Anti-MUC1 Composition and Application Method

Abstract

What is disclosed are antibodies against MUC1, MUC1-CAR compositions, and methods for targeting the MUC1 protein using these antibodies and compositions, wherein cells expressing the MUC1 protein can be targeted and killed, for example, by cytotoxic T cells.

Description

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

[0002] This disclosure relates to molecular biology, and more specifically to antibodies and chimeric antigen receptors that bind to target proteins with high affinity and specificity.

[0003] By referencing the merged sequence list The contents of the file named "POTH-040_001WO_SequenceListing_ST25", which was created on December 15, 2020 and is 412 KB in size, are incorporated here in their entirety by reference. Background Technology

[0004] The development of drugs capable of recognizing and binding to specific target proteins with high affinity and cohesion 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] This disclosure provides isolated antibodies comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence QVQLVQSGAEVKKPGSSVKX1SCKTSGYAFSNFWMNWVX2QX3PGQGLEWIGQIYPGDGDTNYNX4KFKGRX5TLTADKSX6STAYMELSSLRSEX7TAVYFCARSYYRSAWFAYWGQGTLVTVSS (SEQ ID NO:1), 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 wherein SEQ ID NO: X7 of SEQ ID NO: 2 is D or A; the light chain variable region comprises the amino acid sequence EILLTQSPDFQSVTPKEKVTFTCRASQSIGTSIHWYQQKPNQSPKLLIKYASESISGVPSRFSGSGSGTDFTLX1INSX2ESEDIAX3YYCQQSNNWPLTFGQGTKLEIK (SEQ ID NO: 2), wherein X1 of SEQ ID NO: 2 is T or S, wherein X2 of SEQ ID NO: 2 is L or V, and wherein X3 of SEQ ID NO: 2 is T or D.

[0006] The isolated antibody may be humanized. The isolated antibody may be IgG. The isolated antibody may bind to human MUC1-C. The isolated antibody may be a monoclonal antibody, chimeric antibody, single-domain antibody, VHH, VH, single-chain variable fragment (scFv), Fab, or Fab fragment. Preferably, the isolated antibody is scFv.

[0007] The heavy chain variable region contains 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 contains the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.

[0008] scFv may be contained in a linker between the heavy chain variable region and the light chain variable region. In one aspect, the linker contains the amino acid sequence of SEQ ID NO: 59.

[0009] The scFv may contain 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 aspect, the scFv contains the amino acid sequence of SEQ ID NO: 125.

[0010] This disclosure also provides chimeric antigen receptors (CARs) comprising antibodies as disclosed herein. Preferably, the CAR comprises scFv as disclosed herein. The CAR may comprise (a) an extracellular domain containing 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 intracellular domain containing at least one co-stimulatory domain; wherein the scFv comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence QVQLVQSGAEVKKPGSSVKX1SCKTSGYAFSNFWMNWVX2QX3PGQGLEWIGQIYPGDGDTNYNX4KFKGRX5TLTADKSX6STAYMELSSLRSEX7TAVYFCARSYYRSAWFAYWGQGTLVTVSS (SEQ ID NO:1), 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, and X5 of SEQ ID NO:1 is V or I. 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 the amino acid sequence EILLTQSPDFQSVTPKEKVTFTCRASQSIGTSIHWYQQKPNQSPKLLIKYASESISGVPSRFSGSGSGTDFTLX1INSX2ESEDIAX3YYCQQSNNWPLTFGQGTKLEIK (SEQ ID NO: 2), wherein 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.

[0011] The heavy chain variable region of the CAR may contain 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 may contain the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.

[0012] The heavy chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:3, and the light chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:9. The heavy chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:4, and the light chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:9. The heavy chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:5, and the light chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:9. The heavy chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:6, and the light chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:9. The heavy chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:7, and the light chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:9. The heavy chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:8, and the light chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:9. The heavy chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:3, and the light chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:10. The heavy chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:4, and the light chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:10. The heavy chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:5, and the light chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:10. The heavy chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:6, and the light chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:10. The heavy chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:7, and the light chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:10. The heavy chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:8, and the light chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:10. The heavy chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:3, and the light chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:11. The heavy chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:4, and the light chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:11. The heavy chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:5, and the light chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:11.The heavy chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:6, and the light chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:11. The heavy chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:7, and the light chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:11. The heavy chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:8, and the light chain variable region of the CAR may contain the amino acid sequence of SEQ ID NO:11.

[0013] The scFv may contain a linker between the heavy chain variable region and the light chain variable region. Preferably, the linker contains the amino acid sequence of SEQ ID NO: 59. The scFv may contain 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 aspect, the scFv contains the amino acid sequence of SEQ ID NO: 125. The extracellular domain may further contain a signal peptide. Preferably, the signal peptide comprises the amino acid sequence of SEQ ID NO: 57. The CAR may 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 may 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 may comprise a CD3ζ costimulatory domain, a 4-1BB costimulatory domain, or a combination thereof. In one aspect, 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.

[0014] This disclosure also provides a chimeric antigen receptor (CAR) comprising (a) an extracellular domain containing an antigen recognition region, wherein the antigen recognition region contains at least one anti-MUC1 single-chain variable fragment (scFv); (b) a transmembrane domain; and (c) an intracellular domain containing at least one co-stimulatory domain; wherein the scFv comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence QVQLVQSGAEVKKPGSSVKX1SCKTSGYAFSNFWMNWVX2QX3PGQGLEWIGQIYPGDGDTNYNX4KFKGRX5TLTADKSX6STAYMELSSLRSEX7TAVYFCARSYYRSAWFAYWGQGTLVTVSS (SEQ ID NO: 1), 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, and X4 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; the light chain variable region comprises the amino acid sequence EILLTQSPDFQSVTPKEKVTFTCRASQSIGTSIHWYQQKPNQSPKLLIKYASESISGVPSRFSGSGSGTDFTLX1INSX2ESEDIAX3YYCQQSNNWPLTFGQGTKLEIK (SEQ ID NO: 2), wherein X1 of SEQ ID NO: 2 is T or S, X2 of SEQ ID NO: 2 is L or V, and X7 of SEQ ID NO: 2 of X3 is T or D, wherein the scFv contains a linker between the heavy chain variable region and the light chain variable region, wherein the extracellular domain contains a signal peptide, wherein the CAR further contains a hinge region between the antigen recognition region and the transmembrane domain, wherein the transmembrane domain includes a sequence containing a CD8 transmembrane domain; and wherein the at least one costimulatory domain contains 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.

[0015] In some respects, scFv contains the amino acid sequence of SEQ ID NO: 125, the signal peptide contains SEQ ID NO: 57, the hinge region contains SEQ ID NO: 61, the CD8 transmembrane domain contains SEQ ID NO: 63, the 4-1BB co-stimulatory domain contains SEQ ID NO: 65, and the CD3ζ co-stimulatory domain contains SEQ ID NO: 67.

[0016] In some aspects, the CAR contains 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 aspects, the CAR contains the amino acid sequence of SEQ ID NO: 13. In some aspects, 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 aspects, the amino acid sequence of the CAR is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 167.

[0017] This disclosure also provides polynucleotides comprising nucleic acid sequences encoding antibodies as disclosed herein; polynucleotides comprising nucleic acid sequences encoding scFv as disclosed herein; and / or polynucleotides comprising nucleic acid sequences encoding CARs as disclosed herein.

[0018] This disclosure also provides transposons comprising nucleic acids encoding CARs as disclosed herein. In some aspects, the nucleic acid sequence comprises a CAR comprising the amino acid sequence of SEQ ID NO: 13. The transposon may 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 may comprise a DHFR resistance gene. Preferably, the transposon is the piggyBac transposon. In some aspects, the transposon comprises the nucleic acid sequence of SEQ ID NO: 172. This disclosure also provides plasmids or vectors comprising any polynucleotide or transposon disclosed herein.

[0019] This disclosure also provides cells comprising any one of the antibodies, scFvs, CARs, or transposons disclosed herein. This disclosure also provides cell populations wherein multiple populations are modified to express any one of the antibodies, scFvs, CARs, or transposons disclosed herein. In one aspect, the multiple modified cells are multiple modified immune cells. In one aspect, the multiple modified cells are multiple modified T cells. In one aspect, the multiple cell populations comprise 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 a CAR. In one aspect, the CAR comprises the amino acid sequence of SEQ ID NO: 13. In one aspect, at least 50% of the multiple modified T cells express one or more cell surface markers containing CD45RA and CD62L, and do not express one or more cell surface markers containing CD45RO.

[0020] This disclosure also provides compositions 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.

[0021] This disclosure also provides a method for treating a proliferative disease in a subject of need by administering a therapeutically effective amount of any of the antibodies, scFvs, CARs, transposons, cells, cell populations, compositions, or pharmaceutical compositions disclosed herein. In one aspect, the proliferative disease is cancer. In one aspect, the cancer can be MUC1-positive cancer. In one aspect, the cancer is MUC1-C-positive cancer. The cancer can be a primary tumor, metastatic cancer, multi-resistant cancer, progressive tumor, or 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. Attached Figure Description

[0022] The patent or application document contains at least one color drawing. A copy of the patent or patent application publication with color drawings will be provided by the patent office upon request and payment of the necessary fees.

[0023] Figure 1A-1B These are a pair of schematic diagrams depicting the structure of the MUC1 heterodimer. Figure 1A The process by which MUC1 undergoes autoproteolytic cleavage at the SEA domain (sea urchin spermatin, enterokinase, and aggregate protein domains) to generate two subunits that thus form a stable nonvalent heterodimer is described. The nomenclature MUC1-N and MUC1-C is used to specify the location of the subunits after cleavage and to distinguish them from genetic isotypes reclassified using Greek characters. Figure 1B Details of the MUC1-C subunit are provided. The extracellular domain of the 58 amino acids in MUC1-C is located at position 36 (where N is N). 36 The asparagine at the LT site is glycosylated. The amino acid sequence of the extracellular domain (MUC1-C / ECD) at the C-terminus of MUC1-C is shown (SEQ ID NO: 77). The cytoplasmic domain of MUC1-C, consisting of 72 amino acids, interacts with a variety of effectors and is sufficient to induce carcinogenic transformation. Figure 1A-1B Reproduced by Kufe DW, Oncogene, 32(9):1073.

[0024] Figure 2A is a pair of schematic diagrams depicting the banded structure or the predicted structure of the MUC1-C domain of the full-length MUC1 (PDB:2ACM).

[0025] Figure 2B It is a series of diagrams depicting MUC1 expression in different cell types.

[0026] Figure 3 These are a series of flow cytometry plots depicting Muc1 expression in different cancer cell lines.

[0027] Figure 4 This is a schematic diagram depicting 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 into different light-heavy 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 signal transduction (4-1BB) – intracellular signal transduction (CD3ζ).

[0028] Figures 5A-5B This is a diagram illustrating the amino acid sequence alignment of the heavy chain variable region and light chain variable region of the humanized anti-MUC-1C antibody depicting the present disclosure.

[0029] Figure 6 This is a schematic diagram of the piggyBac nanotransposon containing the MUC-1C CAR of this disclosure. The MUC1-C CAR is subcloned into the tricistronic piggyBac transposon. The transposon contains the EF1α promoter – iC9 safety switch – T2A – MUC1-C CAR (L1-linker-H1B scFv) – T2A – DHFR selector gene construct.

[0030] Figure 7 This is a series of flow cytometry images showing the expression of candidate MUC1-C CARs on the surface of T cells. All candidate MUC1-C CARs were expressed and detected on the surface of CAR-T cells generated by piggyBac.

[0031] Figure 8 This graph shows the specific killing of MUC1+ target cells by candidate MUC1-C CAR T cells. For each CAR in the line graph, MDA-MB-468 killing is shown. All CAR+ T cells expressed MUC1+ MDA-MB-468 cells showed specific killing. Transposable T cells did not specifically kill target cells (blue line).

[0032] Figure 9 This graph shows the specific killing of MUC1+ target cells by candidate MUC1-C CAR T cells. For each CAR in the bar graph, the killing is shown for each cell line, and the error bars represent the standard deviation of samples from triplicate runs. All CAR+ T cells expressed specific killing of MUC1+ modified Raji cell lines, but not MUC1-Raji cells. The transposable T cells killed neither Raji nor the modified Raji target cell lines.

[0033] Figure 10 This is a schematic diagram of a study design for the preclinical evaluation of candidate-derived MUC1-C CAR-T cells using a mouse xenograft model at a 'stress' dose.

[0034] All documents referenced herein, including any cross-references or related patents or applications, are incorporated herein in their entirety for all purposes, unless expressly excluded or otherwise limited. Reference to any document does not constitute an admission that it is prior art to any invention disclosed or claimed herein, or that it, alone or in any combination with any other reference, teaches, implies, or discloses any such invention. Furthermore, in the event of any conflict between the meaning or definition of any term in this document and the meaning or definition of the same term in any document incorporated herein by reference, the meaning or definition given to that term in this document shall prevail. Detailed Implementation

[0035] This disclosure provides compositions and methods for using these compositions to recognize and bind specific target proteins, mucin 1, and cell surface-associated (MUC1) proteins with high affinity and cohesion.

[0036] MUC1 MUC1 is a widely expressed protein that is predominantly found in epithelial cells. O - Glycosylated mucin. Secreted and membrane-bound MUC1 forms a physical barrier that protects the apical margin of epithelial cells from damage caused by toxins, microorganisms, and other forms of stress that occur at the interface with the external environment. As found in most cancers, aberrant overexpression of MUC1 confers anchor-independent growth and tumorigenicity. Overexpression of MUC1 confers resistance to apoptosis induced by oxidative stress and genotoxic anticancer agents.

[0037] Human MUC1 is a heterodimeric glycoprotein translated into a single polypeptide in the endoplasmic reticulum and cleaved into N- and C-terminal subunits (MUC1-N and MUC1-C). Cleavage can be mediated by an autocatalytic process. The >250 kDa MUC1 N-terminal (MUC1 N-ter or MUC1-N) subunit contains a variable number of tandem repeats of 20 amino acids, which are incomplete due to highly conserved variation and are cleaved via… O Modification with polysaccharides. MUC1-N is tethered to the cell surface by dimerization with an approximately 23 kDa C-terminal subunit (MUC1 C-ter or MUC1-C), which comprises a 58-amino acid extracellular region, a 28-amino acid transmembrane domain, and a 72-amino acid cytoplasmic domain (CD). Figure 1BMUC1-C contains (SEQ ID NO: 76): SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPPFFSAQSGAG VPGWGIALLVLVCVLVALAIVYLIALAV CQCRRKNYGQLDIFP The amino acid sequence of ARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAATSANL.

[0038] The compositions of this disclosure can bind to the underlined 58 amino acid moiety of MUC1-C / ECD. The bold sequence indicates CD, and the italicized portion is an oligomeric repressive peptide. MUC1 is aberrantly overexpressed in the cytosol and throughout the cell membrane during the transformation from normal epithelium to cancer. Cell membrane-associated MUC1 targets endosomes via clathrin-mediated endocytosis. Furthermore, MUC1-C, but not MUC1-N, targets the nucleus and mitochondria.

[0039] The compositions of this disclosure can selectively bind 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" means one or more amino acids to which the disclosed compositions specifically bind. The one or more amino acids of the epitope of this disclosure can be arranged in a linear, non-linear, continuous, or discontinuous manner. The epitope of this disclosure can be "conformational," meaning that when the amino acid is presented in a conformation of a suitably folded peptide, protein, or protein complex, the protein scaffold binds to one or more amino acids of the epitope with greater affinity or greater selectivity. In some respects, compositions binding to conformational epitopes may not bind to linear epitopes.

[0040] The compositions of this disclosure can selectively bind one or more amino acids of the MUC1-C / extracellular domain (MUC1-C / ECD), said MUC1-C / extracellular domain being defined by the amino acid sequence SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAG (SEQ ID NO: 77) (see [link to SEQ ID NO: 77]). Figure 1BAlternatively or additionally, the composition selectively binds one or more amino acids of the variant MUC1-C / extracellular domain (MUC1-C / ECD). The variant MUC1-C / ECD peptides of this disclosure may 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, as numbered according to SEQ ID NO:76 or SEQ ID NO:77.

[0041] The compositions of this disclosure may selectively bind 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).

[0042] The compositions of this disclosure may selectively incorporate either an alpha3 (α3) helix or an alpha4 (α4) helix in MUC1-C / ED. In some embodiments, MUC1-C / ECD comprises SVVVQLTLAFREGTINVHDVETQFNQYKT EAASRYN The amino acid sequence of LTISDVSVSDVPFPFSAQSGAG (SEQ ID NO: 77); the a3 helix is ​​bold and the a4 helix is ​​bold and italic. The MUC1-C / ED a3 helix may contain the amino acid sequence of VHDVETQFNQ (SEQ ID NO: 81), and is essentially composed of it, or composed of it. The MUC1-C / ED a4 helix may contain EAASRYN The epitope of the compositions of this disclosure may comprise, substantially constitute, or consist of the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 82. The epitope may be linear or conformational. In some aspects, the epitope is discontinuous, optionally comprising, substantially constitute, or consist of two or more discontinuous amino acids of the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 82.

[0043] The compositions disclosed herein This disclosure provides antibodies comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises, is substantially composed of, or is composed of an amino acid sequence, the amino acid sequence being at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between the two) of QVQLVQSGAEVKKPGSSVKX1SCKTSGYAFSNFWMNWVX2QX3PGQGLEWIGQIYPGDGDTNYNX4KFKGRX5TLTADKSX6STAYMELSSLRSEX7TAVYFCARSYYRSAWFAYWGQGTLVTVSS (SEQ ID NO:1), 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, and X6 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 an amino acid sequence, is substantially composed of an amino acid sequence, or is composed of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between the two) of EILLTQSPDFQSVTPKEKVTFTCRASQSIGTSIHWYQQKPNQSPKLLIKYASESISGVPSRFSGSGSGTDFTLX1INSX2ESEDIAX3YYCQQSNNWPLTFGQGTKLEIK (SEQ ID NO: 2), wherein X1 of SEQ ID NO: 2 is T or S, wherein X2 of SEQ ID NO: 2 is L or V, and wherein X3 of SEQ ID NO: 2 is T or D.

[0044] Preferably, the heavy 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, X5 is V or A, X6 is T or S, and X7 is D 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.

[0045] In some aspects, the antibody binds to the human MUC1 polypeptide (UniProt accession number P15941-1), said human MUC1 polypeptide comprising, substantially comprising, or consisting of SEQ ID NO: 160. In one aspect, the antibody binds to the human MUC1-C polypeptide, which comprises, substantially comprising, or consists of SEQ ID NO: 76. In another aspect, the antibody binds to the human MUC1-N polypeptide (a subunit of the MUC1 receptor, also known as the α chain, mature chain), which comprises, substantially comprising, or consists of SEQ ID NO: 161.

[0046] In some aspects, the antibody binds to the human MUC1-C extracellular domain (ED), which comprises, is substantially composed of, or is composed of SEQ ID NO: 77. In some aspects, the antibody binds to MUC1-C ED (SEQ ID NO: 77) with a higher affinity than the full-length human MUC1 polypeptide (SEQ ID NO: 160). In some aspects, the antibody binds to human MUC1-C ED (SEQ ID NO: 77) with a higher affinity than the MUC1-N polypeptide (SEQ ID NO: 161). In some aspects, the antibody binds to MUC1-C ED (SEQ ID NO: 77) and does not bind to the full-length human MUC1 polypeptide (SEQ ID NO: 160). In some aspects, the antibody binds to human MUC1-C ED (SEQ ID NO: 77) and does not bind to the MUC1-N polypeptide (SEQ ID NO: 161).

[0047] In some embodiments, the human variable heavy chain framework receptor comprises, is substantially composed of, or is composed of the polypeptide of IGHV1-69 08 of SEQ ID NO: 162. In some aspects, the human variable light chain framework receptor comprises, is substantially composed of, or is composed of the polypeptide of IGKV6-21 02 of SEQ ID NO: 163.

[0048] The heavy chain variable region includes complementary determination region 1 (CDRH1) containing SEQ ID NO: 69. The heavy chain variable region includes CDRH2 containing SEQ ID NO: 70 or SEQ ID NO: 75. The heavy chain variable region includes CDRH3 containing SEQ ID NO: 71.

[0049] The light chain variable region includes complementary determination region 1 (CDRL1) containing SEQ ID NO: 72. The light chain variable region includes CDRL2 containing SEQ ID NO: 73. The light chain variable region includes CDRL3 containing SEQ ID NO: 74.

[0050] The heavy chain variable region comprises, is substantially composed of, or is composed of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between) 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. In a preferred aspect, the heavy chain variable region comprises, is substantially composed of, or is composed 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.

[0051] The heavy chain variable region is encoded by a polynucleotide comprising, substantially comprising, or consisting of a nucleic acid sequence, that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between) 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. In a preferred aspect, the heavy chain variable region is encoded by a polynucleotide comprising, substantially comprising, or consisting 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.

[0052] The light chain variable region comprises, is substantially composed of, or is composed of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between) equivalent to the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. In a preferred aspect, the light chain variable region comprises, is substantially composed of, or is composed of the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0053] The light chain variable region is encoded by a polynucleotide comprising, substantially comprising, or composed of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between) equivalent to SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 165. In a preferred aspect, the light chain variable region is encoded by a polynucleotide comprising, substantially comprising, or composed of the nucleic acid sequence of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 165.

[0054] The variable region of the heavy chain of the antibody may contain the amino acid sequence of SEQ ID NO: 3, and the variable region of the light chain of the antibody may contain the amino acid sequence of SEQ ID NO: 9. The variable region of the heavy chain of the antibody may contain the amino acid sequence of SEQ ID NO: 4, and the variable region of the light chain of the antibody may contain the amino acid sequence of SEQ ID NO: 9. The variable region of the heavy chain of the antibody may contain the amino acid sequence of SEQ ID NO: 5, and the variable region of the light chain of the antibody may contain the amino acid sequence of SEQ ID NO: 9. The variable region of the heavy chain of the antibody may contain the amino acid sequence of SEQ ID NO: 6, and the variable region of the light chain of the antibody may contain the amino acid sequence of SEQ ID NO: 9. The variable region of the heavy chain of the antibody may contain the amino acid sequence of SEQ ID NO: 7, and the variable region of the light chain of the antibody may contain the amino acid sequence of SEQ ID NO: 9. The variable region of the heavy chain of the antibody may contain the amino acid sequence of SEQ ID NO: 8, and the variable region of the light chain of the antibody may contain the amino acid sequence of SEQ ID NO: 9.

[0055] The variable region of the heavy chain of the antibody may contain the amino acid sequence of SEQ ID NO: 3, and the variable region of the light chain of the antibody may contain the amino acid sequence of SEQ ID NO: 10. The variable region of the heavy chain of the antibody may contain the amino acid sequence of SEQ ID NO: 4, and the variable region of the light chain of the antibody may contain the amino acid sequence of SEQ ID NO: 10. The variable region of the heavy chain of the antibody may contain the amino acid sequence of SEQ ID NO: 5, and the variable region of the light chain of the antibody may contain the amino acid sequence of SEQ ID NO: 10. The variable region of the heavy chain of the antibody may contain the amino acid sequence of SEQ ID NO: 6, and the variable region of the light chain of the antibody may contain the amino acid sequence of SEQ ID NO: 10. The variable region of the heavy chain of the antibody may contain the amino acid sequence of SEQ ID NO: 7, and the variable region of the light chain of the antibody may contain the amino acid sequence of SEQ ID NO: 10. The variable region of the heavy chain of the antibody may contain the amino acid sequence of SEQ ID NO: 8, and the variable region of the light chain of the antibody may contain the amino acid sequence of SEQ ID NO: 10.

[0056] The variable region of the heavy chain of the antibody may contain the amino acid sequence of SEQ ID NO: 3, and the variable region of the light chain of the antibody may contain the amino acid sequence of SEQ ID NO: 11. The variable region of the heavy chain of the antibody may contain the amino acid sequence of SEQ ID NO: 4, and the variable region of the light chain of the antibody may contain the amino acid sequence of SEQ ID NO: 11. The variable region of the heavy chain of the antibody may contain the amino acid sequence of SEQ ID NO: 5, and the variable region of the light chain of the antibody may contain the amino acid sequence of SEQ ID NO: 11. The variable region of the heavy chain of the antibody may contain the amino acid sequence of SEQ ID NO: 6, and the variable region of the light chain of the antibody may contain the amino acid sequence of SEQ ID NO: 11. The variable region of the heavy chain of the antibody may contain the amino acid sequence of SEQ ID NO: 7, and the variable region of the light chain of the antibody may contain the amino acid sequence of SEQ ID NO: 11. The variable region of the heavy chain of the antibody may contain the amino acid sequence of SEQ ID NO: 8, and the variable region of the light chain of the antibody may contain the amino acid sequence of SEQ ID NO: 11.

[0057] In some respects, the antibody contains human or humanized sequences. In some respects, the CDR contains humanized sequences. In some respects, the variable region contains humanized sequences. In some respects, the framework region contains humanized sequences. The framework region may further contain one or more sequence variations to reduce immunogenicity or improve antibody production. In some respects, the antibody is IgG.

[0058] The antibody can 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 aspect, the antibody is an scFv.

[0059] This 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 cohesion. The scFv compositions may comprise heavy chain variable regions and light chain variable regions of an anti-MUC1 antibody.

[0060] scFv may comprise a linker polypeptide between the heavy chain variable region and the light chain variable region. In some embodiments, the linker polypeptide comprises, is substantially composed of, or is composed of the amino acid sequence of SEQ ID NO: 59. The linker polypeptide may be encoded by a polynucleotide comprising, is substantially composed of, or is composed of the nucleic acid sequence of SEQ ID NO: 60.

[0061] The scFv contains, is substantially composed of, or is composed of an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between the two) 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 a preferred aspect, the scFv comprises, is substantially composed of, or is composed of the following amino acid sequences: 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.

[0062] scFv is encoded by a polynucleotide comprising, substantially comprising, or consisting of a nucleic acid sequence, said nucleic acid sequence being equivalent to at least 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between the two) of the following: 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 aspect, the scFv is encoded by a nucleic acid comprising, substantially comprising, or comprising the following nucleic acid sequences: 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.

[0063] This disclosure also provides a chimeric antigen receptor (CAR) comprising an extracellular domain including an antigen recognition region comprising at least one anti-MUC1 single-stranded variable fragment (scFv) of this disclosure; a transmembrane domain; and an intracellular domain including at least one co-stimulatory domain. The CAR may further include a hinge region between the antigen recognition domain and the transmembrane domain. The antigen recognition region may contain at least two anti-MUC1 scFvs. The antigen recognition region may contain at least three anti-MUC1 scFvs. In one aspect, the CAR of this disclosure is a bispecific CAR comprising at least two scFvs that specifically bind to two different antigens.

[0064] The extracellular domain may contain a signal peptide. The signal peptide may contain a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB, or GM-CSFR signal peptide. In a preferred aspect, the signal peptide comprises, is substantially composed of, or is composed of a human CD8α (CD8α) signal peptide (SP) or a portion thereof. The human CD8α SP comprises, is substantially composed of, or is composed of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between both) of SEQ ID NO: 57. Preferably, the human CD8α SP comprises, is substantially composed of, or is composed of the amino acid sequence of SEQ ID NO: 57.

[0065] Human CD8α SP is encoded by a polynucleotide comprising, substantially 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 between the two) of SEQ ID NO: 58. Preferably, human CD8α SP is encoded by a polynucleotide comprising, substantially comprising, or consisting of the amino acid sequence of SEQ ID NO: 58.

[0066] The hinge domain or hinge region may contain human CD8α, IgG4, CD4 sequences, or combinations thereof. In a preferred aspect, the hinge may comprise, consist substantially of, or consist of a human CD8α (CD8α) hinge or a portion thereof. The human CD8α hinge comprises, consists substantially 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 between both) of SEQ ID NO: 61. Preferably, the human CD8α hinge domain comprises, consists substantially of, or consists of the amino acid sequence of SEQ ID NO: 61.

[0067] The human CD8α hinge is encoded by a polynucleotide comprising, substantially comprising, or composed of a nucleic acid sequence equivalent to at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between the two) of SEQ ID NO: 62 or SEQ ID NO: 168. Preferably, the human CD8α hinge domain is encoded by a polynucleotide comprising, substantially comprising, or composed of the nucleic acid sequence of SEQ ID NO: 62 or SEQ ID NO: 168.

[0068] The transmembrane domain may comprise, consist substantially of, or consist of sequences encoding human CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB, or GM-CSFR transmembrane domains. Preferably, the transmembrane domain may comprise, consist substantially of, or consist of a portion of the human CD8α (CD8α) transmembrane domain or thereof. The CD8α transmembrane domain comprises, consists substantially 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 between both) of SEQ ID NO: 63. Preferably, the human CD8α transmembrane domain comprises, consists substantially of, or consists of the amino acid sequence of SEQ ID NO: 63.

[0069] The CD8α transmembrane domain is encoded by a polynucleotide comprising, substantially comprising, or composed of a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between the two) of the nucleic acid sequence of SEQ ID NO: 64 or SEQ ID NO: 169. Preferably, the CD8α transmembrane domain is encoded by a polynucleotide comprising, substantially comprising, or composed of the nucleic acid sequence of SEQ ID NO: 64 or SEQ ID NO: 169.

[0070] At least one costimulatory domain may comprise, substantially consist of, or consist of the following intracellular domains: human 4-1BB, CD28, CD3 zeta (CD3ζ), CD40, ICOS, MyD88, OX-40, or any combination thereof. Preferably, at least one costimulatory domain comprises a CD3ζ, 4-1BB costimulatory domain, or a combination thereof.

[0071] The 4-1BB intracellular domain comprises, is substantially composed of, or is composed of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between the two) of SEQ ID NO: 65. Preferably, the 4-1BB intracellular domain comprises, is substantially composed of, or is composed of the amino acid sequence of SEQ ID NO: 65.

[0072] The 4-1BB intracellular domain is encoded by a polynucleotide comprising, substantially comprising, or composed of a nucleic acid sequence equivalent to at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between the two) of SEQ ID NO: 66 or SEQ ID NO: 170. Preferably, the 4-1BB intracellular domain is encoded by a polynucleotide comprising, substantially comprising, or composed of the nucleic acid sequence of SEQ ID NO: 66 or SEQ ID NO: 170.

[0073] The CD3ζ intracellular domain comprises, is substantially composed of, or is composed of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between the two) of SEQ ID NO: 67. Preferably, the CD3ζ intracellular domain comprises, is substantially composed of, or is composed of the amino acid sequence of SEQ ID NO: 67.

[0074] The CD3ζ intracellular domain is encoded by a polynucleotide comprising, substantially comprising, or composed of a nucleic acid sequence equivalent to at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between the two) of SEQ ID NO: 68 or SEQ ID NO: 171. Preferably, the CD3ζ intracellular domain is encoded by a polynucleotide comprising, substantially comprising, or composed of the nucleic acid sequence of SEQ ID NO: 68 or SEQ ID NO: 171.

[0075] The compositions disclosed herein (e.g., anti-MUC1 scFv, CAR containing anti-MUC1 scFv) can have an affinity K selected from at least one of the following. D Combined with human MUC1: less than or equal to 10 −9 M, less than or equal to 10−10 M, less than or equal to 10 − 11 M, less than or equal to 10 −12 M, less than or equal to 10 −13 M, less than or equal to 10 −14 M, and less than or equal to 10 −15 M. K D It can be determined by any means, including but not limited to surface plasmon resonance.

[0076] Compositions containing anti-MUC1 scFv or CARs containing anti-MUC1 scFv can be incorporated into cell delivery compositions (e.g., transposons or vectors) as described in detail herein, and optionally, can be incorporated into cells.

[0077] Cells modified by contact with and / or incorporation of the compositions of this disclosure (e.g., immune cells and cytotoxic immune cells) can specifically target cells expressing MUC1. A preferred aspect of the methods of this disclosure uses a MUC1-C scFv binder to redirect cytotoxic cell types to mediate the destruction of cells expressing MUC1-C (MUC1-C+ cells). For example, this is used to treat proliferative conditions such as cancer. Modified cells expressing the anti-MUC1 scFv of this disclosure or CARs containing anti-MUC1 scFv have demonstrated improved in vivo persistence and antitumor efficacy. In some aspects, modified cells expressing the anti-MUC1 scFv of this disclosure or CARs containing anti-MUC1 scFv have demonstrated improved potency and reduced immunogenicity compared to cells expressing mouse anti-MUC1 scFv or CARs containing mouse anti-MUC1 scFv. In some aspects, compared with cells expressing scFv containing the heavy chain variable region of SEQ ID NO: 176 and the light chain variable region of SEQ ID NO: 177, or CARs containing scFv, cells expressing the anti-MUC1 scFv of this disclosure or modified CARs containing anti-MUC1 scFv have demonstrated improved potency and reduced immunogenicity. In some aspects, compared with cells expressing scFv containing the heavy chain variable region of SEQ ID NO: 178 and the light chain variable region of SEQ ID NO: 179, or CARs containing scFv, cells expressing the anti-MUC1 scFv of this disclosure or modified CARs containing anti-MUC1 scFv have demonstrated improved potency and reduced immunogenicity. In some respects, compared with cells expressing scFv containing the heavy chain variable region of SEQ ID NO: 180 and the light chain variable region of SEQ ID NO: 181 or CARs containing thereof, cells expressing the anti-MUC1 scFv of this disclosure or modified cells containing CARs of the anti-MUC1 scFv have demonstrated improved potency and reduced immunogenicity.

[0078] The “L1H1” scFv contains 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 contains the amino acid sequence of SEQ ID NO: 124 encoded by the nucleic acid sequence of SEQ ID NO: 142. The “L1H1” CAR contains the amino acid sequence of SEQ ID NO: 12 encoded by the nucleic acid sequence of SEQ ID NO: 30.

[0079] The “L1H1B” scFv contains 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 contains 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 contains 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.

[0080] The “L1H2” scFv contains 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 contains the amino acid sequence of SEQ ID NO: 126 encoded by the nucleic acid sequence of SEQ ID NO: 144. The “L1H2” CAR contains the amino acid sequence of SEQ ID NO: 14 encoded by the nucleic acid sequence of SEQ ID NO: 32.

[0081] The “L1H2B” scFv contains 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 contains the amino acid sequence of SEQ ID NO: 127 encoded by the nucleic acid sequence of SEQ ID NO: 145. The “L1H2B” CAR contains the amino acid sequence of SEQ ID NO: 15 encoded by the nucleic acid sequence of SEQ ID NO: 33.

[0082] The “L1H3” scFv contains a light chain variable region “L1” having an 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 an amino acid sequence of SEQ ID NO: 7 encoded by the nucleic acid sequence of SEQ ID NO: 52. The “L1H3” scFv contains an amino acid sequence of SEQ ID NO: 128 encoded by the nucleic acid sequence of SEQ ID NO: 146. The “L1H3” CAR contains an amino acid sequence of SEQ ID NO: 16 encoded by the nucleic acid sequence of SEQ ID NO: 34.

[0083] The “L1H4” scFv contains 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 contains the amino acid sequence of SEQ ID NO: 129 encoded by the nucleic acid sequence of SEQ ID NO: 147. The “L1H4” CAR contains the amino acid sequence of SEQ ID NO: 17 encoded by the nucleic acid sequence of SEQ ID NO: 35.

[0084] The “L2H1” scFv contains 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 contains the amino acid sequence of SEQ ID NO: 130 encoded by the nucleic acid sequence of SEQ ID NO: 148. The “L2H1” CAR contains the amino acid sequence of SEQ ID NO: 18 encoded by the nucleic acid sequence of SEQ ID NO: 36.

[0085] The “L2H1B” scFv contains 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 contains the amino acid sequence of SEQ ID NO: 131 encoded by the nucleic acid sequence of SEQ ID NO: 149. The “L2H1B” CAR contains the amino acid sequence of SEQ ID NO: 19 encoded by the nucleic acid sequence of SEQ ID NO: 37.

[0086] The “L2H2” scFv contains 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 contains the amino acid sequence of SEQ ID NO: 132 encoded by the nucleic acid sequence of SEQ ID NO: 150. The “L2H2” CAR contains the amino acid sequence of SEQ ID NO: 20 encoded by the nucleic acid sequence of SEQ ID NO: 38.

[0087] The “L2H2B” scFv contains 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 contains the amino acid sequence of SEQ ID NO: 133 encoded by the nucleic acid sequence of SEQ ID NO: 151. The “L2H2B” CAR contains the amino acid sequence of SEQ ID NO: 21 encoded by the nucleic acid sequence of SEQ ID NO: 39.

[0088] The “L2H3” scFv contains 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 contains the amino acid sequence of SEQ ID NO: 134 encoded by the nucleic acid sequence of SEQ ID NO: 152. The “L2H3” CAR contains the amino acid sequence of SEQ ID NO: 22 encoded by the nucleic acid sequence of SEQ ID NO: 40.

[0089] The “L2H4” scFv contains 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 contains the amino acid sequence of SEQ ID NO: 135 encoded by the nucleic acid sequence of SEQ ID NO: 153. The “L2H4” CAR contains the amino acid sequence of SEQ ID NO: 23 encoded by the nucleic acid sequence of SEQ ID NO: 41.

[0090] The “L3H1” scFv contains 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 contains the amino acid sequence of SEQ ID NO: 136 encoded by the nucleic acid sequence of SEQ ID NO: 154. The “L3H1” CAR contains the amino acid sequence of SEQ ID NO: 24 encoded by the nucleic acid sequence of SEQ ID NO: 42.

[0091] The “L3H1B” scFv contains 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 contains the amino acid sequence of SEQ ID NO: 137 encoded by the nucleic acid sequence of SEQ ID NO: 155. The “L3H1B” CAR contains the amino acid sequence of SEQ ID NO: 25 encoded by the nucleic acid sequence of SEQ ID NO: 43.

[0092] The “L3H2” scFv contains 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 contains the amino acid sequence of SEQ ID NO: 138 encoded by the nucleic acid sequence of SEQ ID NO: 156. The “L3H2” CAR contains the amino acid sequence of SEQ ID NO: 26 encoded by the nucleic acid sequence of SEQ ID NO: 44.

[0093] The “L3H2B” scFv contains 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 contains the amino acid sequence of SEQ ID NO: 139 encoded by the nucleic acid sequence of SEQ ID NO: 157. The “L3H2B” CAR contains the amino acid sequence of SEQ ID NO: 27 encoded by the nucleic acid sequence of SEQ ID NO: 45.

[0094] The “L3H3” scFv contains 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 contains the amino acid sequence of SEQ ID NO: 140 encoded by the nucleic acid sequence of SEQ ID NO: 158. The “L3H3” CAR contains the amino acid sequence of SEQ ID NO: 28 encoded by the nucleic acid sequence of SEQ ID NO: 46.

[0095] The “L3H4” scFv contains 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 contains the amino acid sequence of SEQ ID NO: 141 encoded by the nucleic acid sequence of SEQ ID NO: 159. The “L3H4” CAR contains the amino acid sequence of SEQ ID NO: 29 encoded by the nucleic acid sequence of SEQ ID NO: 47.

[0096] Tables 1A and 1B show exemplary MUC-1C CAR components and sequences of this disclosure.

[0097] Table 1. MUC-1C CAR components and amino acid sequences of this disclosure Table 1B. MUC-1C CAR components and nucleic acid sequences of this disclosure MUC1C CAR component SEQ ID NO: "H1" humanized heavy chain variable region 48 "H1B" humanized heavy chain variable region 49、164 "H2" humanized heavy chain variable region 50 "H2B" humanized heavy chain variable region 51 "H3" humanized heavy chain variable region 52 "H4" humanized heavy chain variable region 53 "L1" Humanized Light Chain Variable Region 54、165 "L2" humanized light chain variable region 55 "L3" humanized light chain variable region 56 "L1H1" scFv 142 "L1H1B" scFv 143、166 "L1H2" scFv 144 "L1H2B" scFv 145 "L1H3" scFv 146 "L1H4" scFv 147 "L2H1" scFv 148 "L2H1B" scFv 149 "L2H2" scFv 150 "L2H2B" scFv 151 "L2H3" scFv 152 "L2H4" scFv 153 "L3H1" scFv 154 "L3H1B" scFv 155 "L3H2" scFv 156 "L3H2B" scFv 157 "L3H3" scFv 158 "L3H4" scFv 159 "L1H1" CAR 30 "L1H1B" CAR 31、167 "L1H2" CAR 32 "L1H2B" CAR 33 "L1H3" CAR 34 "L1H4" CAR 35 "L2H1" CAR 36 "L2H1B" CAR 37 "L2H2" CAR 38 "L2H2B" CAR 39 "L2H3" CAR 40 "L2H4" CAR 41 "L3H1" CAR 42 "L3H1B" CAR 43 "L3H2" CAR 44 "L3H2B" CAR 45 "L3H3" CAR 46 "L3H4" CAR 47 CD8a signal peptide 58 Connector sequence 60 CD8a hinge 62、168 CD8a transmembrane domain 64、169 41BB ICS 66、170 CD3z ICS 68、171 "L1H1B" CAR transposon sequence 175 The cells and modified cells disclosed herein The cells and modified cells in this disclosure can be mammalian cells. Preferably, the cells and modified cells are human cells. The cells and modified cells in this disclosure can be immune cells. The immune cells in this disclosure can include lymphoid progenitor cells, natural killer (NK) cells, T lymphocytes (T cells), and stem memory T cells (T cells). SCM ), central memory T cells (T cells) CM Stem cell-like T cells, B lymphocytes (B cells), antigen-presenting cells (APCs), cytokine-induced killer (CIK) cells, myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, macrophages, platelets, erythrocytes, red blood cells (RBCs), megakaryocytes, or osteoclasts.

[0098] Immune progenitor cells can contain any cell capable of differentiating into one or more types of immune cells. Immune progenitor cells can contain pluripotent stem cells capable of self-renewal and developing into immune cells. Immune progenitor cells can contain hematopoietic stem cells (HSCs) or their progeny. Immune progenitor cells can contain precursor cells capable of developing into immune cells. Immune progenitor cells can contain hematopoietic progenitor cells (HPCs).

[0099] Hematopoietic stem cells (HSCs) are pluripotent, self-renewing cells. All differentiated blood cells from lymphoid and myeloid lineages originate from HSCs. HSCs can be found in adult bone marrow, peripheral blood, mobilized peripheral blood, peritoneal dialysis effluent, and umbilical cord blood.

[0100] HSCs can be isolated from or derived from primary or cultured stem cells. HSCs can be isolated from or derived from embryonic stem cells, pluripotent stem cells, multipotent stem cells, adult stem cells, or induced pluripotent stem cells (iPSCs).

[0101] Immune progenitor cells may include HSCs or HSC progeny cells. Non-limiting examples of HSC progeny cells include pluripotent stem cells, lymphoid progenitor cells, natural killer (NK) cells, T lymphocytes (T cells), B lymphocytes (B cells), myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, and macrophages.

[0102] HSCs generated by the disclosed method retain the characteristics of "primitive" stem cells, which share the properties of embryonic stem cells when isolated from or derived from adult stem cells and defined into a single lineage. For example, "primitive" HSCs generated by the disclosed method retain their "stemness" after division and do not differentiate. Therefore, as adoptive cell therapy, "primitive" HSCs generated by the disclosed method not only replenish their number but also expand in vivo. When administered as a single dose, "primitive" HSCs generated by the disclosed method can be therapeutically effective.

[0103] The original HSC can be CD34+. The original HSC can be CD34+ and CD38-. The original HSC can be CD34+, CD38-, and CD90+. The original HSC can be CD34+, CD38-, CD90+, and CD45RA-. The original HSC can be CD34+, CD38-, CD90+, CD45RA-, and CD49f+. The original HSC can be CD34+, CD38-, CD90+, CD45RA-, and CD49f+.

[0104] Primitive HSCs, HSCs, and / or HSC progeny cells can be modified according to the disclosed methods to express exogenous sequences (e.g., chimeric antigen receptors or therapeutic proteins). Modified primitive HSCs, modified HSCs, and / or modified HSC progeny cells can be forward differentiated to generate modified immune cells, including but not limited to modified T cells, modified natural killer cells, and / or modified B cells.

[0105] Modified immune or immune precursor cells can be NK cells. NK cells can be cytotoxic lymphocytes differentiated from lymphoid progenitor cells. Modified NK cells can be derived from modified hematopoietic stem cells and progenitor cells (HSPCs) or modified HSCs. In some respects, inactivated NK cells are derived from CD3-depleted leukapheresis (containing CD14 / CD19 / CD56+ cells).

[0106] Modified immune or immune progenitor cells can be B cells. B cells are a type of lymphocyte that expresses B cell receptors on their cell surface. B cell receptors bind to specific antigens. Modified B cells can be derived from modified hematopoietic stem cells and progenitor cells (HSPCs) or modified HSCs.

[0107] The modified T cells disclosed herein can be derived from modified hematopoietic stem cells and progenitor cells (HSPCs) or modified HSCs. Unlike conventional biologics and chemotherapy agents, the disclosed modified T cells are capable of rapid proliferation upon antigen recognition, potentially avoiding the need for repeated treatment. To achieve this, in some embodiments, the modified T cells not only drive the initial response but also persist in the patient as a stable population of live memory T cells to prevent potential relapse. Alternatively, in some aspects, the modified T cells are not persistently present in the patient when not needed.

[0108] Significant efforts have been focused on the development of antigen receptor molecules that do not lead to T cell depletion through antigen-independent (tetanic) signaling, and on T cells containing early memory, particularly stem cell memory (T cells). SCM The present disclosure describes the development of modified T cell products, specifically stem cell-like T cells. These modified T cells exhibit maximum self-renewal capacity and pluripotency for the derivation of central memory (T cells). CM T cells or T cells CM T-like cells, effector memory (T) EM ) and effector T cells (T E This leads to better tumor eradication and long-term modified T-cell graft implantation. A linear pathway of differentiation may be responsible for generating these cells: naive T cells (T... N ) > T SCM > T CM > T EM > T E > T TE Therefore, T N It directly generates T SCM The parental precursor cells, which then directly produce T cells. CM The T cell compositions of this disclosure may comprise one or more of each parental T cell subset, wherein T... SCM Cells are the most abundant (e.g., T cells) SCM > T CM > T EM > T E > T TE ).

[0109] Immune cell precursors can differentiate into or are capable of differentiating into early memory T cells, stem cells such as T cells, and naive T cells (T cells). N ), T SCM T CM T EM T E or T TEThe precursor immune cells can be the original HSCs, HSCs, or HSC progeny cells described in this disclosure. The immune cells can be early memory T cells, stem cells such as T cells, or naive T cells (T...). N ), T SCM T CM T EM T E or T TE .

[0110] The methods of this disclosure can modify and / or generate modified T cell populations, wherein the population comprises 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 between the two, multiple modified T cells expressing one or more cell surface markers of early memory T cells. The modified early memory T cell population comprises multiple modified stem cell-like T cells. SCM Cells. The modified early memory T cell population contains multiple modified T cells. CM cell.

[0111] The methods of this disclosure can modify and / or generate modified T cell populations, wherein the population comprises 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 between the two, multiple modified T cells expressing one or more cell surface markers of stem cell-like T cells. The modified stem cell-like T cell population comprises multiple modified T cells expressing one or more cell surface markers of stem cell-like T cells. SCM Cells. The modified stem cell-like T cell population contains multiple modified T cells. CM cell.

[0112] In some respects, 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 of modified T cells expressing stem memory T cells (T cells) in the population. SCM ) or T SCMThe cell surface markers include one or more cell surface markers; and one or more of these cell surface markers include 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.

[0113] In some respects, 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 contain multiple modified T cells that express central memory T cells (T cells). CM ) or T CM One or more cell surface markers of the sample cells; and one or more of the cell surface markers include CD45RO and CD62L. The cell surface markers may include one or more of CD45RO, CD95, IL-2Rβ, CCR7 and CD62L.

[0114] The methods of this disclosure can modify and / or generate a modified T cell population, wherein the population contains 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 between the two of a plurality of modified T cells expressing naive T cells (T cells expressing naive T cells). N One or more cell surface markers. Cell surface markers may include one or more of CD45RA, CCR7, and CD62L.

[0115] The methods of this disclosure can modify and / or generate a population of modified T cells, wherein the population comprises 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 between both of the modified T cells, expressing effector T cells (modified T cells). EFF One or more cell surface markers. Cell surface markers may include one or more of CD45RA, CD95, and IL-2Rβ.

[0116] The methods of this disclosure can modify and / or generate a modified T cell population, wherein the population contains 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 between the two of a plurality of modified T cells expressing stem cell-like T cells, stem memory T cells (T cells). SCM ) or central memory T cells (T CM One or more cell surface markers.

[0117] Multiple modified cells in a population contain a transgene or a sequence encoding a transgene (e.g., 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 cells in the population contain a transgene or a sequence encoding a transgene, 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 between the two of the modified T cells in the population express stem cell-like T cells, stem memory T cells (T cells), etc. SCM ) or central memory T cells (T CM One or more cell surface markers. In some aspects, 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.

[0118] Multiple modified cells in a population contain a transgene or a sequence encoding a transgene (e.g., 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 cells in the population contain a transgene or a sequence encoding a transgene, wherein at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91% of the cells in the population contain a transgene or a sequence encoding a transgene. A modified cell population of 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% expressing one or more cell surface markers containing CD34, or a modified cell population of at least about 70% to about 99%, about 75% to about 95%, or about 85% to about 95% expressing one or more cell surface markers containing CD34 (e.g., containing the cell surface marker phenotype CD34+).

[0119] Multiple modified cells in a population contain a transgene or a sequence encoding a transgene (e.g., 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 cells in the population contain 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% of the cells in the population contain a transgene or a sequence encoding a transgene, and ...93% of the cells in the population A modified cell population comprising 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% expressing one or more cell surface markers containing CD34 and not expressing one or more cell surface markers containing CD38, or wherein at least about 45% to about 90%, about 50% to about 80%, or about 65% to about 75% of the modified cell population expresses one or more cell surface markers containing CD34 and not expressing one or more cell surface markers containing CD38 (e.g., containing cell surface marker phenotypes CD34+ and CD38-).

[0120] Multiple modified cells in the population contain a transgene or a sequence encoding a transgene (e.g., 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 cells in the population contain 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 45%, at least 50%, at least 55%, at least 60%, and so on. A modified cell population comprising 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 a cell population expressing one or more cell surface markers comprising CD34 and CD90, and not expressing one or more cell surface markers comprising CD38, or wherein at least about 0.2% to about 40%, about 0.2% to about 30%, about 0.2% to about 2%, or 0.5% to about 1.5% of a cell population expressing one or more cell surface markers comprising CD34 and CD90, and not expressing one or more cell surface markers comprising CD38 (e.g., comprising cell surface marker phenotypes CD34+, CD38-, and CD90+).

[0121] Multiple modified cells in the population contain a transgene or a sequence encoding a transgene (e.g., 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 cells in the population contain 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 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the cells in the population. A modified cell population comprising 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% expressing one or more cell surface markers comprising CD34 and CD90, and not expressing one or more cell surface markers comprising CD38 and CD45RA, or wherein at least about 0.2% to about 40%, about 0.2% to about 30%, about 0.2% to about 2%, or 0.5% to about 1.5% expressing one or more cell surface markers comprising CD34 and CD90, and not expressing one or more cell surface markers comprising CD38 and CD45RA (e.g., comprising cell surface marker phenotypes CD34+, CD38-, CD90+, CD45RA).

[0122] Multiple modified cells in a population contain a transgene or a sequence encoding a transgene (e.g., 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 cells in the population contain a transgene or a sequence encoding a transgene, wherein at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, or 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% A modified cell population of 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% expressing one or more cell surface markers including CD34, CD90, and CD49f, and not expressing CD38 and CD45RA. One or more cell surface markers, or a modified cell population comprising 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%, expressing one or more cell surface markers including CD34, CD90, and CD49f, and not expressing one or more cell surface markers including CD38 and CD45RA (e.g., including cell surface marker phenotypes CD34+, CD38-, CD90+, CD45RA, and CD49f+).

[0123] Multiple modified cells in the population contain a transgene or a sequence encoding a transgene (e.g., 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 cells in the population contain a transgene or a sequence encoding a transgene, wherein at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 3% of the cells in the population contain a transgene or a sequence encoding a transgene. A modified cell population comprising 0%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% of a cell population expressing one or more cell surface markers comprising CD34 and CD90 and not expressing one or more cell surface markers comprising CD45RA, or wherein at least about 0.2% to about 5%, about 0.2% to about 3%, or about 0.4% to about 3% of a modified cell population expressing one or more cell surface markers comprising CD34 and CD90 and not expressing one or more cell surface markers comprising CD45RA (e.g., comprising cell surface marker phenotypes CD34+, CD90+, and CD45RA-).

[0124] Compositions and methods for generating and / or expanding immune cells or immune precursor cells (e.g., the disclosed modified T cells), and buffers for maintaining or enhancing the cell viability level and / or dry-like phenotype of immune cells or immune precursor cells (e.g., the disclosed modified T cells), are disclosed elsewhere herein and in more detail in U.S. Patent No. 10,329,543 and PCT Publication No. WO 2019 / 173636.

[0125] The cells and modified cells disclosed herein can be somatic cells. The cells and modified cells disclosed herein can be differentiated cells. The cells and modified cells disclosed herein can be autologous cells or allogeneic cells. Allogeneic cells are modified to prevent adverse reactions to graft implantation after administration to a subject. Allogeneic cells can be any type of cell. Allogeneic cells can be stem cells or cells derived from stem cells. Allogeneic cells can be differentiated somatic cells.

[0126] Methods for expressing chimeric antigen receptors This disclosure provides a method for expressing CAR on the surface of cells. The method includes (a) obtaining a cell population; (b) contacting the cell population with a composition containing CAR or a sequence encoding CAR under conditions sufficient to transfer CAR across the cell membrane of at least one cell in the cell population, thereby generating a modified cell population; (c) culturing the modified cell population under conditions suitable for integrating the sequence encoding CAR; and (d) expanding and / or selecting at least one cell from the modified cell population expressing CAR on the cell surface.

[0127] In some respects, the cell population may contain leukocytes and / or CD4+ and CD8+ leukocytes. The cell population may contain CD4+ and CD8+ leukocytes at an optimized ratio. This optimized ratio of CD4+ to CD8+ leukocytes does not occur naturally in vivo. The cell population may contain tumor cells.

[0128] In some aspects, conditions sufficient to transfer a CAR or a sequence encoding a CAR, transposon, or vector across the cell membrane of at least one cell in a cell population include the application of one or more electrical pulses at a specific voltage, a buffer solution, and at least one of one or more complementary factors. In some aspects, conditions suitable for integrating a sequence encoding a CAR include a buffer solution and at least one of one or more complementary factors.

[0129] The buffer may contain PBS, HBSS, OptiMEM, BTXpress, Amaxa Nucleofector, human T cell nuclear transfection buffer, or any combination thereof. One or more supplementary factors may contain (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 apoptosis pathways, or combinations thereof; and (e) reagents for modifying or stabilizing one or more nucleic acids. Recombinant human cytokines, chemokines, interleukins, or any combination thereof may 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-γ, IL-1 α / IL-1F1, IL-1 β / IL-1F2, IL-12 p70, IL-12 / IL-35 p35, IL-13, IL-17 / IL-17A, IL-17A / F heterodimer, IL-17F, IL-18 / IL-1F4, IL-23, IL-24, IL-32, IL-32 β, IL-32 γ, IL-33, LAP (TGF-β 1), lymphotoxin-α / TNF-β, TGF-β, TNF-α, TRANCE / TNFSF11 / RANK L or any combination thereof. Salts, minerals, metabolites, or any combination thereof may include HEPES, nicotinamide, heparin, sodium pyruvate, L-glutamine, MEM non-essential amino acid solution, ascorbic acid, nucleosides, FBS / FCS, human serum, serum 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.Cell culture media may contain 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 the following inhibitors: TLR9, MyD88, IRAK, TRAF6, TRAF3, IRF-7, NF-κB, type I interferon, pro-inflammatory cytokines, cGAS, STING, Sec5, TBK1, IRF-3, RNApol III, RIG-1, IPS-1, FADD, RIP1, TRAF3, AIM2, ASC, caspase 1, Pro-IL1B, PI3K, Akt, Wnt3A, glycogen synthase kinase-3β (GSK-3β) inhibitors (e.g., TWS119), or any combination thereof. Examples of such inhibitors may include bafloxacin, chloroquine, quinacrine, AC-YVAD-CMK, Z-VAD-FMK, Z-IETD-FMK, or any combination thereof. Reagents for modifying or stabilizing one or more nucleic acids include pH adjusters, DNA-binding proteins, lipids, phospholipids, CaPO4, net neutral-charged DNA-binding peptides with or without NLS sequences, TREX1 enzymes, or any combination thereof.

[0130] The amplification and selection steps can occur concurrently or sequentially. Amplification can occur before selection. Amplification can occur after selection, and optionally, further (i.e., a second) selection can occur after amplification. Concurrent amplification and selection can occur simultaneously. The amplification and / or selection steps can be performed over a period of 10 to 14 days, including the endpoints.

[0131] Amplification may involve contacting at least one cell of a modified cell population with an antigen to stimulate at least one cell via CAR, thereby generating an expanded cell population. The antigen may be presented on a surface of a substrate. The substrate may have any form, including but not limited to a surface, pores, beads or multiple beads, and a matrix. The substrate may further include paramagnetic or magnetic components. The antigen may be presented on a surface of a substrate, wherein the substrate is magnetic beads, and wherein a magnet can be used to remove or separate the magnetic beads from the modified and expanded cell population. The antigen may be presented on the surface of cells or artificial antigen-presenting cells. Artificial antigen-presenting cells may include, but are not limited to, tumor cells and stem cells.

[0132] In which the transposon or vector contains aspects of a selection gene, the selection step includes contacting at least one cell in the modified cell population with a compound to which the selection gene confers resistance, thereby identifying cells expressing the selection gene as surviving the selection and cells failing to express the selection gene as failing to survive the selection step.

[0133] This disclosure provides compositions comprising modified, expanded, and selected cell populations as described herein.

[0134] A more detailed description of methods for expressing CAR on the surface of cells is disclosed in PCT Publication Nos. WO 2019 / 049816 and PCT / US2019 / 049816.

[0135] This disclosure provides cells or cell populations, wherein the cells constitute a composition comprising (a) an inducible transgene construct containing a sequence encoding an inducible promoter and a sequence encoding a transgene, and (b) a receptor construct containing a sequence encoding a constitutive promoter and a sequence encoding a foreign receptor, such as a CAR, wherein the foreign receptor is expressed after the constructs (a) and (b) are integrated into the genomic sequence of the cell, and wherein the foreign receptor transduces intracellular signals upon binding to a ligand or antigen, the intracellular signals directly or indirectly targeting and regulating the inducible promoter of the expression of the inducible transgene (a) to modify gene expression.

[0136] The composition can modify gene expression by reducing gene expression. The composition can modify gene expression transiently (e.g., for the duration of ligand binding to a foreign receptor). The composition can modify gene expression drastically (e.g., reversible binding of the ligand to the foreign receptor). The composition can modify gene expression permanently (e.g., irreversible binding of the ligand to the foreign receptor).

[0137] Exogenous receptors may include endogenous receptors relative to the genomic sequence of the cell. Exemplary receptors include, but are not limited to, intracellular receptors, cell surface receptors, transmembrane receptors, ligand-gated ion channels, and G protein-coupled receptors.

[0138] Exogenous receptors may include receptors that are not naturally occurring. These receptors may be synthetic, modified, recombinant, mutant, or chimeric. They may contain one or more sequences isolated from or derived from T-cell receptors (TCRs). They may also contain one or more sequences isolated from or derived from scaffold proteins. In some aspects, including those in which the non-naturally occurring receptor does not contain a transmembrane domain, the non-naturally occurring receptor interacts with a second transmembrane, membrane-bound, and / or intracellular receptor that transduces intracellular signals upon contact with the non-naturally occurring receptor. The non-naturally occurring receptor may contain a transmembrane domain. It may interact with intracellular receptors that transduce intracellular signals. It may contain an intracellular signal transduction domain. The non-naturally occurring receptor may be a chimeric ligand receptor (CLR). A CLR may be a chimeric antigen receptor (CAR).

[0139] Sequences encoding inducible promoters include sequences encoding NF-κB promoters, interferon (IFN) promoters, or interleukin-2 promoters. In some respects, IFN promoters are IFNγ promoters. Inducible promoters can be isolated from or derived from promoters of cytokines or chemokines. Cytokines or chemokines may include IL2, IL3, IL4, IL5, IL6, IL10, IL12, IL13, IL17A / F, IL21, IL22, IL23, transforming growth factor β (TGFβ), colony-stimulating factor 2 (GM-CSF), interferon γ (IFNγ), tumor necrosis factor α (TNFα), LTα, perforin, granzyme C (Gzmc), granzyme B (Gzmb), CC motif chemokine ligand 5 (CCL5), CC motif chemokine ligand 4 (Ccl4), CC motif chemokine ligand 3 (Ccl3), XC motif chemokine ligand 1 (Xcl1), or LIF interleukin-6 family cytokines (Lif).

[0140] Inducible promoters can be isolated from or derived from promoters of genes containing surface proteins involved in cell differentiation, activation, depletion, and function. In some respects, these genes include CD69, CD71, CTLA4, PD-1, TIGIT, LAG3, TIM-3, GITR, MHCII, COX-2, FASL, or 4-1BB.

[0141] Inducible promoters can be isolated from or derived from promoters of genes involved in CD metabolism and differentiation. Inducible promoters can be isolated from or derived from promoters of Nr4a1, Nr4a3, Tnfrsf9 (4-1BB), Sema7a, Zfp36l2, Gadd45b, Dusp5, Dusp6, and Neto2.

[0142] In some respects, inducible transgenic constructs include or drive the expression of 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 conferring sensitivity to cancer therapies, and oncogenes or tumor suppressor genes. Non-limiting examples are disclosed in PCT Publication No. WO 2019 / 173636 and PCT Application No. PCT / US2019 / 049816.

[0143] This disclosure provides a method for generating a modified T cell population, comprising, substantially comprising, or comprising the following: introducing a CAR comprising the present disclosure or a composition encoding a sequence thereof into a plurality of primary human T cells to generate a plurality of modified T cells. This disclosure provides a composition comprising a modified T cell population generated by this method. In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population express the CAR of the present disclosure.

[0144] Armored Cells The modified cells of this disclosure (e.g., CAR T cells) can be further modified to enhance their therapeutic potential. Alternatively or additionally, the modified cells can be further modified to make them less sensitive to immunological and / or metabolic checkpoints. This type of modified "armed" cells, after modification, may be referred to herein as "armed" cells (e.g., armed T cells). Armed cells can be generated, for example, by blocking and / or diluting specific checkpoint signals naturally delivered to the cells (e.g., checkpoint inhibition) within a tumor immunosuppressive microenvironment.

[0145] The armed cells of this disclosure can be derived from any cell type, such as T cells, NK cells, hematopoietic progenitor cells, peripheral blood (PB)-derived T cells (including T cells isolated from or derived from G-CSF-mobilized peripheral blood), or umbilical cord blood (UCB)-derived T cells. Armed cells (e.g., armed T cells) may comprise one or more of the following: chimeric ligand receptors (CLRs comprising a protein scaffold, antibody, ScFv, or antibody mimics) / chimeric antigen receptors (CARs comprising a protein scaffold, antibody, ScFv, or antibody mimics), CARTyrin (CARs comprising Centyrin), and / or VCARs (CARs comprising camel VHHs or single-domain VHs). Armed cells (e.g., armed T cells) may comprise inducible pro-apoptotic peptides as disclosed herein. Armed cells (e.g., armed T cells) may comprise exogenous sequences. Exogenous sequences may comprise sequences encoding therapeutic proteins. Exemplary therapeutic proteins may be nuclear, cytoplasmic, intracellular, transmembrane, or cell surface-bound or secreted proteins. Exemplary therapeutic proteins expressed by armed cells (e.g., armed T cells) may modify the activity of the armed cells or may modify the activity of a second cell. Armed cells (e.g., armed T cells) may contain selection genes or selection markers. Armed cells (e.g., armed T cells) may contain synthetic gene expression cassettes (also referred to herein as inducible transgenic constructs).

[0146] The modified cells of this disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding receptors for inhibitory checkpoint signals to produce armed cells (e.g., armed CAR T cells). Receptors for inhibitory checkpoint signals are expressed on the cell surface or in the cytoplasm of the cell. Silencing or reducing the expression of genes encoding receptors for inhibitory checkpoint signals results in the loss of protein expression of the inhibitory checkpoint receptor on the surface of the armed cell or in the cytoplasm. Therefore, armed cells with silenced or reduced expression of one or more genes encoding inhibitory checkpoint receptors are resistant, unacceptable, or insensitive to checkpoint signals. The reduced resistance or sensitivity of armed cells to inhibitory checkpoint signals in the presence of these inhibitory checkpoint signals enhances the therapeutic potential of the armed cells. Non-limiting examples of inhibitory checkpoint signals (and proteins that induce immunosuppression) are disclosed in PCT Publication No. WO 2019 / 173636. Preferred examples of inhibitory checkpoint signals that can be silenced include, but are not limited to, PD-1 and TGFβRII.

[0147] The modified cells of this disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding intracellular proteins involved in checkpoint signaling to produce armed cells (e.g., armed CAR T cells). The activity of the modified cells can be enhanced by targeting any intracellular signaling protein involved in checkpoint signaling pathways, thereby achieving checkpoint inhibition or interference of one or more checkpoint pathways. Non-limiting examples of intracellular signaling proteins involved in checkpoint signaling are disclosed in PCT Publication No. WO 2019 / 173636.

[0148] The modified cells of this disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding transcription factors that inhibit therapeutic efficacy, 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 repressive function) of transcription factors that inhibit therapeutic efficacy. Non-limiting examples of transcription factors that can be modified to silence or reduce expression or repress their function include, but are not limited to, the exemplary transcription factors disclosed in PCT Publication No. WO 2019 / 173636.

[0149] The modified cells of this disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding cell death or apoptosis receptors to produce armed cells (e.g., armed CAR T cells). The interaction of the death receptor with its endogenous ligand leads to the initiation of apoptosis. Disruption of the expression, activity, or interaction of cell death and / or apoptosis receptors and / or ligands makes the modified cells less responsive to death signals, thus making the armed cells more effective in the tumor environment. Non-limiting examples of cell death and / or apoptosis receptors and ligands are disclosed in PCT Publication No. WO 2019 / 173636. A preferred example of a cell death receptor that can be modified is Fas (CD95).

[0150] The modified cells of this disclosure (e.g., CAR T cells) can be further modified to silence or reduce the expression of one or more genes encoding metabolic sensing proteins to produce armed cells (e.g., armed CAR T cells). By disrupting metabolic sensing in the immunosuppressive tumor microenvironment (characterized by low levels of oxygen, pH, glucose, and other molecules) by the modified cells, prolonged preservation of T cell function is achieved, and thus, each cell kills more tumor cells. Non-limiting examples of metabolic sensing genes and proteins are disclosed in PCT Publication No. WO 2019 / 173636. In a preferred example, HIF1a and VHL function as T cells in a hypoxic environment. Armed T cells may have silenced or reduced expression of one or more genes encoding HIF1a or VHL.

[0151] The modified cells (e.g., CAR T cells) of this disclosure can be further modified to silence or reduce the expression of one or more genes encoding proteins that confer sensitivity to cancer therapies, including monoclonal antibodies, to produce armed cells (e.g., armed CAR T cells). Thus, armed cells can function in the presence of cancer therapies (e.g., chemotherapy, monoclonal antibody therapy, or another antitumor therapy), and superior function or efficacy can be demonstrated. Non-limiting examples relating to proteins conferring sensitivity to cancer therapies are disclosed in PCT Publication No. WO2019 / 173636.

[0152] The modified cells (e.g., CAR T cells) of this disclosure can be further modified to silence or reduce the expression of one or more genes encoding growth advantage factors to produce armed cells (e.g., armed CAR T cells). Silencing or reducing the expression of oncogenes can confer a growth advantage on the cells. For example, silencing or reducing the expression of the TET2 gene (e.g., disrupting its expression) during CAR T cell production results in the generation of armed CAR T cells with a significant ability to expand and subsequently eradicate tumors when compared with unarmed CAR T cells lacking such expansion capacity. This strategy can be coupled with a safety switch (e.g., the iC9 safety switch described herein) that allows targeted destruction of armed CAR T cells in the event of an adverse reaction from a subject or uncontrolled growth of armed CAR T cells. Non-limiting examples of growth advantage factors are disclosed in PCT Publication No. WO 2019 / 173636.

[0153] The modified cells of this disclosure (e.g., CAR T cells) can be further modified to express modified / chimeric checkpoint receptors to generate armed T cells of this disclosure.

[0154] Modified / chimeric checkpoint receptors can comprise null receptors, decoy receptors, or dominant-inactive receptors. Null receptors, decoy receptors, or dominant-inactive receptors can be modified / chimeric receptors / proteins. Null receptors, decoy receptors, or dominant-inactive receptors can be truncated for expression of intracellular signal transduction domains. Alternatively or additionally, null receptors, decoy receptors, or dominant-inactive receptors can be mutated at one or more amino acid positions within the intracellular signal transduction domain, said amino acid positions being decisive or essential for effective signal transduction. Truncation or mutation of null receptors, decoy receptors, or dominant-inactive receptors can result in the receptor losing its ability to transmit or transduce checkpoint signals into or within the cell.

[0155] For example, the dilution or blockade of immunosuppressive checkpoint signaling from PD-L1 receptors expressed on the surface of tumor cells can be achieved by expressing modified / chimeric PD-1 null receptors on the surface of armed cells (e.g., armed CAR T cells). These null receptors effectively compete with endogenous (unmodified) PD-1 receptors also expressed on the surface of armed cells to reduce or inhibit the transduction of immunosuppressive checkpoint signals through the endogenous PD-1 receptors of the armed cells. In this non-limiting example, the competition between two different receptors for binding to PD-L1 expressed on tumor cells reduces or attenuates the level of effective checkpoint signaling, thereby enhancing the therapeutic potential of armed cells expressing PD-1 null receptors.

[0156] Modified / chimeric checkpoint receptors may include null receptors, decoy receptors, or dominantly inactivated receptors, which are transmembrane receptors, membrane-associated or membrane-connecting receptors / proteins, or intracellular receptors / proteins. Exemplary null, decoy, or dominantly inactivated intracellular receptors / proteins include, but are not limited to, signal transduction 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 conferring sensitivity to cancer therapies, and oncogenes or tumor suppressor genes. Non-limiting examples of cytokines, cytokine receptors, chemokines, and chemokine receptors are disclosed in PCT Publication No. WO 2019 / 173636.

[0157] Modified / chimeric checkpoint receptors may include switch receptors. Exemplary switch receptors comprise modified / chimeric receptor / proteins in which a native or wild-type intracellular signaling domain is converted or replaced by a different intracellular signaling domain, which may be a non-native and / or non-wild-type domain for the protein. For example, replacing an inhibitory signaling domain with a stimulatory signaling domain converts an immunosuppressive signal into an immunostimulatory signal. Alternatively, replacing an inhibitory signaling domain with a different inhibitory domain may reduce or enhance the level of inhibitory signaling. Expression or overexpression of the switch receptor can lead to dilution and / or blockade of homologous checkpoint signals by competing with endogenous wild-type checkpoint receptors (rather than switch receptors) for binding to homologous checkpoint receptors expressed within an immunosuppressive tumor microenvironment. Armed cells (e.g., armed CAR T cells) may contain sequences encoding switch receptors, resulting in the expression of one or more switch receptors and thus altering the activity of the armed cells. Armed cells (e.g., armed CAR T cells) can express switch receptors that target intracellularly expressed proteins, transcription factors, cytokine receptors, death receptors, metabolic sensing molecules, cancer therapies, oncogenes, and / or tumor suppressor proteins or genes downstream of checkpoint receptors.

[0158] Exemplary switch receptors may comprise or be derived from proteins, including but not limited to signal transduction 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 therapies, and oncogenes or tumor suppressor genes.

[0159] The modified cells of this disclosure (e.g., CAR T cells) can be further modified to express CLR / CARs that mediate conditional gene expression, thereby generating armed T cells. The combination of the CLR / CAR with a conditional gene expression system in the nucleus of the armed T cells constitutes a synthetic gene expression system that is conditionally activated upon binding of a homologous ligand to the CLR or a homologous antigen to the CAR. For example, this system can help ‘arm’ or enhance the therapeutic potential of the modified T cells by reducing or limiting the expression of synthetic genes at or within the ligand or antigen-binding site, in the tumor setting, or within the tumor setting.

[0160] Gene editing compositions and methods Modified cells are produced by introducing transgenes into cells. The introduction step may involve delivering nucleic acid sequences, transgenes, and / or genome editing constructs via a non-transposon delivery system.

[0161] Introducing nucleic acid sequences, transgenic and / or genome-edited constructs into cells in vitro, in vivo, in vitro, or in situ can include one or more of the following methods: local delivery, adsorption, absorption, electroporation, spin-infection, co-culture, transfection, mechanical delivery, acoustic delivery, vibrational delivery, magnetic transfection, or nanoparticle-mediated delivery. Introducing nucleic acid sequences, transgenic and / or genome-edited constructs into cells in vitro, in vivo, in vitro, or in situ can include liposome transfection, calcium phosphate transfection, fugene transfection, and dendritic polymer-mediated transfection. Mechanical transfection of nucleic acid sequences, transgenic and / or genome-edited constructs into cells in vitro, in vivo, in vitro, or in situ can include cell extrusion, cell bombardment, or gene gun technology. Nanoparticle-mediated transfection of nucleic acid sequences, transgenic and / or genome-edited constructs into cells in vitro, in vivo, in vitro, or in situ can include liposome delivery, micelle delivery, and polymerosome delivery.

[0162] The introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells, either in vitro, in vivo, or in situ, may include non-viral vectors. Non-viral vectors may contain nucleic acids. They may contain plasmid DNA, linear double-stranded DNA (dsDNA), linear single-stranded DNA (ssDNA), DoggyBone™ DNA, nanoparticles, small circular DNA, single-stranded oligodeoxynucleotides (ssODN), DDNA oligonucleotides, single-stranded mRNA (ssRNA), and double-stranded mRNA (dsRNA). Non-viral vectors may contain transposons as described herein.

[0163] Introducing nucleic acid sequences, transgenes, and / or genome editing constructs into cells, either in vitro, in vivo, or in situ, can include viral vectors. Viral vectors can be non-integrating, non-chromosomal vectors. Non-limiting examples of non-integrating, non-chromosomal vectors include adeno-associated viruses (AAVs), adenoviruses, and herpesviruses. Viral vectors can also be integrated chromosomal vectors. Non-limiting examples of integrated chromosomal vectors include adeno-associated vectors (AAVs), lentiviruses, and gamma retroviruses.

[0164] The introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells, whether in vitro, in vivo, or in situ, can include combinations of vectors. Non-limiting examples of vector combinations include viral and non-viral vectors, multiple non-viral vectors, or multiple viral vectors. Non-limiting examples of vector combinations include combinations of DNA-derived and RNA-derived vectors, combinations of RNA and reverse transcriptase, combinations of transposons and transposases, combinations of non-viral vectors and endonucleases, and combinations of viral vectors and endonucleases.

[0165] Genome modification can involve introducing nucleic acid sequences, transgenes, and / or genome editing constructs into cells, either in vitro, in vivo, ex vivo, or in situ, to achieve stable integration, transient integration, site-specific integration, or biased integration of nucleic acid sequences. The nucleic acid sequence can be a transgene.

[0166] Genome modification can involve the in vitro, in vivo, in vitro, or in situ introduction of nucleic acid sequences, transgenes, and / or genome editing constructs into cells to stably integrate the nucleic acid sequences. Stable chromosomal integration can be random, site-specific, or biased. Site-specific integration can be unhelpful or helper-assisted. Helper-specific integration is co-delivered with a site-directed nuclease. The site-directed nuclease contains a transgene with 5' and 3' nucleotide sequence extensions containing a percentage of homology to the upstream and downstream regions of the genome integration site. Transgenes with homologous nucleotide extensions enable genome integration via homologous recombination, microhomology-mediated end joining, or non-homologous end joining. Site-specific integration can occur at safe harbor sites. Genome safe harbor sites accommodate the integration of new genetic material in a manner that ensures the newly inserted genetic element functions reliably (e.g., expressed at therapeutically effective expression levels) and does not cause harmful alterations to the host genome that pose a risk to the host organism. Non-limiting examples of potential genomic safe harbors include intron sequences of the human albumin gene, adeno-associated virus site 1 (AAVS1), naturally occurring integration sites of AAV viruses on chromosome 19, sites of the chemokine receptor 5 (CCR5) gene, and sites of human orthologs of the mouse Rosa26 locus.

[0167] Site-specific transgene integration can occur at sites that disrupt the expression of target genes. Disruption of target gene expression can occur through site-specific integration at introns, exons, promoters, genetic elements, enhancers, repressors, start codons, stop codons, and response elements. Non-restricted examples of target genes targeted by site-specific integration include TRAC, TRAB, PDI, any immunosuppressive gene, and genes involved in allogeneic rejection.

[0168] Site-specific transgene integration can occur at sites that lead to enhanced expression of the target gene. Enhanced target gene expression can occur through site-specific integration at introns, exons, promoters, genetic elements, enhancers, repressors, start codons, stop codons, and response elements.

[0169] Enzymes can be used to create strand breaks in the host genome to facilitate the delivery or integration of transgenes. Enzymes can produce single-strand or double-strand breaks. Non-restrictive examples of break-inducible enzymes include transposases, integrases, endonucleases, CRISPR-Cas9, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), Cas-CLOVER™, and CPF1. Break-inducible enzymes can be encoded by DNA, by mRNA, as proteins, or as nucleoprotein complexes with guide RNA (gRNA) delivered to cells.

[0170] Site-specific transgene integration can be controlled by vector-mediated integration site bias. Vector-mediated integration site bias can be controlled by the selected lentiviral vector or the selected gamma retroviral vector.

[0171] Site-specific transgene integration sites can be unstable chromosomal insertions. Integrated transgenes can become silent, removed, excised, or further modified. Genomic modification can lead to unstable transgene integration. Unstable integration can be transient nonchromosomal integration, semi-stable nonchromosomal integration, semi-persistent nonchromosomal insertion, or unstable chromosomal insertion. Transient nonchromosomal insertions can be extrachromosomal or cytoplasmic. In one aspect, transient nonchromosomal insertions of transgenes do not integrate into the chromosome, and the modified genetic material does not replicate during cell division.

[0172] Genome modifications can enable semi-stable or persistent non-chromosomal integration of transgenes. DNA vectors encode scaffold / matrix attachment region (S-MAR) modules that bind to nuclear matrix proteins for attachability retention in non-viral vectors, allowing autonomous replication in the nucleus of dividing cells.

[0173] Genomic modifications can enable the unstable chromosomal integration of transgenes. Integrated transgenes can be silenced, removed, excised, or further modified.

[0174] Genome modification via transgenic insertion can occur through: host cell-guided double-strand break repair via homologous recombination (HR) (homology-mediated repair), microhomology-mediated end joining (MMEJ), non-homologous end joining (NHEJ), transposase-mediated modification, integrase-mediated modification, endonuclease-mediated modification, or recombinase-mediated modification. Genome modification via transgenic insertion can also occur via CRISPR-Cas9, TALEN, ZFN, Cas-CLOVER™, and cpf1.

[0175] In gene editing systems involving the insertion of new or existing nucleotides / nucleic acids, in addition to cutting enzymes (e.g., nucleases, recombinases, integrases, or transposases), an insertion tool (e.g., a DNA template vector, a transposable element (transposon or retrotransposon)) must be delivered to the cell. Examples of such insertion tools for recombinases may include DNA vectors. Other gene editing systems require the delivery of integrases along with the insertion vector, transposases along with transposons / retrotransposons, etc. An example of a recombinase that can be used as a cutting enzyme is the CRE recombinase. Non-limiting examples of integrases that can be used in insertion tools include virus-based enzymes derived from any of a variety of viruses (including AAV, gamma retroviruses, and lentiviruses). Examples of transposons / retrotransposons that can be used in insertion tools are described in more detail herein.

[0176] Cells with genomic modifications, whether in vitro, in vivo, or in situ, can be germline cells or somatic cells. Modified cells can be human, non-human, mammalian, rat, mouse, or canine cells. Modified cells can be differentiated, undifferentiated, or immortalized. Modified undifferentiated cells can be stem cells. Modified undifferentiated cells can be induced pluripotent stem cells. Modified cells can be immune cells. Modified cells can be T cells, hematopoietic stem cells, natural killer cells, macrophages, dendritic cells, monocytes, megakaryocytes, or osteoclasts. Modified cells can be modified at rest, in an activated state, quiescent, interphase, prophase, metaphase, anaphase, or telophase. Modified cells can be fresh, cryopreserved, bulk, sorted into subsets, derived from whole blood, derived from leukocyte removal, or derived from immortalized cell lines. Detailed descriptions of methods for separating cells from leukocyte removal products or blood are disclosed in PCT Publication Nos. WO 2019 / 173636 and PCT / US2019 / 049816.

[0177] This disclosure provides gene editing compositions and / or cells comprising gene editing compositions. The gene editing compositions may comprise 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 comprise a DNA sequence, an RNA sequence, or a combination thereof. The nuclease or its nuclease domain may comprise one or more of CRISPR / Cas proteins, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and endonucleases.

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

[0179] This disclosure provides compositions comprising a small Cas9 (Cas9) operatively linked to an effector. This disclosure provides fusion proteins comprising, substantially comprising, or consisting of an effector molecule, comprising a DNA localization component and an effector molecule, wherein the effector comprises a small Cas9 (Cas9). Small Cas9 constructs of this disclosure may comprise effectors comprising an IIS-type endonuclease. Staphylococcus aureus Cas9 with an active catalytic site comprises the amino acid sequence of SEQ ID NO: 83.

[0180] This disclosure provides compositions comprising an inactivated small Cas9 (dSaCas9) operatively linked to an effector. This disclosure provides fusion proteins comprising, substantially comprising, or consisting of an effector molecule, wherein the effector comprises a small inactivated Cas9 (dSaCas9). The small inactivated Cas9 (dSaCas9) constructs of this disclosure may comprise an effector comprising an IIS-type endonuclease. dSaCas9 comprises the amino acid sequence of SEQ ID NO: 84, including D10A and N580A mutations to inactivate the catalytic site.

[0181] This disclosure provides compositions comprising inactivated Cas9 (dCas9) operatively linked to an effector. This disclosure also provides fusion proteins comprising, substantially comprising, or consisting of an effector molecule, wherein the effector comprises inactivated Cas9 (dCas9). The inactivated Cas9 (dCas9) constructs of this disclosure may include effectors comprising an IIS-type endonuclease.

[0182] dCas9 can be isolated from or derived from Streptococcus pyogenes ( Streptococcus pyogenes dCas9 may be comprised of dCas9 with substitutions at amino acid positions 10 and 840, which inactivate the catalytic site. In some aspects, these substitutions are D10A and H840A. dCas9 may comprise the amino acid sequence of SEQ ID NO: 85 or SEQ ID NO: 86.

[0183] The exemplary Clo051 nuclease domain contains, is substantially composed of, or is composed of the amino acid sequence of SEQ ID NO: 87.

[0184] An exemplary dCas9-Clo051 (Cas-CLOVER) fusion protein may comprise, consist substantially of, or be composed of the amino acid sequence of SEQ ID NO: 88. An exemplary dCas9-Clo051 fusion protein may be encoded by a polynucleotide comprising, consist substantially of, or be composed of the nucleic acid sequence of SEQ ID NO: 89. The nucleic acid encoding the dCas9-Clo051 fusion protein may be DNA or RNA.

[0185] An exemplary dCas9-Clo051 (Cas-CLOVER) fusion protein may comprise, consist substantially of, or be composed of the amino acid sequence of SEQ ID NO: 90. An exemplary dCas9-Clo051 fusion protein may be encoded by a polynucleotide comprising, consist substantially of, or be composed of the nucleic acid sequence of SEQ ID NO: 91. The nucleic acid encoding the dCas9-Clo051 fusion protein may be DNA or RNA.

[0186] Cells containing the gene-editing composition can stably or transiently express the gene-editing composition. Preferably, the gene-editing composition is expressed transiently. The guide RNA may contain a sequence complementary to a target sequence within the genomic DNA sequence. The target sequence within the genomic DNA sequence may be a target sequence within a safe harbor site of the genomic DNA sequence.

[0187] Gene editing compositions, including Cas-CLOVER, and methods of using these compositions for gene editing are described in detail in U.S. Patent Publications 2017 / 0107541, 2017 / 0114149, 2018 / 0187185 and U.S. Patent No. 10,415,024.

[0188] One or more poly(histidine)-based micelles can also be used to deliver gene-editing tools to cells. Poly(histidine) (e.g., poly(L-histidine)) is a pH-sensitive polymer due to the imidazole ring providing a lone pair of electrons on the unsaturated nitrogen. That is, poly(histidine) is amphoteric through protonation-deprotonation. In particular, at certain pH values, poly(histidine)-containing triblock copolymers can assemble into micelles with positively charged poly(histidine) units on their 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 performing desired gene modifications. In particular, such micelle-based delivery systems offer significant flexibility regarding charged materials, as well as large payload capacity and targeted release of nanoparticle payloads. In one example, the use of poly(histidine)-based micelles to deliver nucleases enables site-specific cleavage of double-stranded DNA. To avoid being bound by a specific theory, it is believed that in micelles formed by various triblock copolymers, hydrophobic blocks aggregate to form a core, leaving hydrophilic blocks and poly(histidine) blocks at the ends to form one or more enclosing layers.

[0189] In one aspect, this disclosure provides triblock copolymers made of hydrophilic blocks, hydrophobic blocks, and charged blocks. In some aspects, the hydrophilic blocks may be poly(ethylene oxide) (PEO), and the charged blocks may be poly(L-histidine). An example triblock copolymer that can be used is PEO-b-PLA-b-PHIS, wherein the variable number of repeating units in each block varies depending on the design.

[0190] Diblock copolymers, which can be used as intermediates for the preparation of triblock copolymers, can have hydrophilic, biocompatible poly(ethylene oxide) (PEO) (chemically synonymous with PEG) coupled 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(glycolic acid) (PLGA), poly(lactic-co-glycolic acid) (PLGA), poly(ε-caprolactone) (PCL), and poly(trimethylene carbonate) (PTMC). Polymer micelles composed of a 100% polyethylene glycol-modified surface exhibit improved in vitro chemical stability, enhanced in vivo bioavailability, and prolonged circulating half-life.

[0191] Polymer vesicles, polymeric vesicles, and poly(histidine)-based micelles, including those comprising triblock copolymers, and methods for preparing the same, are described in further detail in U.S. Patent Nos. 7,217,427; 7,868,512; 6,835,394; 8,808,748; 10,456,452; U.S. Publication Nos. 2014 / 0363496; 2017 / 0000743; and 2019 / 0255191; and PCT Publication No. WO 2019 / 126589.

[0192] Transposer and carrier composition This disclosure provides compositions and methods for delivering antibodies (e.g., scFv) or CARs (e.g., containing scFv) into cells or cell populations. Non-limiting examples of compositions for delivering the compositions of this disclosure into cells or cell populations include transposons or vectors. Therefore, this disclosure provides transposons containing antibodies (e.g., scFv) or CARs (e.g., containing scFv), or vectors containing antibodies (e.g., scFv) or CARs (e.g., containing scFv).

[0193] Transposons or vectors containing the CAR of this disclosure may further contain a sequence encoding an inducible pro-apoptotic polypeptide. Alternatively or additionally, a transposon or vector may contain the CAR of this disclosure, while a second transposon or vector may contain a sequence encoding an inducible pro-apoptotic polypeptide of this disclosure. The inducible pro-apoptotic polypeptide is described in more detail herein.

[0194] Transposons of CARs containing the present disclosure or vectors containing CARs containing the present disclosure may further include a sequence encoding a chimeric stimulatory receptor (CSR). Alternatively or additionally, a transposon or vector may include a CAR containing the present disclosure, while a second transposon or vector may include a sequence encoding a CSR containing the present disclosure. Chimeric stimulatory receptors are described in more detail herein.

[0195] The transposon or vector of the CAR containing this disclosure may further contain a sequence encoding a recombinant HLA-E polypeptide. Alternatively or additionally, a transposon or vector may contain the CAR of this disclosure, while a second transposon or vector may contain a sequence encoding a recombinant HLA-E polypeptide. The recombinant HLA-E polypeptide is described in more detail herein.

[0196] Transposons of CARs containing this disclosure or vectors of CARs containing this disclosure may further include a selection gene. The selection gene may encode a gene product essential for cell viability and survival. When challenged by selective cell culture conditions, the selection gene may encode a gene product essential for cell viability and survival. Selective cell culture conditions may contain compounds detrimental to cell viability or survival, and the gene product confers resistance to the compounds. Non-limiting examples of selection genes include... neo (Conferring resistance to neomycin), DHFR (encoding dihydrofolate reductase and conferring resistance to methotrexate), TYMS (encoding thymidylate synthase), MGMT (encoding O(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encoding aldehyde dehydrogenase 1 family member A1), FRANCF, RAD51C (encoding RAD51 paralog C), GCS (encoding glucose ceramide synthase), NKX2.2 (encoding NK2 homeobox 2), or any combination thereof.

[0197] In a preferred aspect, the selected gene encodes a DHFR mutant protease. The DHFR mutant protease comprises, is substantially composed of, or is composed of the amino acid sequence of SEQ ID NO: 92. The DHFR mutant protease is encoded by a polynucleotide comprising, is substantially composed of, or is composed of the nucleic acid sequence of SEQ ID NO: 93 or SEQ ID NO: 174. The amino acid sequence of the DHFR mutant protease may further include mutations at one or more positions at positions 80, 113, or 153. The amino acid sequence of the DHFR mutant protease may include one or more of the following: 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.

[0198] The transposon of the CAR containing this disclosure or the vector of the CAR containing this disclosure may further contain at least one self-cleaving peptide. For example, the self-cleaving peptide may be located between the CAR (e.g., containing scFv) and an inducible pro-apoptotic peptide; or, the self-cleaving peptide may be located between the CAR (e.g., containing scFv) and a protein encoded by a selected gene.

[0199] The transposon of the CAR containing this disclosure or the vector of the CAR containing this disclosure may further comprise at least two self-cleaving peptides. For example, the first self-cleaving peptide is located upstream or immediately upstream of the CAR, while the second self-cleaving peptide is located downstream or immediately downstream of the CAR; or, the first and second self-cleaving peptides are adjacent to the CAR. For example, the first self-cleaving peptide is located upstream or immediately upstream of an inducible pro-apoptotic peptide, while the second self-cleaving peptide is located downstream or immediately downstream of the inducible pro-apoptotic peptide; or, the first and second self-cleaving peptides are adjacent to an inducible pro-apoptotic peptide. For example, the first self-cleaving peptide is located upstream or immediately upstream of a protein encoded by a selection gene, while the second self-cleaving peptide is located downstream or immediately downstream of a protein encoded by a selection gene; or, the first and second self-cleaving peptides are adjacent to a protein encoded by a selection gene.

[0200] 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. The T2A peptide comprises, is substantially composed of, or is composed of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between both) of SEQ ID NO: 94. The GSG-T2A peptide comprises, is substantially composed of, or is composed of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between both) of SEQ ID NO: 95. The GSG-T2A peptide is encoded by a polynucleotide comprising or consisting of a nucleic acid sequence, the nucleic acid sequence being at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between both) of SEQ ID NO: 96. The E2A peptide comprises, consists substantially of, or consists of an amino acid sequence, the amino acid sequence being at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between both) of SEQ ID NO: 97. The GSG-E2A peptide comprises, consists substantially of, or consists of an amino acid sequence, the amino acid sequence being at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between both) of SEQ ID NO: 98. The F2A peptide comprises, is substantially composed of, or is composed of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between both) of SEQ ID NO: 99. The GSG-F2A peptide comprises, is substantially composed of, or is composed of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between both) of SEQ ID NO: 100. The P2A peptide comprises, is substantially composed of, or is composed of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between both) of SEQ ID NO: 101.The GSG-P2A peptide comprises an amino acid sequence, is substantially composed of an amino acid sequence, or is composed of an amino acid sequence that is equivalent to at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between the two) of SEQ ID NO: 102.

[0201] In some aspects, the transposon of this disclosure comprises a nucleic acid encoding a CAR of SEQ ID NO: 13. In some aspects, the nucleic acid encoding the CAR is flanked by a nucleic acid encoding a T2A peptide of SEQ ID NO: 95. In some aspects, the transposon further comprises a nucleic acid encoding a DHFR enzyme of SEQ ID NO: 92. In some aspects, the transposon further comprises a nucleic acid encoding an IC9 safety switch peptide of SEQ ID NO: 172.

[0202] In some respects, the transposons of this disclosure comprise, consist substantially of, or consist of nucleic acid sequences that are at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between the two) of SEQ ID NO: 175.

[0203] Rotary seat system This disclosure provides transposons comprising protein scaffolds as disclosed herein, or transposons comprising antibodies (e.g., scFv) or CARs (e.g., containing scFv) as disclosed herein. In a preferred aspect, the transposon is a plasmid DNA transposon comprising a nucleotide sequence encoding scFv or CAR (e.g., containing scFv) as disclosed herein, flanked by two cis-regulatory insulator elements. This disclosure also provides compositions comprising transposons. In a preferred aspect, the composition comprising transposons further comprises a plasmid comprising a nucleotide sequence encoding a transposase. The nucleotide sequence encoding the transposase may be a DNA sequence or an RNA sequence. Preferably, the sequence encoding the transposase is an mRNA sequence.

[0204] The transposon disclosed herein may be a piggyBac™ (PB) transposon. In some aspects, when the transposon is a PB transposon, the transposase is a piggyBac™ (PB) transposase, a piggyBac-like (PBL) transposase, or a Super piggyBac™ (SPB) transposase. The sequence encoding the SPB transposase is an mRNA sequence.

[0205] Non-restricted examples of PB transposons and PB, PBL, and SPB transposases are described in detail in the following: U.S. Patent Nos. 6,218,182, 6,962,810, 8,399,643, and PCT Publication No. WO 2010 / 099296.

[0206] PB, PBL, and SPB transposases recognize transposon-specific inverted terminal repeat (ITR) sequences at the ends of transposons and insert contents between ITRs at sequence 5'-TTAT-3' within the chromosomal locus (TTAT target sequence) or sequence 5'-TTAA-3' within the chromosomal locus (TTAA target sequence). The target sequence of the PB or PBL transposon may contain or consist of the following: 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'T GAA-3', 5'-AAAT-3', 5'-AATC-3', 5'-ACAA-3', 5'-ACAT-3', 5'-ACTC-3', 5'-AGTG-3', 5'-ATAG-3', 5'-CAAA-3', 5'-CACA-3', 5'-CATA-3', 5'-CCAG-3', 5'-CCCA-3', 5'-CGTA-3', 5'-GTCC-3', 5'-TAAG-3', 5'-TCTA-3', 5'-TGAG-3', 5'-TGTT-3', 5'-TTCA-3', 5'-TTCT-3', and 5'-TTTT-3'. PB or PBL transposon systems have no payload limitations for the target gene that can be included between ITRs.

[0207] Exemplary amino acid sequences of one or more PB, PBL, and SPB transposases are disclosed in U.S. Patent Nos. 6,218,185, 6,962,810, and 8,399,643. In a preferred aspect, the PB transposase comprises or is composed of an amino acid sequence that is equivalent to at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between both) of SEQ ID NO: 103.

[0208] The PB or PBL transposase may comprise or consist of an amino acid sequence having amino acid substitutions at positions 30, 165, 282, or 538 of the sequence in SEQ ID NO: 103, consisting of two or more, three or more, or each of these amino acid substitutions. The transposase may be an SPB transposase comprising or consisting of the amino acid sequence of SEQ ID NO: 103, wherein the amino acid substitution at position 30 may be valine (V) replacing isoleucine (I), the amino acid substitution at position 165 may be serine (S) replacing glycine (G), the amino acid substitution at position 282 may be valine (V) replacing methionine (M), and the amino acid substitution at position 538 may be lysine (K) replacing asparagine (N). In a preferred aspect, the SPB transposase comprises or consists of an amino acid sequence that is equivalent to at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between the two) of SEQ ID NO: 104.

[0209] In which the transposase comprises some aspect of the aforementioned mutations at positions 30, 165, 282, and / or 538, the PB, PBL, and SPB transposases may further comprise amino acid substitutions at one or more of the 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 in SEQ ID NO: 103 or SEQ ID NO: 104, as described in PCT Publication No. WO It is described in more detail in 2019 / 173636 and PCT / US2019 / 049816.

[0210] As described in more detail in PCT Publications WO 2019 / 173636 and PCT / US2019 / 049816, PB, PBL, or SPB transposases can be isolated from or derived from insects, vertebrates, crustaceans, or chordates. In a preferred embodiment, PB, PBL, or SPB transposases are isolated from or derived from the insect *Spodoptera litura* (also known as the white-spotted armyworm). Trichoplusia ni (GenBank login number AAA87375) or silkworm ( Bombyx mori (GenBank login number BAD11135).

[0211] Highly active PB or PBL transposases are transposases that are more active than their naturally occurring variants. In a preferred aspect, highly active PB or PBL transposases are isolated from or derived from silkworms or Xenopus laevis (…). Xenopus tropicalis Examples of highly active PB or PBL transposases are disclosed in U.S. Patent Nos. 6,218,185, 6,962,810, 8,399,643, and WO 2019 / 173636. A list of highly active amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[0212] In some respects, PB or PBL transposases are integration-deficient. Integration-deficient PB or PBL transposases are transposases that can cleave their respective transposons but integrate the cleaved transposons at a lower frequency than the corresponding wild-type transposases. Examples of integration-deficient PB or PBL transposases are disclosed in U.S. Patent Nos. 6,218,185, 6,962,810, 8,399,643, and WO 2019 / 173636. A list of integration-deficient amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[0213] In some respects, PB or PBL transposases are fused with nuclear localization signals. Examples of PB or PBL transposases fused with nuclear localization signals are disclosed in U.S. Patent Nos. 6,218,185, 6,962,810, 8,399,643, and WO 2019 / 173636.

[0214] The transposon of this disclosure may be a Sleeping Beauty transposon. In some aspects, when the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase (e.g., disclosed in U.S. Patent No. 9,228,180) or a highly active Sleeping Beauty (SB100X) transposase. In a preferred aspect, 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% (or any percentage between both) of SEQ ID NO: 105. In a preferred aspect, the highly active 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 between both) of SEQ ID NO: 106.

[0215] The transposons of this disclosure may be Helraiser transposons. Exemplary Helraiser transposons include Helibat1, which 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 between both) equivalent to SEQ ID NO: 107. In some aspects, when the transposon is a Helraiser transposon, the transposase is a Helitron transposase (e.g., as disclosed in WO 2019 / 173636). In a preferred aspect, the Helitron 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 between both) equivalent to SEQ ID NO: 108.

[0216] The transposon of this disclosure may be a Tol2 transposon. An exemplary Tol2 transposon includes an inverted repeat, a subterminal sequence, and a Tol2 transposase, 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 between both) equivalent to SEQ ID NO: 109. In some aspects, when the transposon is a Tol2 transposon, the transposase is a Tol2 transposase (e.g., as disclosed in WO 2019 / 173636). In a preferred aspect, the Tol2 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 between both) equivalent to SEQ ID NO: 110.

[0217] The transposons in this disclosure may be TcBuster transposons. In some aspects, when the transposon is a TcBuster transposon, the transposase is a TcBuster transposase or a highly active TcBuster transposase (e.g., as disclosed in WO 2019 / 173636). The TcBuster transposase may comprise or consist of a naturally occurring amino acid sequence or a non-naturally occurring amino acid sequence, or be composed of such sequences. In a preferred aspect, 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% (or any percentage between both) of SEQ ID NO: 111. The polynucleotide encoding the TcBuster transposase may comprise or consist of a naturally occurring nucleic acid sequence or a non-naturally occurring nucleic acid sequence, or be composed of such sequences. In a preferred aspect, the TcBuster transposase is encoded by a polynucleotide comprising or consisting of a nucleic acid sequence that is equivalent to at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between the two) of SEQ ID NO:112.

[0218] In some respects, when compared with wild-type TcBuster transposases as described in more detail in PCT Publications WO 2019 / 173636 and PCT / US2019 / 049816, mutant TcBuster transposases contain one or more sequence variations.

[0219] Transposons can be nanotransposons. Nanotransposons may comprise, substantially consist of, or consist of: (a) a sequence encoding a transposon insert comprising a sequence encoding a first inverted terminal repeat (ITR), a sequence encoding a second inverted terminal repeat (ITR), and an intra-ITR sequence; (b) a sequence encoding a backbone, wherein the backbone sequence comprises a sequence having 1 to 450 nucleotides (including the endpoints) encoding an origin of replication, and a sequence having 1 to 200 nucleotides (including the endpoints) encoding an optional marker; and (c) an inter-ITR sequence. In some aspects, the inter-ITR sequence of (c) comprises the sequence of (b). In some aspects, the intra-ITR sequence of (a) comprises the sequence of (b).

[0220] The sequence encoding the backbone can contain 1 to 600 nucleotides, including the endpoints. In some respects, the sequence encoding the backbone consists of the following ranges: 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, and 550 to 600 nucleotides, each range including the endpoints.

[0221] Inter-ITR sequences can contain 1 to 1000 nucleotides, including the endpoints. In some aspects, inter-ITR sequences consist of the following ranges: 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 1000 nucleotides, each range including the endpoints.

[0222] Nanotransposons can be short nanotransposons (SNTs), wherein the inter-ITR sequence comprises 1 to 200 nucleotides, including the endpoints. The inter-ITR sequence can consist of 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 range including the endpoints.

[0223] Selectable markers having 1 to 200 nucleotides (including endpoints) may include sequences encoding sucrose selectable markers. Sequences encoding sucrose selectable markers may include sequences encoding RNA-OUT sequences. RNA-OUT sequences may contain 137 base pairs (bp) or consist of 137 base pairs (bp). Selectable markers having 1 to 200 nucleotides (including endpoints) may include sequences encoding fluorescent markers. Selectable markers having 1 to 200 nucleotides (including endpoints) may include sequences encoding cell surface markers.

[0224] A sequence encoding an origin of replication of 1 to 450 nucleotides (including the endpoints) may contain a sequence encoding a micro-origin of replication. In some aspects, a sequence encoding an origin of replication of 1 to 450 nucleotides (including the endpoints) may contain a sequence encoding an R6K origin of replication. An R6K origin of replication may contain an R6K γ origin of replication. An R6K origin of replication may contain an R6K micro-origin of replication. An R6K γ micro-origin of replication may consist of 281 base pairs (bp) or be composed of 281 base pairs (bp).

[0225] In some aspects, the sequence encoding the backbone does not contain recombination sites, excision sites, linkage sites, or combinations thereof. In some aspects, neither the nanotransposon nor the sequence encoding the backbone contains products of recombination sites, excision sites, linkage sites, or combinations thereof. In some aspects, neither the nanotransposon nor the sequence encoding the backbone is derived from recombination sites, excision sites, linkage sites, or combinations thereof.

[0226] In some aspects of nanotransposons, the recombination site contains a sequence that originates from a recombination event. In some aspects, the recombination site contains a sequence that is a product of the recombination event. In some aspects, the recombination event includes the activity of a recombinase (e.g., a recombinase site).

[0227] In some aspects of nanotransposons, the sequence encoding the backbone does not further include a sequence encoding foreign DNA.

[0228] In some aspects of nanotransposons, the inter-ITR sequence does not contain a recombination site, excision site, linkage site, or a combination thereof. In some aspects, the inter-ITR sequence does not contain the product of a recombination event, excision event, linkage event, or a combination thereof. In some aspects, the inter-ITR sequence is not derived from a recombination event, excision event, linkage event, or a combination thereof. In some aspects, the inter-ITR sequence contains a sequence encoding foreign DNA. In some aspects, the intra-ITR sequence contains at least one sequence encoding an insulator and a sequence encoding a promoter capable of expressing the foreign sequence in mammalian cells. The mammalian cell can be a human cell. In some aspects, the intra-ITR sequence contains a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing the foreign sequence in mammalian cells, and a second sequence encoding an insulator. In some aspects, the intra-ITR sequence contains a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing the foreign sequence in mammalian cells, a polyadenosine (polyA) sequence, and a second sequence encoding an insulator. In some respects, the ITR inner sequence contains a first sequence encoding an insulator, a sequence encoding a promoter capable of expressing a foreign sequence in mammalian cells, at least one foreign sequence, a polyadenosine (polyA) sequence, and a second sequence encoding an insulator.

[0229] Nanotransposons are described in more detail in PCT / US2019 / 067758.

[0230] carrier system The vectors used in this disclosure can be viral vectors or recombinant vectors. Viral vectors may contain sequences isolated from or derived from retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or any combination thereof. Viral vectors may contain sequences isolated from or derived from adeno-associated viruses (AAVs). Viral vectors may contain recombinant AAVs (rAAVs). Exemplary adeno-associated viruses and recombinant adeno-associated viruses contain two or more inverted terminal repeat (ITR) sequences cis-positioned immediately adjacent to the scFv or CAR encoding this 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 AAVs (scAAVs) and AAV heterozygotes containing the genome of one serotype and the capsid of another serotype (e.g., AAV2 / 5, AAV-DJ, and AAV-DJ8). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, rAAV-LK03.

[0231] The carriers of this disclosure can be nanoparticles. Non-limiting examples of nanoparticle carriers include nucleic acids (e.g., RNA, DNA, synthetic nucleotides, modified nucleotides, or any combination thereof), amino acids (L-amino acids, D-amino acids, synthetic amino acids, modified amino acids, or any combination thereof), polymers (e.g., polymer vesicles), 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 carriers can be passively or actively transported across cell membranes.

[0232] The cell delivery compositions disclosed herein (e.g., transposons, vectors) may contain nucleic acids encoding therapeutic proteins or therapeutic agents. Examples of therapeutic proteins include those disclosed in PCT Publications WO 2019 / 173636 and PCT / US2019 / 049816.

[0233] Inducible pro-apoptotic peptides The inducible pro-apoptotic peptide disclosed herein is superior to existing inducible peptides because it has significantly lower immunogenicity. The inducible pro-apoptotic peptide is a recombinant peptide and therefore not naturally occurring. Furthermore, the sequence is recombined to produce the inducible pro-apoptotic peptide, which does not contain non-human sequences that the host's human immune system can recognize as "non-self," and thus induces an immune response in a subject receiving the inducible pro-apoptotic peptide, cells containing the inducible pro-apoptotic peptide, or a composition containing the inducible pro-apoptotic peptide or cells containing the inducible pro-apoptotic peptide.

[0234] This 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 contain a non-human sequence. In some aspects, the non-human sequence contains a restriction site. In some aspects, the ligand-binding region may be a multimeric ligand-binding region. In some aspects, the pro-apoptotic peptide is a caspase polypeptide. Non-limiting examples of caspase polypeptides include caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, and caspase 14. Preferably, the caspase polypeptide is a caspase 9 polypeptide. The caspase 9 polypeptide may be a truncated caspase 9 polypeptide. The inducible pro-apoptotic polypeptide may be non-naturally occurring. When caspase is caspase 9 or a truncated caspase 9, the inducible pro-apoptotic peptide can also be called the "iC9 safety switch".

[0235] The inducible caspase polypeptide may comprise (a) a ligand-binding region, (b) a linker, and (c) a caspase polypeptide, wherein the inducible pro-apoptotic polypeptide does not contain non-human sequences. In some aspects, the inducible caspase polypeptide comprises (a) a ligand-binding region, (b) a linker, and (c) a truncated caspase 9 polypeptide, wherein the inducible pro-apoptotic polypeptide does not contain non-human sequences.

[0236] The ligand-binding region may comprise an FK506-binding protein 12 (FKBP12) polypeptide. The amino acid sequence of the ligand-binding region comprising the FK506-binding protein 12 (FKBP12) polypeptide may include a modification at position 36 of the sequence. This modification may be a substitution of phenylalanine (F) for valine (V) at position 36 (F36V). The FKBP12 polypeptide may comprise, consist substantially 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 between both) of SEQ ID NO: 113. The FKBP12 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 between both) of SEQ ID NO: 114.

[0237] The adapter region may comprise, consist substantially 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 between both) of SEQ ID NO: 115, or the adapter region 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 between both) of SEQ ID NO: 116. In some aspects, the nucleic acid sequence encoding the adapter does not contain restriction sites.

[0238] The truncated caspase 9 polypeptide may comprise an amino acid sequence that does not contain arginine (R) at position 87 of the sequence. Alternatively or additionally, the truncated caspase 9 polypeptide may comprise an amino acid sequence that does not contain alanine (A) at position 282 of the sequence. The truncated caspase 9 polypeptide may comprise, consist substantially 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 between the two) of 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 between the two) of SEQ ID NO: 118.

[0239] In some respects, when the polypeptide comprises a truncated caspase 9 polypeptide, the inducible pro-apoptotic polypeptide comprises, is substantially composed of, or is composed of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between the two) of SEQ ID NO: 119, or the inducible pro-apoptotic polypeptide is encoded by a polynucleotide that comprises or is composed of a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between the two) of SEQ ID NO: 120.

[0240] In some respects, when the polypeptide comprises a truncated caspase 9 polypeptide, the inducible pro-apoptotic polypeptide comprises, is substantially composed of, or is composed of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between the two) of SEQ ID NO: 172, or the inducible pro-apoptotic polypeptide is encoded by a polynucleotide that comprises or is composed of a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between the two) of SEQ ID NO: 173.

[0241] Inducible pro-apoptotic peptides can be expressed in cells under the transcriptional regulation of any promoter known in the art, which can initiate and / or regulate the expression of the inducible pro-apoptotic peptide in the cell.

[0242] Activation of inducible pro-apoptotic peptides can be accomplished, for example, by chemically induced dimerization (CID) mediated by an inducer, to produce conditionally controlled proteins or peptides. Due to degradation of unstable dimerizing agents or the application of monomeric competitive inhibitors, pro-apoptotic peptides are not only inducible, but the induction of these peptides is also reversible.

[0243] In some respects, when the ligand-binding region comprises an FKBP12 polypeptide with valine (V) substituted for phenylalanine (F) at position 36 (F36V), the inducer may comprise AP1903, a synthetic drug (CAS index name: 2-piperidinic acid, 1-[(2S)-1-oxo-2-(3,4,5-trimethoxyphenyl)butyl]-, 1,2-ethylenedimethylbis[imino(2-oxo-2,1-ethylenedimethyl)oxy-3,1-phenylene[(1R)-3-(3,4-dimethoxyphenyl)propylene]] 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 No.: 195514-80-8 and Molecular Formula: C82H107N5O20) or AP20187 analogues, such as AP1510. As used herein, inducers AP20187, AP1903 and AP1510 are interchangeable.

[0244] Inducible pro-apoptotic peptides and methods for inducing these peptides are described in detail in U.S. Patent Publication No. WO 2019 / 0225667 and PCT Publication No. WO2018 / 068022.

[0245] Chimeric stimulatory receptors and recombinant HLA-E peptides For adoptive cell compositions to be "generally" safe for any patient, a significant reduction or elimination of allogeneic reactivity is required. To this end, the cells of this disclosure (e.g., allogeneic cells) can be modified to disrupt the expression or function of the T-cell receptor (TCR) and / or a major histocompatibility complex (MHC). TCR mediates graft-versus-host (GvH) responses, while MHC mediates host-versus-graft (HvG) responses. In a preferred aspect, any expression and / or function of the TCR is eliminated to prevent T-cell-mediated GvH, which can cause death in the subject. Therefore, in a preferred aspect, this disclosure provides a pure TCR-negative allogeneic T-cell composition (e.g., each cell in the composition is expressed at such a low level that it is undetectable or absent).

[0246] The expression and / or function of MHC class I (MHC-I, specifically HLA-A, HLA-B, and HLA-C) are reduced or eliminated to prevent HvG and thus improve cell graft implantation in subjects. Improved graft implantation leads to longer cell persistence and therefore a larger therapeutic window for subjects. Specifically, the expression and / or function of the MHC-1 structural element β-2-microglobulin (B2M) are reduced or eliminated.

[0247] The above strategy presents further challenges. T cell receptor (TCR) knockout (KO) in T cells results in the loss of expression of CD3-ζ (CD3z or CD3ζ), a component of the TCR complex. The loss of CD3ζ in TCR-KO T cells drastically reduces the ability to optimally activate and expand these cells using standard stimulatory / activating agents (including, but not limited to, the agonist anti-CD3 mAb). When the expression or function of any component of the TCR complex is interrupted, all components of the complex are lost, including TCR-α (TCRα), TCR-β (TCRβ), CD3-γ (CD3γ), CD3-ε (CD3ε), CD3-δ (CD3δ), and CD3-ζ (CD3ζ). Both CD3ε and CD3ζ are required for T cell activation and expansion. The agonist anti-CD3 mAb typically recognizes CD3ε and another possible protein within the complex, which in turn signals CD3ζ. CD3ζ provides the primary stimulus (along with secondary co-stimulatory signals) for T cell activation, leading to optimal activation and expansion. Under normal conditions, complete T cell activation depends on the binding of the TCR to a second signal mediated by one or more co-stimulatory receptors (e.g., CD28, CD2, 4-1BBL), which enhances the immune response. However, when the TCR is absent, T cell expansion is severely reduced upon stimulation with standard activating / stimulating agents (including the agonist antiCD3 mAb). In fact, T cell expansion is reduced to only 20-40% of normal expansion levels when stimulated with standard activating / stimulating agents (including the agonist antiCD3 mAb).

[0248] Therefore, this disclosure provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an extracellular domain containing an activation component, wherein the activation component is isolated from or derived from a first protein; (b) a transmembrane domain; and (c) an intracellular domain containing at least one signal transduction domain, wherein the at least one signal transduction domain is isolated from or derived from a second protein; wherein the first protein and the second protein are not equivalent.

[0249] The activating component may include one or more of the following: a component of the T cell receptor (TCR), a component of the TCR complex, a component of the TCR co-receptor, a component of the TCR co-stimulatory protein, a component of the TCR repressor protein, a cytokine receptor, and a chemokine receptor to which the agonist of the activating component binds. The activating component may include an extracellular domain of CD2 or a portion thereof to which the agonist binds.

[0250] The signal transduction domain may contain one or more of the following: components of a human signal transduction domain, a T cell receptor (TCR), a component of the TCR complex, a component of a TCR co-receptor, a component of a TCR co-stimulatory protein, a component of a TCR repressor protein, a cytokine receptor, and a chemokine receptor. The signal transduction domain may contain a CD3 protein or a portion thereof. The CD3 protein may contain a CD3ζ protein or a portion thereof.

[0251] The intracellular domain may further include a cytoplasmic domain. The cytoplasmic domain may be isolated from or derived from a third protein. The first and third proteins may be equivalent. The extracellular domain may further include a signal peptide. The signal peptide may be derived from a fourth protein. The first and fourth proteins may be equivalent. The transmembrane domain may be isolated from or derived from a fifth protein. The first and fifth proteins may be equivalent.

[0252] In some respects, the activating component does not bind to naturally occurring molecules. In some respects, the activating component binds to naturally occurring molecules, but the CSR does not transduce the signal after the activating component binds to a naturally occurring molecule. In some respects, the activating component binds to non-naturally occurring molecules. In some respects, the activating component does not bind to naturally occurring molecules, but rather to non-naturally occurring molecules. The CSR can selectively transduce the signal after the activating component binds to a non-naturally occurring molecule.

[0253] In a preferred aspect, this disclosure provides a non-naturally occurring chimeric stimulatory receptor (CSR) comprising: (a) an extracellular domain comprising a signal peptide and an activation component, wherein the signal peptide comprises a CD2 signal peptide or a portion thereof, and wherein the activation component comprises a CD2 extracellular domain or a portion thereof to which an agonist binds; (b) a transmembrane domain comprising a CD2 transmembrane domain or a portion thereof; and (c) an intracellular domain comprising a cytoplasmic domain and at least one signal transduction domain, wherein the cytoplasmic domain comprises a CD2 cytoplasmic domain or a portion thereof, and wherein the at least one signal transduction domain comprises a CD3ζ protein or a portion thereof. In some aspects, the non-natural CSR comprises an amino acid sequence equivalent to at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between both) of SEQ ID NO: 121. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 121.

[0254] This disclosure also provides a non-naturally occurring chimeric stimulatory receptor (CSR) wherein the extracellular domain contains modifications. The modifications may include mutations or truncations of the amino acid sequence of the activating component or the first protein when compared to the wild-type sequence of the activating component or the first protein. Mutations or truncations of the amino acid sequence of the activating component may include mutations or truncations of the CD2 extracellular domain to which the agonist binds, or a portion thereof. Mutations or truncations of the CD2 extracellular domain may reduce or eliminate binding to naturally occurring CD58. In some aspects, the mutated or truncated CD2 extracellular domain contains an amino acid sequence equivalent to at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between both) of SEQ ID NO: 122. In a preferred aspect, the mutated or truncated CD2 extracellular domain contains the amino acid sequence of SEQ ID NO: 122.

[0255] In a preferred aspect, this disclosure provides a non-natural chimeric stimulatory receptor (CSR) comprising: (a) an extracellular domain comprising a signal peptide and an activation component, wherein the signal peptide comprises a CD2 signal peptide or a portion thereof, and wherein the activation component comprises an agonist-binding CD2 extracellular domain or a portion thereof, wherein the agonist-binding CD2 extracellular domain or a portion thereof comprises a mutation or truncation; (b) a transmembrane domain comprising a CD2 transmembrane domain or a portion thereof; and (c) an intracellular domain comprising a cytoplasmic domain and at least one signal transduction domain, wherein the cytoplasmic domain comprises a CD2 cytoplasmic domain or a portion thereof, and wherein the at least one signal transduction domain comprises a CD3ζ protein or a portion thereof. In some aspects, the non-natural CSR comprises an amino acid sequence equivalent to at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage between both) of SEQ ID NO: 123. In a preferred aspect, the non-naturally occurring CSR comprises the amino acid sequence of SEQ ID NO: 123.

[0256] This disclosure provides nucleic acid sequences encoding any of the CSRs disclosed herein. This disclosure also provides transposons or vectors containing nucleic acid sequences encoding any of the CSRs disclosed herein.

[0257] This disclosure provides cells comprising any CSR disclosed herein. This disclosure provides cells comprising a nucleic acid sequence encoding any CSR disclosed herein. This disclosure provides cells comprising a vector containing a nucleic acid sequence encoding any CSR disclosed herein. This disclosure provides cells comprising a transposon containing a nucleic acid sequence encoding any CSR disclosed herein.

[0258] The modified cells disclosed herein can be allogeneic cells or autologous cells. In some preferred aspects, the modified cells are allogeneic cells. In some aspects, the modified cells are autologous T cells or modified autologous CAR T cells. In some preferred aspects, the modified cells are allogeneic T cells or modified allogeneic CAR T cells.

[0259] This disclosure provides compositions comprising any CSR disclosed herein. This disclosure provides compositions comprising a nucleic acid sequence encoding any CSR disclosed herein. This disclosure provides compositions comprising a vector containing a nucleic acid sequence encoding any CSR disclosed herein. This disclosure provides compositions comprising a transposon containing a nucleic acid sequence encoding any CSR disclosed herein. This disclosure provides compositions comprising cells with modifications disclosed herein, or compositions comprising multiple modifications disclosed herein.

[0260] This disclosure provides modified T lymphocytes (T cells) comprising: (a) a modification of an endogenous sequence encoding a T cell receptor (TCR), wherein the modification reduces or eliminates the expression or activity level of the TCR; and (b) a chimeric stimulatory receptor (CSR) comprising: (i) an extracellular domain containing an activation component, wherein the activation component is isolated from or derived from a first protein; (ii) a transmembrane domain; and (iii) an intracellular domain containing at least one signal transduction domain, wherein the at least one signal transduction domain is isolated from or derived from a second protein; wherein the first protein and the second protein are not equivalent.

[0261] The modified T cells may further contain induced pro-apoptotic peptides. The modified T cells may further contain modifications encoding an endogenous sequence of β-2-microglobulin (B2M), wherein said modifications reduce or eliminate the expression or activity levels of major histocompatibility complex (MHC) class I (MHC-I).

[0262] The modified T cells may further comprise a non-naturally occurring polypeptide containing the HLA class I histocompatibility antigen, α-chain E (HLA-E) polypeptide. The non-naturally occurring polypeptide containing the HLA-E polypeptide may further comprise a B2M signal peptide. The non-naturally occurring polypeptide containing the HLA-E polypeptide may further comprise a B2M polypeptide. The non-naturally occurring polypeptide containing the HLA-E polypeptide may further comprise a linker positioned between the B2M polypeptide and the HLA-E polypeptide. The non-naturally occurring polypeptide containing the HLA-E polypeptide may further comprise a peptide and a B2M polypeptide. The non-naturally occurring polypeptide containing HLA-E may 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.

[0263] Modified T cells may further include non-naturally occurring antigen receptors, sequences encoding therapeutic peptides, or combinations thereof. Non-naturally occurring antigen receptors may include chimeric antigen receptors (CARs).

[0264] CSRs can be transiently expressed in modified T cells. CSRs can be stably expressed in modified T cells. Peptides containing HLA-E peptides can be transiently expressed in modified T cells. Peptides containing HLA-E peptides can be stably expressed in modified T cells. Inducible pro-apoptotic peptides can be transiently expressed in modified T cells. Inducible pro-apoptotic peptides can be stably expressed in modified T cells. Non-naturally occurring antigen receptors or sequences encoding therapeutic proteins can be transiently expressed in modified T cells. Non-naturally occurring antigen receptors or sequences encoding therapeutic proteins can be stably expressed in modified T cells.

[0265] 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 and reduce or eliminate the expression of endogenous T-cell receptors. In a preferred aspect, the gene editing compositions target and delete genes, portions of genes, or regulatory elements (e.g., promoters) encoding endogenous T-cell receptors. Non-limiting examples of primers (including T7 promoters, genomic target sequences, and gRNA scaffolds) for generating guide RNA (gRNA) templates are disclosed in PCT application PCT / US2019 / 049816, the guide RNA templates being used to target and delete TCR-α (TCR-α), target and delete TCR-β (TCR-β), and target and delete β-2-microglobulin (β2M).

[0266] Gene editing compositions, including but not limited to RNA-guided fusion proteins containing dCas9-Clo051, can be used to target and reduce or eliminate the expression of endogenous MHC1, MHCII, or MHC activators. In a preferred aspect, the gene editing composition targets and deletes a gene, a portion of a gene, or a regulatory element (e.g., a promoter) encoding one or more components of an endogenous MHC1, MHCII, or MHC activator. Non-limiting examples of guide RNA (gRNA) for targeting and deleting MHC activators are disclosed in PCT application PCT / US2019 / 049816.

[0267] A detailed description of a non-naturally occurring chimeric stimulatory receptor, genetic modifications of the endogenous sequences encoding TCR-α (TCR-α), TCR-β (TCR-β) and / or β-2-microglobulin (β2M), and a non-naturally occurring polypeptide containing a polypeptide of HLA class I histocompatibility antigen α chain E (HLA-E) is disclosed in PCT application number PCT / US2019 / 049816.

[0268] Formulation, dosage and administration method This disclosure provides formulations, dosages, and methods for administering the compositions described herein.

[0269] The disclosed compositions and pharmaceutical compositions may further comprise at least one of any suitable excipients, such as, but not limited to, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, etc. Pharmaceutically acceptable excipients are preferred. Non-limiting examples and methods of preparing such sterile solutions are well known in the art, for example, but not limited to, Gennaro, ed., Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Co. (Easton, Pa.) 1990, and "Physician's Desk Reference", 52nd edition, Medical Economics (Montvale, NJ) 1998. As is well known in the art or as described herein, pharmaceutically acceptable carriers suitable for the administration modality, solubility, and / or stability of protein scaffold, fragment, or variant compositions may be conventionally selected.

[0270] Non-limiting examples of suitable pharmaceutical excipients and additives include proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; derived sugars, such as sugar alcohols, aldonic acids, esterified sugars, etc.; and polysaccharides or sugar polymers), which may be present alone or in combination, constituting 1-99.99% by weight or volume. Non-limiting examples of protein excipients include serum albumin, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, etc. Representative amino acid / protein components that may also function in terms of buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, etc. A preferred amino acid is glycine.

[0271] Non-limiting examples of suitable carbohydrate excipients include monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbitol, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melitriose, maltodextrin, dextran, starch, etc.; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucol), inositol, etc. Preferably, the carbohydrate excipient is mannitol, trehalose, and / or raffinose.

[0272] The composition may also include a buffer or pH adjuster; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts, such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers. Preferred buffers are organic acid salts, such as citrate.

[0273] Additionally, the disclosed compositions may include polymeric excipients / additives such as polyvinylpyrrolidone, sucrose (polymeric sugar), glucose dextrorates (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).

[0274] Many known and developed modes of administration can be used to administer therapeutically effective amounts of the compositions or pharmaceutical compositions disclosed herein. Non-limiting examples of modes of administration include bolus, buccal, infusion, intra-articular, intrabronchial, intra-abdominal, intra-sacral, intra-cartilaginous, intracavitary, intra-body cavity, intracerebellum, intravenous, colonic, intracervical, gastric, intrahepatic, intralesional, intramuscular, intracardiac, intranasal, intraocular, intraosseous, intraosseous, intraosseous, intrapelvic, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, rectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intratumoral, intravenous, intravesical, intravesical, oral, extra-gastric, rectal, sublingual, subcutaneous, percutaneous, or vaginal means.

[0275] The compositions disclosed herein can be prepared for parenteral (subcutaneous, intramuscular, or intravenous) or any other administration, particularly in the form of a liquid solution or suspension; for vaginal or rectal administration, particularly in semi-solid forms such as, but not limited to, creams and suppositories; for buccal or sublingual administration, such as, but not limited to, tablets or capsules; or for intranasal administration, such as, but not limited to, powders, nasal drops, or aerosols or certain pharmaceutical preparations; or for transdermal administration, such as, but not limited to, gels, ointments, lotions, suspensions, or patch delivery systems having chemical enhancers such as dimethyl sulfoxide to modify skin structure or increase drug concentration in transdermal patches (Junginger et al., in “Drug Permeation Enhancement;” Hsieh, DS, ed., pp. 59-90 (Marcel Dekker, Inc., New York 1994), or having oxidizing agents that enable the application of formulations containing proteins and peptides to the skin (WO). 98 / 53847), or applying an electric field to create a transient transport pathway, such as electroporation, or increasing the fluidity of charged drugs through the skin, such as iontophoresis, or applying ultrasound, such as ultrasound delivery (US Patent Nos. 4,309,989 and 4,767,402) (the foregoing disclosures and patents are incorporated herein by reference in their entirety).

[0276] For parenteral administration, any composition disclosed herein may be formulated as a solution, suspension, emulsion, granules, powder, or lyophilized powder provided in combination with or separately from a pharmaceutically acceptable parenteral medium. Formulations for parenteral administration may contain sterile water or saline, polyalkylene glycols such as polyethylene glycol, plant-derived oils, hydrogenated naphthalene, etc., as common excipients. Aqueous or oily suspensions for injection may be prepared by known methods using appropriate emulsifiers or humectants and suspending agents. Pharmaceuticals for injection may be non-toxic, non-oral diluents, such as aqueous solutions in a solvent, sterile injectable solutions, or suspensions. Water, Ringer's solution, isotonic saline, etc., are permitted as usable mediators or solvents; sterile non-volatile oils may be used as common solvents or suspending agents. For these purposes, any kind of non-volatile oil and fatty acid may be used, including natural or synthetic or semi-synthetic fatty oils or fatty acids; natural or synthetic or semi-synthetic monoglycerides, diglycerides, or triglycerides. Parenteral administration is known in the art and includes, but is not limited to, conventional injection methods, such as the pneumatic needleless injection device described in U.S. Patent No. 5,851,198, and the laser perforation device described in U.S. Patent No. 5,839,446.

[0277] For oral administration, formulations may involve the co-administration of adjuvants (e.g., resorcinol and nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether) to artificially increase intestinal wall permeability, and the co-administration of enzyme inhibitors (e.g., pancreatic trypsin inhibitors, diisopropyl fluorophosphate (DFF), and trasylol) to inhibit enzymatic degradation. U.S. Patent No. 6,309,663 describes formulations for delivering hydrophilic pharmaceutical agents comprising a combination of proteins and protein scaffolds and at least two surfactants, intended for oral, buccal, mucosal, nasal, pulmonary, vaginal, transmembrane, or rectal administration. The active ingredient compound in solid dosage forms intended for oral administration may be mixed with at least one additive, said additive including sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, arginine, chitin, deacetylated chitosan, pectin, gum arabic, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, and glycerides. These dosage forms may also contain other types of additives, such as inert diluents, lubricants (e.g., magnesium stearate, parabens), preservatives (e.g., sorbic acid, ascorbic acid, α-tocopherol), antioxidants (e.g., cysteine), disintegrants, binders, thickeners, buffers, sweeteners, flavorings, and fragrances.

[0278] Tablets and pills can be further processed into enteric-coated formulations. Liquid formulations for oral administration include emulsions, syrups, elixirs, suspensions, and solutions permissible for medical use. These formulations may contain inert diluents, such as water, commonly used in the aforementioned fields. Liposomes have also been described as drug delivery systems for insulin and heparin (US Patent No. 4,239,754). Recently, microspheres of artificial polymers of mixed amino acids (protein-like substances) have been used for drug delivery (US Patent No. 4,925,673). Furthermore, carrier compounds described in US Patent Nos. 5,879,681 and 5,871,753 for oral delivery of bioactive agents are known in the art.

[0279] For pulmonary administration, preferably, the compositions or pharmaceutical compositions described herein are delivered at a particle size that allows for effective access to the lower airways or sinuses of the lungs. The compositions or pharmaceutical compositions can be delivered using any of a variety of inhalation or nasal devices known in the art for administration via inhalation of a therapeutic agent. These devices capable of depositing aerosolized formulations into the sinuses or alveoli of a patient include metered-dose inhalers, nebulizers (e.g., jet nebulizers, ultrasonic nebulizers), dry powder generators, nebulizers, and the like. All such devices can use formulations suitable for administration to dispense the compositions or pharmaceutical compositions described herein as an aerosol. This aerosol can consist of a solution (aqueous and non-aqueous) or solid particles. Additionally, a spray comprising 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 (MDIs), the propellant, the compositions or pharmaceutical compositions described herein, and any excipients or other additives are contained in a canister as a mixture comprising liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol, which preferably contains particles in the size range of less than about 10 μm, preferably about 1 μm to about 5 μm, and most preferably about 2 μm to about 3 μm. A more detailed description of the pulmonary administration, formulation, and related device is disclosed in PCT Publication No. WO 2019 / 049816.

[0280] For absorption via mucosal surfaces, the composition comprises an emulsion containing multiple submicron particles, mucosal adhesion macromolecules, bioactive peptides, and an aqueous continuous phase, which promotes absorption via mucosal surfaces by achieving mucosal adhesion of the emulsion particles (US Patent No. 5,514,670). Suitable mucosal surfaces for applying the emulsions of this disclosure may include cornea, conjunctiva, buccal, sublingual, nasal, vaginal, lung, gastric, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, such as suppositories, may contain excipients such as polyalkylene glycols, petrolatum, cocoa butter, etc. Formulations for intranasal administration may be solid and contain excipients such as lactose, or may be an aqueous or oily solution for nasal drops. For buccal administration, excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch, etc. (US Patent No. 5,849,695). A more detailed description of the mucosal application and formulation is disclosed in PCT Publication No. WO 2019 / 049816.

[0281] For transdermal administration, the compositions or pharmaceutical compositions disclosed herein are encapsulated in a delivery device, such as liposomes or polymeric nanoparticles, microparticles, microcapsules, or microspheres (collectively referred to as microparticles unless otherwise specified). Many suitable devices are known, including microparticles made of synthetic and natural polymers, such as polyhydroxy acids, like polylactic acid, polyglycolic acid, and copolymers thereof, polyorthoesters, polyanhydrides, and polyphosphazenes, and natural polymers such as collagen, polyamino acids, albumin and other proteins, alginates and other polysaccharides, and combinations thereof (US Patent No. 5,814,599). A more detailed description of transdermal administration, formulations, and suitable devices is disclosed in PCT Publication No. WO 2019 / 049816.

[0282] It is expected that the disclosed compound can be delivered to the subject over an extended period, such as from one week to one year from the start of a single administration. Various slow-release, reservoir, or implantable dosage forms can be utilized. For example, the dosage form may contain a pharmaceutically acceptable, non-toxic salt of a compound having low solubility in bodily fluids, such as (a) an acid addition salt of a polybasic acid, such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pyric acid, alginic acid, polyglutamic acid, naphthalene monosulfonic acid or naphthalene disulfonic acid, polygalacturonic acid, etc.; (b) a salt of a polyvalent metal cation, such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, etc., or a salt of an organic cation, such as N,N'-dibenzylethylenediamine or ethylenediamine; or (c) a combination of (a) and (b), such as zinc tannate. Additionally, the disclosed compounds, or preferably relatively insoluble salts, such as those just described, can be formulated in gels suitable for injection, such as aluminum monostearate gel containing, for example, sesame oil. Particularly preferred salts are zinc salts, zinc tannate salts, dihydroxynaphthyl salts, etc. Another type of slow-release reservoir formulation for injection will contain compounds or salts dispersed in a slowly degrading, non-toxic, non-antigenic polymer, such as the polylactic acid / polyglycolic acid polymer described in U.S. Patent No. 3,773,919. Compounds, or preferably relatively insoluble salts, such as those described above, can also be formulated in cholesterol-based silicone rubber pellets, particularly for use in animals. Other slow-release, reservoir, or implantable formulations, such as gaseous or liquid liposomes, are known in the literature (U.S. Patent No. 5,770,222 and “Sustained and Controlled Release Drug Delivery Systems,” edited by JR Robinson, Marcel Dekker, Inc., NY, 1978).

[0283] Appropriate dosage is well known in the art. See, for example, Wells et al., eds., Pharmacotherapy Handbook, 2nd ed., Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, deluxe edition, Tarascon Publishing, Loma Linda, Calif. (2000); Nursing 2001 Handbook of Drugs, 21st ed., Springhouse Corp., Springhouse, Pa., 2001; Health Professional's Drug Guide 2001, eds., Shannon, Wilson, Stang, Prentice-Hall, Inc., Upper Saddle River, NJ. Preferred doses may optionally include about 0.1-99 and / or 100-500 mg / kg per administration, or any range, value, or fraction thereof, or achieve a serum concentration of about 0.1-5000 μg / ml per single or multiple administrations, or any range, value, or fraction thereof. A preferred dose 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 of the subject's body weight.

[0284] Alternatively, the dosage may vary depending on known factors such as the pharmacodynamic properties of the particular agent, and its mode and route of administration; the recipient's age, health, and weight; the nature and severity of symptoms, the type of concurrent treatment, the frequency of treatment, and the desired effect. Typically, the dosage of the active ingredient can be from about 0.1 to 100 mg / kg body weight. Typically, 0.1 to 50, and preferably 0.1 to 10 mg / kg / administration, or in a sustained-release form, is effective in achieving the desired result.

[0285] As a non-limiting example, treatment in humans or animals may be provided as a single, infusion, or repeated dose, for at least one day during days 1-40, or alternatively or additionally, for at least one week during weeks 1-52, or alternatively or additionally, for at least one year during years 1-20, or any combination thereof, as a single or periodic dose of the compositions or pharmaceutical compositions disclosed herein, from about 0.1 to 100 mg / kg or any range, value, or fraction per day.

[0286] Dosage forms suitable for internal administration generally contain about 0.001 mg to about 500 mg of the active ingredient per unit or container. In these pharmaceutical compositions, the active ingredient is typically present in an amount of about 0.5-99.999% by weight, based on the total weight of the composition.

[0287] An effective amount may comprise about 0.001 to about 500 mg / kg per single (e.g., bolus), multiple or consecutive administration to achieve a serum concentration of 0.01-5000 μg / ml per single, multiple or consecutive administration, or any effective range or value thereof, as performed and determined using known methods, as described herein or known in the relevant field.

[0288] The composition to be administered to subjects in need is a modified cellular aspect as disclosed herein, and can be administered at approximately 1 x 10⁻⁶ units. 3 Up to 1x10 15 1 x 10 cells; approximately 1 x 10 4 Up to 1x10 12 1 x 10 cells; approximately 1 x 10 5 Up to 1x10 10 1 x 10 cells; approximately 1 x 10 6 Up to 1x10 9 1 x 10 cells; approximately 1 x 10 6 Up to 1x10 8 1 x 10 cells; approximately 1 x 10 6 Up to 1x10 7 1 x 10 cells; or approximately 1 x 10 6 Up to 25x10 6 Cells per cell. In one application, approximately 5 x 10- cells are used. 6 Up to 25x10 6 A cell of 100 cells.

[0289] 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. WO 2019 / 049816.

[0290] Methods of using the compositions disclosed herein This disclosure provides for the use of the disclosed compositions or pharmaceutical compositions in treating a disease or condition in cells, tissues, organs, animals, or subjects, as known in the art or as described herein, by applying or contacting the disclosed compositions or pharmaceutical compositions with cells, tissues, organs, animals, or subjects in a therapeutically effective amount. In one aspect, the subject is a mammal. Preferably, the subject is a human. The terms “subject” and “patient” are used interchangeably herein.

[0291] This disclosure provides methods for modulating or treating at least one malignant disease or condition in cells, tissues, organs, animals, or subjects. Preferably, the malignant disease is cancer. Non-limiting examples of malignant diseases or conditions include leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphoblastic leukemia, B-cell, T-cell, or FAB. ALL, Acute Myeloid Leukemia (AML), Acute Granulocytic 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 / Hypercalcemia of Malignant Tumors, Solid Tumors, Bladder Cancer, Breast Cancer, Colorectal Cancer, Endometrial Cancer, Head Cancer, Neck Cancer, Hereditary Nonpolyposis Cancer, Hodgkin's Lymphoma, Liver Cancer, Lung Cancer, Non-Small Cell Lung Cancer, Ovarian Cancer, Pancreatic Cancer, Prostate Cancer, Renal Cell Carcinoma, Testicular Cancer, Adenocarcinoma, Sarcoma, Malignant Melanoma, Hemangioma, Metastatic Disease, Cancer-Related Bone Resorption, Cancer-Related Bone Pain, etc.

[0292] In a preferred aspect, treatment of malignant diseases or conditions includes adoptive cell therapy. For example, in one aspect, this disclosure provides modified cells expressing at least one disclosed antibody (e.g., scFv) and / or a CAR containing the antibody (e.g., scFv), which have been selected and / or expanded for administration to subjects in need. 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 subjects from sterile packaging. The modified cells can be formulated in a pharmaceutically acceptable carrier having indicators of cell viability and / or CAR expression levels to ensure minimum levels of cell function and CAR expression. The modified cells can be formulated in a pharmaceutically acceptable carrier at a prescribed density using one or more reagents to inhibit further expansion and / or prevent cell death.

[0293] Any method may include administering an effective amount of any of the compositions or pharmaceutical compositions disclosed herein to cells, tissues, organs, animals, or subjects in need of such modulation, treatment, or therapy. Such methods may optionally further include co-administration or combination therapy for treating such diseases or conditions, wherein the administration of any of the compositions or pharmaceutical compositions disclosed herein further includes administering at least one chemotherapeutic agent (e.g., alkylating agents, mitotic inhibitors, radiopharmaceuticals) before, during, and / or after treatment.

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

[0295] In some respects, the therapeutically effective dose is a single dose. In some respects, a single dose is one of a number of doses simultaneously manufactured, including 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 between both. In some respects, when the composition is autologous or allogeneic cells, the dose is an amount sufficient to allow cell implantation and / or to persist for a sufficient time to treat the disease or condition.

[0296] In one instance, this disclosure provides a method of treating cancer in a subject in need, comprising administering to the subject a composition comprising an antibody (e.g., scFv) or a CAR comprising an antibody (e.g., scFv) that specifically binds to an antigen on tumor cells. In aspects wherein the composition comprises modified cells or cell populations, the cells or cell populations may be autologous or allogeneic.

[0297] In some aspects of the treatment methods described herein, treatment can be modified or terminated. Specifically, in aspects where the composition used for treatment comprises an inducible pro-apoptotic peptide, apoptosis can be selectively induced in the cells by contacting the cells with the inducer. Treatment can be modified or terminated in response to, for example, signs of recovery or signs of reduced disease severity / progression, signs of disease remission / cessation, and / or the occurrence of adverse events. In some aspects, the method includes the step of administering an inhibitor of the inducer to inhibit the modification of the cell therapy, thereby restoring the function and / or efficacy of the cell therapy (e.g., when signs or symptoms of disease reappear, or when increased severity and / or adverse events are resolved).

[0298] Antibody / scFv production, screening and purification At least one antibody of this disclosure (e.g., monoclonal antibody, chimeric antibody, single-domain antibody, VHH, VH, single-chain variable fragment (scFv), antigen-binding fragment (Fab), or Fab fragment) may optionally be generated from a cell line, a hybrid cell line, immortalized cells, or a clonal population of immortalized cells, as is well known in the art. See, for example, Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987–2001); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor, NY (1989); Harlow and Lane, Antibodies, a Laboratory Manual, ColdSpring Harbor, NY (1989); Colligan et 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).

[0299] Amino acids from scFv may be altered, added, and / or deleted to reduce immunogenicity, or to reduce, enhance, or modify binding, affinity, binding rate, dissociation rate, specificity, half-life, stability, solubility, or any other suitable property, as known in the art.

[0300] Optionally, scFv can be modified with the retention of high affinity for antigens and other favorable biological properties. To achieve this, scaffold proteins can be optionally prepared by analyzing the parental sequence and various conceptually modified products using three-dimensional models of the parental and modified sequences. Three-dimensional models are widely available and familiar to those skilled in the art. Computer programs are available that illustrate and demonstrate possible three-dimensional conformations of selected candidate sequences and can measure possible immunogenicity (e.g., the Immunofilter program of Xencor, Inc., Monrovia, Calif.). These demonstrations allow analysis of the possible roles of residues in the function of the candidate sequence, i.e., analysis of residues affecting the ability of the candidate scFv to bind its antigen. In this way, residues can be selected and combined from the parental and reference sequences to achieve the desired properties, such as affinity for the target antigen. Alternatively, or in addition to the procedures described above, other suitable modification methods can be used.

[0301] Nucleotide (DNA or RNA display) or peptide display libraries can be used, for example in in vitro display, to conveniently screen scFvs that specifically bind to similar proteins or fragments. This method involves screening large sets of peptides for individual members having the desired function or structure. The length of the displayed nucleotide or peptide sequence can be 3 to 5000 or more nucleotides or amino acids, frequently 5-100 amino acids long, and often about 8 to 25 amino acids long. 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 bacterial phages or cells. Each bacterial phage or cell contains a nucleotide sequence encoding a specific displayed peptide sequence. Such methods are described in PCT patent publications WO 91 / 17271, WO 91 / 18980, WO 91 / 19818, and WO 93 / 08278.

[0302] Other systems for generating peptide libraries 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. Also see U.S. patents 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, for example, 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; ​​5,223,409, 5,403,484, 5,571,698, and 5,837,500 assigned to Dyax; 5,427,908 and 5,580,717 assigned to Affymax; and 5,427,908 assigned to Cambridge Antibody. 5,885,793 to Technologies; 5,750,373 to Genentech; to Xoma, Colligan, ibid.; Ausubel, ibid.; or Sambrook, ibid. 5,618,920, 5,595,898, 5,576,195, 5,698,435, 5,693,493, 5,698,417.

[0303] The scFv of this disclosure can bind human or other mammalian proteins with a wide range of affinity (KD). In a preferred aspect, at least one scFv of this disclosure can optionally bind to the target protein with high affinity, for example, having a KD equal to or less than about 10. −7 M, for example, but not limited to, 0.1–9.9 (or any range or value thereof) x 10 −8 10 −9 10 −10 10 −11 10 −12 10 −13 10 −14 10 −15 Or any range or value thereof, such as that determined by surface plasmon resonance or the Kinexa method, as practiced by those skilled in the art.

[0304] The affinity or cohesion of scFv to an antigen can be experimentally determined using any suitable method. (See, for example, Berzofsky et al., “Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, WE, ed., Raven Press: New York, NY (1984); Kuby, Janis Immunology, WH Freeman and Company: New York, NY (1992); and the methods described herein). The affinity of a particular scFv-antigen interaction measured can vary depending on the conditions (e.g., salt concentration, pH). Therefore, measurements of affinity and other antigen-binding parameters (e.g., KD, Kon, Koff) are preferably performed using a protein scaffold and a standardized solution of the antigen, and a standardized buffer such as those described herein.

[0305] Competitive assays can be performed using the scFv of this disclosure to determine which proteins, antibodies, and other antagonists compete with the scFv of this disclosure for binding to target proteins and / or sharing epitope regions. These assays, as readily known to those skilled in the art, assess competition between antagonists or ligands for a limited number of binding sites on a protein. The protein and / or antibody are fixed or insoluble before or after the competition, and the sample bound to the target protein is separated from the unbound sample, for example, by decanting (where the protein / antibody is pre-dissolved) or by centrifugation (where the protein / antibody is precipitated after the competitive reaction). Furthermore, competitive binding can be determined by whether the function is altered by the binding of the scFv to the target protein or by the lack thereof; for example, whether the scFv molecule inhibits or enhances, for example, the activity of a labeled enzyme. ELISA and other functional assays, as well as those well known in the art, can be used.

[0306] Nucleic acid molecules The nucleic acid molecule encoding scFv in this disclosure can be in the form of RNA, such as mRNA, hnRNA, tRNA, or any other form, or in the form of DNA, including but not limited to cDNA and genomic DNA obtained by cloning or synthesized, or any combination thereof. DNA can be triple-stranded, double-stranded, or single-stranded, or any combination thereof. Any portion of at least one strand of DNA or RNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also known as the antisense strand.

[0307] The isolated nucleic acid molecules of this disclosure may include nucleic acid molecules containing an open reading frame (ORF), optionally having one or more introns, such as, but not limited to, at least one designated portion of at least one scFv; nucleic acid molecules containing a coding sequence for a protein scaffold or loop region that binds to a target protein; and nucleic acid molecules containing nucleotide sequences that are substantially different from those described above, but still encode a protein scaffold as described herein and / or as known in the art due to the degeneracy of the genetic code. Of course, the genetic code is well known in the art. Therefore, it would be conventional for those skilled in the art to generate such degenerate nucleic acid variants encoding a specific scFv of this disclosure. See, for example, Ausubel et al., ibid., and such nucleic acid variants are included in this disclosure.

[0308] As indicated herein, nucleic acid molecules comprising nucleic acids encoding scFv may include, but are not limited to, those nucleic acid molecules whose amino acid sequence encodes the scFv fragment itself; coding sequences of the entire protein scaffold or a portion thereof; coding sequences of scFv, fragments, or a portion thereof; and additional sequences, such as coding sequences of at least one signal leader region or fusion peptide, with or without the aforementioned additional coding sequences, such as at least one intron, together with additional non-coding sequences, including but not limited to non-coding 5' and 3' sequences, such as transcriptional non-translation sequences that function in transcription, mRNA processing including splicing and polyadenylation signals (e.g., ribosome binding and mRNA stability); and additional coding sequences encoding additional amino acids, such as those providing additional functionality. Therefore, sequences encoding protein scaffolds can be fused with marker sequences, such as sequences encoding peptides, which facilitate the purification of fused protein scaffolds containing fragments or portions of the protein scaffold.

[0309] Multinucleotides that selectively hybridize with multinucleotides as described herein This disclosure provides isolated nucleic acids that hybridize with the polynucleotides disclosed herein under selective hybridization conditions. Therefore, polynucleotides can be used to isolate, detect, and / or quantify nucleic acids containing such polynucleotides. For example, the polynucleotides of this disclosure can be used to identify, isolate, or amplify partial or full-length clones in a library. The polynucleotides can be genomic or cDNA sequences isolated from a human or mammalian nucleic acid library, or genomic or cDNA sequences otherwise complementary to cDNA from a human or mammalian nucleic acid library.

[0310] Preferably, the cDNA library contains at least 80% of the full-length sequence, more preferably at least 85% or 90% of the full-length sequence, and more preferably at least 95% of the full-length sequence. The cDNA library can be normalized to increase the representation of rare sequences. Low or moderate stringency hybridization conditions are typically, but not exclusively, used for sequences with reduced sequence identity relative to their complementary sequences. Moderate and high stringency conditions can optionally be used for sequences with higher identity. Low stringency conditions allow selective hybridization of sequences with approximately 70% sequence identity and can be used to identify orthologous or paralogous sequences.

[0311] Optionally, the polynucleotide encodes 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 with the polynucleotide encoding the protein scaffold of this disclosure. See, for example, Ausubel, ibid.; Colligan, ibid., each incorporated herein by reference in its entirety.

[0312] Nucleic acid construction The isolated nucleic acids of this disclosure can be prepared using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques and / or (d) combinations thereof, as is well known in the art.

[0313] Nucleic acids can conveniently contain nucleotide sequences other than the polynucleotides described herein. For example, a multiple cloning site containing one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in the isolation of polynucleotides. Additionally, a translatable sequence can be inserted to aid in the isolation of polynucleotides translated according to this disclosure. For example, a hexahistine marker sequence provides a convenient means of purifying the proteins of this disclosure. The nucleic acids of this disclosure, excluding coding sequences, are optionally vectors, adaptors, or linkers for cloning and / or expressing polynucleotides of this disclosure.

[0314] Additional sequences may 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, adaptors, and adapters is well known in the art. (See, for example, Ausubel, ibid.; or Sambrook, ibid.).

[0315] Recombinant methods for constructing nucleic acids The isolated nucleic acid compositions of this disclosure, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methods known to those skilled in the art. In some aspects, under stringent conditions, oligonucleotide probes that selectively hybridize with polynucleotides of this disclosure are used to identify desired sequences in cDNA or genomic DNA libraries. The isolation of RNA and the construction of cDNA and genomic libraries are well known to those skilled in the art (see, for example, Ausubel, ibid.; or Sambrook, ibid.).

[0316] Nucleic acid screening and isolation methods Probes based on the polynucleotide sequences of this disclosure can be used to screen cDNA or genomic libraries. The probes can be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes from the same or different organisms. Those skilled in the art will understand that various degrees of stringency can be employed in the assay; and both the hybridization and washing media can be stringent. As the conditions used for hybridization become more stringent, a greater degree of complementarity must exist between the probe and the target for duplex formation to occur. The degree of stringency can be controlled by one or more of temperature, ionic strength, pH, and the presence of a partially denaturing solvent such as formamide. For example, the stringency of the hybridization can be conveniently altered by manipulating the formamide concentration in the range of 0% to 50% to change the polarity of the reactant solution. The degree of complementarity (sequence identity) required for detectable binding will vary depending on the stringency of the hybridization and / or washing media. The optimal degree of complementarity is 100%, or 70-100%, or any range or value therein. However, it should be understood that minor sequence variations in the probe and primers can be compensated for by reducing the stringency of the hybridization and / or washing media.

[0317] Methods for amplifying RNA or DNA are well known in the art and can be used in accordance with this disclosure without excessive experimentation, based on the teachings and instructions presented herein.

[0318] Known DNA or RNA amplification methods include, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (see, for example, U.S. Patent Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188 to Mullis et al.; 4,795,699 and 4,921,794 to Tabor et al.; 5,142,033 to Innis; 5,122,464 to Wilson et al.; and Innis...). U.S. Patent No. 5,091,310 to Gyllensten et al.; 5,066,584 to Gyllensten et al.; 4,889,818 to Gellensten et al.; 4,994,370 to Silver et al.; 4,766,067 to Biswas; and 4,656,134 to Ringold), and RNA-mediated amplification using antisense RNA targeting a target sequence as a template for double-stranded DNA synthesis (U.S. Patent No. 5,130,238 to Malek et al., under the trade name NASBA), the entire contents of which are incorporated herein by reference. (See, for example, Ausubel, ibid.; or Sambrook, ibid.) For example, polymerase chain reaction (PCR) technology can be used to directly amplify the polynucleotides and related gene sequences of this disclosure from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods can also be useful, for example, to clone nucleic acid sequences encoding proteins to be expressed, to prepare nucleic acids for use as probes to detect the presence of desired mRNAs in samples, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to guide a person skilled in the art to perform in vitro amplification methods can be found in: Berger, ibid., Sambrook, ibid., and Ausubel, ibid., and Mullis et al., U.S. Patent No. 4,683,202 (1987); and Innis et al., PCR Protocols: A Guide to Methods and Applications, editor, 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, the T4 gene 32 protein (Boehringer Mannheim) can be used to improve the yield of long PCR products.

[0319] Synthetic methods for constructing nucleic acids The isolated nucleic acids described in this disclosure can also be prepared by direct chemical synthesis via known methods (see, for example, Ausubel et al., ibid.). Chemical synthesis generally produces single-stranded oligonucleotides, which can be converted into double-stranded DNA by hybridization with complementary sequences or by polymerization with a DNA polymerase using a single strand as a template. Those skilled in the art will recognize that while the chemical synthesis of DNA can be limited to sequences of about 100 or more bases, longer sequences can be obtained by ligating shorter sequences.

[0320] Recombinant expression cassette This disclosure further provides recombinant expression cassettes containing nucleic acids of this disclosure. The nucleic acid sequences of this disclosure, such as cDNA or genomic sequences encoding a protein scaffold of this disclosure, can be used to construct recombinant expression cassettes that can be introduced into at least one desired host cell. The recombinant expression cassette typically contains a polynucleotide of this disclosure operatively linked to a transcription initiation regulatory sequence that will direct the transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct the expression of the nucleic acids of this disclosure.

[0321] In some respects, isolated nucleic acids that act as promoters, enhancers, or other elements can be introduced into appropriate locations (upstream, downstream, or introns) in non-heterologous forms of the polynucleotides of this disclosure in order to upregulate or downregulate the expression of the polynucleotides of this disclosure. For example, endogenous promoters can be altered in vivo or in vitro by mutation, deletion, and / or substitution.

[0322] Expression vectors and host cells This disclosure also relates to vectors containing isolated nucleic acid molecules, genetically modified host cells using recombinant vectors, and the production of at least one protein scaffold via recombinant technology, as is well known in the art. See, for example, Sambrook et al., ibid.; Ausubel et al., ibid., each incorporated herein by reference in its entirety.

[0323] Optionally, polynucleotides can be linked to vectors containing selectable markers for replication in the host. Generally, plasmid vectors are introduced in precipitates such as calcium phosphate precipitates or complexes containing charged lipids. If the vector is a virus, it can be packaged in vitro using appropriate packaging cell lines and then transduced into host cells.

[0324] The DNA insert should be operatively linked to a suitable promoter. The expression construct will further contain sites for transcription initiation and termination, as well as ribosome-binding sites for translation within the transcription region. The coding portion of the mature transcript expressed by the construct will preferably include a translation initiation codon at the beginning and a stop codon (e.g., UAA, UGA, or UAG) appropriately placed at the end of the mRNA to be translated, wherein UAA and UAG are preferably used for mammalian or eukaryotic cell expression.

[0325] The expression vector will preferably, but optionally, include at least one optional marker. Such markers include, for example, but not limited to, ampicillin for eukaryotic cell culture, zeocin (… Sh bla Gene), puromycin ( pac Gene), Hygromycin B ( hygB Gene), G418 / genetic mycin ( neo Genes), DHFR (encoding dihydrofolate reductase and conferring resistance to methotrexate), mycophenolic acid or glutamine synthase (GS, US patents 5,122,464; 5,770,359; 5,827,739), and blast fungicides ( bsd (Gene) resistance genes, and ampicillin and zeocin (used for culturing in Escherichia coli and other bacteria or prokaryotes) Sh bla Gene), puromycin ( pac Gene), Hygromycin B ( hygB Gene), G418 / genetic mycin ( neo Genes for resistance to kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline (the above patents are incorporated herein by reference in their entirety). Suitable culture media and conditions for the aforementioned host cells are known in the art. Suitable vectors will be apparent to those skilled in the art. The introduction of vector constructs into host cells can be achieved by calcium phosphate transfection, DEAE-glucan-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other known methods. Such methods are described in the art, for example, Sambrook, ibid., Chapters 1-4 and 16-18; Ausubel, ibid., Chapters 1, 9, 13, 15, and 16.

[0326] The expression vector will preferably, but optionally, include at least one optional cell surface marker for isolating cells modified by the compositions and methods of this disclosure. The optional cell surface markers of this disclosure comprise surface proteins, glycoproteins, or proteomes that distinguish a cell or subset of cells from another defined subset of cells. Preferably, the optional cell surface markers distinguish those cells modified by the compositions or methods of this disclosure from those not modified by the compositions or methods of this disclosure. Such cell surface markers include, for example, but not limited to, “designated cluster” or “taxonomic cluster” proteins (often abbreviated as “CD”), such as truncated or full-length forms of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof. Cell surface markers further include the suicide gene marker RQR8 (Philip B et al. Blood. 2014 Aug 21; 124(8):1277-87).

[0327] The expression vector will preferably, but optionally, include at least one optional drug resistance marker for isolating cells modified by the compositions and methods of this disclosure. The optional drug resistance markers of this disclosure may include wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

[0328] At least one protein scaffold of this disclosure can be expressed in a modified form, such as a fusion protein, and can include not only secretion signals but also additional heterologous functional regions. For example, regions of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the protein scaffold to improve stability and durability in host cells, during purification, or during subsequent processing and storage. Additionally, peptide moieties can be added to the protein scaffold of this disclosure to facilitate purification. Such regions can be removed prior to the final preparation of the protein scaffold or at least one fragment thereof. Such methods are described in numerous standard laboratory manuals, such as Sambrook, ibid., Chapters 17.29–17.42 and 18.1–18.74; Ausubel, ibid., Chapters 16, 17, and 18.

[0329] Those skilled in the art are familiar with numerous expression systems that can be used to express nucleic acids encoding the proteins of this disclosure. Alternatively, the nucleic acids of this disclosure can be expressed in host cells by being turned on (by manipulation) in host cells containing endogenous DNA encoding the protein scaffold of this 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, which are incorporated herein by reference in their entirety.

[0330] Examples of cell cultures that can be used to generate protein scaffolds, their specified portions, or variants are bacterial, yeast, and mammalian cells as known in the art. Mammalian cell systems 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 complete 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, etc., which are readily available from, for example, the American Type Culture Collection, Manassas, Va. (www.atcc.org). Preferred host cells include lymphoid cells, such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC accession number CRL-1580) and SP2 / 0-Ag14 cells (ATCC accession number CRL-1851). In a preferred aspect, the recombinant cells are P3X63Ab8.653 or SP2 / 0-Ag14 cells.

[0331] Expression vectors used in these cells may include one or more of the following expression control sequences, such as, but not limited to, origin of replication; promoters (e.g., late or early SV40 promoters, CMV promoters (US Patent Nos. 5,168,062; 5,385,839), HSV tk promoters, pgk (phosphoglycerate kinase) promoters, EF-1 α promoters (US Patent No. 5,266,491), at least one human promoter); enhancers and / or processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites (e.g., SV40 large T Ag polyA addition sites), and transcription terminator sequences. See, for example, Ausubel et al., ibid.; Sambrook et al., ibid. Other cells that can be used to produce the nucleic acids or proteins of this disclosure are known, and / or are available, for example, from the U.S. Center for Type Culture Collection's catalogue of cell lines and hybridomas (www.atcc.org) or other known or commercial sources.

[0332] When using eukaryotic host cells, polyadenylated flower or transcription terminator sequences are typically introduced into the vector. An example of a terminator sequence is a polyadenylated sequence from the bovine growth hormone gene. Sequences for precise splicing of the transcript may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague et al., J. Virol. 45:773-781 (1983)). Additionally, as is known in the art, gene sequences controlling replication in host cells can be introduced into the vector.

[0333] scFv purification scFv can be recovered and purified from recombinant cell cultures using well-known methods, including but not limited to protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High-performance liquid chromatography (“HPLC”) can also be used for purification. See, for example, Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997–2001), e.g., Chapters 1, 4, 6, 8, 9, and 10, each incorporated herein by reference in its entirety.

[0334] The scFv of this disclosure includes purified products, products of chemical synthesis procedures, and products generated from prokaryotic or eukaryotic hosts via recombinant technology, including, for example, *Escherichia coli*, yeast, higher plants, insects, and mammalian cells. Depending on the host used in the recombinant production procedure, the protein scaffold of this disclosure may be glycosylated or non-glycosylated. Such methods are described in numerous standard laboratory manuals, such as Sambrook, ibid., sections 17.37–17.42; Ausubel, ibid., chapters 10, 12, 13, 16, 18, and 20; and Colligan, Protein Science, ibid., chapters 12–14, all of which are incorporated herein by reference in their entirety.

[0335] Amino acid code The amino acids constituting the protein scaffold of this disclosure are often abbreviated. Amino acid nomenclature can be indicated by specifying the amino acid via its single-letter code, its three-letter code, name, or trinucleotide codon, as is well known in the art (see Alberts, B. et al., Molecular Biology of The Cell, 3rd ed., Garland Publishing, Inc., New York, 1994). The protein scaffold of this disclosure may include one or more amino acid substitutions, deletions, or additions, as specified herein, resulting from spontaneous or mutated and / or artificially manipulated processes. The amino acids essential for function in the protein scaffold of this disclosure can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (e.g., Ausubel, ibid., Chapters 8 and 15; Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces a single alanine mutation at each residue in the molecule. The resulting mutant molecule is then tested for biological activity, such as, but not limited to, at least one neutralizing activity. Key sites for protein scaffold binding can also be identified by structural analysis, such as crystallization, nuclear magnetic resonance, or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos et al., Science 255:306-312 (1992)).

[0336] As those skilled in the art will understand, this disclosure includes at least one bioactive protein scaffold of this disclosure. The bioactive protein scaffold has a specific activity of at least 20%, 30%, or 40%, preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95%-99% or more, that of natural (non-synthetic), endogenous, or related and known protein scaffolds. Methods for determining and quantifying enzymatic activity and substrate specificity are well known to those skilled in the art.

[0337] In another aspect, this disclosure relates to protein scaffolds and fragments as described herein, which are modified by covalent attachment of an organic portion. Such modifications can produce protein scaffold fragments with improved pharmacokinetic properties, such as increased serum half-life in vivo. The organic portion can be a linear or branched hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. In a particular aspect, the hydrophilic polymer group can have a molecular weight of about 800 to about 120,000 Daltons and can be a polyalkane glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), a carbohydrate polymer, an amino acid polymer, or polyvinylpyrrolidone, and the fatty acid or fatty acid ester group can contain about eight to about forty carbon atoms.

[0338] The modified protein scaffolds and fragments of this disclosure may comprise one or more organic moieties that are covalently bonded to an antibody, directly or indirectly. Each organic moiety bonded to the protein scaffold or fragment of this disclosure may independently be a hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. As used herein, the term "fatty acid" encompasses both monocarboxylic and dicarboxylic acids. As used herein, "hydrophilic polymer group" refers to an organic polymer that is more soluble in water than in octane. For example, polylysine is more soluble in water than in octane. Therefore, protein scaffolds modified by covalent attachment of polylysine are covered by this disclosure. The hydrophilic polymers suitable for modifying the protein scaffolds of this disclosure can be linear or branched, and include, for example, polyalkane glycols (e.g., PEG, 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 this 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 subscripts are the average molecular weight of the polymer in Daltons. The hydrophilic polymer groups can be substituted with 1 to 6 alkyl, fatty acid, or fatty acid ester groups. Hydrophilic polymers substituted with fatty acid or fatty acid ester groups can be prepared by employing suitable methods. For example, polymers containing amine groups can be coupled to carboxylic esters of fatty acids or fatty acid esters, and activated carboxylic esters on fatty acids or fatty acid esters (e.g., activated with N,N-carbonyldiimidazole) can be coupled to hydroxyl groups on the polymer.

[0339] The fatty acids and fatty acid esters suitable for modifying the protein scaffolds of this disclosure may be saturated or may contain one or more unsaturated units. Suitable fatty acids for modifying the protein scaffolds of this disclosure include, for example, dodecanoic acid (C12, lauric acid), tetradecanoic acid (C14, myristic acid), stearic acid (C18, stearic acid), icosanoic acid (C20, arachidic acid), icosanoic acid (C22, benzanoic acid), triacontanoic acid (C30), tetradecanoic acid (C40), cis-Δ9-octadecanoic acid (C18, oleic acid), all cis-Δ5,8,11,14-eicosatetraenoic acids (C20, arachidonic acid), octanoic acid, tetradecanoic acid, octadecanoic acid, docosanoic acid, etc. Suitable fatty acid esters include dicarboxylic acid monoesters containing straight-chain or branched lower alkyl groups. The lower alkyl group may contain 1 to about 12, preferably 1 to about 6 carbon atoms.

[0340] Modified protein scaffolds and fragments can be prepared using suitable methods, such as by reacting with one or more modifiers. As used herein, a “modifier” refers to a suitable organic group (e.g., a 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 to form a covalent bond between the modifier and the second chemical group. For example, amine-reactive activating groups include electrophilic groups such as toluenesulfonates, methanesulfonates, halogens (chlorine, bromine, fluorine, iodine), N-hydroxysuccinimide esters (NHS), etc. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acryloyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), etc. Aldehyde functional groups can be coupled to molecules containing amines or hydrazides, and azide groups can react with trivalent phosphorus groups to form aminophosphate or phosphorimide bonds. Suitable methods for introducing an activating group into a molecule are known in the art (see, for example, Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)). The activating group can be directly bonded to an organic group (e.g., a hydrophilic polymer, fatty acid, fatty acid ester) or via a linker portion, where one or more carbon atoms can be replaced by a heteroatom such as a divalent C1-C12 group of oxygen, nitrogen, or sulfur. Suitable linker portions 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—. Modifiers containing the linker portion can be generated, for example, by reacting a mono-Boc-alkyl diamine (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 ester. The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine that can couple to another carboxylic ester, as described, or it can be reacted with maleic anhydride and the resulting product cyclized to produce an activated maleimide derivative of the fatty acid. (See, for example, Thompson et al., WO 92 / 16221, the entire teachings of which are incorporated herein by reference.) The modified protein scaffolds of this disclosure can be produced by reacting a protein scaffold or fragment with a modifier. For example, the organic portion can be bonded to the protein scaffold in a non-site-specific manner using an amine-reactive modifier, such as the NHS ester of PEG. Modified protein scaffolds and fragments containing organic moieties (which are bound to specific sites of the protein scaffolds of this disclosure) can be prepared using suitable methods, such as reverse enzymatic digestion (Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci. 6 (10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68 (1996); Capellas et al., Biotechnol. Bioeng., 56 (4):456-463 (1997)), and the methods described in Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996).

[0341] definition As used throughout this disclosure, the singular forms “a,” “an,” and “the” include the plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “method” includes a variety of such methods, and reference to “dosage” includes reference to one or more dosages and their equivalents known to those skilled in the art, etc.

[0342] The terms “about” or “approximately” mean within an acceptable margin of error for a particular value, as determined by a person skilled in the art, which will depend in part on how the value is measured or determined, for example, the limitations of the measurement system. For example, “about” may mean within one or more standard deviations. Alternatively, “about” may mean a range of a given value of up to 20%, or up to 10%, or up to 5%, or up to 1%. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude of the value, preferably within 5 times, and more preferably within 2 times. When a particular value is described in this application and claims, unless otherwise stated, the term “about” should be assumed to mean within an acceptable margin of error for the particular value.

[0343] This disclosure provides isolated or substantially purified polynucleotide or protein compositions. The “isolated” or “purified” polynucleotide or protein, or its biologically active portion, is substantially or substantially free of components typically associated with or interacting with the polynucleotide or protein, as found in its natural environment. Therefore, the isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. Preferably, the “isolated” polynucleotide does not contain the sequence of the naturally flanked polynucleotide (preferably the protein-coding sequence) (i.e., the sequence located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various aspects, the isolated polynucleotide may contain a nucleotide sequence of less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb, whose naturally flanked polynucleotide is derived from the genomic DNA of the cell from which it is derived. Proteins that are essentially free of cellular material include protein formulations containing less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating proteins. When recombinantly producing the proteins of this disclosure or their bioactive portions, the culture medium preferably represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-target protein chemicals.

[0344] This disclosure provides disclosed DNA sequences and fragments and variants of proteins encoded by these DNA sequences. As used throughout this 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 therein. A fragment of a DNA sequence containing a coding sequence may encode a protein fragment that retains the biological activity of the native protein and therefore retains the DNA recognition or binding activity to a target DNA sequence as described herein. Alternatively, fragments of DNA sequences that can be used as hybridization probes generally do not encode proteins that retain biological activity or promoter activity. Therefore, fragments of DNA sequences can range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide of this disclosure.

[0345] The nucleic acids or proteins of this disclosure can be constructed using a modular approach, which includes pre-assembling monomeric units and / or repeating units in a target vector, which can then be assembled into a final target vector. The peptides of this disclosure can contain repeating monomers of this disclosure and can be constructed using a modular approach by pre-assembling repeating units in a target vector, which can then be assembled into a final target vector. This disclosure provides peptides generated by this method and nucleic acid sequences encoding these peptides. This disclosure provides host organisms and cells containing nucleic acid sequences encoding peptides generated by this modular approach.

[0346] The term "antibody" is used in its broadest sense and specifically covers monoclonal antibodies (including agonist and antagonist antibodies) and antibody compositions with multi-epitope specificity. Natural or synthetic analogs, mutants, variants, alleles, homologs, and orthologs of antibodies as defined herein (collectively referred to herein as "analytes") are also within its scope. Therefore, according to one aspect, the term "antibody of" also covers such analogs in its broadest sense. Generally, in such analogs, one or more amino acid residues may have been substituted, deleted, and / or added compared to antibodies as defined herein.

[0347] As used herein, “antibody fragment” and all its grammatical variations are defined as a portion of a complete antibody containing the antigen-binding site or variable region of the complete antibody, wherein the portion does not contain the constant heavy chain domain (i.e., CH2, CH3, and CH4, depending on the antibody isotype) of the Fc region of the complete antibody. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; biantibodies; any antibody fragment of a polypeptide having a primary structure consisting of a continuous sequence of adjacent 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 containing only one light chain variable domain, or fragments thereof containing three CDRs of the light chain variable domain without the associated heavy chain portion, and (3) single-chain polypeptides containing only one heavy chain variable region, or fragments thereof containing three CDRs of the heavy chain variable region without the associated light chain portion; and multispecific or multivalent structures formed by antibody fragments. In antibody fragments comprising one or more heavy chains, the heavy chains may contain any constant domain sequence found in the non-Fc region of the intact antibody (e.g., CHI in an IgG isotype), and / or may contain any hinge region sequence found in the intact antibody, and / or may contain a leucine zipper sequence fused to or located therein with the hinge region sequence or constant domain sequence of the heavy chain. The term further includes single-domain antibodies (“sdAB”), which generally refers to antibody fragments having a single monomeric variable antibody domain (e.g., from camels). Such antibody fragment types will be readily understood by those skilled in the art.

[0348] "Binding" refers to sequence-specific, non-covalent interactions between macromolecules (e.g., between proteins and nucleic acids). Not all components of a binding interaction need to be sequence-specific (e.g., in contact with phosphate residues in the DNA backbone), as long as the interaction as a whole is sequence-specific.

[0349] The term "comprising" is intended to mean that a composition and method include the described elements, but does not exclude other elements. When used to define compositions and methods, "consisting substantially of..." should mean excluding other elements that are of any significance to the composition when used for the intended purpose. Therefore, a composition consisting substantially of elements as defined herein will not exclude trace impurities or inert carriers. "Constitutes..." should mean excluding other components and basic method steps beyond the trace elements. The aspects defined by these transitional terms are within the scope of this disclosure.

[0350] The term "epitaxy" refers to the antigenic determinant of a polypeptide. An epitope may contain three amino acids in a spatial conformation that are unique to the epitope. Generally, an epitope consists of at least 4, 5, 6, or 7 such amino acids, and more commonly, at least 8, 9, or 10 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.

[0351] As used herein, “expression” refers to the process by which a polynucleotide is transcribed into mRNA, and / or the process by which transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression can include mRNA splicing in eukaryotic cells.

[0352] "Gene expression" refers to the conversion of information contained in a gene into a gene product. Gene products can be direct transcription products of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozymes, shRNA, microRNA, structural RNA, or any other type of RNA) or proteins produced through the translation of mRNA. Gene products also include RNA modified through processes such as capping, polyadenylation, methylation, and editing, and proteins modified through processes such as methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0353] The "regulation" or "control" of gene expression refers to changes in gene activity. Regulation of expression can include, but is not limited to, gene activation and gene repression.

[0354] The term “operatively linked” or its equivalent (e.g., “linked operatively”) means that two or more molecules are positioned relative to each other such that they can interact to achieve a function attributable to one or two molecules or a combination thereof.

[0355] Non-covalently linked components and methods for preparing and using non-covalently linked components are disclosed. Various components can take various forms as described herein. For example, non-covalently linked (i.e., operatively linked) proteins can be used to allow temporary interactions, which avoid one or more problems in the art. Non-covalently linked components, for example, possess the ability to bind and dissociate proteins, achieving functional binding only or primarily where such binding is required for the desired activity. The bond can have a duration sufficient to allow the desired effect.

[0356] A method for directing proteins to specific loci in the genome of an organism is disclosed. The method may include the steps of providing a DNA positioning component and providing an effector molecule, wherein the DNA positioning component and the effector molecule are operably linked via non-covalent bonding.

[0357] The term "scFv" refers to a single-chain variable fragment. A scFv is a fusion protein consisting of variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin linked by a adaptor peptide. The adaptor peptide can be approximately 5 to 40 amino acids long, or approximately 10 to 30 amino acids long, or approximately 5, 10, 15, 20, 25, 30, 35, or 40 amino acids long. Single-chain variable fragments lack the constant Fc region found in intact antibody molecules and therefore lack common binding sites (such as protein G) used for antibody purification. The term further includes scFvs that are intracellular antibodies (antibodies stable in the cytoplasm of cells) and can bind to intracellular proteins.

[0358] 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 typically peptide chains of about 110 amino acids in length, containing a variable domain (VH) of heavy-chain antibodies or common IgG. They generally have similar affinity for the antigen as intact antibodies, but are more heat-resistant and more stable against detergents and high concentrations of urea. Examples are antibodies derived from camels or fish. Alternatively, single-domain antibodies can be made from common mouse or human IgG, which has four chains.

[0359] 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 specific antigen present in a homogeneous mixture of different antigens. In some respects, specific binding interactions will distinguish between desired and undesired antigens in a sample. In some respects, this binding is more than about 10 to 100 times or more (e.g., more than about 1,000 times or 10,000 times). “Specific” refers to the ability of an immunoglobulin or immunoglobulin fragment, such as a nanobody, to preferentially bind to one antigen target relative to different antigen targets and does not necessarily imply high affinity.

[0360] A "target site" or "target sequence" is a portion of the nucleic acid to which a binding molecule binds, provided that sufficient conditions exist for binding.

[0361] The terms "nucleic acid," "oligonucleotide," or "polynucleotide" refer to at least two nucleotides covalently linked together. The description of a single strand also defines the sequence of its complementary strand. Therefore, nucleic acid can also encompass the complementary strand of the described single strand. Nucleic acid in this disclosure also encompasses substantially equivalent nucleic acids and their complements that retain the same structure or encode the same protein.

[0362] The probes disclosed herein may comprise single-stranded nucleic acids capable of hybridizing with a target sequence under stringent hybridization conditions. Therefore, the nucleic acid in this disclosure may refer to a probe that hybridizes under stringent hybridization conditions.

[0363] The nucleic acids disclosed herein can be single-stranded or double-stranded. Even when the majority of the molecule is single-stranded, the nucleic acids of this disclosure may contain double-stranded sequences. Even when the majority of the molecule is double-stranded, the nucleic acids of this disclosure may contain single-stranded sequences. The nucleic acids of this disclosure may include genomic DNA, cDNA, RNA, or hybrids thereof. The nucleic acids of this disclosure may contain combinations of deoxyribonucleotides and ribonucleotides. The nucleic acids of this disclosure may contain combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. The nucleic acids of this disclosure may be synthesized to include non-natural amino acid modifications. The nucleic acids of this disclosure may be obtained by chemical synthesis or by recombinant methods.

[0364] The nucleic acids disclosed herein, or their entire sequence, or any part thereof, may be non-natural. The nucleic acids disclosed herein may contain one or more mutations, substitutions, deletions, or insertions that are not naturally occurring, resulting in a non-natural nucleic acid sequence. The nucleic acids disclosed herein may contain one or more duplicate, inverted, or repeating sequences, the resulting sequences of which are not naturally occurring, resulting in a non-natural nucleic acid sequence. The nucleic acids disclosed herein may contain modified, artificial, or synthetic nucleotides that are not naturally occurring, resulting in a non-natural nucleic acid sequence.

[0365] Given the redundancy of the genetic code, multiple nucleotide sequences can encode any particular protein. This article considers all such nucleotide sequences.

[0366] 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 positioned 5' (upstream) or 3' (downstream) of the gene it controls. The distance between the promoter and the gene can be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. Variations in the distance between the promoter and the gene can be accommodated without loss of promoter function.

[0367] As used throughout this disclosure, the term "promoter" refers to a synthetic or naturally derived molecule capable of conferring, activating, or enhancing nucleic acid expression in a cell. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance its expression and / or alter spatial and / or temporal expression. A promoter may also contain distal enhancers or repressor elements, which may be located up to several thousand base pairs from the transcription start site. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters may constitutively or differentially regulate the expression of genomic molecules in relation to the cell, tissue, or organ in which expression occurs, or in relation to the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include the bacterial phage T7 promoter, bacterial phage T3 promoter, SP6 promoter, lac operon-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, EF-1 α promoter, CAG promoter, SV40 early promoter or SV40 late promoter and CMV IE promoter.

[0368] As used throughout this disclosure, the term "substantially complementary" means that in 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, the complement of the first sequence to the second sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% equivalent, or that the two sequences hybridize under strict hybridization conditions.

[0369] As used throughout this disclosure, the term "substantially equivalent" means that in 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, the first and second sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% equivalent, or, in terms of nucleic acids, if the complementarities of the first and second sequences are substantially complementary.

[0370] As used throughout this disclosure, when used to describe nucleic acids, the term "variant" means (i) a portion or fragment of a reference nucleotide sequence; (ii) a complement to a portion of a reference nucleotide sequence; (iii) a nucleic acid substantially equivalent to a reference nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions to a reference nucleic acid, its complement, or a sequence substantially equivalent thereto.

[0371] As used throughout this disclosure, the term "vector" refers to a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, a bacterial 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, and is preferably a DNA plasmid. A vector can contain amino acids and a DNA sequence, an RNA sequence, or a combination of both DNA and RNA sequences.

[0372] As used throughout this disclosure, when describing a peptide or polypeptide, the term "variant" refers to a peptide or polypeptide that differs in its amino acid sequence by the insertion, deletion, or conserved substitution of amino acids, but retains at least one biological activity. A variant can also mean a protein having an amino acid sequence that is substantially equivalent to a reference protein having an amino acid sequence that retains at least one biological activity.

[0373] Conservative substitution of amino acids, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is generally considered in the art to involve minor changes. These minor changes can be partially identified by taking into account the hydrophilicity index of the amino acid. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydrophilicity index of an amino acid is based on considerations of its hydrophobicity and charge. Amino acids with similar hydrophilicity indices can be substituted and still retain protein function. In one aspect, amino acids with a hydrophilicity index of ±2 are substituted. The hydrophilicity of an amino acid can also be used to reveal substitutions that will result in the retention of the protein's biological function. Consideration of the hydrophilicity of amino acids in the context of a peptide allows for the calculation of the peptide's maximum local average hydrophilicity, a useful metric reported to be well correlated with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, which is incorporated herein by reference in its entirety.

[0374] Substitution of amino acids with similar hydrophilicity values ​​can result in the retention of biological activity, such as immunogenic peptides. Substitution can be performed using amino acids with hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with this observation, biologically compatible amino acid substitutions are understood to depend on the relative similarity of the amino acids, and especially their side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0375] As used herein, “conservative” amino acid substitutions can be defined as set forth in Tables A, B, or C below. In some respects, fusion polypeptides and / or nucleic acids encoding such fusion polypeptides include conserved substitutions introduced by modifying polynucleotides encoding polypeptides of this disclosure. Amino acids can be classified according to their physical properties and their contribution to secondary and tertiary protein structures. A conserved substitution is the substitution of one amino acid for another amino acid that has similar properties. Exemplary conserved substitutions are set forth in Table A.

[0376] Table A -- Conservative Substitution I Alternatively, as illustrated in Table B, conserved amino acids can be grouped as described in Lehninger, (Biochemistry, 2nd edition; Worth Publishers, Inc. NY, NY (1975), pp. 71-77).

[0377] Table B -- Conservative Substitution II Alternatively, exemplary conservative alternatives are presented in Table C.

[0378] Table C -- Conservative Substitution III Original residues Exemplary replacement Ala (A) Val Leu Ile Met Arg (R) Lys His Asn (N) Gln Asp (D) Glu Cys (C) Ser Thr Gln (Q) Asn Glu (E) Asp Gly (G) Ala Val Leu Pro His (H) Lys Arg Ile (I) Leu Val Met Ala Phe Leu (L) Ile Val Met Ala Phe Lys (K) Arg His Met (M) Leu Ile Val Ala Phe (F) Trp Tyr Ile Pro (P) Gly Ala Val Leu Ile Ser (S) Thr Thr (T) Ser Trp (W) Tyr Phe Ile Tyr (Y) Trp Phe Thr Ser Val (V) Ile Leu Met Ala It should be understood that the polypeptides of this disclosure are intended to include polypeptides with one or more insertions, deletions, or substitutions of amino acid residues, or any combination thereof, as well as polypeptides modified in addition to the insertion, deletion, or substitution of amino acid residues. The polypeptides or nucleic acids of this disclosure may contain one or more conserved substitutions.

[0379] As used throughout this disclosure, the term "more than one" 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 stated amino acid substitutions. The term "more than one" can also refer to 2, 3, 4, or 5 of the stated amino acid substitutions.

[0380] The polypeptides and proteins disclosed herein, or their entire sequence or any part thereof, may be non-natural. The polypeptides and proteins disclosed herein may contain one or more mutations, substitutions, deletions, or insertions that are not naturally occurring, resulting in a non-natural amino acid sequence. The polypeptides and proteins disclosed herein may contain one or more duplicate, inverted, or repeated sequences, the resulting sequences of which are not naturally occurring, resulting in a non-natural amino acid sequence. The polypeptides and proteins disclosed herein may contain modified, artificial, or synthetic amino acids that are not naturally occurring, resulting in a non-natural amino acid sequence.

[0381] As used throughout this disclosure, “sequence identity” can be determined by using a separate executable BLAST engine program to align (blast) two sequences (bl2seq), a program retrieved from the National Center for Biotechnology Information (NCBI) FTP site using default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; which are incorporated herein by reference in their entirety). When used in the context of two or more nucleic acid or polypeptide sequences, the term “equivalent” or “identity” refers to a specified percentage of identical residues in a specified region of each sequence. The percentage can be calculated by: optimally aligning the two sequences, comparing the two sequences in a specified region, determining the number of positions where equivalent residues appear in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. In cases where two sequences have different lengths, or where the alignment produces one or more staggered ends and the specified comparison region includes only a single sequence, the residues of the single sequence are included in the denominator rather than the numerator in the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identification can be performed manually or using computer sequence algorithms such as BLAST or BLAST 2.0.

[0382] 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 the host cell into which it is introduced.

[0383] As used throughout this disclosure, the term “exogenous” refers to a nucleic acid or protein sequence that is not naturally associated with the target gene or the host cell into which it is introduced, including naturally occurring nucleic acids such as non-natural multiple copies of a DNA sequence or naturally occurring nucleic acid sequences located in non-natural genomic locations.

[0384] This disclosure provides a method for introducing a polynucleotide construct comprising a DNA sequence into a host cell. "Introduction" means presenting the polynucleotide construct to the cell in such a manner that the construct gains access to the interior of the host cell. The method of this disclosure does not rely on a specific method for introducing the polynucleotide construct into the host cell, but only on the polynucleotide construct gaining access to the interior of one type of host cell. 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

[0385] Example 1: Generation of humanized MUC1-C scFv antibody and chimeric antigen receptor (CAR) Generate a chimeric antigen receptor (CAR) with an antigen recognition region comprising a single-chain antibody that specifically binds to an epitope of MUC1-C. Figure 1A and 1B ).

[0386] As a preliminary study, MUC1 expression was evaluated in different cell types (Figures 2A and 2B), including K562 cells (immortalized human chronic myeloid leukemia cells), Raji cells (an artificial 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). MUC1 expression in these cell types was evaluated by staining with an anti-MUC1-N antibody. For K562 cells, the staining control peak appeared to the left of the anti-MUC1-N Ab peak. For Raji cells, the staining control peak overlapped with the anti-MUC1-N Ab peak; however, the anti-MUC1-N Ab peak was higher. For activated T cells, the staining control peak appeared to the left of the anti-MUC1-N Ab peak. For RPMI8226 cells, the staining control peak appeared to the left of the anti-MUC1-N Ab peak. MUC-1C was also expressed in experimental groups of different cancer cell lines, including breast cancer (MDA-MB 468), cervical cancer, pancreatic cancer, lung cancer, leukemia, and multiple myeloma. Figure 3 ).

[0387] To generate a humanized MUC1-C scFv CAR, computational (in silico) complementarity-determining region (CDR) transplantation humanization of the variable region of a mouse monoclonal antibody recognizing MUC1-C was performed. Computational modeling of the variable region (both variable heavy (VH) and variable light (VL)) was performed, and the most suitable human VH / VL framework receptors were identified without altering the CDR region; the most suitable human VH and VL framework receptors were IGHV1-69 08 and IGKV6-21 02, respectively. During the humanization process, the mouse CDR was transplanted into the human framework receptor. Residues in the human framework that differed from those in the mouse framework were investigated. Reverse mutations from human residues to mouse residues in the receptor framework were designed if new contacts were generated, if old contacts were lost, if canonical mouse residues were prepared, or if instability within the antigen-binding region was predicted. All obtained heavy chain (4 + 2 variants (H1B and H2B)) and light chain (3) sequences are presented together with their alignments. Figure 5A and 5B ).

[0388] An exemplary mouse CDR region used to generate a humanized MUC1-C scFv CAR contains the following amino acid sequence: 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 is mutated to generate a humanized MUC1-C scFv CAR. This includes CDRH2 containing the amino acid sequence QIYPGDGDTNYNAKFKG (SEQ ID NO: 75).

[0389] An exemplary diagram of a 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 signal transduction (4-1BB) – intracellular signal transduction (CD3ζ).

[0390] Example 2 - Functional analysis of humanized MUC1-C scFv CAR-T cells The MUC1-C candidate CAR was subcloned into the tricistronic piggyBac transposon (EF1α promoter - iC9 safety switch - T2A - MUC1-C CAR - T2A - DHFR selector gene), and CAR-T cells were generated using pan-T cells from normal human donors as described herein. Figure 6 Nineteen days after transposon delivery, expression of each candidate CAR on the surface of piggyBac-modified cells was confirmed by FACS staining using His-tagged p62 / p58 MUC1 protein, followed by anti-His secondary antibody. Figure 6 Specifically, cell surface expression of CARs on simulated transposons (blue) or anti-CD3 / CD28 bead-reactivated (red) cells (activated for 48 hours) was examined by flow cytometry, and the data were displayed as superimposed histograms; the numbers represent the percentage of cells expressing CARs on their surface. All candidate MUC1-C CARs were expressed on the surface of CAR-T cells generated by piggyBac and detected. Figure 7 Table 2 shows the mean fluorescence intensity (MFI) of CAR expression on the surface of inactive and activated T cells.

[0391] Table 2. Surface expression of CAR candidate MUC1-C CAR-T cells.

[0392] As described in this article, candidate MUC1-C CAR-T cells were generated using pan-T cells from healthy human blood donors. CAR+ T cells were subjected to FACS staining for expression of CD45RA, CD45RO, and CD62L on their surface 19 days after transposon delivery to define T cells. 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-). The results are shown in Table 3. All piggyBac-generated candidate MUC1-C CAR-T cell populations were predominantly composed of abnormally high levels of favorable T cells. SCM and T CM Cellular composition.

[0393] Table 3. Proportions of TEFF, TSCM, TCM, and TEM in CD8+ candidate MUC-1C CAR-T cells on day 19. All piggyBac-produced candidate MUC1-C CAR-T cell populations were predominantly composed of high levels of T cells. SCM and T CM Cellular composition.

[0394] As described in this article, candidate MUC1-C CAR-T cells were generated using pan-T cells from normal human blood donors. The CAR-T cells were co-cultured for 24 hours 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, 0.313:1). Figure 8 Reporting molecular signals were measured to determine cytotoxicity. MDA-MB-468 killing is shown for each CAR in the line plot. All CAR+ T cells expressed specific killing of MUC1+ MDA-MB-468. Transposable T cells did not specifically kill target cells (grey dashed line). The area under the curve (AUC) for killing MDA-MB-468 in the E:T range for each CAR, accompanied by SEM (standard error of the mean of triplicate runs), is shown in Table 4. All CAR+ T cells expressed specific killing of MUC1+ MDA-MB-468.

[0395] Table 4. Area under the curve of specific killing of MDA-MB-468 (MUC1+ target cells) by candidate MUC1-C CAR-T cells As described herein, candidate MUC1-C CAR-T cells were generated using pan-T cells from normal human blood donors. The CAR-T cells were co-cultured for 24 hours at a 10:1 E:T ratio with either the Raji cell line (MUC1-; blue bar) or a Raji cell line modified to express the human MUC1 isotype 10 GenBank NP_001191215.1 (MUC1+; red bar), both cell lines expressing green fluorescent protein (GFP). Figure 9Reporting molecular signals were measured to determine cytotoxicity. The bar charts for each CAR show the killing effect for each cell line, and the error bars represent the standard deviation of samples from triplicate runs. All CAR+ T cells expressed specific killing effects against MUC1+ modified Raji cell lines, but not against MUC1-Raji cells. The transposable T cells killed neither Raji nor the modified Raji target cell lines.

[0396] Example 3 - Using a mouse xenograft model, candidate-derived MUC1-C CAR-T cells were synthesized under stress. Preclinical assessment A schematic diagram of the study design for preclinical evaluation of candidate-derived MUC1-C CAR-T cells using a mouse xenograft model at a 'stress' dose is shown below. Figure 10 In the middle. Using 5x10 6 A mouse xenograft model was established by subcutaneous (SC) injection of luciferase-expressing MDA-MB-468.lucGFP (MDA-MB-468) cell lines into female NSG mice at a dose of 4 x 10⁻⁶ cells per cell line. This model was used to evaluate the effects of 'stress' at doses (4 x 10⁻⁶ cells per cell line). 6 The in vivo antitumor efficacy of candidate MUC1-C CAR-T cells was evaluated. For this study, the experimental group containing total candidate CAR-T cells was selected. 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 via the axilla and tumor establishment (100–200 mm as measured by calipers) was observed. 3 Treatment was administered at the specified time. Table 5 shows the preclinical evaluation of candidate-derived MUC1-C CAR-T cells at 'stress' doses using a mouse xenograft model. Tumor volume was evaluated according to caliper measurements for all treated animals, standardized against measurements in the untreated group. Table 5 shows tumor volume according to caliper measurements in both the vector and treatment groups.

[0397] Table 5. Tumor volume as measured by area under the curve (AUC) after treatment with candidate MUC1-C CAR-T cells. Mice were administered an IV dose of 'stress' (4 x 10) of the drug. 6 The candidate P-MUC1-C-101 CAR-T was used for treatment to detect potential efficacy and functional differences among different CAR candidates at a higher resolution. Total T cells in the blood of both the vector and treated mice were measured by TruCount staining. The area under the curve (AUC) of T cells in the blood of both the vector and treated mice was determined by blood sampling, and the results are shown in Table 6.

[0398] Table 6. Area under the curve of T cell (hCD45+) proliferation in the blood after treatment with the MUC1-C CART-T cell candidate.

[0399] The CD8+ T cell phenotypes of the vector and treated mice were determined. Blood CD8+ T cell phenotypes were measured for all animals by FACS staining, and percentages are listed as group means, with error bars as SEM. Cells were stained for expression of surface CD45RA, CD45RO, and CD62L to define T cell phenotypes. 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, T cells were observed on day 1, day 12, and day 19 before infusion. SCM and T CM Phenotypic ratio T EM and T EFF The phenotypic diversity is greater. sequence list <110> Posida Therapy Company <120> Anti-MUC1 Compositions and Methods of Use <130> POTH-040 / 001WO 325002-2400 <150> 62 / 951,257 <151> 2019-12-20 <160> 181 <170> PatentIn version 3.5 <210> 1 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (20)..(20) <223> X is Val or Ile <220> <221> MISC_FEATURE <222> (38)..(38) <223> X is either Arg or Lys <220> <221> MISC_FEATURE <222> (40)..(40) <223> X is either Ala or Arg <220> <221> MISC_FEATURE <222> (62)..(62) <223> X is Gly or Ala <220> <221> MISC_FEATURE <222> (68)..(68) <223> X is Val or Ala <220> <221> MISC_FEATURE <222> (76)..(76) <223> X is Thr or Ser <220> <221> MISC_FEATURE <222> (90)..(90) <223> X is Asp or Ala <400> 1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Xaa Ser Cys Lys Thr Ser Gly Tyr Ala Phe Ser Asn Phe 20 25 30 Trp Met Asn Trp Val Xaa Gln Xaa Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Xaa Lys Phe 50 55 60 Lys Gly Arg Xaa Thr Leu Thr Ala Asp Lys Ser Xaa Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Xaa Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ser Tyr Tyr Arg Ser Ala Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 2 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (74)..(74) <223> X is Thr or Ser <220> <221> MISC_FEATURE <222> (78)..(78) <223> X is Leu or Val <220> <221> MISC_FEATURE <222> (85)..(85) <223> X is Thr or Asp <400> 2 Glu Ile Leu Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Phe Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Ser 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Asn Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Xaa Ile Asn Ser Xaa Glu Ser 65 70 75 80 Glu Asp Ile Ala Xaa Tyr Tyr Cys Gln Gln Ser Asn Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 3 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide - "H1" humanized heavy chain variable region <400> 3 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Ser Gly Tyr Ala Phe Ser Asn Phe 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ser Tyr Tyr Arg Ser Ala Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 4 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide - "H1B" humanized heavy chain variable region <400> 4 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Ser Gly Tyr Ala Phe Ser Asn Phe 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Ala Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ser Tyr Tyr Arg Ser Ala Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 5 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide - "H2" humanized heavy chain variable region <400> 5 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Ser Gly Tyr Ala Phe Ser Asn Phe 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Ala Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ser Tyr Tyr Arg Ser Ala Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 6 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide - "H2B" humanized heavy chain variable region <400> 6 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Ser Gly Tyr Ala Phe Ser Asn Phe 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Ala Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Ala Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ser Tyr Tyr Arg Ser Ala Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 7 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide - "H3" humanized heavy chain variable region <400> 7 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Ser Gly Tyr Ala Phe Ser Asn Phe 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Ala Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ser Tyr Tyr Arg Ser Ala Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 8 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide - "H4" humanized heavy chain variable region <400> 8 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Ser Gly Tyr Ala Phe Ser Asn Phe 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Ala Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ser Tyr Tyr Arg Ser Ala Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 9 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide - "L1" humanized light chain variable region <400> 9 Glu Ile Leu Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Phe Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Ser 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Asn Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ser 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 10 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide - "L2" humanized light chain variable region <400> 10 Glu Ile Leu Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Phe Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Ser 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Asn Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser Asn Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 11 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide - "L3" humanized light chain variable region <400> 11 Glu Ile Leu Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Phe Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Ser 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Asn Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser Asn Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 12 <211> 485 <212> PRT <213> artificial sequence <220> <223> Synthesis - "L1H1" CAR <400> 12 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Ile Leu Leu Thr Gln Ser Pro Asp Phe Gln 20 25 30 Ser Val Thr Pro Lys Glu Lys Val Thr Phe Thr Cys Arg Ala Ser Gln 35 40 45 Ser Ile Gly Thr Ser Ile His Trp Tyr Gln Gln Lys Pro Asn Gln Ser 50 55 60 Pro Lys Leu Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95 Asn Ser Leu Glu Ser Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser 100 105 110 Asn Asn Trp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 130 135 140 Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser 145 150 155 160 Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Ala Phe Ser Asn Phe Trp 165 170 175 Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly 180 185 190 Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys 195 200 205 Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met 210 215 220 Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys Ala 225 230 235 240 Arg Ser Tyr Tyr Arg Ser Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 245 250 255 Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr 260 265 270 Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala 275 280 285 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe 290 295 300 Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val 305 310 315 320 Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys 325 330 335 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 340 345 350 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 355 360 365 Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 370 375 380 Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 385 390 395 400 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 405 410 415 Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 420 425 430 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 435 440 445 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 450 455 460 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 465 470 475 480 Ala Leu Pro Pro Arg 485 <210> 13 <211> 485 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide - "L1H1B" CAR <400> 13 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Ile Leu Leu Thr Gln Ser Pro Asp Phe Gln 20 25 30 Ser Val Thr Pro Lys Glu Lys Val Thr Phe Thr Cys Arg Ala Ser Gln 35 40 45 Ser Ile Gly Thr Ser Ile His Trp Tyr Gln Gln Lys Pro Asn Gln Ser 50 55 60 Pro Lys Leu Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95 Asn Ser Leu Glu Ser Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser 100 105 110 Asn Asn Trp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 130 135 140 Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser 145 150 155 160 Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Ala Phe Ser Asn Phe Trp 165 170 175 Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly 180 185 190 Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Ala Lys Phe Lys 195 200 205 Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met 210 215 220 Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys Ala 225 230 235 240 Arg Ser Tyr Tyr Arg Ser Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 245 250 255 Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr 260 265 270 Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala 275 280 285 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe 290 295 300 Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val 305 310 315 320 Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys 325 330 335 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 340 345 350 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 355 360 365 Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 370 375 380 Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 385 390 395 400 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 405 410 415 Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 420 425 430 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 435 440 445 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 450 455 460 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 465 470 475 480 Ala Leo Pro Pro Arg 485 <210> 14 <211> 485 <212> PRT <213> artificial sequence <220> <223> Synthesis - "L1H2" CAR <400> 14 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Ile Leu Leu Thr Gln Ser Pro Asp Phe Gln 20 25 30 Ser Val Thr Pro Lys Glu Lys Val Thr Phe Thr Cys Arg Ala Ser Gln 35 40 45 Ser Ile Gly Thr Ser Ile His Trp Tyr Gln Gln Lys Pro Asn Gln Ser 50 55 60 Pro Lys Leu Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95 Asn Ser Leu Glu Ser Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser 100 105 110 Asn Asn Trp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 130 135 140 Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser 145 150 155 160 Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Ala Phe Ser Asn Phe Trp 165 170 175 Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly 180 185 190 Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys 195 200 205 Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met 210 215 220 Glu Leu Ser Ser Leu Arg Ser Glu Ala Thr Ala Val Tyr Phe Cys Ala 225 230 235 240 Arg Ser Tyr Tyr Arg Ser Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 245 250 255 Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr 260 265 270 Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala 275 280 285 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe 290 295 300 Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val 305 310 315 320 Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys 325 330 335 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 340 345 350 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 355 360 365 Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 370 375 380 Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 385 390 395 400 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 405 410 415 Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 420 425 430 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 435 440 445 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 450 455 460 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 465 470 475 480 Ala Leo Pro Pro Arg 485 <210> 15 <211> 485 <212> PRT <213> artificial sequence <220> <223> Synthesis - "L1H2B" CAR <400> 15 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Ile Leu Leu Thr Gln Ser Pro Asp Phe Gln 20 25 30 Ser Val Thr Pro Lys Glu Lys Val Thr Phe Thr Cys Arg Ala Ser Gln 35 40 45 Ser Ile Gly Thr Ser Ile His Trp Tyr Gln Gln Lys Pro Asn Gln Ser 50 55 60 Pro Lys Leu Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95 Asn Ser Leu Glu Ser Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser 100 105 110 Asn Asn Trp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 130 135 140 Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser 145 150 155 160 Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Ala Phe Ser Asn Phe Trp 165 170 175 Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly 180 185 190 Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Ala Lys Phe Lys 195 200 205 Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met 210 215 220 Glu Leu Ser Ser Leu Arg Ser Glu Ala Thr Ala Val Tyr Phe Cys Ala 225 230 235 240 Arg Ser Tyr Tyr Arg Ser Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 245 250 255 Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr 260 265 270 Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala 275 280 285 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe 290 295 300 Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val 305 310 315 320 Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys 325 330 335 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 340 345 350 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 355 360 365 Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 370 375 380 Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 385 390 395 400 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 405 410 415 Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 420 425 430 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 435 440 445 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 450 455 460 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 465 470 475 480 Ala Leu Pro Pro Arg 485 <210> 16 <211> 485 <212> PRT <213> artificial sequence <220> <223> Synthetic peptide - "L1H3" CAR <400> 16 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Ile Leu Leu Thr Gln Ser Pro Asp Phe Gln 20 25 30 Ser Val Thr Pro Lys Glu Lys Val Thr Phe Thr Cys Arg Ala Ser Gln 35 40 45 Ser Ile Gly Thr Ser Ile His Trp Tyr Gln Gln Lys Pro Asn Gln Ser 50 55 60 Pro Lys Leu Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95 Asn Ser Leu Glu Ser Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser 100 105 110 Asn Asn Trp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 130 135 140 Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser 145 150 155 160 Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Ala Phe Ser Asn Phe Trp 165 170 175 Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 180 185 190 Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys 195 200 205 Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met 210 215 220 Glu Leu Ser Ser Leu Arg Ser Glu Ala Thr Ala Val Tyr Phe Cys Ala 225 230 235 240 Arg Ser Tyr Tyr Arg Ser Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr 245 250 255 Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr 260 265 270 Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala 275 280 285 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe 290 295 300 Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val 305 310 315 320 Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys 325 330 335 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 340 345 350 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 355 360 365 Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 370 375 380 Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 385 390 395 400 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 405 410 415 Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 420 425 430 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 435 440 445 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 450 455 460 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 465 470 475 480 Ala Leu Pro Pro Arg 485 <210> 17 <211> 485 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide - "L1H4" CAR <400> 17 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Ile Leu Leu Thr Gln Ser Pro Asp Phe Gln 20 25 30 Ser Val Thr Pro Lys Glu Lys Val Thr Phe Thr Cys Arg Ala Ser Gln 35 40 45 Ser Ile Gly Thr Ser Ile His Trp Tyr Gln Gln Lys Pro Asn Gln Ser 50 55 60 Pro Lys Leu Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95 Asn Ser Leu Glu Ser Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser 100 105 110 Asn Asn Trp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 130 135 140 Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser 145 150 155 160 Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Ala Phe Ser Asn Phe Trp 165 170 175 Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 180 185 190 Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys 195 200 205 Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser T...

Claims

1. A chimeric antigen receptor (CAR) comprising: (a) An extracellular domain containing an antigen recognition region, wherein the antigen recognition region contains at least one anti-MUC1 single-stranded variable fragment (scFv); (b) Transmembrane domains, and (c) An intracellular domain containing at least one co-stimulatory domain; The scFv contains a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 4; The light chain variable region contains the amino acid sequence of SEQ ID NO:

9.

2. The CAR of claim 1, wherein scFv comprises a joint between the heavy chain variable region and the light chain variable region.

3. The CAR of claim 2, wherein the adapter comprises the amino acid sequence of SEQ ID NO:

59.

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

125.

5. The CAR of claim 1, wherein the extracellular domain further comprises a signal peptide.

6. The CAR of claim 5, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO:

57.

7. The CAR of any one of claims 1-6, wherein the CAR further comprises a hinge region between the antigen recognition region and the transmembrane structural domain.

8. The CAR of claim 7, wherein the hinge region comprises the amino acid sequence of SEQ ID NO:

61.

9. The CAR of claim 1, wherein the transmembrane domain comprises a sequence encoding a CD8 transmembrane domain.

10. The CAR of claim 9, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO:

63.

11. The CAR of claim 1, wherein the intracellular domain comprises the CD3ζ intracellular signal transduction domain, the 4-1BB co-stimulatory domain, or a combination thereof.

12. The CAR of claim 1, wherein the intracellular domain comprises a CD3ζ intracellular signal transduction domain and a 4-1BB co-stimulatory domain, and wherein the 4-1BB co-stimulatory domain is located between the transmembrane domain and the CD3ζ intracellular signal transduction domain.

13. The CAR of claim 12, wherein the 4-1BB co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:

65.

14. The CAR of claim 12, wherein the CD3ζ intracellular signal transduction domain comprises the amino acid sequence of SEQ ID NO:

67.

15. The CAR of claim 1, wherein The scFv contains a joint between the heavy chain variable region and the light chain variable region. The extracellular domain contains a signal peptide. The CAR further includes a hinge region between the antigen recognition region and the transmembrane structural domain. The transmembrane domain includes a sequence containing a CD8 transmembrane domain; and The intracellular domain comprises a CD3ζ intracellular signal transduction domain and a 4-1BB co-stimulatory domain, wherein the 4-1BB co-stimulatory domain is located between the transmembrane domain and the CD3ζ intracellular signal transduction domain.

16. The CAR of claim 15, wherein The scFv contains the amino acid sequence of SEQ ID NO: 125; The signal peptide described herein comprises SEQ ID NO: 57; The hinge region therein includes SEQ ID NO: 61; The CD8 transmembrane domain includes SEQ ID NO: 63; The 4-1BB co-stimulatory domain includes SEQ ID NO: 65; and The CD3ζ intracellular signal transduction domain described therein includes SEQ ID NO:

67.

17. The CAR of claim 15, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

13.

18. The CAR of claim 15, wherein the amino acid sequence of the CAR is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 31 or SEQ ID NO:

167.

19. The CAR of claim 18, wherein the amino acid sequence of the CAR is encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:

167.

20. A polynucleotide comprising a nucleic acid sequence encoding the CAR of claim 1 or the CAR of claim 15.

21. A transposon comprising a nucleic acid encoding the CAR of claim 1 or the CAR of claim 15.

22. The transposon of claim 21, wherein the nucleic acid encodes a CAR comprising the amino acid sequence of SEQ ID NO:

13.

23. The transposon of claim 21, wherein the transposon further comprises a nucleic acid encoding an inducible caspase polypeptide, a nucleic acid encoding a selection gene, or a combination thereof.

24. The transposon of claim 23, wherein the selection gene comprises a DHFR resistance gene.

25. The transposon of claim 21, wherein the transposon is a piggyBac transposon.

26. The transposon of claim 21, wherein the transposon comprises the nucleic acid sequence of SEQ ID NO:

172.

27. A vector comprising the polynucleotide of claim 20.

28. A carrier comprising the transposon of claim 21.

29. A non-plant cell comprising the CAR of claim 1.

30. A non-plant cell comprising the transposon of claim 21.

31. A non-plant cell population wherein multiple cells are modified to express the CAR of claim 1.

32. The non-plant cell population of claim 31, wherein the plurality of modified cells are plurality of modified immune cells.

33. The non-plant cell population of claim 31, wherein the plurality of modified cells are plurality of modified T cells.

34. The non-plant cell population of claim 31, wherein the plurality of cells comprises at least 75% cells expressing the CAR of claim 1.

35. The non-plant cell population of claim 34, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

13.

36. The non-plant cell population of claim 33, wherein at least 50% of the plurality of modified T cells express one or more cell surface markers comprising CD45RA and CD62L, and do not express one or more cell surface markers comprising CD45RO.

37. A composition comprising the CAR of claim 1.

38. A composition comprising the cells of claim 29.

39. A composition comprising the cell population of claim 31.

40. A pharmaceutical composition comprising the composition of any one of claims 37-39 and a pharmaceutically acceptable carrier.

41. Use of the composition of any one of claims 38-39 in the preparation of a medicament for treating cancer in a subject in need, wherein said 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, or esophageal cancer.

42. Use of a pharmaceutical composition comprising the composition of claim 38 or 39 and a pharmaceutically acceptable carrier in the preparation of a medicament for treating cancer in a subject of need, wherein said 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, or esophageal cancer.

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