated herein by reference in their entireties).

[0299] For parenteral administration, any composition disclosed herein can be formulated as a solution, suspension, emulsion, granules, powder, or lyophilized powder, or a pharmaceutically acceptable parenteral vehicle can be provided separately. Typical excipients for parenteral administration preparations include sterilized water or saline, polyalkylene glycols (such as polyethylene glycol), oils of plant origin, hydrogenated naphthalenes, etc. Aqueous or oily suspensions for injection can be prepared by using appropriate emulsifiers or wetting agents and suspending agents according to known methods. For injections, non-toxic, parenterally administrable diluents (such as aqueous solutions, sterile injectable solutions or suspensions in solvents) are acceptable. Usable vehicles or solvents include water, Ringer's solution, isotonic saline, etc. Sterile, fixed oils can be used as common solvents or suspending media. For these purposes, any type of fixed oil and fatty acid can be used, including natural, synthetic, or semi-synthetic fatty oils or fatty acids, natural, synthetic, or semi-synthetic monoglycerides, diglycerides, or triglycerides. Parenteral administration is known in the art, and non-limiting examples include conventional injection means, the gas-pressure 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 (incorporated herein by reference in their entireties).

[0300] 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, and enzyme inhibitors (e.g., pancreatic trypsin inhibitor, diisopropylfluorophosphate (DFF) and trasylol) to inhibit enzymatic degradation. Formulations for delivery of hydrophilic substances, including proteins and protein scaffolds, and combinations of at least two surfactants for oral, buccal, mucosal, nasal, pulmonary, transvaginal, or rectal administration are described in U.S. Patent No. 6,309,663. The active ingredient compound in a solid dosage form for oral administration can be mixed with at least one additive, including sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, and glycerides. These dosage forms can also contain other types of additives, such as inert diluents, lubricants such as magnesium stearate, preservatives such as parabens, sorbic acid, ascorbic acid, α-tocopherol, antioxidants such as cysteine, disintegrants, binders, thickeners, buffers, sweeteners, flavorings, flavoring agents, etc.

[0301] Tablets and pills can be further processed into enteric-coated preparations. Liquid preparations for oral administration include emulsions, syrups, elixirs, suspensions, and solution preparations that are medically acceptable. These preparations can contain inert diluents commonly used in the art, 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). Furthermore, carrier compounds used for orally delivering biologically active agents, as described in U.S. Pat. Nos. 5,879,681 and 5,871,753, are known in the art.

[0302] For pulmonary administration, the compositions or pharmaceutical compositions described herein are preferably delivered in a particle size effective to reach the lower respiratory tract of the lungs or paranasal sinuses. The compositions or pharmaceutical compositions can be delivered by any of a variety of inhalation or nasal devices known in the art for administering therapeutic agents by inhalation. These devices, which can deposit aerosolized formulations in a patient's paranasal sinuses or alveoli, include metered-dose inhalers, nebulizers (e.g., jet nebulizers, ultrasonic nebulizers), dry powder generators, sprayers, and the like. All such devices can employ formulations suitable for administration for dispensing the compositions or pharmaceutical compositions described herein into aerosols. Such aerosols can be composed of either solutions (both aqueous and non-aqueous) or solid particles. Furthermore, sprays containing the compositions or pharmaceutical compositions described herein can be produced by forcing a suspension or solution of at least one protein scaffold through a nozzle under pressure. In metered-dose inhalers, the propellant, the compositions or pharmaceutical compositions described herein, and any excipients or other additives are contained in a canister as a mixture with a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol containing particles preferably 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 PCT Publication No. 2019 / 049816.

[0303] For absorption through mucosal surfaces, the composition comprises an emulsion containing a plurality of submicron particles, a mucoadhesive polymer, a bioactive peptide, and an aqueous continuous phase that promotes absorption through mucosal surfaces by achieving mucoadhesion of the emulsion particles (U.S. Pat. No. 5,514,670). Mucous surfaces suitable for application of the emulsions of the present disclosure may include the cornea, conjunctiva, oral cavity, sublingual, nasal, vaginal, pulmonary, gastric, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, such as suppositories, may contain excipients such as polyalkylene glycols, vaseline, cocoa butter, etc. Formulations for intranasal administration are solid and may contain excipients such as lactose, or may be aqueous or oil solutions for nasal sprays. For buccal administration, excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch, etc. (U.S. Pat. No. 5,849,695). A more detailed description of mucosal administration and formulations is disclosed in PCT Publication WO2019 / 049816.

[0304] For transdermal administration, the compositions or pharmaceutical compositions disclosed herein are encapsulated in a delivery device such as a liposome or polymer nanoparticle, microparticle, microcapsule, or microsphere (collectively referred to as microparticles unless otherwise specified). Many suitable devices are known, including microparticles made of synthetic polymers such as polyhydroxy acids (e.g., polylactic acid, polyglycolic acid, and their copolymers), polyorthoesters, polyanhydrides, and polyphosphazenes, as well as natural polymers such as collagen, polyamino acids, albumin, and other proteins, alginate 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 PCT Publication No. 2019 / 049816.

[0305] It is desirable to deliver the disclosed compounds to a subject for an extended period of time, for example, one week to one year from a single administration. Various sustained-release, depot, or implant dosage forms can be used. For example, the dosage form can contain a pharmaceutically acceptable, non-toxic salt of a compound that is poorly soluble in body 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 mono- or disulfonic acid, or polygalacturonic acid; (b) a salt with a polyvalent metal cation, such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, or cadmium, or a salt with an organic cation, such as formed from N,N'-dibenzylethylenediamine or ethylenediamine; or (c) a combination of (a) and (b), such as zinc tannate. Furthermore, the disclosed compounds, or preferably relatively insoluble salts such as those just described, can be formulated into gels suitable for injection, such as aluminum monostearate gels containing 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 would contain the compound or salt dispersed for encapsulation in a slowly degrading, non-toxic, non-antigenic polymer, such as a polylactic acid / polyglycolic acid polymer, as described in U.S. Pat. No. 3,773,919. The compound or preferably a relatively insoluble salt, such as those described above, can also be formulated into cholesterol-matrix silastic pellets, particularly for use in animals. Additional sustained-release, depot, or implant formulations, such as gas 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).

[0306] Suitable dosage is well known in the art.See, for example, Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, California (2000); Nursing 2001 Handbook of Drugs, 21st Edition, Springhouse Corp., Springhouse, Pa., 2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc., Upper Saddle River, NJ. Preferred doses can optionally include about 0.1-99 and / or 100-500 mg / kg / dose, or any range, value, or fraction thereof, or achieve a serum concentration of about 0.1-5000 μg / ml, or any range, value, or fraction thereof, per single or multiple dose. A preferred dosage range for the compositions or pharmaceutical compositions disclosed herein is about 1 mg / kg, up to about 3, about 6, or about 12 mg / kg body weight of the subject.

[0307] Alternatively, the administered dosage may vary depending on known factors, such as the pharmacodynamic properties of the particular drug, its mode and route of administration; the recipient's age, health, and weight; the nature and severity of the condition; the type of concurrent treatment; the frequency of treatment; and the desired effect. Typically, the dosage of the active ingredient may be about 0.1 to 100 milligrams per kilogram of body weight. To achieve the desired results, a dosage of 0.1 to 50, preferably 0.1 to 10 milligrams per kilogram, or sustained-release form, is usually effective.

[0308] By way of non-limiting example, human or animal treatment can be provided as a single or periodic dose of a composition or pharmaceutical composition disclosed herein, at least about 0.1 to 100 mg / kg per day, or any range, value, or rate thereof, for 1 to 40 days, or alternatively or additionally at least once every 1 to 52 weeks, or alternatively or additionally at least once every 1 to 20 years, or any combination thereof, in single, infusion, or repeated doses.

[0309] Dosage forms suitable for internal use generally contain from about 0.001 milligrams to about 500 milligrams of active ingredient per unit or container. In these pharmaceutical compositions, the active ingredient will usually be present in an amount of from about 0.5 to 99.999% by weight based on the total weight of the composition.

[0310] An effective amount may include an amount of about 0.001 to about 500 mg / kg per single (e.g., bolus), multiple, or continuous administration, or may achieve a serum concentration of 0.01 to 5000 μg / ml per single, multiple, or continuous administration, or any effective range or value therein, as performed and determined using methods described herein or known in the relevant art.

[0311] In embodiments in which the composition administered to a subject is a modified cell as disclosed herein, the cells are about 1 x 10 3 and 1 x 10 15 Cells; approx. 1 x 10 4 and 1 x 10 12 Cells; approx. 1 x 10 5 and 1 x 10 10 Cells; approx. 1 x 10 6 and 1 x 10 9 Cells; approx. 1 x 10 6 and 1 x 10 6 Cells; approx. 1 x 10 6 and 1 x 10 7 cells; or approximately 1 x 10 6 and 25 x 10 6 In one embodiment, the cells can be administered between about 5 x 10 6 ~25×10 6administered between cells.

[0312] A more detailed description of the pharmaceutically acceptable excipients, formulations, dosages, and methods of administration of the disclosed compositions and pharmaceutical compositions is disclosed in PCT Publication No. 2019 / 049816.

[0313] Methods of Using the Disclosed Compositions The present disclosure provides uses of the disclosed compositions and pharmaceutical compositions to treat a disease or disorder in a cell, tissue, organ, animal, or subject, for example, by administering or contacting a therapeutically effective amount of the composition or pharmaceutical composition to the cell, tissue, organ, animal, or subject, using the disclosed compositions and pharmaceutical compositions as known in the art or as described herein. In one embodiment, the subject is a mammal. Preferably, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0314] The present disclosure provides methods 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. These include 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 sarcoma, colorectal cancer, pancreatic cancer, nasopharyngeal carcinoma, malignant histiocytosis, paraneoplastic syndrome / malignant hypercalcemia, solid tumors, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head cancer, neck cancer, hereditary non-polyposis carcinoma, Hodgkin's lymphoma, liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, testicular cancer, adenocarcinoma, sarcoma, malignant melanoma, hemangioma, metastatic disease, cancer-related bone resorption, and cancer-related bone pain.

[0315] In a preferred embodiment, the treatment of a malignant disease or disorder involves adoptive cell therapy. For example, in one embodiment, the present disclosure provides modified cells expressing at least one disclosed antibody (e.g., scFv) and / or CAR, including selected and / or expanded antibodies (e.g., scFv), for administration to a subject in need thereof. The modified cells can be formulated for storage at any temperature, including room temperature and body temperature. The modified cells can be formulated for cryopreservation and subsequent thawing. The modified cells can be formulated in a pharmaceutically acceptable carrier for direct administration to a subject from sterile packaging. The modified cells can be formulated in a pharmaceutically acceptable carrier with indicators of cell viability and / or CAR expression level to ensure a minimum level of cell function and CAR expression. The modified cells can be formulated in a pharmaceutically acceptable carrier at a predetermined density with one or more reagents to inhibit further expansion and / or prevent cell death.

[0316] Any of the methods can 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 such modulation, treatment, or therapy. Such methods can optionally further include co-administration or combination therapy for treating such a disease or disorder, wherein the administration of any of the compositions or pharmaceutical compositions disclosed herein further includes administering at least one chemotherapeutic agent (e.g., alkylating agent, mitotic inhibitor, radiopharmaceutical) before and / or after the co-administration.

[0317] 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 can be by any means known in the art and described in detail herein. Preferably, systemic administration is by intravenous injection or infusion. In one embodiment, administration is local. Local administration can be by any means known in the art and described in detail herein. Preferably, local administration is by intratumoral, intrathecal, intraventricular, intraocular, or intraosseous injection or infusion.

[0318] In some embodiments, the therapeutically effective dose is a single dose. In some embodiments, the single dose is 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 produced simultaneously. In some embodiments, where the composition is autologous or allogeneic cells, the amount administered is sufficient for the cells to engraft and / or persist for a sufficient time to treat the disease or disorder.

[0319] In one example, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an antibody (e.g., an scFv) or a CAR comprising the antibody (e.g., an scFv), wherein the antibody or CAR specifically binds to an antigen on a tumor cell. In embodiments where the composition comprises modified cells or cell populations, the cells or cell populations can be autologous or allogeneic.

[0320] In some embodiments of the therapeutic methods described herein, treatment may be modified or terminated. Specifically, in embodiments in which the composition used in treatment comprises an inducible apoptosis-inducing polypeptide, apoptosis can be selectively induced in cells by contacting the cells with the inducer. Treatment may be modified or terminated, for example, 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 adverse events. In some embodiments, the method includes administering an inhibitor of the inducer to inhibit modification of the cell therapy, thereby restoring the function and / or efficacy of the cell therapy (e.g., if signs or symptoms of disease return or increase in severity and / or adverse events disappear).

[0321] Antibody / scFv production, screening and purification At least one antibody of the disclosure (e.g., a monoclonal antibody, a chimeric antibody, a single domain antibody, a VHH, a VH, a single-chain variable fragment (scFv), an antigen-binding fragment (Fab) or a Fab fragment) can be produced by a cell line, a mixed cell line, an immortalized cell, or a clonal population of immortalized cells, as desired, as is well known in the art. For example, Ausubel et al., editors, 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. al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY (1997-2001).

[0322] Amino acids from the scFv may be altered, added and / or deleted to reduce immunogenicity or to reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, stability, solubility or other suitable properties known in the art.

[0323] If desired, scFvs can be engineered with high affinity for the antigen and retention of other favorable biological properties. To this end, scaffold proteins can optionally be prepared by a process of analysis of the parental sequences and various conceptually engineered products using three-dimensional models of the parental and engineered sequences. Three-dimensional models are publicly available and familiar to those skilled in the art. Computer programs are available (e.g., Xencor's Immunofilter program, Monrovia, CA) that illustrate and display the likely three-dimensional conformational structures of selected candidate sequences and measure their potential immunogenicity. By inspecting these displays, the possible role of residues in the function of the candidate sequence can be analyzed, i.e., the analysis of residues that affect the ability of the candidate scFv to bind to its antigen. In this way, residues can be selected and combined from the parental and reference sequences to achieve desired characteristics, such as affinity for the target antigen. Alternatively, or in addition to the above procedures, other suitable engineering methods can be used.

[0324] Screening for scFvs that specifically bind 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 large collections of peptides for individual members with the desired function or structure. The displayed nucleotide or peptide sequences can be 3 to 5,000 or more nucleotides or amino acids in length, often 5 to 100 amino acids in length, often about 8 to 25 amino acids in length. In addition to direct chemical synthesis methods for generating peptide libraries, several recombinant DNA methods have been described. One type involves displaying peptide sequences on the surface of bacteriophage or cells, each of which contains a nucleotide sequence encoding a particular displayed peptide sequence. Such methods are described in PCT Patent Publications WO 91 / 17271, WO 91 / 18980, WO 91 / 19818, and WO 93 / 08278.

[0325] Other systems for generating libraries of peptides incorporate aspects of both in vitro chemical synthesis and recombinant methods. See PCT Patent Publications WO 92 / 05258, WO 92 / 14843, and WO 96 / 19256. See also U.S. Patent Nos. 5,658,754 and 5,643,768. Peptide display libraries, vectors, and screening kits are commercially available from suppliers 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 assigned to Enzon; ​​U.S. Patent Nos. 5,223,409, 5,403,484, 5,571,698, and 5,837,500 assigned to Dyax; U.S. Patent Nos. 5,427,908 and 5,580,717 assigned to Affymax; and Cambridge Antibody See U.S. Patent No. 5,885,793 assigned to Genentech; U.S. Patent No. 5,750,373 assigned to Genentech; U.S. Patent Nos. 5,618,920, 5,595,898, 5,576,195, 5,698,435, 5,693,493, and 5,698,417 assigned to Xoma; Colligan, supra; Ausubel, supra; or Sambrook, supra.

[0326] The scFvs 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 scFv of the present disclosure binds with high affinity, e.g., about 10, as determined by surface plasmon resonance or Kinexa methods, as performed by one of skill in the art. -7KD of 0.1 to 9.9 (or any range or value therein) x 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , 10 -15 Or, it can bind to the target protein at any range or value therebetween.

[0327] The affinity or avidity of an scFv for an antigen can be experimentally determined using any suitable method (see, for example, Berzofsky et al., "Antibody-Antigen Interactions," In Fundamental Immunology, edited by Paul, WE, Raven Press: New York, NY (1984); Kuby, Janis, Immunology, WH Freeman and Company: New York, NY (1992); and methods described therein). The measured affinity of a particular scFv-antigen interaction may vary when measured under different conditions (e.g., salt concentration, pH). Therefore, measurements of affinity and other antigen binding parameters (e.g., KD, Kon, Koff) are preferably performed using standard solutions of the protein scaffold and antigen, as well as standardized buffers such as those described herein.

[0328] Competition assays can be performed using the scFvs of the present disclosure to determine which proteins, antibodies, and other antagonists compete with the scFvs of the present disclosure for binding to a target protein and / or share epitope regions. These assays assess competition between antagonists or ligands for a limited number of binding sites on a protein, as is readily known to those of skill in the art. The protein and / or antibody is 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 was pre-insolubilized) or centrifugation (if the protein / antibody was precipitated after the competition reaction). Competitive binding can also be determined by whether function is altered by binding or lack of binding of the scFv to the target protein, for example, whether the scFv molecule inhibits or enhances, for example, the enzymatic activity of a label. ELISAs and other functional assays can be used, as are well known in the art.

[0329] nucleic acid molecule The disclosed nucleic acid molecule encoding an scFv 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, cloned or synthetically produced cDNA and genomic DNA, 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 called the antisense strand.

[0330] Isolated nucleic acid molecules of the present disclosure can include nucleic acid molecules comprising an open reading frame (ORF), optionally with one or more introns, e.g., at least one specific portion of at least one scFv; nucleic acid molecules comprising a coding sequence for a protein scaffold or loop region that binds to a target protein; and nucleic acid molecules that comprise a nucleotide sequence substantially different from those described above, but that, due to the degeneracy of the genetic code, still encode a protein scaffold as described herein and / or known in the art. Of course, the genetic code is well known in the art. Thus, it would be routine for one of skill in the art to generate such degenerate nucleic acid variants encoding specific scFvs of the present disclosure. See, e.g., Ausubel et al., supra; such nucleic acid variants are included in the present disclosure.

[0331] As provided herein, nucleic acid molecules of the present disclosure, including nucleic acids encoding scFvs, can include, but are not limited to, those encoding the amino acid sequence of an scFv fragment alone; coding sequences for an entire protein scaffold or a portion thereof; coding sequences for an scFv, fragment, or portion, 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, and 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 mRNA processing (e.g., ribosome binding and mRNA stability), including transcription, splicing, and polyadenylation signals; and additional coding sequences encoding additional amino acids, such as those that provide additional functions. In this manner, the protein scaffold-encoding sequence can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of a fusion protein scaffold comprising the protein scaffold fragment or portion.

[0332] Polynucleotides that selectively hybridize to the polynucleotides described herein The present disclosure provides isolated nucleic acids that hybridize to the polynucleotides disclosed herein under selective hybridization conditions. Thus, the polynucleotides can be used to isolate, detect, and / or quantify nucleic acids containing such polynucleotides. For example, the polynucleotides of the present disclosure can be used to identify, isolate, or amplify partial or full-length clones in a deposited library. The polynucleotides may be genomic or cDNA sequences isolated from a human or mammalian nucleic acid library, or may be complementary to cDNAs from a human or mammalian nucleic acid library.

[0333] Preferably, the cDNA library contains at least 80% full-length sequences, preferably at least 85% or 90% full-length sequences, and more preferably at least 95% full-length sequences. The cDNA library can be normalized to increase the representation of rare sequences. Low or moderate stringency hybridization conditions are typically used with sequences having reduced sequence identity to complementary sequences, but are not limited to these. Moderate and high stringency conditions can optionally be used for sequences with greater identity. Low stringency conditions allow selective hybridization of sequences with approximately 70% sequence identity and can be used to identify orthologous or paralogous sequences.

[0334] Optionally, the polynucleotide will encode at least a portion of the protein scaffold encoded by the polynucleotide described herein. The polynucleotide includes a nucleic acid sequence that can be used for selective hybridization to a polynucleotide encoding a protein scaffold of the present disclosure. See, e.g., Ausubel, supra; Colligan, supra.

[0335] Nucleic acid construction 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 is well known in the art.

[0336] A nucleic acid can advantageously contain nucleotide sequences in addition to the polynucleotides of the present disclosure. For example, a multiple cloning site containing one or more endonuclease restriction sites can be inserted into the nucleic acid to facilitate isolation of the polynucleotide. Also, a translatable sequence can be inserted to facilitate isolation of the translated polynucleotides of the present disclosure. For example, a hexa-histidine marker sequence provides a convenient means for purifying the proteins of the present disclosure. A nucleic acid of the present disclosure, excluding the coding sequence, is optionally a vector, adapter, or linker for cloning and / or expression of the polynucleotides of the present disclosure.

[0337] Additional sequences can be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, to aid in the isolation of polynucleotides, or to improve the introduction of polynucleotides into cells. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art (see, e.g., Ausubel, supra; or Sambrook, supra).

[0338] Recombinant methods for constructing nucleic acids The isolated nucleic acid compositions of the present disclosure, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methods known to those of skill in the art. In some embodiments, oligonucleotide probes that selectively hybridize under stringent conditions to the polynucleotides of the present disclosure are used to identify the desired sequence in a cDNA or genomic DNA library. The isolation of RNA and the construction of cDNA and genomic libraries are well known to those of skill in the art (see, e.g., Ausubel, supra; or Sambrook, supra).

[0339] Nucleic Acid Screening and Isolation Methods cDNA or genomic libraries can be screened using probes based on the sequences of the polynucleotides disclosed herein. Probes 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 understand that various degrees of hybridization stringency can be used in the assay, and that either the hybridization or wash medium can be made more stringent. The more stringent the hybridization conditions, the greater the degree of complementarity required between the probe and target for duplex formation to occur. The degree of stringency can be controlled by one or more of temperature, ionic strength, pH, and the presence of a partially denaturing solvent such as formamide. For example, hybridization stringency can be conveniently varied by changing the polarity of the reactant solution, e.g., by manipulating the concentration of formamide within the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding varies according to the stringency of the hybridization medium and / or wash medium. The degree of complementarity is optimally 100%, or 70-100%, or any range or value therein, however, it should be understood that minor sequence variations in the probes and primers can be compensated for by reducing the stringency of the hybridization and / or wash medium.

[0340] Methods for amplifying RNA or DNA are well known in the art and can be used in accordance with the disclosure without undue experimentation, based on the teachings and guidance presented herein.

[0341] Known methods of DNA or RNA amplification include, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (e.g., U.S. Patent Nos. 4,683,195, 4,683,202, 4,800,159, and 4,965,188 to Mullis et al.; U.S. Patent Nos. 4,795,699 and 4,921,794 to Tabor et al.; U.S. Patent No. 5,142,033 to Innis; U.S. Patent No. 5,122,464 to Wilson et al.; U.S. Patent No. 5,091,310 to Innis; U.S. Patent No. 5,066, No. 584 by Gelfand et al.; U.S. Pat. No. 4,889,818 by Gelfand et al.; U.S. Pat. No. 4,994,370 by Silver et al.; U.S. Pat. No. 4,766,067 by Biswas; and U.S. Pat. No. 4,656,134 by Ringold), and RNA-mediated amplification (U.S. Pat. No. 5,130,238 by Malek et al., trade name NASBA), which uses antisense RNA against a target sequence as a template for double-stranded DNA synthesis, the entire contents of which are incorporated herein by reference (see Ausubel, supra; or Sambrook, supra).

[0342] For example, polymerase chain reaction (PCR) techniques can be used to amplify the sequences of the disclosed polynucleotides and related genes directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods can be useful, for example, for cloning nucleic acid sequences encoding expressed proteins, for generating nucleic acids for use as probes to detect the presence of desired mRNA in a sample, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to guide those skilled in the art with in vitro amplification methods can be found in Berger, supra; Sambrook, supra; Ausubel, supra; Mullis et al., U.S. Pat. No. 4,683,202 (1987); 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, the Advantage-GC Genomic PCR Kit (Clontech). Additionally, for example, T4 gene 32 protein (Boehringer Mannheim) can be used to improve yields of long PCR products.

[0343] Nucleic acid synthesis method The isolated nucleic acids of the present disclosure can also be prepared by direct chemical synthesis using 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. Those skilled in the art will recognize that while chemical synthesis of DNA may be limited to sequences of about 100 or more bases, longer sequences can be obtained by ligating shorter sequences.

[0344] Recombinant Expression Cassette The present disclosure further provides recombinant expression cassettes comprising the nucleic acids of the present disclosure. The nucleic acid sequences of the present disclosure, for example, cDNA or genomic sequences encoding the protein scaffolds of the present disclosure, can be used to construct recombinant expression cassettes that can be introduced into at least one desired host cell. Recombinant expression cassettes typically comprise a polynucleotide of the present disclosure operably linked to a transcription initiation regulatory sequence that directs transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct expression of the nucleic acids of the present disclosure.

[0345] In some embodiments, isolated nucleic acids that function as promoters, enhancers, or other elements can be introduced into a non-heterologous form of the disclosed polynucleotide at an appropriate location (upstream, downstream, or intron) to up- or down-regulate expression of the disclosed polynucleotide. For example, endogenous promoters can be modified in vivo or in vitro by mutation, deletion, and / or substitution.

[0346] Expression vectors and host cells 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. See, e.g., Sambrook et al., supra; Ausubel et al., supra.

[0347] Polynucleotide can be optionally linked to a vector that contains a selectable marker for propagation in a host.Generally, plasmid vector is introduced into a precipitate such as calcium phosphate precipitate or in a complex with charged lipid.If vector is virus, it can be packaged in vitro using suitable packaging cell line, and then transduced into host cell.

[0348] The DNA insert must be operably linked to a suitable promoter. The expression construct will further contain 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 will preferably include a stop codon (e.g., UAA, UGA, or UAG) appropriately positioned for translation to begin at the beginning and the end of the mRNA to be translated, with UAA and UAG being preferred for mammalian or eukaryotic expression.

[0349] Expression vectors will preferably, but optionally, contain 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, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739), blasticidin (bsd gene), eukaryotic cell culture resistance genes, and the like, including ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / geneticin (neo gene), DHFR (encoding dihydrofolate reductase and conferring resistance to methotrexate), mycophenolic acid, or glutamine synthetase (GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739), blasticidin (bsd gene), eukaryotic cell culture resistance genes, and the like. Examples of suitable host cell resistance genes include those for resistance to erythromycin (bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline for culturing in E. coli and other bacteria or prokaryotes (the above patents are incorporated herein by reference in their entireties). Appropriate culture media and conditions for the above host cells are known in the art. Suitable vectors will be readily apparent to those skilled in the art. Introduction of vector constructs into host cells can be accomplished by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other known methods. Such methods are described in the art, e.g., Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, and 16.

[0350] The expression vector preferably, but optionally, will include at least one selectable cell surface marker for isolation of cells modified by the disclosed compositions and methods. Selectable cell surface markers of the present disclosure include surface proteins, glycoproteins, or groups of proteins that distinguish a cell or subset of cells from another defined subset of cells. Preferably, the selectable cell surface marker distinguishes those cells modified by the disclosed compositions or methods from those cells not modified by the disclosed compositions or methods. Such cell surface markers include, for example, but are not limited to, "designated cluster" or "classification determinant" proteins (often abbreviated as "CD"), such as truncated or full-length forms of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof. Further cell surface markers include the suicide gene marker RQR8 (Philip B et al., Blood. 2014 Aug 21;124(8):1277-87).

[0351] The expression vector will preferably, but optionally, include at least one selectable drug resistance marker for isolation of cells modified by the disclosed compositions and methods. The selectable 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.

[0352] At least one of the disclosed protein scaffolds can be expressed in modified forms, such as fusion proteins, which can contain not only secretion signals but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the protein scaffold to improve stability and persistence in host cells, during purification, or during subsequent handling and storage. Peptide moieties can also be added to the disclosed protein scaffolds to facilitate purification. Such regions can be removed prior to final preparation of the protein scaffold or at least one fragment thereof. Such methods are described in many standard laboratory manuals, e.g., Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17, and 18.

[0353] Those skilled in the art are familiar with the many expression systems available for expressing nucleic acids encoding the proteins of the present disclosure. Alternatively, the nucleic acids of the present disclosure can be expressed in host cells by turning on (by engineering) in host cells containing 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 entire contents of which are incorporated herein by reference.

[0354] Examples of cell cultures useful for producing protein scaffolds, specific portions or variants thereof are bacterial, yeast, and mammalian cells known in the art. Mammalian cell lines are often in the form of monolayers of cells, although mammalian cell suspensions or bioreactors can also be used. Many 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.

[0355] Expression vectors for these cells can include, but are not limited to, one or more of the following expression control sequences, such as 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-1 alpha promoter (U.S. Pat. No. 5,266,491), at least one human promoter; an enhancer, and / or processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., the SV40 large T antigen polyA addition site), and a transcription terminator sequence. See, e.g., Ausubel et al., supra; Sambrook et al., supra. Other cells useful for producing the nucleic acids or proteins of the disclosure can be found, for example, in the American Type Culture Collection Catalogue of Cells. Lines and Hybridomas (www.atcc.org) or other known or commercial sources.

[0356] When eukaryotic host cells are used, a polyadenylation or transcription terminator sequence is typically incorporated into the vector. An example of a terminator sequence is the polyadenylation sequence derived from the bovine growth hormone gene. Sequences for accurate splicing of the transcript can also be included. An example of a splicing sequence is the VP1 intron derived from SV40 (Sprague et al., J. Virol. 45:773-781 (1983)). Furthermore, gene sequences that control replication in host cells can be incorporated into vectors, as is known in the art.

[0357] scFv purification scFvs 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, hydroxylapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification. See, e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), e.g., chapters 1, 4, 6, 8, 9, and 10, each of which is incorporated herein by reference in its entirety.

[0358] The scFvs of the present disclosure include purified products, products of chemical synthesis methods, and products produced by recombinant techniques from prokaryotic or eukaryotic hosts, including, for example, E. coli, yeast, higher plants, insects, and mammalian cells. Depending on the host used in a recombinant production procedure, the disclosed protein backbones can be glycosylated or non-glycosylated. Such methods are described in many standard laboratory manuals, see, e.g., Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18, and 20; Colligan, Protein Science, supra, Chapters 12-14, which are incorporated herein by reference in their entirety.

[0359] Amino acid code The amino acids comprising the protein backbone of the present disclosure are often abbreviated. The designation of an amino acid can be indicated by its single-letter code, its three-letter code, its name, or by designating the amino acid by its three-nucleotide codon, as is well understood in the art (see Alberts, B., et al., Molecular Biology of The Cell, Third Ed., Garland Publishing, Inc., New York, 1994). The protein backbone of the present disclosure can contain one or more amino acid substitutions, deletions, or additions, either naturally occurring or resulting from mutations and / or human manipulation, as specified herein. Amino acids in the protein backbone 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, Chapters 8, 15; Cunningham and Wells, Science 244:1081-1085 (1989)). The latter method introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as, but not limited to, at least one neutralizing activity. Sites important for protein backbone binding can also be identified by structural analysis, such as crystallization, nuclear magnetic resonance, or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos et al., Science 255:306-312 (1992)).

[0360] As will be appreciated by those skilled in the art, the present disclosure includes at least one biologically active protein scaffold of the present disclosure. A biologically active protein scaffold has a specific activity that is at least 20%, 30%, or 40%, preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95% to 99% or greater of the specific activity of a natural (non-synthetic), endogenous, or related known protein scaffold. Methods for assaying and quantifying measures of enzymatic activity and substrate specificity are well known to those skilled in the art.

[0361] In another aspect, the present disclosure relates to protein scaffolds and fragments, as described herein, modified by the covalent attachment of organic moieties. Such modifications can produce protein scaffold fragments with improved pharmacokinetic properties (e.g., increased in vivo serum half-life). The organic moieties can be linear or branched hydrophilic polymer groups, fatty acid groups, or fatty acid ester groups. In particular, the hydrophilic polymer groups can have a molecular weight of about 800 to about 120,000 daltons and can be polyalkane glycols (e.g., polyethylene glycol), 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.

[0362] The modified protein scaffolds and fragments of the present disclosure can include one or more organic moieties covalently attached directly or indirectly to an antibody. Each organic moiety attached to a protein scaffold or fragment of the present disclosure can independently be a hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. As used herein, the term "fatty acid" encompasses monocarboxylic and dicarboxylic acids. The term "hydrophilic polymer group" as used herein refers to an organic polymer that is more soluble in water than octane. For example, polylysine is more soluble in water than octane. Thus, protein scaffolds modified by the covalent attachment of polylysine are encompassed by the present disclosure. Hydrophilic polymers suitable for modifying the protein scaffolds of the present disclosure can be linear or branched and include, for example, polyalkane glycols (e.g., PEG, monomethoxypolyethylene 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 daltons as separate molecular entities. For example, PEG5000 and PEG20,000 can be used, where the subscript is the average molecular weight of the polymer in daltons. The hydrophilic polymer group can be substituted with one to about six alkyl, fatty acid, or fatty acid ester groups. Hydrophilic polymers substituted with fatty acid or fatty acid ester groups can be prepared by suitable methods. For example, a polymer containing an amine group can be coupled to the carboxylate of a fatty acid or fatty acid ester, and an activated carboxylate on the fatty acid or fatty acid ester (e.g., activated with N,N-carbonyldiimidazole) can be coupled to a hydroxyl group on the polymer.

[0363] Fatty acids and fatty acid esters suitable for modifying the protein scaffolds of the present disclosure may be saturated or may contain one or more unsaturated units. Fatty acids suitable for modifying the protein scaffolds 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, arachidonic acid), octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like. Suitable fatty acid esters include monoesters of dicarboxylic acids containing a straight or branched chain lower alkyl group, which can contain from 1 to about 12, preferably from 1 to about 6, carbon atoms.

[0364] Modified protein scaffolds and fragments can be prepared using suitable methods, such as 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) containing an activating group. An "activating group" is a chemical moiety or functional group that can react with a second chemical group under appropriate 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 (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl ester (NHS), and the like. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol, and the like. Aldehyde functional groups can be coupled to amine hydrazide-containing molecules, and azide groups can react with trivalent phosphate groups to form phosphoramidate or phosphorimide bonds. Suitable methods for introducing activating groups into molecules are known in the art (see, e.g., 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 via linker moieties, e.g., divalent C1-C12 groups in which one or more carbon atoms can be replaced with heteroatoms 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 can be prepared, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylic acid.The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA), allowing the primary amine to be coupled with another carboxylate as described in, or reacted with maleic anhydride and the resulting product cyclized to form an activated maleimide derivative of a fatty acid (see, e.g., Thompson et al., WO 92 / 16221, the entire disclosure of which is incorporated herein by reference).

[0365] The modified protein scaffolds of the present disclosure can be prepared by reacting a protein scaffold or fragment with a modifying agent. For example, organic moieties can be attached to the protein backbone in a non-site-specific manner using amine-reactive modifying agents, such as NHS esters of PEG. Modified protein scaffolds and fragments comprising organic moieties attached to specific sites on 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).

[0366] definition As used throughout this disclosure, the singular forms "a," "an," and "the" include plural references 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 a reference to one or more doses and equivalents thereof known to those skilled in the art.

[0367] The terms "about" or "approximately" mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within one or more standard deviations. Alternatively, "about" can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. When specific values ​​are described in the application and claims, unless otherwise specified, the term "about" meaning within an acceptable error range for the particular value should be assumed.

[0368] 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 naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, and substantially free of chemical precursors or other chemicals when 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 can 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. Protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When the disclosed proteins or biologically active portions thereof are recombinantly produced, optimal culture media represent less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein chemicals of interest.

[0369] The present disclosure provides fragments and variants of the disclosed DNA sequences and proteins encoded by these DNA sequences. As used throughout the disclosure, the term "fragment" refers to a portion of a DNA sequence or a portion of an amino acid sequence, and thus the protein encoded thereby. Fragments of DNA sequences, including coding sequences, retain the biological activity of the native protein and can therefore encode protein fragments that retain DNA recognition or target DNA sequence binding activity as described herein. Alternatively, fragments of DNA sequences useful as hybridization probes generally do not encode proteins that retain biological activity or do not retain promoter activity. Thus, fragments of DNA sequences can range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotides of the present disclosure.

[0370] The nucleic acids or proteins of the present disclosure can be constructed by a modular approach that involves preassembling monomeric and / or repeating units in a target vector that can be assembled into a final destination vector. Polypeptides of the present disclosure may comprise repeating monomers of the present disclosure and can be constructed by a modular approach by preassembling repeating units in a target vector that can then be assembled into a final destination vector. The present disclosure provides polypeptides produced by the present methods, as well as nucleic acid sequences encoding these polypeptides. The present disclosure provides host organisms and cells containing nucleic acid sequences encoding the polypeptides produced by this modular approach.

[0371] The term "antibody" is used in the broadest sense and specifically encompasses 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 herein defined herein. Thus, according to one aspect of the present specification, the term "antibodies herein" in its broadest sense also encompasses such analogs. Generally, such analogs may have one or more amino acid residues substituted, deleted, and / or added compared to the antibodies herein defined herein.

[0372] As used herein, the term "antibody fragment," and all grammatical variations thereof, is defined as a portion of an intact antibody comprising the antigen-binding site or variable region of the intact antibody, which portion 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; and any antibody fragment having a primary structure consisting of a single contiguous sequence of consecutive amino acid residues (referred to herein as a "single-chain antibody fragment" or "single-chain polypeptide"), including, but not limited to, (1) single-chain Fv (scFv) molecules; (2) single-chain polypeptides comprising one light-chain variable domain, or fragments comprising the three CDRs of the light-chain variable domain without any associated heavy chain portions; and (3) single-chain polypeptides comprising only one heavy-chain variable region, or fragments thereof comprising the three CDRs of the heavy-chain variable region without any associated light chain portions; as well as multispecific or multivalent structures formed from antibody fragments. In antibody fragments comprising one or more heavy chains, the heavy chain may include any constant domain sequence found in the non-Fc region of an intact antibody (e.g., CHI in an IgG isotype), and / or may include any hinge region sequence found in an intact antibody, and / or may include a leucine zipper sequence fused to or located at 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 types will be readily understood by those skilled in the art.

[0373] "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 of phosphate residues in a DNA backbone), provided that the interaction as a whole is sequence-specific.

[0374] The term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" shall mean excluding other elements that are essential to the combination when used for the intended purpose. Thus, a composition consisting essentially of the elements defined herein would not exclude trace contaminants or inert carriers. "Consisting of" refers to excluding more than trace elements of other components and substantial method steps. Aspects defined by each of these transition terms are within the scope of this disclosure.

[0375] The term "epitope" refers to an antigenic determinant of a polypeptide. An epitope can contain three amino acids having a spatial conformation unique to the epitope. Generally, an epitope consists of at least four, five, six, or seven such amino acids, and more usually, at least eight, nine, or ten such 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.

[0376] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which 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.

[0377] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, microRNA, structural RNA, or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0378] "Modulation" or "regulation" of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression.

[0379] The term "operably linked" or its equivalents (e.g., "operably linked") means that two or more molecules are positioned relative to one another so that they can interact in a manner that affects a function attributed to one or both molecules, or a combination thereof.

[0380] Non-covalently linked components and methods for making and using non-covalently linked components are disclosed. The various components can take a variety of different forms, as described herein. For example, non-covalently linked (i.e., operably linked) proteins can be used to allow for temporary interactions, avoiding 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 when, or under circumstances where, such association is required primarily for the desired activity. Association may be of sufficient duration to allow for the desired effect.

[0381] 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 can be operably linked via a non-covalent bond.

[0382] The term "scFv" refers to a single-chain variable fragment. An scFv is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin linked to a linker peptide. The linker peptide can be about 5 to 40 amino acids, or about 10 to 30 amino acids, or about 5, 10, 15, 20, 25, 30, 35, or 40 amino acids in length. Single-chain variable fragments lack the constant Fc region found in intact antibody molecules and thus lack the common binding site (e.g., protein G) used to purify antibodies. The term also includes scFvs that are in vivo, antibodies that are stable in the cytoplasm of a cell and can bind to intracellular proteins.

[0383] 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 generally peptide chains about 110 amino acids in length, containing one variable domain (VH) of a heavy-chain antibody or common IgG; they generally have a similar affinity for antigen as the whole antibody, but are more heat-resistant and stable to detergents and high concentrations of urea. Examples are derived from camel or fish antibodies. Alternatively, single-domain antibodies can be generated from common mouse or human IgG, which has four chains.

[0384] 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 homogeneous mixture of different antigens. In some embodiments, a specific binding interaction will distinguish between desired and undesired antigens in a sample. In certain embodiments, it is about 100-fold or greater (e.g., about 1000-fold or greater or 10,000-fold or greater). "Specificity" refers to the ability of an immunoglobulin or immunoglobulin fragment, such as a nanobody, to bind preferentially to one antigen target over different antigen targets, and does not necessarily imply high affinity.

[0385] 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 when sufficient conditions for binding exist.

[0386] The terms "nucleic acid" or "oligonucleotide" or "polynucleotide" refer to at least two nucleotides covalently linked to each other. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid can also encompass the complementary strand of a depicted single strand. Nucleic acids of the present disclosure also include substantially identical nucleic acids and their complements that retain the same structure or encode the same protein.

[0387] A probe of the present disclosure can 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 can refer to a probe that hybridizes under stringent hybridization conditions.

[0388] The nucleic acids of the present disclosure can be single-stranded or double-stranded. The nucleic acids of the present disclosure can comprise a double-stranded sequence even if the majority of the molecule is single-stranded. The nucleic acids of the present disclosure can comprise a single-stranded sequence even if the majority of the molecule is double-stranded. The nucleic acids of the present disclosure can include genomic DNA, cDNA, RNA, or hybrids thereof. The nucleic acids of the present disclosure can include a combination of deoxyribonucleotides and ribonucleotides. The nucleic acids of the present disclosure can include a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. The nucleic acids of the present disclosure can be synthesized to include unnatural amino acid modifications. The nucleic acids of the present disclosure can be obtained by chemical synthesis or recombinant methods.

[0389] The nucleic acids of the present disclosure, their entire sequences, or any portion thereof, may not be naturally occurring. The nucleic acids of the present disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not occur in nature, making the entire nucleic acid sequence non-naturally occurring. The nucleic acids of the present disclosure may contain one or more duplications, inversions, or repeats, resulting in a sequence that does not occur in nature, making the entire nucleic acid sequence non-naturally occurring. The nucleic acids of the present disclosure may contain modified, artificial, or synthetic nucleotides that do not occur in nature, making the entire nucleic acid sequence non-naturally occurring.

[0390] Given the redundancy of the genetic code, multiple nucleotide sequences may encode any particular protein, and all such nucleotide sequences are contemplated herein.

[0391] As used throughout this disclosure, the term "operably linked" refers to the expression of a gene under the control of a promoter to which it is spatially linked. The promoter can be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene is approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. Changes in the distance between the promoter and the gene can be accommodated without loss of promoter function.

[0392] As used throughout this disclosure, the term "promoter" refers to a synthetic or naturally occurring molecule capable of conferring, activating, or enhancing expression of a nucleic acid in a cell. A promoter can contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter spatial and / or temporal expression of the same. A promoter can also contain distal enhancer or repressor elements located as far away as several thousand base pairs from the transcription start site. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters can constitutively or differentially regulate expression of genetic components with respect to the cell, tissue or organ in which expression occurs, or 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-1 alpha promoter, a CAG promoter, an SV40 early promoter or an SV40 late promoter, and a CMV IE promoter.

[0393] The term "substantially complementary," as used throughout this disclosure, refers to a first sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

[0394] The term "substantially identical," as used throughout this disclosure, refers to first and second sequences that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids, or across nucleic acids, where the first sequence is substantially complementary to the complement of the second sequence.

[0395] As used throughout the disclosure, the term "variant," when used to describe a nucleic acid, refers to: (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of the referenced nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to the referenced nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, its complement, or a sequence substantially identical thereto.

[0396] As used throughout the disclosure, the term "vector" refers to a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector, preferably a DNA plasmid. A vector can contain a DNA sequence, an RNA sequence, or a combination of amino acids with both DNA and RNA sequences.

[0397] As used throughout the disclosure, the term "variant," when used to describe a peptide or polypeptide, refers to a peptide or polypeptide that differs in amino acid sequence by amino acid insertions, deletions, or conservative substitutions, but retains at least one biological activity. Variant can also refer to a protein having an amino acid sequence substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity.

[0398] Conservative amino acid substitutions, i.e., substitutions of amino acids with different amino acids having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are generally recognized in the art as resulting in minor changes. These minor changes can be identified, in part, by considering the hydropathic index of the amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. Amino acids with similar hydropathic indices can be substituted while retaining protein function. In one embodiment, amino acids with hydropathic indices of ±2 are substituted. Amino acid hydrophilicity can also be used to identify substitutions that result in proteins that retain biological function. Consideration of amino acid hydrophilicity in the context of a peptide allows for calculation of the peptide's greatest local average hydrophilicity, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101 is incorporated herein by reference in its entirety.

[0399] Substitution of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, e.g., immunogenicity. Substitutions can be made with amino acids with hydrophilicity values ​​within ±2 of each other. The hydrophobicity index and hydrophilicity of an amino acid are influenced by the specific side chain of that amino acid. Consistent with this observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of the amino acids, particularly their side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0400] As used herein, "conservative" amino acid substitutions can be defined as shown in Tables A, B, or C below. In some embodiments, fusion polypeptides and / or nucleic acids encoding such fusion polypeptides include conservative substitutions by modifying the polynucleotide encoding the polypeptide of the present disclosure. Amino acids can be classified by their physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is the substitution of one amino acid for another amino acid with similar properties. Exemplary conservative substitutions are shown in Table A.

[0401] [Table 3]

[0402] Alternatively, conserved amino acids can be grouped as described in Lehninger (Biochemistry, Second Edition; Worth Publishers, Inc. NY, NY (1975), pp. 71-77) (listed in Table B).

[0403] [Table 4]

[0404] Alternatively, exemplary conservative substitutions are shown in Table C.

[0405] [Table 5]

[0406] It should be understood that 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 other than insertions, deletions, or substitutions of amino acid residues. A polypeptide or nucleic acid of the present disclosure may include one or more conservative substitutions.

[0407] As used throughout this disclosure, the term "more than one" of the foregoing amino acid substitutions refers to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more of the listed amino acid substitutions. The term "more than one" can refer to 2, 3, 4, or 5 of the listed amino acid substitutions.

[0408] The polypeptides and proteins of the present disclosure, their entire sequences, or any portion thereof, may not be naturally occurring. The polypeptides and proteins of the present disclosure may contain one or more non-naturally occurring mutations, substitutions, deletions, or insertions, making the entire amino acid sequence non-naturally occurring. 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 and making the entire amino acid sequence non-naturally occurring. The polypeptides and proteins of the present disclosure may contain non-naturally occurring modified, artificial, or synthetic amino acids, making the entire amino acid sequence non-naturally occurring.

[0409] As used throughout the disclosure, "sequence identity" can be determined by using a standalone executable BLAST engine program to blast two sequences (bl2seq), 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 specified percentage of residues that are identical over a specified region of each sequence. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions where identical residues occur in both sequences, yielding the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. If the two sequences are of different lengths, or if the alignment results in one or more kinked ends and the designated region of comparison contains only a single sequence, the residues of the single sequence are included in the denominator but not in the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identification can be performed manually or using a computer sequencing algorithm such as BLAST or BLAST 2.0.

[0410] As used throughout this disclosure, the term "endogenous" refers to a nucleic acid or protein sequence that is naturally associated with the target gene or host cell into which it is introduced.

[0411] As used throughout the disclosure, the term "exogenous" refers to a nucleic acid or protein sequence that is not naturally associated with the target gene or host cell into which it is introduced, including a naturally occurring nucleic acid, e.g., a DNA sequence, or non-naturally occurring multiple copies of a naturally occurring nucleic acid sequence located in a non-naturally occurring genomic location.

[0412] The present disclosure provides methods for introducing a polynucleotide construct containing a DNA sequence into a host cell. By "introducing" it is intended to mean presenting the polynucleotide construct to the cell in such a way that the construct gains access to the interior of the host cell. The methods of the present disclosure do not depend on a particular method for introducing the polynucleotide construct into the host cell, simply by allowing the polynucleotide construct to gain 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, including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. [Example]

[0413] Example 1: Generation of humanized MUC1-C scFv antibody and chimeric antigen receptor (CAR) We generated a chimeric antigen receptor (CAR) with an antigen recognition region comprising a single-chain antibody that specifically binds to an epitope on MUC1-C (Figures 1A and 1B).

[0414] In initial studies, MUC1 expression was assessed in different cell types, including K562 cells (immortalized human chronic myeloid leukemia cells), Raji cells (a human hematopoietic cell line used as a cancer model), Raji cells modified to express MUC1-C, activated T cells, and RPMI8226 cells (a human peripheral blood B-cell plasmacytoma / myeloma cell line) (Figures 2A and 2B). MUC1 expression in each of these cells was assessed by staining with anti-MUC1-N antibody. For K562 cells, the staining control peak appears to the left of the anti-MUC1-N Ab peak. In Raji cells, the staining control peak overlaps with the anti-MUC1-N Ab peak, but the anti-MUC1-N Ab peak is higher. In Raji cells modified to express MUC1-C, the staining control peak overlaps with the anti-MUC1-N Ab peak, but the anti-MUC1-N Ab peak is higher. In activated T cells, the staining control peak appears to the left of the anti-MUC1-N Ab peak. For RPMI8226 cells, the staining control peak appears to the left of the anti-MUC1-N Ab peak. MUC-1C is also expressed on a panel of various cancer cell lines, including breast or adenocarcinoma (MDA-MB 468, cervical cancer, pancreatic cancer, lung cancer, leukemia, and multiple myeloma) (Figure 3).

[0415] To generate a humanized MUC1-C scFv CAR, in silico complementarity-determining region (CDR) grafting humanization of the variable region of a murine monoclonal antibody recognizing MUC1-C was performed. Computational modeling of the variable region (both variable heavy (VH) and variable light (VL) regions) was performed to identify the most suitable human VH / VL framework acceptors without altering the CDR regions; the most suitable human VH and VL framework acceptors were IVGV1-69 08 and IVGKV6-21 02, respectively. During the humanization process, the murine CDRs were grafted onto the human framework acceptors. Residues in the human framework that differed from the murine framework were investigated. Backmutations from human to murine residues within the acceptor framework were designed when new contacts were made, old contacts were lost, canonical murine residues were created, or instability was predicted within the antigen-binding region. All obtained heavy chain (4+2 variants (H1B and H2B)) and light chain (3) sequences are displayed together with their alignment (Figures 5A and 5B).

[0416] Exemplary murine CDR regions used to generate humanized MUC1-C scFv CARs comprise the following amino acid sequences: CDRH1-NFWMN (SEQ ID NO: 69); CDRH2-QIYPGDGDTNYNGKFKG (SEQ ID NO: 70); CDRH3-SYYRSAWFAY (SEQ ID NO: 71); CDRL1-RASQSIGTSIH (SEQ ID NO: 72); CDRL2-YASESIS (SEQ ID NO: 73); CDRL3-QQSNNWPLT (SEQ ID NO: 74). In some cases, the CDRH2 region was mutated to generate a humanized MUC1-C scFv CAR, which contains a CDRH2 comprising the amino acid sequence of QIYPGDGDTNYNAKFKG (SEQ ID NO: 75).

[0417] A diagram of an exemplary humanized MUC1-C-scFv CAR is shown in Figure 4. The following humanized MUC1-C CAR structure was used: signal peptide (CD8α)-light chain-linker-heavy chain-hinge (CD8α)-transmembrane (CD8α)-intracellular signaling (4-1BB)-intracellular signaling (CD3ζ).

[0418] Example 2: Functional analysis of humanized MUC1-C scFv CAR-T cells MUC1-C candidate CARs were subcloned into a tricistronic piggyBac transposon (EF1α promoter-iC9 safety switch-T2A-MUC1-C-CAR-T2A-DHFR selection gene), and CAR-T cells were generated using pan-T cells derived from normal human blood donors as described herein (Figure 6). Expression of each candidate CAR on the surface of piggyBac-modified cells was confirmed by FACS staining 19 days after transposon delivery using His-tagged p62 / p58 MUC1 protein followed by an anti-His secondary antibody (Figure 6). Specifically, cells were examined by flow cytometry for surface expression of CAR in either mock (blue) or anti-CD3 / CD28 bead-reactivated (red) cells (48 h activation) that received the CAR-encoding transposon. Data are shown as overlay histograms; numbers represent the percentage of cells expressing CAR on the cell surface. All candidate MUC1-C CARs were expressed and detected on the surface of piggyBac-producing CAR-T cells (Figure 7). Table 2 shows the mean fluorescence intensity (MFI) of CAR expression on the surface of resting and activated T cells.

[0419] [Table 6]

[0420] Candidate MUC1-C CAR-T cells were generated using pan-T cells from normal human blood donors as described herein. CAR+ T cells were FACS stained 19 days after transposon delivery for expression of surface CD45RA, CD45RO, and CD62L. SCM , T CM , T EM , and T EFF cell;T SCM (CD45RA+ / CD45RO- / CD62L+), T CM (CD45RA- / CD45RO+ / CD62L+), TEM (CD45RA- / CD45RO+ / CD62L-), T EFF (CD45RA+ / CD45RO- / CD62L-) was defined. The results are shown in Table 3. All piggyBac-producing candidate MUC1-C CAR-T cell populations contained predominantly high levels of favorable TOA and TOB cells.

[0421] [Table 7]

[0422] All piggyBac-producing candidate MUC1-C CAR-T cell populations predominantly expressed high levels of T SCM and TOB cells.

[0423] Candidate MUC1-C CAR-T cells were generated using pan-T cells derived from normal human blood donors as described herein. CAR-T cells were co-cultured with the triple-negative breast cancer cell line MDA-MB-468.lucGFP (MDA-MB-468 expressing luciferase (luc) and green fluorescent protein (GFP)) at various E:T ratios (40:1, 20:1, 10:1, 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.313:1) for 24 hours (Figure 8). Reporter signals were measured to determine cytotoxicity. MDA-MB-468 cell killing is shown for each CAR in the line graph. All CAR+ T cells expressed specific killing of MUC1+ MDA-MB-468. Mock T cells did not specifically kill target cells (gray dotted line). The area under the curve with SEM (standard error of the mean measurement for samples in triplicate) for killing MDA-MB-468 at a range of E:Ts is shown for each CAR in Table 4. All CAR+ T cells demonstrated specific killing of MUC1+ MDA-MB-468.

[0424] [Table 8]

[0425] Candidate MUC1-C CAR-T cells were generated using pan-T cells from normal human blood donors as described herein. CAR-T cells were engineered with human MUC1 isoform 10 (GenBank NP) expressing green fluorescent protein (GFP). The CAR+ T cells were co-cultured with a Raji cell line (MUC1-; blue bars) or a Raji cell line engineered to express 001191215.1 (MUC1+; red bars) at an E:T ratio of 10:1 for 24 hours (Figure 9). Reporter signals were measured to determine cytotoxicity. Killing of each cell line is shown for each CAR in the bar graphs, and error bars represent the standard deviation of samples performed in triplicate. All CAR+ T cells expressed specific killing of the MUC1+ engineered Raji cell line, but not MUC1- Raji cells. Mock transposon T cells did not kill either the Raji target cell line or the engineered Raji target cell line.

[0426] Example 3: Preclinical evaluation of candidate humanized MUC1-C CAR-T cells at stress doses using a mouse xenograft model A schematic of the study design for preclinical evaluation of candidate humanized MUC1-C CAR-T cells at a "stress" dose using a mouse xenograft model is shown in Figure 10. Female NSG mice were injected subcutaneously (SC) with 5 x 10 6 A mouse xenograft model was used with the luciferase-expressing MDA-MB-468.lucGFP (MDA-MB-468) cell line at a "stress" dose (4 x 10 6 The in vivo antitumor efficacy of candidate MUC1-C CAR-T cells was evaluated in mice. A panel of candidate CAR-T cells was selected for this study. All CAR-T cells were generated using piggyBac (PB) delivery of the candidate P-MUC1-C-101 transposon as described herein. Mice were injected with MDA-MB-468 in the axilla and treated when tumors were established (100–200 mm by caliper measurement). 3). Table 5 shows the preclinical evaluation of candidate humanized MUC1-C CAR-T cells at "stress" doses using a mouse xenograft model. For all treated animals, tumor volume assessment by caliper measurement was normalized to measurements from the untreated group. Table 5 shows tumor volumes by caliper in the vehicle and treatment groups.

[0427] [Table 9]

[0428] Mice were treated with a "stress" dose (4 × 10) of the P-MUC1-C-101 CAR-Ts candidate to gain greater resolution in detecting functional differences in potential efficacy between different CAR candidates. 6 ) intravenously. Total T cells in the blood of vehicle and treated mice were measured by TruCount staining. The area under the curve (AUC) of T cells in the blood of vehicle and treated mice was measured from blood draws and the results are shown in Table 6.

[0429] [Table 10]

[0430] The CD8+ T cell phenotype of vehicle and treated mice was determined. The phenotype of CD8+ T cells in the blood was determined by FACS staining for all animals, and percentages were listed as group means with error bars as SEM. Cells were stained for surface CD45RA, CD45RO, and CD62L expression, and T SCM , T CM , T EM , and T EFF cell;T SCM (CD45RA+ / CD45RO- / CD62L+), T CM (CD45RA- / CD45RO+ / CD62L+), T EM (CD45RA- / CD45RO+ / CD62L-), T EFF (CD45RA+CD45RO- / CD62L-). For all MUC-1C CAR-T cells tested, TSCM and T CM The phenotype was assessed on the day before infusion, on day 12, and on day 19. EM and T EFF It is more abundant than the phenotype.

Claims

1. Amino acid sequence: 【Chemical 1】 [X1 in SEQ ID NO: 1 is V or I, X2 of SEQ ID NO: 1 is R or K; X3 of SEQ ID NO: 1 is A or R; X4 in SEQ ID NO: 1 is G or A; X5 of SEQ ID NO: 1 is V or A; X6 of SEQ ID NO: 1 is T or S, and X7 in SEQ ID NO: 1 is D or A. a heavy chain variable region comprising: Amino acid sequence: 【Chemistry 2】 [X1 in SEQ ID NO: 2 is T or S, X2 of SEQ ID NO: 2 is L or V, and X3 of SEQ ID NO: 2 is T or D. Light chain variable region containing or a chimeric antigen receptor (CAR) comprising a single-chain variable fragment (scFv).

2. (a) an ectodomain comprising an antigen recognition region, wherein the antigen recognition region comprises at least one anti-MUC1 single-chain variable fragment (scFv); (b) a transmembrane domain, and (c) an endodomain comprising at least one costimulatory domain; A chimeric antigen receptor (CAR) comprising: wherein the scFv has the amino acid sequence: 【Chemistry 3】 [X1 in SEQ ID NO: 1 is V or I, X2 of SEQ ID NO: 1 is R or K; X3 of SEQ ID NO: 1 is A or R; X4 in SEQ ID NO: 1 is G or A; X5 of SEQ ID NO: 1 is V or A; X6 of SEQ ID NO: 1 is T or S, and X7 in SEQ ID NO: 1 is D or A. a heavy chain variable region comprising: Amino acid sequence: 【Chemistry 4】 [X1 in SEQ ID NO: 2 is T or S; X2 of SEQ ID NO: 2 is L or V, and X3 of SEQ ID NO: 2 is T or D. a light chain variable region comprising A chimeric antigen receptor (CAR) comprising:

3. The CAR according to claim 1 or 2, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO:

8.

4. The CAR according to any one of claims 1 to 3, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:

11.

5. (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 3 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 9; (ii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 4 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 9; (iii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:5 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:9; (iv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 6 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 9; (v) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 9; (vi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 9; (vii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 3 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 10; (viii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 4 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 10; (ix) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 5 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 10; (x) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 6 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 10; (xi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 10; (xii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 10; (xiii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 3 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 11; (xiv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 4 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 11; (xv) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:5 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:11; (xvi) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 6 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 11; (xvii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 11; or (xviii) The CAR according to any one of claims 1 to 4, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

11.

6. The CAR according to any one of claims 1 to 5, wherein the scFv comprises a linker between the heavy chain variable region and the light chain variable region.

7. The CAR of claim 6, wherein the linker comprises the amino acid sequence of SEQ ID NO:

59.

8. The CAR of claim 1 or 2, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140 or 141.

9. The CAR of claim 1 or 2, wherein the scFv comprises the amino acid sequence of SEQ ID NO:

125.

10. The CAR according to any one of claims 2 to 9, wherein the ectodomain further comprises a signal peptide.

11. The CAR of claim 10, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO:

57.

12. The CAR according to any one of claims 2 to 11, further comprising a hinge region between the antigen recognition region and the transmembrane domain.

13. The CAR of claim 12, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:

61.

14. The CAR according to any one of claims 2 to 13, wherein the transmembrane domain comprises a sequence encoding a CD8 transmembrane domain.

15. The CAR of claim 14, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO:

63.

16. The CAR according to any one of claims 2 to 15, wherein the at least one costimulatory domain comprises a CD3ζ costimulatory domain, a 4-1BB costimulatory domain, or a combination thereof.

17. The CAR according to any one of claims 2 to 16, wherein the at least one costimulatory domain comprises a CD3ζ costimulatory domain and a 4-1BB costimulatory domain, and the 4-1BB costimulatory domain is located between the transmembrane domain and the CD3ζ costimulatory domain.

18. The CAR of claim 16 or 17, wherein the 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 65, and / or the CD3ζ costimulatory domain comprises the amino acid sequence of SEQ ID NO:

67.

19. The scFv comprises a linker between the heavy chain variable region and the light chain variable region; the ectodomain comprises a signal peptide, the CAR further comprises a hinge region between the antigen recognition region and the transmembrane domain; the transmembrane domain comprises a sequence comprising a CD8 transmembrane domain; The CAR according to any one of claims 2 to 18, wherein the at least one costimulatory domain comprises a CD3ζ costimulatory domain and a 4-1BB costimulatory domain, and the 4-1BB costimulatory domain is located between the transmembrane domain and the CD3ζ costimulatory domain.

20. the scFv comprises the amino acid sequence of SEQ ID NO: 125; the signal peptide comprises SEQ ID NO:57; the hinge region comprises SEQ ID NO: 61; the CD8 transmembrane domain comprises SEQ ID NO: 63; the 4-1BB costimulatory domain comprises SEQ ID NO:65; and The CAR of claim 19, wherein the CD3ζ costimulatory domain comprises SEQ ID NO:

67.

21. 20. The CAR of claim 19, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO:

29.

22. The CAR of claim 19, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

13.

23. The CAR of claim 19, wherein the amino acid sequence of the CAR is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, or SEQ ID NO:

167.

24. The CAR of claim 19, wherein the amino acid sequence of the CAR is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:

167.

25. A polynucleotide comprising a nucleic acid sequence encoding the CAR according to any one of claims 2 to 24.

26. A vector comprising the polynucleotide of claim 25.

27. A cell comprising the CAR according to any one of claims 2 to 24.

28. A cell population, wherein a plurality of cell populations are modified to express the CAR according to any one of claims 2 to 24.

29. 29. The cell population of claim 28, wherein the plurality of modified cells is a plurality of modified immune cells or a plurality of modified T cells.

30. A cell population, wherein a plurality of cell populations comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of cells that express the CAR of any one of claims 1 to 24.

31. A composition comprising the CAR according to any one of claims 2 to 24, or the cell according to claim 27, or the cell population according to any one of claims 28 to 30.

32. 32. A pharmaceutical composition comprising the composition of claim 31 and a pharmaceutically acceptable carrier.

33. 33. The composition of claim 31 or the pharmaceutical composition of claim 32 for use in a method of treating cancer in a subject in need thereof, wherein a therapeutically effective amount is the amount administered to the subject.

34. The composition or pharmaceutical composition for use according to claim 33, wherein the cancer is a MUC1-positive cancer or a MUC1-C-positive cancer.

35. 35. The composition or pharmaceutical composition for use according to claim 33 or 34, wherein the cancer is a primary tumor, a metastatic cancer, a multidrug resistant cancer, an advanced tumor or a recurrent cancer.

36. The composition or pharmaceutical composition for use according to any one of claims 33 to 35, wherein the cancer is a solid tumor.

37. The composition or pharmaceutical composition for use according to any one of claims 33 to 36, wherein the cancer is 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 or esophageal cancer.

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