Cancer vaccines and methods of use thereof

Abstract

The present disclosure provides modified human cancer cells that express exogenous human leukocyte antigen (HLA) alleles.The present disclosure also provides expression vectors for the co-expression of one or more HLA alleles.A method is provided for using the modified human cancer cells of the present disclosure as a whole cell cancer vaccine for treating cancer in a subject. TIFF2025507939000062.tif85159

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 316,251, filed March 3, 2022, and U.S. Provisional Application No. 63 / 415,475, filed October 12, 2022, the disclosures of which are incorporated by reference in their entireties herein for all purposes. [Background technology]

[0002] background Generating an effective immune response is essential for cancer immunotherapy as well as vaccine development. Adoptive immune responses begin with antigen presentation by professional antigen-presenting cells (APCs). These cells typically express class II major histocompatibility complex (MHC) proteins along with costimulatory molecules such as CD80 and CD86. It is generally assumed that to be effective, whole-cell immunotherapies must express the immunogenic antigens that are the target of the immune response, and that APCs such as dendritic cells (DCs) must present such antigens to T cells after uptake of the immunogenic proteins. Thus, there is also a need for improved active immunotherapies for cancer and related diseases. The present disclosure meets these needs and provides related advantages as well. Summary of the Invention

[0003] overview In one aspect, the disclosure provides modified human cancer cells comprising: (a) one or more recombinant polynucleotides each encoding an allele of a human leukocyte antigen (HLA) class I gene; and (b) one or more recombinant polynucleotides each encoding an allele of an HLA class II gene, wherein one or more HLA alleles endogenous to the cell are inactivated.

[0004] In a related aspect, the disclosure provides modified human cancer cells that express class I and II MHC proteins, wherein the modified human cancer cells comprise: (a) one or more recombinant polynucleotides that each encode an allele of an HLA class I gene (e.g., for expression of a recombinant class I MHC protein in the cell); and (b) one or more recombinant polynucleotides that each encode an allele of an HLA class II gene (e.g., for expression of a recombinant class II MHC protein in the cell), wherein one or more HLA alleles endogenous to the cell are inactivated.

[0005] In some embodiments, the modified human cancer cells comprise (a) a first recombinant polynucleotide encoding a first allele of an HLA class I gene and a second recombinant polynucleotide encoding a second allele of an HLA class I gene, or (a) a first recombinant polynucleotide encoding a first allele of an HLA class I gene and a second recombinant polynucleotide encoding an allele of a second HLA class I gene. In some embodiments, the modified human cancer cells further comprise (b) a first recombinant polynucleotide encoding a first allele of an HLA class II gene and a second recombinant polynucleotide encoding an allele of a second HLA class II gene, or (b) a first recombinant polynucleotide encoding a first allele of an HLA class II gene and a second recombinant polynucleotide encoding a second allele of an HLA class II gene.

[0006] In some embodiments, the HLA class I genes include an HLA-A gene, an HLA-B gene, an HLA-C gene, an HLA-E gene, an HLA-F gene, an HLA-G gene, a beta-2-microglobulin (B2M) gene, or a combination thereof. In some embodiments, alleles of the HLA-A gene include an HLA-A*01:01 allele, an HLA-A*02:01 allele, an HLA-A*02:06 allele, an HLA-A*03:01 allele, an HLA-A*11:01 allele, an HLA-A*23:01 allele, an HLA-A*24:02 allele, an HLA-A*26:01 allele, an HLA-A*29:02 allele, an HLA-A*31:01 allele, an HLA-A*32:01 allele, an HLA-A*33:03 allele, an HLA-A*68:01 allele, or a combination thereof.

[0007] In some embodiments, the alleles of the HLA-A gene include (i) an HLA-A*01:01 allele and an HLA-A*68:01 allele; (ii) an HLA-A*02:01 allele and an HLA-A*11:01 allele; (iii) an HLA-A*03:01 allele and an HLA-A*23:01 allele; or (iv) an HLA-A*33:03 allele and, optionally, an HLA-A*24:02 allele.

[0008] In various embodiments, the HLA class II genes include HLA class II alpha subunit genes, HLA class II beta subunit genes, or combinations thereof. In some embodiments, the HLA class II genes include HLA-DP genes, HLA-DM genes, HLA-DO genes, HLA-DQ genes, HLA-DR genes, or combinations thereof. In some embodiments, the HLA-DM genes include HLA-DMA genes, HLA-DMB genes, or combinations thereof. In some embodiments, the HLA-DR genes include HLA-DRA genes, HLA-DRB1 genes, HLA-DRB3 genes, HLA-DRB4 genes, HLA-DRB5 genes, or combinations thereof.

[0009] In some embodiments, the HLA-DR gene is an HLA-DRB3 gene comprising an HLA-DRB3*01:01 allele, an HLA-DRB3*02:02 allele, an HLA-DRB3*03:01 allele, or a combination thereof. In some embodiments, the HLA-DR gene is an HLA-DRB4 gene comprising an HLA-DRB4*01:01 allele, an HLA-DRB4*01:03 allele, or a combination thereof. In some embodiments, the HLA-DR gene is an HLA-DRB5 gene comprising an HLA-DRB5*01:01 allele, an HLA-DRB5*01:02 allele, an HLA-DRB5*02:02 allele, or a combination thereof.

[0010] In certain embodiments, the alleles of the HLA-DR gene include (i) an HLA-DRB3*02:02 allele and an HLA-DRB5*01:01 allele; (ii) an HLA-DRB4*01:01 allele and an HLA-DRB3*01:01 allele; (iii) an HLA-DRB3*03:01 allele and an HLA-DRB5*01:02 allele; or (iv) an HLA-DRB5*02:02 allele and an HLA-DRB3*01:01 allele.

[0011] In certain other embodiments, the alleles of the HLA-DR gene include (i) an HLA-DRB3*02:02 allele and an HLA-DRB4*01:01 allele; (ii) an HLA-DRB4*01:01 allele and an HLA-DRB3*03:01 allele; (iii) an HLA-DRB3*01:01 allele and an HLA-DRB5*01:02 allele; or (iv) an HLA-DRB5*01:01 allele and an HLA-DRB5*02:02 allele.

[0012] In certain embodiments, the modified human cancer cells comprise one or more recombinant polynucleotides encoding alleles of a human leukocyte antigen (HLA) class I gene that have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity, respectively, to the polynucleotide sequence of any one of SEQ ID NOs: 22-29. In certain embodiments, the modified human cancer cells comprise one or more recombinant polynucleotides encoding alleles of a human leukocyte antigen (HLA) class II gene that have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity, respectively, to the polynucleotide sequence of any one of SEQ ID NOs: 30-35. In certain embodiments, the modified human cancer cells comprise (a) one or more recombinant polynucleotides encoding alleles of a human leukocyte antigen (HLA) class I gene having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polynucleotide sequence of any one of SEQ ID NOs:22-29, respectively, and (b) one or more recombinant polynucleotides encoding alleles of a human leukocyte antigen (HLA) class II gene having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the polynucleotide sequence of any one of SEQ ID NOs:30-35, respectively.

[0013] In certain embodiments, the modified human cancer cells comprise one or more recombinant polynucleotides encoding each of the alleles of a human leukocyte antigen (HLA) class I gene having a polynucleotide sequence of any one of SEQ ID NOs:22-29. In certain embodiments, the modified human cancer cells comprise one or more recombinant polynucleotides encoding each of the alleles of a human leukocyte antigen (HLA) class II gene having a polynucleotide sequence of any one of SEQ ID NOs:30-35. In certain embodiments, the modified human cancer cells comprise (a) one or more recombinant polynucleotides encoding each of the alleles of a human leukocyte antigen (HLA) class I gene having a polynucleotide sequence of any one of SEQ ID NOs:22-29, and (b) one or more recombinant polynucleotides encoding each of the alleles of a human leukocyte antigen (HLA) class II gene having a polynucleotide sequence of any one of SEQ ID NOs:30-35.

[0014] In another aspect, the present disclosure provides a recombinant polynucleotide encoding at least one of: (a) an HLA-A*01:01 allele, an HLA-A*68:01 allele, an HLA-DRB3*02:02 allele, and an HLA-DRB5*01:01 allele; (b) an recombinant polynucleotide encoding at least one of an HLA-A*02:01 allele, an HLA-A*11:01 allele, an HLA-DRB4*01:01 allele, and an HLA-DRB3*01:01 allele; (c) an HLA-A*03:01 allele, an HLA-A*2 and / or (d) a recombinant polynucleotide encoding at least one of an HLA-A*24:02 allele, an HLA-A*33:03 allele, an HLA-DRB5*02:02 allele, and an HLA-DRB3*01:01 allele, wherein one or more HLA alleles endogenous to the cell are inactivated. In some embodiments, the modified human cancer cells comprise: (a) a recombinant polynucleotide encoding an HLA-A*01:01 allele, an HLA-A*68:01 allele, an HLA-DRB3*02:02 allele, and an HLA-DRB5*01:01 allele; (b) a recombinant polynucleotide encoding an HLA-A*02:01 allele, an HLA-A*11:01 allele, an HLA-DRB4*01:01 allele, and an HLA-DRB3*01:01 allele; (c) a recombinant polynucleotide encoding an HLA-A*03:01 allele. and / or (d) a recombinant polynucleotide encoding an HLA-A*24:02 allele, an HLA-A*33:03 allele, an HLA-DRB5*02:02 allele, and an HLA-DRB3*01:01 allele, wherein one or more HLA alleles endogenous to the cell are inactivated.In certain embodiments, the recombinant polynucleotide comprises one or more codon-optimized sequences encoding the alleles described herein (eg, SEQ ID NOs:22-35).

[0015] In yet another aspect, the disclosure provides a recombinant polynucleotide encoding at least one of: (a) an HLA-A*01:01 allele, an HLA-A*68:01 allele, an HLA-DRB3*02:02 allele, and an HLA-DRB4*01:01 allele; (b) an recombinant polynucleotide encoding at least one of an HLA-A*02:01 allele, an HLA-A*11:01 allele, an HLA-DRB4*01:01 allele, and an HLA-DRB3*03:01 allele; (c) an recombinant polynucleotide encoding at least one of an HLA-A*03:01 allele, and an HLA-A*03:01 allele. and / or (d) a recombinant polynucleotide encoding at least one of an HLA-A*33:03 allele, an HLA-DRB5*01:01 allele, and an HLA-DRB5*02:02 allele, wherein one or more HLA alleles endogenous to the cell are inactivated. In some embodiments, the modified human cancer cells comprise: (a) a recombinant polynucleotide encoding an HLA-A*01:01 allele, an HLA-A*68:01 allele, an HLA-DRB3*02:02 allele, and an HLA-DRB4*01:01 allele; (b) a recombinant polynucleotide encoding an HLA-A*02:01 allele, an HLA-A*11:01 allele, an HLA-DRB4*01:01 allele, and an HLA-DRB3*03:01 allele; (c) a recombinant polynucleotide encoding an HLA-A* and / or (d) a recombinant polynucleotide encoding an HLA-A*33:03 allele, an HLA-DRB5*01:01 allele, and an HLA-DRB5*02:02 allele, wherein one or more HLA alleles endogenous to the cell are inactivated. The modified human cancer cell of (d) may further comprise an HLA-A*24:02 allele.In certain embodiments, the recombinant polynucleotide comprises one or more codon-optimized sequences encoding the alleles described herein (eg, SEQ ID NOs:22-35).

[0016] In various embodiments, the modified human cancer cells further comprise a recombinant polynucleotide encoding a cytokine. In some embodiments, the cytokine comprises a chemokine, an interferon, an interleukin, a tumor necrosis factor, or a combination thereof. In some embodiments, the recombinant polynucleotide encoding a cytokine is an early T cell activation antigen-1 (ETA-1), lymphocyte activating factor (LAF), an interleukin-1 family member (IL-1α, IL-β, IL-1Ra, IL-18, IL-33, IL-36Ra, IL-36α, IL-36β, IL-36γ, IL-37, IL-38), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), or a combination thereof. 5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-19 (IL-20), interleukin-21 (IL-22), interleukin-23 (IL-24), interleukin-25 (IL-26), interleukin-27 (IL-28), interleukin-29 (IL-29), interleukin-30 (IL-30), interleukin-31 (IL-31), interleukin-32 (IL-32), interleukin-33 (IL-33), interleukin-34 (IL-34), interleukin-35 (IL-35), interleukin-36 (IL-36), interleukin-37 (IL-37), interleukin-38 (IL-38), interleukin-39 (IL-39), interleukin-40 (IL-39), interleukin-41 (IL-39), interleukin-42 (IL-39), interleukin-43 (IL-39), interleukin-44 (IL-39), interleukin-45 (IL-39), interleukin-46 (IL-39), interleukin-47 (IL-39), interleukin-48 (IL-39), interleukin-49 (IL-39), interleukin-49 (IL-39), interleukin-41 (IL-39), interleukin-42 (IL-39), interleukin-43 (IL Interleukin-21 (IL-21), interleukin-23 (IL-23), interleukin-25 (IL-25), interleukin-33 (IL-33), interferon alpha (IFN-α), interferon lambda 1 (IFNλ1 (IL-29)), interferon lambda 2 (IFNλ2 (IL-28A)), interferon lambda 3 (IFNλ3 (IL-28B)), interferon lambda 4 (IFNλ 4), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage CSF (CSF-1), macrophage migration inhibitory factor (MIF), CD40L molecule (CD40L), RANTES molecule (RANTES), monocyte chemotactic protein (MCP-1), monocyte inflammatory proteins (MIP-1α, MIP-1β), lymphotactin, fractalkine, or a combination thereof. In some embodiments, the cytokine comprises granulocyte macrophage colony stimulating factor (GM-CSF).In some embodiments, the cytokine comprises interferon alpha (IFN-α), such as IFN-α2a or IFN-α2b. In some embodiments, the cytokine comprises GM-CSF and (IFN-α), preferably IFN-α2a or IFN-α2b. In some embodiments, the IFN-α is an exogenous protein comprising pegylated IFN-α2a.

[0017] In some embodiments, the cytokine comprises granulocyte macrophage colony stimulating factor (GM-CSF) encoded by a polynucleotide having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:39. In some embodiments, the cytokine comprises interferon alpha (IFN-α) encoded by a polynucleotide having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:40. In some embodiments, the cytokine comprises GM-CSF and IFN-α, such as IFN-α2a or IFN-α2b. In some embodiments, the recombinant polynucleotide encoding CM-CSF comprises SEQ ID NO:39. In some embodiments, the recombinant polynucleotide encoding IFN-α comprises SEQ ID NO:40. In some embodiments, the cytokine comprises interleukin-7 (IL-7). In some embodiments, the cytokine comprises IL-7 encoded by a polynucleotide having at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:41. In some embodiments, the recombinant polynucleotide encoding IL-7 comprises SEQ ID NO:41. In some embodiments, the cytokine comprises IL-7 encoded by a polynucleotide having at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO:42. In some embodiments, the recombinant polynucleotide encoding IL-7 comprises SEQ ID NO:42. In some embodiments, the cytokine comprises interleukin-12 (IL-12). In some embodiments, the cytokine comprises a bioactive form of IL-12 containing subunits p40 and p35.In some embodiments, the cytokine comprises IL-12 encoded by a polynucleotide having at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 44. In some embodiments, the recombinant polynucleotide encoding IL-12 comprises SEQ ID NO:44.

[0018] In various embodiments, the modified human cancer cells further comprise a recombinant polynucleotide encoding a costimulatory molecule. In some embodiments, the recombinant polynucleotide encoding the costimulatory molecule comprises a recombinant polynucleotide encoding a CD86 molecule (CD86), a CD80 molecule (CD80), a 4-1BB ligand molecule (also known as 4-1BBL, TNFSF9, or CD137L), an ICOS ligand molecule (ICOS-L), a CD70 molecule (CD70, also known as CD27L), a CD40 molecule (CD40), an OX40 ligand molecule (OX40L), a GITR ligand molecule (GITRL), a TIM-4 molecule (TIM-4), a LIGHT molecule (LIGHT), an ICAM1 molecule (ICAM1), an LFA3 molecule (LFA3), a CD30 molecule (CD30), or a combination thereof.

[0019] In some aspects, the modified human cancer cells further comprise a recombinant polynucleotide encoding an antigen of a pathogen, a tumor-associated antigen, a neoantigen, an allergen, an antigen that is a target of an autoimmune response, a fragment thereof, or a combination thereof.

[0020] In some embodiments, one or more HLA-A and / or HLA-DR alleles endogenous to the modified human cancer cells are inactivated. In some embodiments, the one or more HLA-A alleles comprise HLA-A*24:02 allele and / or HLA-A*11:01 allele. In some embodiments, the one or more HLA-DR alleles comprise one or more HLA-DRB3 alleles. In some embodiments, the one or more HLA-DRB3 alleles comprise HLA-DRB3*01:01 allele and / or HLA-DRB3*02:02 allele.

[0021] In any of the above embodiments, one or more of the recombinant polynucleotides present in the modified human cancer cell are present in a vector in the cell. In some embodiments, one or more recombinant polynucleotides encoding each of the alleles of the HLA class I gene and one or more recombinant polynucleotides encoding each of the alleles of the HLA class II gene are present in the same vector in the cell. In some embodiments, the vector comprises two recombinant polynucleotides encoding each of the alleles of the HLA class I gene. In other embodiments, the vector comprises two recombinant polynucleotides encoding each of the alleles of the HLA class II gene. In other embodiments, the vector comprises two recombinant polynucleotides encoding each of the alleles of the HLA class I gene and two recombinant polynucleotides encoding each of the alleles of the HLA class II gene. In still other embodiments, one or more recombinant polynucleotides encoding each of the alleles of the HLA class I gene and one or more recombinant polynucleotides encoding each of the alleles of the HLA class II gene are present in separate vectors in the cell. In yet other embodiments, the one or more recombinant polynucleotides encoding each of the two alleles of an HLA class I gene and the one or more recombinant polynucleotides encoding each of the two alleles of an HLA class II gene are present on separate vectors within the cell.

[0022] In some embodiments, one or more of the recombinant polynucleotides present in the modified human cancer cells comprise a sequence having at least 85% identity to any one of SEQ ID NOs:22-35. In some embodiments, one or more of the recombinant polynucleotides present in the modified human cancer cells comprise a sequence that encodes a polypeptide having at least 85% identity to a polypeptide encoded by any one of SEQ ID NOs:22-35. In some embodiments, one or more of the recombinant polynucleotides present in the modified human cancer cells comprise a sequence of any one of SEQ ID NOs:22-35.

[0023] In some embodiments, the human cancer cell is a human cancer cell line. In certain embodiments, the human cancer cell line is a breast cancer (e.g., SV-BR-1), prostate cancer (e.g., PC-3, LNCaP), melanoma (e.g., SK-MEL-24), or lung cancer (e.g., NCI-H2228) cell line.

[0024] In various embodiments, the one or more recombinant polynucleotides present in the modified human cancer cells each comprise a heterologous polynucleotide sequence between a polynucleotide sequence encoding a first HLA allele and a polynucleotide sequence encoding a second HLA allele. In some embodiments, the heterologous polynucleotide sequence encodes one or more self-cleaving peptides. In some embodiments, the self-cleaving peptide is located at the 3' end of the first HLA allele and at the 5' end of the second HLA allele. In some embodiments, the self-cleaving peptide comprises T2A, P2A, E2A, and / or a combination thereof. In some embodiments, the heterologous polynucleotide sequence is a non-coding sequence.

[0025] In another aspect, the disclosure provides a method for the treatment of a disease comprising administering to a patient a therapeutically effective amount of at least one of the following: (a) one or more vectors each comprising a recombinant polynucleotide encoding at least one gene selected from GM-CSF, IFN-α, CD86, IL-12, CD80, HLA-DRA, IL-7, and 4-1BBL; (b) at least one HLA-A allele selected from the HLA-A*01:01 allele, the HLA-A*68:01 allele, the HLA-A*02:01 allele, the HLA-A*11:01 allele, the HLA-A*03:01 allele, the HLA-A*23:01 allele, the HLA-A*24:02, and the HLA-A*33:03 allele. and / or (c) one or more vectors each comprising a recombinant polynucleotide encoding at least one HLA-DR allele selected from HLA-DRB3*02:02, HLA-DRB5*01:01, HLA-DRB4*01:01, HLA-DRB3*01:01, HLA-DRB3*03:01, HLA-DRB5*01:02, and / or HLA-DRB5*02:02, wherein one or more HLA alleles endogenous to the cell are inactivated.

[0026] In certain embodiments, the modified human cancer cells comprise (a) four vectors each comprising a recombinant polynucleotide encoding two different genes selected from GM-CSF, IFN-α, CD86, IL-12, CD80, HLA-DRA, IL-7, and 4-1BBL. In certain other embodiments, the modified human cancer cells comprise (b) one vector comprising a recombinant polynucleotide encoding the HLA-A*01:01 allele and the HLA-A*68:01 allele, or the HLA-A*02:01 allele and the HLA-A*11:01 allele, or the HLA-A*03:01 allele and the HLA-A*23:01 allele, or the HLA-A*24:02 allele and the HLA-A*33:03 allele; and / or or (c) a vector comprising a recombinant polynucleotide encoding the HLA-DRB3*02:02 allele and the HLA-DRB5*01:01 allele, or the HLA-DRB4*01:01 allele and the HLA-DRB3*01:01 allele, or the HLA-DRB3*03:01 allele and the HLA-DRB5*01:02 allele, or the HLA-DRB5*02:02 allele and the HLA-DRB3*01:01 allele.

[0027] In certain embodiments, the modified human cancer cells comprise: (a) four vectors each comprising a recombinant polynucleotide encoding two different genes selected from GM-CSF, IFN-α, CD86, IL-12, CD80, HLA-DRA, IL-7, and 4-1BBL; (b) one vector comprising a recombinant polynucleotide encoding an HLA-A*01:01 allele and an HLA-A*68:01 allele; and (c) one vector comprising a recombinant polynucleotide encoding an HLA-DRB3*02:02 allele and an HLA-DRB5*01:01 allele.

[0028] In certain embodiments, the modified human cancer cells comprise: (a) four vectors each comprising a recombinant polynucleotide encoding two different genes selected from GM-CSF, IFN-α, CD86, IL-12, CD80, HLA-DRA, IL-7, and 4-1BBL; (b) one vector comprising a recombinant polynucleotide encoding an HLA-A*02:01 allele and an HLA-A*11:01 allele; and (c) one vector comprising a recombinant polynucleotide encoding an HLA-DRB4*01:01 allele and an HLA-DRB3*01:01 allele.

[0029] In certain embodiments, the modified human cancer cells comprise: (a) four vectors each comprising a recombinant polynucleotide encoding two different genes selected from GM-CSF, IFN-α, CD86, IL-12, CD80, HLA-DRA, IL-7, and 4-1BBL; (b) one vector comprising a recombinant polynucleotide encoding an HLA-A*03:01 allele and an HLA-A*23:01 allele; and (c) one vector comprising a recombinant polynucleotide encoding an HLA-DRB3*03:01 allele and an HLA-DRB5*01:02 allele.

[0030] In certain embodiments, the modified human cancer cells comprise: (a) four vectors each comprising a recombinant polynucleotide encoding two different genes selected from GM-CSF, IFN-α, CD86, IL-12, CD80, HLA-DRA, IL-7, and 4-1BBL; (b) one vector comprising a recombinant polynucleotide encoding an HLA-A*24:02 allele and an HLA-A*33:03 allele; and (c) one vector comprising a recombinant polynucleotide encoding an HLA-DRB5*02:02 allele and an HLA-DRB3*01:01 allele.

[0031] In various embodiments, one or more HLA-A and / or HLA-DR alleles endogenous to the cell are inactivated. In some embodiments, one or more inactivated endogenous HLA-A alleles in the modified human cancer cell comprise HLA-A*24:02 allele and / or HLA-A*11:01 allele. In some embodiments, one or more inactivated endogenous HLA-DR alleles in the modified human cancer cell comprise one or more HLA-DRB3 alleles. In some embodiments, one or more inactivated endogenous HLA-DRB3 alleles comprise HLA-DRB3*01:01 allele and / or HLA-DRB3*02:02 allele.

[0032] In some embodiments, one or more of the recombinant polynucleotides comprises a sequence having at least 85% identity to any one of SEQ ID NOs: 22-44. In some embodiments, one or more of the recombinant polynucleotides comprises the sequence of any one of SEQ ID NOs: 22-44.

[0033] In some embodiments, the human cancer cell is a human cancer cell line. In certain embodiments, the human cancer cell line is a breast cancer (e.g., SV-BR-1), prostate cancer (e.g., PC-3, LNCaP), melanoma (e.g., SK-MEL-24), or lung cancer (e.g., NCI-H2228) cell line.

[0034] In some embodiments, the modified human cancer cells described herein are replication-incompetent modified human cancer cells. In some embodiments, the modified human cancer cells are replication-incompetent by irradiation, freeze-thawing, and / or mitomycin C treatment. In some embodiments, the modified human cancer cells are human cancer cell lines. In some embodiments, the modified human cancer cells further comprise an inactivated CD47 molecule. In some embodiments, the modified human cancer cells further comprise an inactivated Ii (CD74) molecule.

[0035] In another aspect, the present disclosure provides a composition comprising the modified human cancer cells described herein. In some embodiments, the composition comprises at least 1,000,000 cells. In some embodiments, the composition comprising the modified human cancer cells described herein is formulated as a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises a cryoprotectant.

[0036] In another aspect, the present disclosure provides a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. In some embodiments, the pharmaceutical composition is administered weekly. In some embodiments, the pharmaceutical composition is administered at a dose of at least 1,000,000 cells. In some embodiments, the pharmaceutical composition is administered intradermally to the upper back or thigh of the subject. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of cyclophosphamide at least 2 days prior to administration of the pharmaceutical composition. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of IFN-α2a, pegylated IFN-α2a, or IFN-α2b at least 1 hour and at least 1 day after administration of the pharmaceutical composition.

[0037] In another aspect, the present disclosure provides a kit comprising the pharmaceutical composition described herein for treating a subject having cancer and in need thereof.In some embodiments, the kit further comprises a therapeutically effective amount of IFN-α2b.The kit may comprise instructions for treating the subject using any of the methods described herein.

[0038] In another aspect, the present disclosure provides a recombinant polynucleotide encoding an HLA-A*02:01 allele, an HLA-A*11:01 allele, an HLA-DRB4*01:01 allele, and an HLA-DRB3*03:01 allele; (b) a recombinant polynucleotide encoding an HLA-A*01:01 allele, an HLA-A*68:01 allele, an HLA-DRB3*02:02 allele, and an HLA-DRB4*01:01 allele; (c) a recombinant polynucleotide encoding an HLA-A*03:01 allele, an HLA-A*23:01 allele, and an HLA-DRB4*01:01 allele. and / or (d) a vector comprising a recombinant polynucleotide encoding an HLA-A*33:03 allele, an HLA-DRB5*01:01 allele, and an HLA-DRB5*02:02 allele, wherein each recombinant polynucleotide in (a)-(d) further comprises a polynucleotide encoding a self-cleaving peptide between two HLA alleles. In some embodiments, the vector in (d) further comprises a recombinant polynucleotide encoding an HLA-A*24:02 allele. In some embodiments, the polynucleotide encoding the self-cleaving peptide is located at the 3' end of the first HLA allele and at the 5' end of the second HLA allele. In some embodiments, the self-cleaving peptide comprises T2A, P2A, E2A, and / or a combination thereof.

[0039] In various embodiments, the recombinant polynucleotide present in any of (a)-(d) in the vector described herein comprises at least one of a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs:22-35. In some embodiments, the recombinant polynucleotide present in any of (a)-(d) in the vector comprises a sequence encoding a polypeptide having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide encoded by any one of SEQ ID NOs:22-35. In some embodiments, the recombinant polynucleotide comprises at least one of any one of SEQ ID NOs:22-35. In some embodiments, the recombinant polynucleotide comprises the MNDU3 promoter.

[0040] In some embodiments, a recombinant polynucleotide present in any of (a)-(d) in a vector described herein comprises an HLA class I gene having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 22-29. In some embodiments, a recombinant polynucleotide present in any of (a)-(d) in a vector described herein comprises an HLA class II gene having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 30-35. In some embodiments, the recombinant polynucleotide present in any of (a)-(d) in a vector described herein comprises an HLA class I gene having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs:22-29, and an HLA class II gene having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs:30-35.

[0041] In some embodiments, the recombinant polynucleotide present in any of (a)-(d) in the vectors described herein comprises an HLA class I gene having a polynucleotide sequence of any one of SEQ ID NOs:22-29. In some embodiments, the recombinant polynucleotide present in any of (a)-(d) in the vectors described herein comprises an HLA class II gene having a polynucleotide sequence of any one of SEQ ID NOs:30-35. In some embodiments, the recombinant polynucleotide present in any of (a)-(d) in the vectors described herein comprises an HLA class I gene having a polynucleotide sequence of any one of SEQ ID NOs:22-29 and an HLA class II gene having a polynucleotide sequence of any one of SEQ ID NOs:30-35.

[0042] In various embodiments, the vector further comprises a recombinant polynucleotide encoding a cytokine. In some embodiments, the cytokine comprises a chemokine, an interferon, an interleukin, a tumor necrosis factor, or a combination thereof. In some embodiments, the recombinant polynucleotide encoding the cytokine is an early T cell activation antigen-1 (ETA-1), lymphocyte activating factor (LAF), an interleukin-1 family member (IL-1α, IL-β, IL-1Ra, IL-18, IL-33, IL-36Ra, IL-36α, IL-36β, IL-36γ, IL-37, IL-38), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-14), interleukin-14 (IL-15), interleukin-15 (IL-16), interleukin-16 (IL-17), interleukin-17 (IL-18), interleukin-18 (IL-19), interleukin-19 (IL-20), interleukin-19 (IL-21), interleukin-11 (IL-22), interleukin-12 (IL-23), interleukin-13 (IL-24), interleukin-14 (IL-25), interleukin-15 (IL-26), interleukin-16 (IL-27), interleukin-17 (IL-28), interleukin-18 (IL-29), interleukin-19 (IL Interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interleukin-17 (IL-17), interleukin Interferon-18 (IL-18), interleukin-21 (IL-21), interleukin-23 (IL-23), interleukin-25 (IL-25), interleukin-33 (IL-33), interferon alpha (IFN-α), interferon lambda 1 (IFNλ1 (IL-29)), interferon lambda 2 (IFNλ2 (IL-28A)), interferon lambda 3 (IFNλ3 (IL-28B)), interferon The cytokines include recombinant polynucleotides encoding lambda 4 (IFNλ4), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage CSF (CSF-1), macrophage migration inhibitory factor (MIF), CD40L molecule (CD40L), RANTES molecule (RANTES), monocyte chemotactic protein (MCP-1), monocyte inflammatory proteins (MIP-1α, MIP-1β), lymphotactin, fractalkine, or combinations thereof. In some embodiments, the cytokines include granulocyte macrophage colony stimulating factor (GM-CSF).In some embodiments, the cytokine comprises interferon alpha (IFN-α), such as, for example, IFN-α2a or IFN-α2b. In some embodiments, the cytokine comprises GM-CSF and IFN-α, preferably IFN-α2a or IFN-α2b.

[0043] In some embodiments, the vector further comprises a recombinant polynucleotide encoding a costimulatory molecule. In some embodiments, the recombinant polynucleotide encoding the costimulatory molecule comprises a recombinant polynucleotide encoding a CD86 molecule (CD86), a CD80 molecule (CD80), a 4-1BB ligand molecule (4-1BBL, also known as CD137L), an ICOS ligand molecule (ICOS-L), a CD70 molecule (CD70, also known as CD27L), a CD40 molecule (CD40), an OX40 ligand molecule (OX40L), a GITR ligand molecule (GITRL), a TIM-4 molecule (TIM-4), a LIGHT molecule (LIGHT), an ICAM1 molecule (ICAM1), an LFA3 molecule (LFA3), a CD30 molecule (CD30), or a combination thereof.

[0044] In some aspects, the vector further comprises a recombinant polynucleotide encoding an antigen of a pathogen, a tumor-associated antigen, a neoantigen, an allergen, an antigen that is the target of an autoimmune response, a fragment thereof, or a combination thereof.

[0045] In another aspect, the present disclosure provides a recombinant polynucleotide comprising a sequence encoding an allele of an HLA class I gene having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs:22-29, and optionally at least one heterologous polynucleotide sequence. In some embodiments, the recombinant polynucleotide comprises a sequence of any one of SEQ ID NOs:22-29. In some embodiments, the recombinant polynucleotide comprises a sequence encoding a polypeptide having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide encoded by any one of SEQ ID NOs:22-29. In some embodiments, the recombinant polynucleotide encoding an allele of an HLA class I gene comprises a sequence of any one of SEQ ID NOs:22-29. In some embodiments, the recombinant polynucleotide further comprises a recombinant polynucleotide encoding a second allele of an HLA class I gene or a second allele of an HLA class I gene.

[0046] In some embodiments, the recombinant polynucleotide further comprises a recombinant polynucleotide encoding an allele of an HLA class II gene. In some embodiments, the recombinant polynucleotide encoding an allele of an HLA class II gene has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 30-35. In some embodiments, the recombinant polynucleotide comprises a sequence of any one of SEQ ID NOs: 30-35. In some embodiments, the recombinant polynucleotide comprises a sequence encoding a polypeptide having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide encoded by any one of SEQ ID NOs: 30-35. In some embodiments, the recombinant polynucleotide encoding an allele of an HLA class II gene comprises a sequence of any one of SEQ ID NOs: 30-35.

[0047] In various embodiments, the recombinant polynucleotide comprises a heterologous polynucleotide sequence encoding an amino acid sequence of a cytokine. In some embodiments, the cytokine comprises a chemokine, an interferon, an interleukin, a tumor necrosis factor, or a combination thereof. In some embodiments, the heterologous polynucleotide sequence encoding a cytokine is an early T-cell activation antigen-1 (ETA-1), lymphocyte activating factor (LAF), interleukin-1 family members (IL-1α, IL-β, IL-1Ra, IL-18, IL-33, IL-36Ra, IL-36α, IL-36β, IL-36γ, IL-37, IL-38), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-13), interleukin-13 (IL-14), interleukin-14 (IL-15), interleukin-15 (IL-16), interleukin-16 (IL-17), interleukin-17 (IL-18), interleukin-18 (IL-19), interleukin-19 (IL-20), interleukin-19 (IL-21), interleukin-11 (IL-22), interleukin-12 (IL-23), interleukin-13 (IL-14), interleukin-14 (IL-15), interleukin-15 (IL-16), interleukin-16 (IL-17), interleukin-17 (IL-18), interleukin-19 (IL- Interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interleukin-17 (IL-17), interleukin Interferon-18 (IL-18), interleukin-21 (IL-21), interleukin-23 (IL-23), interleukin-25 (IL-25), interleukin-33 (IL-33), interferon alpha (IFN-α), interferon lambda 1 (IFNλ1 (IL-29)), interferon lambda 2 (IFNλ2 (IL-28A)), interferon lambda 3 (IFNλ3 (IL-28B)), interferon lambda The heterologous polynucleotide sequence encoding muda 4 (IFNlambda 4), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage CSF (CSF-1), macrophage migration inhibitory factor (MIF), CD40L molecule (CD40L), RANTES molecule (RANTES), monocyte chemotactic protein (MCP-1), monocyte inflammatory proteins (MIP-1α, MIP-1β), lymphotactin, fractalkine, or combinations thereof. In some embodiments, the cytokine comprises granulocyte macrophage colony stimulating factor (GM-CSF).In some embodiments, the cytokine comprises interferon alpha (IFN-α), such as IFN-α2a or IFN-α2b. In some embodiments, the cytokine comprises GM-CSF and IFN-α, preferably IFN-α2a or IFN-α2b. In some embodiments, the IFN-α is an exogenous protein comprising pegylated IFN-α2a.

[0048] In various embodiments, the recombinant polynucleotide comprises a heterologous polynucleotide sequence encoding one or more self-cleaving peptides between two HLA alleles. In some embodiments, the recombinant polynucleotide comprises a heterologous polynucleotide sequence encoding one or more self-cleaving peptides between an HLA allele and another HLA allele, a cytokine, a costimulatory molecule, or a combination thereof. In some embodiments, the self-cleaving peptide is located at the 3' end of the first HLA allele and at the 5' end of the second HLA allele. In some embodiments, the self-cleaving peptide comprises T2A, P2A, E2A, or a combination thereof.

[0049] In some aspects, the recombinant polynucleotide comprises a heterologous polynucleotide sequence comprising a promoter. In some aspects, the promoter comprises the MNDU3 promoter or the EF1α promoter.

[0050] In various embodiments, the recombinant polynucleotide comprises a heterologous polynucleotide sequence encoding the amino acid sequence of a costimulatory molecule. In some embodiments, the costimulatory molecule comprises a CD86 molecule (CD86), a CD80 molecule (CD80), a 4-1BB ligand molecule (4-1BBL, also known as CD137L), an ICOS ligand molecule (ICOS-L), a CD70 molecule (CD70, also known as CD27L), a CD40 molecule (CD40), an OX40 ligand molecule (OX40L), a GITR ligand molecule (GITRL), a TIM-4 molecule (TIM-4), a LIGHT molecule (LIGHT), an ICAM1 molecule (ICAM1), an LFA3 molecule (LFA3), a CD30 molecule (CD30), or a combination thereof.

[0051] In some embodiments, the recombinant polynucleotide comprises a heterologous polynucleotide sequence comprising a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 36-44. In some embodiments, the heterologous polynucleotide comprises a sequence encoding a polypeptide having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide encoded by any one of SEQ ID NOs: 36-44. In some embodiments, the heterologous polynucleotide sequence comprises the sequence of any one of SEQ ID NOs: 36-44.

[0052] In some aspects, the heterologous polynucleotide sequence encodes the amino acid sequence of an antigen of a pathogen, a tumor-associated antigen, a neoantigen, an allergen, an antigen that is the target of an autoimmune response, a fragment thereof, or a combination thereof. [Brief description of the drawings]

[0053] [Figure 1] Schematic diagram of BC1.68.3.4 (BC1). Plasmid backbone pCC1-c with inserted four HLA alleles including HLA-A*01:01, HLA-A*68:01, HLA-DRB3*02:02 and HLA-DRB4*01:01. The plasmid contains a cytomegalovirus (CMV) enhanced promoter at the 5' end (CMV enhanced 5' LTR). AMP: ampicillin resistance gene. PSI: packaging signal. RRE: Rev responsive element. cPPT: central polypurine tract. EF1a: EF1 alpha promoter. MNDU3: MNDU3 promoter. T2A, P2A, and E2A: 2A self-splicing peptides. [Diagram 2]Schematic diagram of BC2.11.4.3 (BC2). Plasmid backbone, pCC1-c, with insertion of four HLA alleles including HLA-A*02:01, HLA-A*11:01, HLA-DRB4*01:01 and HLA-DRB3*03:01. The plasmid contains a cytomegalovirus (CMV) enhanced promoter at the 5' end (CMV enhanced 5' LTR). AMP: ampicillin resistance gene. PSI: packaging signal. RRE: Rev responsive element. cPPT: central polypurine tract. EF1a: EF1 alpha promoter. MNDU3: MNDU3 promoter. T2A, P2A, and E2A: 2A self-splicing peptides. [Diagram 3] Schematic diagram of BC3.23.3.5 (BC3). Plasmid backbone, pCC1-c, with insertion of four HLA alleles including HLA-A*03:01, HLA-A*23:01, HLA-DRB3*01:01 and HLA-DRB5*01:02. The plasmid contains a cytomegalovirus (CMV) enhanced promoter at the 5' end (CMV enhanced 5' LTR). AMP: ampicillin resistance gene. PSI: packaging signal. RRE: Rev responsive element. cPPT: central polypurine tract. EF1a: EF1 alpha promoter. MNDU3: MNDU3 promoter. T2A, P2A, and E2A: 2A self-splicing peptides. [Figure 4] Schematic diagram of BC24.33.5.5 (BC4). Plasmid backbone, pCC1-c, with insertion of four HLA alleles including HLA-A*24:02, HLA-A*33:03, HLA-DRB5*01:01 and HLA-DRB5*02:02. The plasmid contains a cytomegalovirus (CMV) enhanced promoter at the 5' end (CMV enhanced 5' LTR). AMP: ampicillin resistance gene. PSI: packaging signal. RRE: Rev responsive element. cPPT: central polypurine tract. EF1a: EF1 alpha promoter. MNDU3: MNDU3 promoter. T2A, P2A, and E2A: 2A self-splicing peptides. [Diagram 5]Shown are shared costimulatory molecules and their ligands presented on T cells and dendritic cells (DCs), respectively. [Figure 6] We demonstrate the dual mechanism of action of Bria-IMT and Bria-OTS therapeutics. SV-BR-1-GM cells secrete GM-CSF, which supports antigen presentation by DCs. Cancer cell antigens are taken up by DCs after cell degradation and presented to CD4+ and CD8+ T cells, thereby inducing an immune response against the tumor. SV-BR-1-GM cells can also directly activate T cells to stimulate the fight against cancer as an additional boost to the immune response. [Figure 7A] The experimental strategy for generating Bria-OTS cell lines is shown. (A) Endogenous HLA-A / DRB genes are first KO'd by CRISPR / Cas9 to generate Bria-KO cells. Then GM-CSF and other cytokines and costimulatory molecules are overexpressed by sequential lentiviral transduction of four unique constructs to generate Bria-APTC. Finally, two HLA-A and two HLA-DRB alleles are overexpressed by lentiviral transduction to generate a collection of four cell lines (Bria-OTS-1 / 2 / 3 / 4) per parental tumor cell. (B) Lentiviral vectors used to express two distinct genes from independent promoters. Cytokines and costimulatory molecules used to generate Bria-APTC (table on the left). HLA-A and HLA-DRB alleles used to generate the final Bria-OTS product (table on the right). [Figure 7B] Please see the legend to FIG. 7A. [Figure 8] Expression of GM-CSF and cytokines / costimulatory molecules in SV-BR-1-APTC. SV-BR-1-KO clone 17 was cloned by sequential transduction with lentiviruses expressing eight different cytokines and costimulatory molecules. (A) Levels of secreted GM-CSF, IFN-α, IL-12, and IL-7 were measured by ELISA. (B) Expression of surface receptors CD86 and CD80 in SV-BR-1-APTC as measured by flow cytometry. [Figure 9] Validation of SV-BR-1-APTC using a modified MLR assay. (A) Bria-APTC or SV-BR-1KO cells are incubated with PMBCs at different ratios. T cell proliferation and secretion of IFNγ, IL-2, and IL-4 are measured on days 1, 2, 3, and 7 to assess T cell activation. (B) 10,000 cells / well of SV-BR-1-KO or APTC in 96-well plates were co-cultured with PBMCs from healthy donors at the indicated ratios. After 24 h of incubation, secreted IFNγ was measured by ELISA. N=3. [Figure 10] Functional characterization of prostate-derived Bria-APTC. PC3-APTC or PC3KO cells (10,000 cells / well in 96-well plates) were incubated alone or with PBMCs at the indicated ratios for 24 hours. IFNγ levels in the supernatants were measured by ELISA. [Figure 11] Expression of GM-CSF and cytokine / costimulatory molecules in PC3-APTC and expression of HLA-A alleles in PC3-Bria-OTS are shown. PC3-KO clone A4 was sequentially transduced with lentiviruses expressing eight different cytokines and costimulatory molecules and cloned. (A) Expression of surface receptors CD86 and CD80 in PC3-APTC as measured by flow cytometry. (B) Expression of HLA-A alleles in PC3-Bria-OTS cell line as measured by flow cytometry. (C) Levels of secreted IL-7, GM-CSF, and IL-12 in PC3-APTC were measured by ELISA. [Figure 12] KO of HLA alleles in H2228 and SK-MEL-24 cells. HLA-DR expression was measured in single clones of H2228-KO and SK-MEL-24-KO cells compared to wild-type (WT) cells by flow cytometry. [Figure 13] 1 shows validation of HLA functionality using TCR transgenic Jurkat cells. [Figure 14] Validation of HLA functionality in PC3-Bria-OTS using a T cell activation bioassay (NFAT). The assay consists of engineered Jurkat T cell lines in which the endogenous TCR alpha and beta chains have been knocked out using clustered regularly interspaced short interspaced repeats (CRISPR)-Cas9. These cells also express a luciferase reporter driven by a TCR pathway-dependent promoter. (A) Jurkat cells were transduced with a TCR specific for the WT1 peptide (HLA-A*24 restricted) and incubated with the indicated cell lines and the appropriate WT1 peptide. After 6 hours, bioluminescent signals were quantified using standard luminometers such as the Bio-Glo-NL™ Luciferase Assay System and the GloMax® Discover System. Transgenic HLA induces T cell activation at a rate comparable to endogenous HLA. (B) Flow cytometry analysis showing expression of HLA-A*24 in the indicated cell lines. (C) Jurkat cells were transduced with TCR (HLA-A*01 restricted) specific for MAGE-A3 peptide and incubated with the indicated cell lines and appropriate WT1 peptide. After 6 hours, bioluminescent signal was quantified using standard luminometers such as Bio-Glo-NL™ Luciferase Assay System and GloMax® Discover System. Transgenic HLA induces cell activation at a higher rate than endogenous HLA. (D) Flow cytometry analysis showing expression of HLA-A*01 in the indicated cell lines. [Figure 15]Validation of HLA functionality in SV-BR-1-Bria-OTS using a T cell activation bioassay (NFAT) is shown. The assay consists of genetically engineered Jurkat T cell lines in which the endogenous TCR alpha and beta chains have been knocked out using CRISPR-Cas9. These cells also express a luciferase reporter driven by a TCR pathway-dependent promoter. Jurkat cells were transduced with a TCR (HLA-A*24 restricted) specific for the WT1 peptide and incubated with the indicated cell lines and the appropriate WT1 peptide. After 6 hours, bioluminescence signals were quantified using a standard luminometer such as the Bio-Glo-NL™ Luciferase Assay System and the GloMax® Discover System. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] Detailed Description I. Introduction Induction of effective immune response by whole cell immunotherapy is an effective approach for treating and preventing diseases such as cancer.To be effective, it is generally assumed that cancer vaccines must express immunogenic antigens that are co-expressed in the patient's tumor cells, and that antigen-presenting cells (APCs), such as dendritic cells (DCs), must cross-present such antigens after uptake of fragments of vaccine cells.

[0055] The transformation technique at the core of this disclosure is the development of therapeutic strategies that address the current gap in safe and reliable treatment for patients suffering from cancer.The whole cell immunotherapy described herein significantly improves previous attempts to develop whole cell therapeutic vaccines, as the disclosed approach works by two distinct but complementary mechanisms to stimulate the patient's immune system.In particular, the modified human cancer cells described herein have the characteristics of both cancer cells (expressing tumor-associated antigens) and dendritic cells (presenting tumor-associated antigens to T cells). In certain aspects, the modified human cancer cells described herein (e.g., Bria-OTS cell line) express one or more immunomodulatory cytokines, one or more costimulatory molecules, one or more human major histocompatibility complex-I (MHC-I) (e.g., HLA-A, HLA-B, HLA-C) molecules, and one or more MHC-II (e.g., HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DRA) molecules. The modified human cancer cells described herein express CD4 + They can directly activate T cells in an antigen-specific, HLA-restricted manner and function as APCs. In certain aspects, the modified human cancer cells described herein advantageously provide the following features: (a) a dual mechanism of action to achieve robust clinical activity by both producing and presenting tumor-associated antigens to induce an immune response in the subject; (b) precision therapy by tailoring therapies to patients based on HLA antigens; and (c) improved safety by reducing or eliminating the use of current chemotherapy and hormone-based therapies associated with severe adverse events that can be life-threatening. Thus, the modified human cancer cells described herein can provide a rapid and cost-effective treatment of cancer. In some cases, the modified human cancer cells can be used as "off-the-shelf" cell lines that do not require personalized manufacturing and can be administered immediately after HLA genotyping of the patient or as a cell mixture to cover approximately 70% to over 99% of the cancer patient population.

[0056] In certain aspects, the modified human cancer cells described herein are HER-2 / neu positive allogeneic whole cell breast cancer (BC) cell lines that secrete granulocyte macrophage colony stimulating factor (GM-CSF) in situ and enhance dendritic cell activity for breast cancer immunotherapy.In addition, the modified human cancer cells described herein can be engineered to express multiple HLA alleles, for example, to match at least 90% or more than 99% of breast cancer patient population.For example, HLA-A and HLA-DRB3 / 4 / 5 alleles are the least polymorphic HLA loci, and the fewest number of alleles can match the greatest number of patients.

[0057] Described herein are compositions of modified human cancer cells or cell lines for targeted immunotherapy of cancer. Additionally, kits and methods containing "off-the-shelf" cell lines for preventing or treating cancer in subjects in need thereof are provided.

[0058] II. Definition Unless otherwise specifically indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.In addition, any method or material similar or equivalent to the method or material described herein can be used to implement this disclosure.For purposes of this disclosure, the following terms are defined.

[0059] As used herein, the terms "a," "an," or "the" include aspects having one member as well as aspects having more than one member. For example, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "cell" includes a plurality of such cells, reference to an "agent" includes reference to one or more agents known to those of skill in the art, and so forth.

[0060] The terms "about" and "approximately" shall generally mean an acceptable degree of error for the measured quantity, taking into account the nature or accuracy of the measurement. A typical exemplary degree of error is within 20 percent (%), preferably within 10%, more preferably within 5% of a given value or range of values. Alternatively, particularly in biological systems, the terms "about" and "approximately" may mean values ​​within an order of magnitude, preferably within 5-fold, more preferably within 2-fold of a given value. The quantities given herein are approximate unless otherwise stated, meaning that the term "about" or "approximately" can be inferred when not expressly stated.

[0061] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, mice, rats, monkeys, humans, farm animals, sports animals, and pets. Also included are tissues, cells, and the progeny thereof of biological entities obtained in vivo or cultured in vitro.

[0062] The term "administering" as used herein includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intratumoral, intradermal, intralymphatic, intrathecal, intranasal, or subcutaneous administration to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other delivery modes include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like.

[0063] The term "treat" refers to an approach to obtain beneficial or desired results, including but not limited to therapeutic benefit and / or prophylactic benefit. Therapeutic benefit refers to any therapeutically relevant improvement or effect in one or more diseases, conditions or symptoms being treated. Therapeutic benefit can also refer to causing a cure of one or more diseases, conditions or symptoms being treated.

[0064] The term "effective amount" or "sufficient amount" refers to an amount of modified cancer cells or other compositions sufficient to cause beneficial or desired results. The therapeutically effective amount may vary depending on one or more of the subject and condition being treated, the subject's weight and age, the severity of the condition, the manner of administration, etc., and can be easily determined by those skilled in the art. The specific amount may vary depending on one or more of the individual agent selected, the target cell type, the location of the target cell in the subject, the dosing regimen to be followed, whether it is co-administered with other compounds, the timing of administration, and the bodily delivery system in which it is delivered.

[0065] For the purposes herein, effective amount is determined by such considerations as may be known in the art.Amount must be effective to achieve desired therapeutic effect in the subject suffering from cancer.Desired therapeutic effect may include, for example, ameliorating the undesired symptoms associated with cancer, preventing the onset of such symptoms before they occur, slowing down the progression of symptoms associated with cancer, slowing down or limiting any irreversible damage caused by cancer, reducing the severity or curing cancer, or improving survival rate or providing faster recovery from cancer.

[0066] Effective amount depends on, among other things, the type and severity of the disease to be treated and the treatment regimen.Effective amount is typically determined in a properly designed clinical trial (dose range test), and those skilled in the art know how to properly carry out such a test to determine effective amount.As is generally known, effective amount depends on various factors, including the distribution profile of therapeutic agent (e.g. whole cell cancer vaccine) or composition in the body, the relationship between various pharmacological parameters (e.g. half-life in the body) and undesired side effects, and other factors such as age and sex.

[0067] The term "pharmaceutically acceptable carrier" refers to a substance that aids in the administration of an active agent to a cell, organism, or subject. "Pharmaceutically acceptable carrier" refers to a carrier or excipient that can be included in the composition of the present disclosure and does not cause significant adverse toxic effects to the subject. Non-limiting examples of pharmaceutically acceptable carriers include water, sodium chloride, saline, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavorings and colorants, liposomes, dispersion media, microcapsules, cationic lipid carriers, isotonicity agents and absorption delaying agents, etc. Carriers can also be substances that provide stability, sterility, and isotonicity to the formulation (e.g., antimicrobial preservatives, antioxidants, chelating agents, and buffers), substances that prevent the action of microorganisms (e.g., antimicrobial and antifungal agents such as parabens, chlorobutanol, sorbic acid, etc.), or substances that impart edible flavor to the formulation, etc. In some cases, the carrier is an agent that facilitates delivery of the modified cancer cells to the target cells or tissues. Those of skill in the art will recognize that other pharmaceutical carriers are useful in the present disclosure.

[0068] The term "nucleic acid" or "nucleotide" as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides, either in single-stranded or double-stranded form, including DNA, RNA, and hybrids thereof. DNA can be in the form of, for example, antisense molecules, plasmid DNA, DNA-DNA duplexes, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. RNA can be in the form of small interfering RNA (siRNA), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include synthetic, natural and non-natural nucleic acids that contain known nucleotide analogs or modified backbone residues or bonds and have similar binding properties to the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).Unless otherwise specified, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid.Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).A "nucleotide" comprises a sugar, deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate group. "Base" includes purines and pyrimidines, which further include the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and naturally occurring analogues, as well as synthetic derivatives of purines and pyrimidines, including but not limited to modifications that place new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0069] The term "gene" refers to a DNA segment involved in producing a polypeptide chain. The DNA segment may include regions preceding and following the coding region (leader and trailer) involved in the transcription / translation of the gene product and regulation of transcription / translation, as well as intervening sequences (introns) between individual coding segments (exons).

[0070] The terms "vector" and "expression vector" refer to a recombinant or synthetically produced nucleic acid construct that has a series of specific nucleic acid elements that allow transcription of a particular polynucleotide sequence in a host cell. An expression vector can be part of a plasmid, a viral genome, or a nucleic acid fragment. Typically, an expression vector contains a polynucleotide to be transcribed, which is operably linked to a promoter. The term "promoter" is used herein to refer to an array of nucleic acid control sequences that direct the transcription of a nucleic acid. A promoter as used herein includes a necessary nucleic acid sequence near the start site of transcription, such as a TATA element in the case of a polymerase II type promoter. A promoter also optionally includes a distal enhancer or repressor element, which can be located several thousand base pairs from the transcription start site. Other elements that can be present in an expression vector include elements that enhance transcription (e.g., enhancers) and elements that terminate transcription (e.g., terminators). In the context of the present disclosure, co-expression of multiple genes (e.g., polynucleotides terminating HLA class I and / or HLA class II alleles) can be achieved by co-transfection of two or more vectors, use of multiple promoters or bidirectional promoters, or creation of bicistronic or multicistronic vectors. Co-expression of genes can be driven by using a plasmid with multiple separate expression cassettes. Generally, each promoter creates a unique mRNA transcript for each gene expressed. Bicistronic or multicistronic vectors simultaneously express two or more separate proteins from the same mRNA. Bicistronic vectors may contain an internal ribosome entry site (IRES) to allow translation initiation from an internal region of the mRNA. Multicistronic vectors containing one or more self-cleaving 2A peptides are advantageous as they allow gene co-expression from the same cassette. In some cases, multicistronic vectors are preferred when only a portion of the plasmid is packaged for viral delivery or when the relative expression levels between two or more genes are important.

[0071] The terms "self-cleaving peptide" and "self-cleaving 2A peptide" refer to short peptides that can produce equimolar levels of multiple genes from the same mRNA. These peptides were first discovered in picornaviruses. Self-cleaving peptides are thought to function by causing the ribosome to skip synthesis of the peptide bond at the C-terminus of the 2A element, resulting in a separation between the end of the 2A sequence and the next downstream peptide. "Cleavage" occurs between the glycine and proline residues found at the C-terminus, meaning that the upstream cistron typically has a few additional residues added to its end, while the downstream cistron typically begins with a proline. Non-limiting examples of self-cleaving peptides include T2A, P2A, E2A, and F2A.

[0072] "Recombinant" refers to a genetically modified polynucleotide, polypeptide, cell, tissue, or organism. For example, a recombinant polynucleotide (or a copy or complement of a recombinant polynucleotide) is a polynucleotide that has been engineered using well-known methods. A recombinant expression cassette that includes a promoter operably linked to a second polynucleotide (e.g., a coding sequence) can include a promoter that is heterologous to the second polynucleotide as a result of human manipulation (e.g., by the methods described in Sambrook et al., Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). A recombinant expression cassette (or expression vector) typically includes polynucleotides in combinations not found in nature. For example, human-engineered restriction sites or plasmid vector sequences can flank or separate the promoter from other sequences. A recombinant protein is a protein expressed from a recombinant polynucleotide, and recombinant cells, tissues, and organisms are those that contain recombinant sequences (polynucleotides and / or polypeptides). A recombinant cell is a cell that has been modified (e.g., transfected or transformed) with a recombinant nucleotide, expression vector or cassette, etc.

[0073] The term "amino acid" refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. Amino acids include the naturally occurring α-amino acids and their stereoisomers as well as unnatural (non-natural) amino acids and their stereoisomers. A "stereoisomer" of a given amino acid refers to isomers that have the same molecular formula and intramolecular bonds but differ in the three-dimensional arrangement of bonds and atoms (e.g., an L-amino acid and the corresponding D-amino acid).

[0074] Naturally occurring amino acids are those encoded by the genetic code as well as those amino acids that are subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. The stereoisomers of natural α-amino acids are D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), and D-leucine (D-Lys). D-amino acids include, but are not limited to, D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0075] Unnatural (non-natural) amino acids include, but are not limited to, amino acid analogs in either the L- or D-configuration, amino acid mimetics, synthetic amino acids, N-substituted glycines, and N-methyl amino acids that function similarly to natural amino acids. For example, an "amino acid analog" can be an unnatural amino acid that has the same basic chemical structure as a natural amino acid (i.e., a hydrogen, a carboxyl group, and a carbon attached to an amino group) but has a modified side group or a modified peptide backbone, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. An "amino acid mimetic" refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but functions similarly to a natural amino acid. Amino acids may be referred to by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0076] The terms "identity", "substantial identity", "similarity", "substantial similarity", "homology" and related terms and expressions used in connection with the description of amino acid sequences refer to a sequence having at least 60% sequence identity with a reference sequence. Examples include at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity when compared to a reference sequence using a program for comparing amino acid sequences, such as BLAST using standard parameters. For sequence comparison, typically, one sequence serves as a reference sequence and a test sequence is compared to it. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. Default (standard) program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the program parameters. A "comparison window" includes reference to any segment of the number of contiguous positions (20-600, usually about 50-200, more usually about 100-150) within which a sequence can be compared to a reference sequence of the same number of contiguous positions after optimally aligning the two sequences. Methods for aligning sequences for comparison are well known. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, 1981, the homology alignment algorithm of Needleman and Wunsch, 1970, the search for similarity method of Pearson and Lipman, 1988, computer implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.

[0077] Suitable algorithms for determining percent sequence identity and percent sequence similarity include the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990 and Altschul et al., 1977, respectively. Software for performing BLAST analysis is publicly available at the National Center for Biotechnology Information (NCBI) website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet some positive-valued threshold score T when aligned with words of the same length in the database sequence. T is called the neighborhood word score threshold. These initial neighborhood word hits serve as seeds to initiate searches to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is stopped when the cumulative alignment score falls off its maximum achieved value by an amount X, or when the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as default values ​​a word size (W) of 28, expectation (E) of 10, M=1, N=-2, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1989). The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (Karlin and Altschul, 1993).

[0078] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to natural and non-natural amino acid polymers, as well as to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding natural amino acids. As used herein, these terms encompass any length of amino acid chain, including full-length proteins (i.e., alleles), in which the amino acid residues are linked by covalent peptide bonds. As used herein, the amino acid sequence of a polypeptide is presented from the N-terminus to the C-terminus. In other words, when describing the amino acid sequence of a polypeptide, the first amino acid at the N-terminus is referred to as the "first amino acid".

[0079] When used in the context of describing a partner of a recombinant polypeptide, the term "heterologous" refers to the relationship of one polynucleotide fusion partner to another polynucleotide fusion partner: the manner in which the fusion partner is present in the recombinant polynucleotide is not one that can be found in the native polynucleotide or in the polynucleotide encoding the native protein. For example, a "heterologous polynucleotide" fused with an HLA class I allele and / or an HLA class II allele to form a fusion polynucleotide can be derived from a protein other than an HLA class I protein or an HLA class II protein, such as granulocyte macrophage colony stimulating factor (GM-CSF), a costimulatory molecule (e.g., CD80), or a 2A self-splicing peptide (e.g., T2A, P2A, E2A). On the other hand, a "heterologous polynucleotide" can be derived from another part of an HLA class I or HLA class II protein that is not directly adjacent to the HLA class I or HLA class II allele. A "heterologous polynucleotide" can encode a peptide that includes a modification of the native protein sequence or a portion thereof, such as a deletion, addition, or substitution of one or more amino acid residues.

[0080] The terms "gene editing", "genome editing", "genome engineering" and "genome manipulation" are used interchangeably. These terms refer to a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Genome editing can be site-specific. Non-limiting examples of genome editing techniques include the use of nucleases such as clustered regularly interspaced short interspaced repeats / Cas9 (CRISPR / Cas9) nucleases, meganucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs). Viral vectors such as integrase-deficient lentiviral vectors (IDLV), adenoviruses and adeno-associated viruses (AAVs) are typically used to deliver DNA for genome editing. Delivery techniques for genome editing are known in the art, and any approach can be used to inactivate specific HLA alleles in the modified human cancer cells described herein (see, e.g., review by Yin et al., 2017. "Delivery technologies for genome editing." Nature Review Drug Discovery. 16, 387-399).

[0081] The term "cancer" is intended to include any member of the class of diseases characterized by the uncontrolled growth of abnormal cells. The term includes all known cancers and tumor conditions, whether characterized as malignant, benign, recurrent, soft tissue, or solid, and all stages and grades of cancer, including advanced, pre-metastatic, and post-metastatic cancers. Examples of different types of cancer include gynecological cancers (e.g. ovarian, cervical, uterine, vaginal, and vulvar cancer); lung cancer (e.g. non-small cell lung cancer, small cell lung cancer, mesothelioma, carcinoid tumor, lung adenocarcinoma); breast cancer (e.g. triple-negative breast cancer, ductal carcinoma in situ, invasive ductal carcinoma, tubular carcinoma, medullary carcinoma, mucinous carcinoma, papillary carcinoma, cribriform carcinoma, invasive lobular carcinoma, inflammatory breast cancer, lobular carcinoma in situ, Paget's disease, phyllodes tumor); gastrointestinal-gastrointestinal cancers (e.g. stomach cancer), colorectal cancer, gastrointestinal stromal tumor (GIST), gastrointestinal carcinoid tumor, colon cancer, rectal Cancers of the intestine, anus, bile duct, small intestine, and esophagus; thyroid cancer; gallbladder cancer; liver cancer; pancreatic cancer; appendix cancer; prostate cancer (e.g., prostate adenocarcinoma); kidney cancer (e.g., renal cell carcinoma); central nervous system cancer (e.g., glioblastoma, neuroblastoma, medulloblastoma); skin cancer (e.g., melanoma); bone and soft tissue sarcoma (e.g., Ewing's sarcoma); lymphoma; choriocarcinoma; urinary tract cancer (e.g., urinary bladder cancer); head and neck cancer; and bone marrow and blood cancer (e.g., acute leukemia, chronic leukemia (e.g., chronic lymphocytic leukemia), lymphoma, multiple myeloma). As used herein, a "tumor" includes one or more types of cancerous cells.

[0082] The term "allele" typically refers to a particular form or variant of a gene. Alleles can result, for example, from nucleotide substitutions, additions, or deletions, or can represent a variable number of short nucleotide repeats. In the context of human leukocyte antigen (HLA) genes, HLA alleles are named by the World Health Organization Nomenclature Committee for the factors of the HLA system. According to this system, the HLA gene name is followed by a series of numeric segments. A minimum of two numeric digit segments are included. As a non-limiting example, HLA-A*02:101 refers to a particular allele of the HLA-A gene. The first segment, separated from the gene name by an asterisk, refers to the allele group. The second segment, separated from the first segment by a colon, refers to the particular HLA protein produced. In some cases, longer names are used (e.g., HLA-A*02:101:01:02N). In this example, the third numbered section refers to whether synonymous DNA substitutions are present in the coding region, and the fourth numbered section refers to the difference between alleles present in the non-coding region. In some other examples, the HLA allele name includes one letter at the end. According to the HLA allele naming system, "N" means that the allele is a null allele (i.e., the allele produces a non-functional protein), "L" means that the allele results in lower than normal cell surface expression of a particular HLA protein, "S" means that the allele produces a soluble protein that is not found on the cell surface, "Q" means that the allele is questionable (i.e., an allele that may not affect normal expression), "C" means that the allele produces a protein that is present in the cytoplasm but not on the cell surface, and "A" means that the allele results in abnormal expression (i.e., it is uncertain whether a particular HLA protein is expressed). Those skilled in the art are familiar with the various alleles and their naming conventions.

[0083] The term "allelic profile" refers to the collection of alleles of one or more genes in a particular sample. The sample may be obtained from a subject, a particular cell or cell type (e.g., breast cells or breast cancer cells), or an engineered cell (e.g., a cancer cell engineered to express one or more proteins). In some cases, the allelic profile may describe the alleles of a single gene present in a sample (e.g., cells obtained from a subject or cancer vaccine cells), or may describe the alleles present for two or more genes in a sample. As a non-limiting example, the allelic profile may describe the alleles present for an HLA-A gene in a particular sample. For a diploid cell, only one allele may be present (e.g., when both chromosomes contain the same allele, such as HLA-A*02:01). Alternatively, two different alleles may be present (e.g., the allelic profile includes HLA-A*02:01 and HLA-A*24:02, or HLA-A*02:01 and HLA-A*03:01). In other cases, an allelic profile lists the alleles present for two or more genes. As a non-limiting example, an allelic profile may describe the alleles of the HLA-A and HLA-DRB3 genes present in a patient sample.

[0084] For illustrative purposes only, the allelic profile of the subject may indicate that the HLA-A*02:01 and HLA-A*24:02 alleles of the HLA-A gene are present, and the HLA-DRB3*03:01 allele of the HLA-DRB3 gene is present. Furthermore, the allelic profiles can be compared. As a non-limiting example, the subject can have an allelic profile that includes the HLA-A*02:01 and HLA-A*24:02 alleles, whereas the cancer vaccine cells can have a profile that includes the HLA-A*02:01 and HLA-A*03:01 alleles. In this example, when comparing two allelic profiles, there is a partial match between the profiles (i.e., the HLA-A*02:01 allele is present in both profiles). As another non-limiting example, if the vaccine cells have an allelic profile that includes the HLA-A*02:01 and HLA-A*24:02, the profiles of the subject and the vaccine cells are fully matched with respect to this particular gene.

[0085] The term "human leukocyte antigen (HLA)" refers to the gene complex that encodes the human major histocompatibility complex (MHC) proteins, a set of cell surface proteins essential for the recognition of foreign molecules by the adaptive immune system. The HLA complex is found within a 3 Mbp stretch of chromosome 6p21. Class I MHC proteins, which present peptides from inside the cell, are encoded by the HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G genes. The HLA-A, HLA-B, and HLA-C genes are more polymorphic, whereas the HLA-E, HLA-F, and HLA-G genes are less polymorphic. HLA-K and HLA-L are also known to exist as pseudogenes. In addition, beta-2-microglobulin is an MHC class I protein encoded by the (B2M) gene. Non-limiting examples of HLA-A nucleotide sequences are shown under GenBank reference numbers NM_001242758 and NM_002116. Non-limiting examples of HLA-B nucleotide sequences are shown under GenBank reference numbers NM_005514. Non-limiting examples of HLA-C nucleotide sequences are shown under GenBank reference numbers NM_001243042 and NM_002117. Non-limiting examples of HLA-E nucleotide sequences are shown under GenBank reference numbers NM_005516. Non-limiting examples of HLA-F nucleotide sequences are shown under GenBank reference number NM_018950. Non-limiting examples of HLA-G nucleotide sequences are shown under GenBank reference number NM_002127. Non-limiting examples of B2M nucleotide sequences are shown under GenBank reference number NM_004048.

[0086] Class II MHC proteins, which present antigens from outside the cell to T lymphocytes, are encoded by HLA-DP, HLA-DM, HLA-DO, HLA-DQ, and HLA-DR genes. HLA-DM genes include HLA-DMA and HLA-DMB. HLA-DO genes include HLA-DOA and HLA-DOB. HLA-DP genes include HLA-DPA1 and HLA-DPB1. HLA-DQ genes include HLA-DQA1, HLA-DQA2, HLA-DQB1, and HLA-DQB2. HLA-DR genes include HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5. Non-limiting examples of HLA-DMA and HLA-DMB nucleotide sequences are shown in GenBank reference numbers NM_006120 and NM_002118, respectively. Non-limiting examples of HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 nucleotide sequences are set forth in GenBank Reference Nos. NM_01911, NM_002124, NM_022555, NM_021983, NM_002125, respectively.

[0087] The term "vaccine" refers to a biological composition that, when administered to a subject, has the ability to generate adaptive immunity against a particular pathogen or disease in the subject. Typically, one or more antigens or fragments of antigens associated with the pathogen or disease of interest are administered to the subject. Vaccines can include, for example, inactivated or attenuated organisms (e.g., bacteria or viruses), cells, proteins expressed from or on cells (e.g., cell surface proteins), proteins produced by organisms (e.g., toxins), or parts of organisms (e.g., viral envelope proteins). In some cases, cells are engineered to express proteins that, when administered as a vaccine, enhance the ability of the subject to acquire immunity against a particular cell type (e.g., enhance the ability of the subject to acquire immunity against cancer cells). As used herein, the term "vaccine" or "whole cell cancer vaccine" includes, but is not limited to, modified cancer cells of the present disclosure.

[0088] The term "cytokine" refers to small proteins released by cells that have specific effects on cell-cell interactions and signaling. Cytokines are generally known as lymphokines (e.g., cytokines made by lymphocytes), monokines (e.g., cytokines made by monocytes), or chemokines (e.g., cytokines made by one white blood cell that act on other white blood cells). Cytokines may act on the cell that secretes them (e.g., autocrine action), on nearby cells (e.g., paracrine action), or on distant cells (e.g., endocrine action). In the context of the present disclosure, cytokines may include chemokines, interferons, interleukins, and / or tumor necrosis factors (TNF).Examples of cytokines include early T cell activation antigen-1 (ETA-1), lymphocyte-activating factor (LAF), interleukin-1 family members (IL-1α, IL-β, IL-1Ra, IL-18, IL-33, IL-36Ra, IL-36α, IL-36β, IL-36γ, IL-37, IL-38), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-14), interleukin-14 (IL-15), interleukin-15 (IL-16), interleukin-15 (IL-17), interleukin-16 (IL-18), interleukin-17 (IL-19), interleukin-18 (IL-20), interleukin-19 (IL-21), interleukin-19 (IL-22), interleukin-10 (IL-23), interleukin-11 (IL-24), interleukin-12 (IL-25), interleukin-13 (IL-26), interleukin-14 (IL-27), interleukin-15 (IL-28), interleukin-15 (IL-29), interleukin-16 (IL-29), interleukin-17 (IL-29), interleukin-18 (IL-29), interleukin-19 (IL-20), interleukin-19 (IL-20), interleukin-1 -Leukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interleukin-17 (IL-17), interleukin-18 (IL-18) , interleukin-23 (IL-21), interleukin-23 (IL-23), interleukin-25 (IL-25), interleukin-33 (IL-33), type I interferon family members (IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-ω), type II interferon family members (IFN-γ), type III interferon family members (IFNλ1 (IL-29), IFNλ2 (IL- 28A), IFNλ3 (IL-28B), IFNλ4), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage CSF (CSF-1), macrophage migration inhibitory factor (MIF), CD40L molecule (CD40L), RANTES molecule (RANTES), monocyte chemotactic protein (MCP-1), monocyte inflammatory proteins (MIP-1α, MIP-1β), lymphotactin, and / or fractalkine.

[0089] The term "granulocyte-macrophage colony-stimulating factor (GM-CSF)" refers to a monomeric glycoprotein, also known as "colony-stimulating factor (CSF2)", secreted by cells such as macrophages, T cells, mast cells, natural killer (NK) cells, endothelial cells, and fibroblasts. GM-CSF functions as a cytokine that affects several cell types, particularly macrophages and eosinophils. As part of the immune / inflammatory cascade, GM-CSF stimulates stem cells to produce granulocytes (i.e., neutrophils, eosinophils, and basophils) and monocytes. Monocytes subsequently mature into macrophages and dendritic cells after tissue infiltration. A non-limiting example of a CSF2 nucleotide sequence in humans (the gene encoding GM-CSF) is shown in GenBank reference number NM_000758.

[0090] The term "interferon" refers to cytokines produced in response to infection or other inflammatory stimuli. Interferons are signaling proteins synthesized and released by host cells in response to pathogens (e.g., viruses, bacteria, parasites, tumor cells). Interferons are classified into three subgroups: type I interferons, type II interferons (IFNγ), and type III interferons. Functionally, these cytokines modulate immune cell function. Although type III interferons are structurally distinct from type I interferons, they have overlapping functions, and both signal via the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway to induce transcription of interferon-stimulated genes (ISGs) and promote immune responses. (See, e.g., Goel et al. (2021). Interferon lambda in inflammation and autoimmune rheumatic diseases. Nat Rev Rheumatol 17, 349-362). Type I interferon proteins include IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-τ, IFN-δ, IFN-ζ, IFN-ω, and IFN-ν. Interferon alpha proteins are produced by white blood cells and are primarily involved in innate immune responses. Genes encoding IFN-α proteins include IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21. Non-limiting examples of human nucleotide sequences for IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21 are set forth in Gene Bank Reference Nos. NM_024013, NM_000605, NM_021068, NM_002169, NM_021002, NM_021057, NM_002170, NM_002171, NM_006900, NM_002172, NM_002173, NM_021268, and NM_002175, respectively.As an example, the gene IFNA2 encodes the IFN-α2a, IFN-α2b, and IFN-α2c variants. As used herein, the terms "IFN-α" and "IFN-α2" are used interchangeably and refer to the interferon proteins IFN-α2a or IFN-α2b. Type III interferon proteins include interferon lambda 1 (IFNλ1 (IL-29), interferon lambda 2 (IFNλ2 (IL-28A)), interferon lambda 1 (IFNλ3 (IL-28B)), and interferon lambda 4 (IFNλ4). Interferon lambda family members signal via the common IL-10 receptor subunit 2 (IL-10R2). Human interferon lambda proteins are encoded by four IFNL genes, IFNL1 (IL29), IFNL2 (IL28A), IFNL3 (IL28B), and IFNL4.

[0091] The term "costimulatory molecule" refers to a cell surface molecule that amplifies or counteracts the initial activation signal provided by the T cell receptor (TCR) to T cells after its interaction with antigen / major histocompatibility complex (MHC). Costimulatory molecules can generally affect the differentiation and fate of T cells. Costimulatory molecules belong to three major families: the immunoglobulin (Ig) superfamily, the tumor necrosis factor (TNF)-TNF receptor (TNFR) superfamily, and the T cell Ig and mucin (TIM) domain family. (See, for example, Rodriguez-Manzanet, Roselynn et al. "The costimulatory role of TIM molecules." Immunological reviews vol. 229,1 (2009): 259-70). Exemplary costimulatory molecules and ligands include, but are not limited to, CD28 and ligand B7-1 (CD80), CTLA-4, PDL-1, or B7-2 (CD86), CTLA-4 and ligand B7-1 (CD80) or B7-2 (CD86), ICOS and ligand ICOS-L, CD27 and ligand CD70, CD30 and ligand CD30L, CD40 and ligand CD40L (also known as CD154), OX40 and ligand OX40L, GITR and ligand GITRL, TIM-1 and ligand TIM-1, TIM-4, IgA, or phosphatidylserine (PtdSer), TIM-2 and ligand H-ferritin or semaphorin 4A (Sem4A), and TIM-4 and ligand phosphatidylserine (PtdSer).In the context of the present disclosure, costimulatory molecules can include a CD86 molecule (CD86), a CD80 molecule (CD80), a 4-1BB ligand molecule (4-1BBL, also known as CD137L), an ICOS ligand molecule (ICOS-L), a CD70 molecule (CD70, also known as CD27L), a CD40 molecule (CD40), an OX40 ligand molecule (OX40L), a GITR ligand molecule (GITRL), a TIM-4 molecule (TIM-4), a LIGHT molecule (LIGHT), an ICAM1 molecule (ICAM1), an LFA3 molecule (LFA3), a CD30 molecule (CD30), and combinations thereof (see, e.g., FIG. 5).

[0092] The term "tumor antigen" refers to an antigenic substance produced in tumor cells that can induce an immune response in the host. Tumor antigens generally refer to tumor-associated antigens (TAA) or tumor-specific antigens (TSA). Typically, TSAs are found only in cancer cells and not in healthy (e.g., non-cancerous) cells. TSAs can result from oncogenic driver mutations that generate novel peptide sequences (e.g., neo-antigens). A non-limiting example of a TSA is alpha-fetoprotein (AFP), which is expressed in germ cell tumors and hepatocellular carcinoma. TAA can have high levels in tumor cells and low levels in healthy cells. A non-limiting example of a TAA is melanoma-associated antigen (MAGE), which is expressed in the testis along with malignant melanoma.

[0093] The terms "CD47 molecule", "integrin-associated protein (IAP)", "MER6" and "OA3" are used interchangeably herein. CD47 generally refers to a variably glycosylated transmembrane protein of the immunoglobulin superfamily (IgSF). It contains an extracellular domain (ECD) with a single Ig-like domain, five transmembrane domains with a short intervening loop, and a C-terminal 34 amino acid cytoplasmic tail. CD47 is overexpressed on the surface of many types of cancer cells. CD47 forms a signaling complex with signal regulatory protein alpha (SIRPα) on macrophages, neutrophils, and T lymphocytes, allowing these cancer cells to escape from macrophage-mediated phagocytosis. SIRPα is expressed on myeloid cells, including macrophages and dendritic cells (DCs). Without being bound to any particular theory, CD47 typically provides a "do not eat me" signal to macrophages by binding to SIRPα to prevent phagocytosis. (See, e.g., Li et al., "Vaccination with CD47 deficient tumor cells elicits an antitumor immune response in mice." Nat Commun 11, 581 (2020)). Cells lacking CD47 are rapidly removed from the bloodstream by red pulp macrophages. (See, e.g., Oldenborg et al., "Role of CD47 as a marker of self on red blood cells." Science 288, 2051 (2000)). By way of example, in the context of cancer prophylactic or therapeutic agents, vaccination with CD47-deficient tumor cells can induce antitumor immunity. (See, Li et al., supra).

[0094] The terms "Ii (CD74) molecule", "CD74 molecule", "Cluster of Differentiation 74", "Major Histocompatibility Complex (MHC) class II-associated invariant chain (Ii)", "HLA class II histocompatibility antigen gamma chain", and "HLA-DR antigen-associated invariant chain (Ii)" refer to the protein encoded by the CD74 gene in humans. Invariant chain (Ii) is a polypeptide present on professional antigen-presenting cells, where it mediates peptide loading onto MHC class II molecules and CD4 + Regulates the peptidome presented to T lymphocytes. Generally, CD4 + Activation of T cells requires the delivery of an antigen-specific signal consisting of a peptide bound to MHC II molecules, in addition to costimulatory signals. T cell activation is initiated by CD4+ cells, which endocytose exogenously synthesized antigen, process it, and present it in association with their own MHC II molecules. + T cells are typically activated. This processing and presentation process requires the invariant chain (Ii), a molecule that is coordinately synthesized with MHC II molecules and prevents the binding and presentation of endogenous peptides encoded by APCs. Tumor-reactive CD4 + T cells are typically activated to tumor peptides generated by the antigen processing machinery of professional APCs rather than to peptides generated by tumor cells. Without being bound to any particular theory, tumor cells lacking Ii (CD74) may present a novel repertoire of MHC II-restricted tumor peptides that are not presented by professional APCs and may therefore be highly immunogenic. After activation, CD4 + T cells produce IFNγ and CD8 + MHC II vaccines provide help to T cells that do not have to react with native tumor cells. Thus, MHC II vaccines target CD4 T cells that facilitate antitumor immunity. +They have the potential to activate Th1 cells. For example, Ii(CD74)-negative breast cancer cells can present unique peptides that activate tumor-specific T cells from breast cancer patients. (See, e.g., Chornoguz et al., 2012. "Major histocompatibility complex class II+ invariant chain negative breast cancer cells present unique peptides that activate tumor-specific T cells from breast cancer patients." Molecular & Cellular Proteomics (11)11:1457-67 (2012)). For example, in the context of cancer preventive or therapeutic drugs, vaccination with Ii(CD74)-deficient tumor cells can induce anti-tumor immunity. (See, Chornoguz et al., supra).

[0095] The term "survival" refers to the period of time after diagnosis of a disease and / or after initiating or completing a particular course of treatment for a disease (e.g., cancer). The term "overall survival" includes clinical endpoints that describe a patient surviving for a given period of time after being diagnosed or treated for a disease, such as cancer. The term "disease-free survival" includes the period of time that a patient survives without signs of disease (e.g., without known recurrence) after treatment for a particular disease (e.g., cancer). In certain embodiments, disease-free survival is a clinical parameter used to determine the effectiveness of a particular therapy, and is usually measured in 1 or 5 year increments. The term "progression-free survival" includes the period of time that a patient lives with the disease without additional disease symptoms during and after treatment for a particular disease (e.g., cancer). In some embodiments, survival is expressed as a median or mean value.

[0096] III. DETAILED DESCRIPTION OF EMBODIMENTS The present disclosure is based in part on the inventors' discovery that modified human cancer cells (e.g., modified human cancer cell lines) containing selected HLA alleles can induce robust immune responses in subjects. Here, the inventors engineered targeting constructs containing selected HLA-A and HLA-DRB3 / 4 / 5 alleles. To eliminate the possibility of competition between endogenous HLA alleles and exogenous HLA alleles introduced as part of the delivery construct in the modified cancer cells, the inventors inactivated endogenous HLA class I and / or HLA class II alleles (e.g., HLA-A and / or HLA-DRB3 alleles) by genome editing. The engineered delivery constructs containing selected HLA alleles are transfected / electroporated or transduced into human cancer cells with HLA knock-in constructs to generate clonal cell lines.

[0097] In one aspect, the present disclosure provides modified human cancer cells comprising (a) one or more recombinant polynucleotides encoding alleles of human leukocyte antigen (HLA) class I genes, respectively; and (b) one or more recombinant polynucleotides encoding alleles of HLA class II genes, respectively, wherein one or more HLA alleles endogenous to the cells are inactivated. In some embodiments, the modified human cancer cells express at least one HLA class I allele and at least one HLA class II allele. Without being bound by any theory, the presence of both HLA class I and HLA class II alleles favors the activation of CD4+ T cells by class II molecules to provide help to and boost the activity of CD8+ T cells activated by class I HLA molecules. It has also been noted that CD8+ T cells can make cytokines that can provide help to CD4+ T cells, and CD4+ T cells can be cytotoxic. Expression of both class I and class II molecules on the same modified cancer cells provides a self-perpetuating feedback loop that elicits an immune response in the subject receiving the modified cancer cells.

[0098] In some embodiments, the recombinant polynucleotide is integrated into the cell genome. In other embodiments, the recombinant polynucleotide is present in one or more vectors in the cell. In some cases, all of the recombinant polynucleotides can be present in the same vector. In other examples, each recombinant polynucleotide can be present in a separate vector. In still other examples, two, three, four, five, six, or more recombinant polynucleotides can be present in the same vector. Any number of combinations of recombinant polynucleotides on a single vector and any number of vectors in a cell are permitted. In certain examples, all of the recombinant polynucleotides encoding HLA class I alleles can be present in one vector, and all of the recombinant polynucleotides encoding HLA class II alleles can be present in another vector. In certain embodiments, each vector in the cell comprises two recombinant polynucleotides. As a non-limiting example, recombinant polynucleotides encoding two unique HLA class I alleles (e.g., HLA-A alleles) can be present in the same vector. As another non-limiting example, recombinant polynucleotides encoding two unique HLA class II alleles (e.g., HLA-DRB3, HLA-DRB4, and / or HLA-DRB5 alleles) can be present on the same vector. As a further non-limiting example, recombinant polynucleotides encoding two unique cytokines and costimulatory molecules (e.g., pairwise combinations of GM-CSF, IFN-α, CD80, CD86, IL-12, IL-7, HLA-DRA, and 4-1BBL) can be present on the same vector.In certain embodiments, the cells comprise: (a) a vector comprising a recombinant polynucleotide encoding two unique HLA class I alleles (e.g., HLA-A alleles); (b) a vector comprising a recombinant polynucleotide encoding two unique HLA class II alleles (e.g., HLA-DRB3, HLA-DRB4, and / or HLA-DRB5 alleles); and (c) one or more vectors (e.g., 1, 2, 3, 4, or more vectors) each comprising a recombinant polynucleotide encoding two unique cytokines and costimulatory molecules (e.g., pairwise combinations of GM-CSF, IFN-α, CD80, CD86, IL-12, IL-7, HLA-DRA, and 4-1BBL).

[0099] The recombinant polynucleotides related to the present disclosure may include polynucleotides encoding at least one HLA-A class I allele and at least one HLA-A class II allele. Generally, HLA alleles are selected based on their profile. Without being bound to any particular theory, HLA alleles are highly polymorphic. Less polymorphic HLA alleles may be present in a greater proportion of the population. In some embodiments, the HLA (e.g., HLA-A and / or HLA-DRB) alleles selected for the modified human cancer cells described herein have low polymorphism and have at least one match with 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%, or at least 99% of the population. As a non-limiting example, the HLA-A alleles selected for the modified human cancer cells described herein match with about 92% of the population with HLA-A alleles. As another non-limiting example, the HLA-DRB (e.g., HLA-DRB3, HLA-DRB4, and / or HLA-DRB5) alleles selected for the modified human cancer cells described herein are compatible with the HLA-DRB alleles in about 98% of a population. In some embodiments, the population is a cancer patient population.

[0100] In some embodiments, the HLA class I genes are HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, or B2M genes. In other embodiments, the HLA class I genes are a combination of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, and / or B2M genes. In some embodiments, the modified human cancer cells comprise recombinant polynucleotides encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) alleles of HLA class I genes.

[0101] Examples of suitable HLA-A alleles include, but are not limited to, HLA-A*11:01, HLA-A*01:01, HLA-A*02:01, HLA-A*03:01, HLA-A*26:01, HLA-A*29:02, HLA-A*32:01, HLA-A*24:02, HLA-A*33:03, HLA-A*68:01, HLA-A*31:01, and HLA-A*02:06. The modified human cancer cells of the present invention can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more) recombinant polynucleotides encoding HLA-A alleles. In some embodiments, the one or more HLA-A alleles are each present in a population at a median frequency of at least about 2%. In other embodiments, the one or more HLA-A alleles are each present in a population at a maximum frequency of at least about 5%. In still other embodiments, the one or more HLA-A alleles are each present in a population at a median frequency of at least about 2% and a maximum frequency of at least about 5%.

[0102] Examples of suitable HLA-B alleles include, but are not limited to, HLA-B*13:02, HLA-B*41:01, HLA-B*18:03, HLA-B*44:02, HLA-B*07:02, HLA-B*35:01, HLA-B*40:01, HLA-B*35:08, HLA-B*55:01, HLA-B*51:01, HLA-B*44:03, HLA-B*58:01, HLA-B*08:01, HLA-B*18:01, HLA-B*15:01, and HLA-B*52:01. The modified human cancer cells of the present disclosure can include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more) recombinant polynucleotides encoding HLA-B alleles. In some embodiments, the one or more HLA-B alleles are each present in a population at a median frequency of at least about 2%. In other embodiments, the one or more HLA-B alleles are each present in a population at a maximum frequency of at least about 5%. In yet other embodiments, the one or more HLA-B alleles are each present in a population at a median frequency of at least about 2% and a maximum frequency of at least about 5%.

[0103] Examples of suitable HLA-C alleles include, but are not limited to, HLA-C*04:01, HLA-C*07:02, HLA-C*07:01, HLA-C*06:02, HLA-C*03:04, HLA-C*01:02, HLA-C*02:02, HLA-C*08:02, HLA-C*15:02, HLA-C*03:03, HLA-C*05:01, HLA-C*08:01, HLA-C*16:01, HLA-C*12:03, and HLA-C*14:02. The modified human cancer cells of the present disclosure can include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) recombinant polynucleotides encoding HLA-C alleles. In some embodiments, the one or more HLA-C alleles are present in the population at a median frequency of at least about 2%. In other embodiments, the one or more HLA-C alleles are present in the population at a maximum frequency of at least about 5%. In yet other embodiments, the one or more HLA-C alleles are present in the population at a median frequency of at least about 2% and a maximum frequency of at least about 5%.

[0104] In some embodiments, the HLA class II gene is an HLA class II alpha subunit gene. In other embodiments, the HLA class II gene is an HLA class II beta subunit gene. In certain embodiments, the HLA class II gene is a combination of an HLA class II alpha subunit gene and an HLA class II beta subunit gene.

[0105] In other embodiments, the HLA class II genes are HLA-DP, HLA-DM, HLA-DO, HLA-DQ, and / or HLA-DR genes. In some cases, the HLA-DO genes are HLA-DOA genes. In other cases, the HLA-DO genes are HLA-DOB genes. In certain cases, the modified human cancer cells contain recombinant nucleotides encoding both HLA-DOA and HLA-DOB gene alleles. In some cases, the HLA-DM genes are HLA-DMA genes. In other cases, the HLA-DM genes are HLA-DMB genes. In certain cases, the modified human cancer cells contain recombinant nucleotides encoding both HLA-DMA and HLA-DMB gene alleles.

[0106] In some embodiments, the HLA-DR gene is an HLA-DRA gene, an HLA-DRB1 gene, an HLA-DRB3 gene, an HLA-DRB4 gene, and / or an HLA-DRB5 gene. In certain embodiments, the modified human cancer cell comprises a recombinant polynucleotide encoding one or more (e.g., 1, 2, 3, 4, 5, or more) alleles of the HLA-DR gene.

[0107] Examples of suitable HLA-DRB3 alleles include, but are not limited to, HLA-DRB3*02:02, HLA-DRB3*01:01, and HLA-DRB3*03:01. Examples of suitable HLA-DRB4 alleles include, but are not limited to, HLA-DRB4*01:01 and HLA-DRB4*01:03. Examples of suitable HLA-DRB5 alleles include, but are not limited to, HLA-DRB5*01:02, HLA-DRB5*01:01, and HLA-DRB5*02:02. The modified human cancer cells of the present disclosure can include one or more (e.g., 1, 2, 3, 4, 5, or more) recombinant polynucleotides encoding HLA-DRB3 / 4 / 5 alleles. In some embodiments, one or more HLA-DRB3 / 4 / 5 alleles are present in the population at a median frequency of at least about 2% each. In other embodiments, one or more HLA-DRB3 / 4 / 5 alleles are present in the population at a maximum frequency of at least about 5% each. In yet other embodiments, one or more HLA-DRB3 / 4 / 5 alleles are present in the population at a median frequency of at least about 2% and a maximum frequency of at least about 5% each.

[0108] In some embodiments, the modified human cancer cells comprise at least one recombinant polynucleotide encoding one or more HLA class I genes selected from any one of class I genes listed in Table 1, codon optimized versions, mutants, or fragments thereof (SEQ ID NOs: 1-13, 22-29). For example, the modified human cancer cells may comprise recombinant polynucleotides encoding one, two, three, four, or more HLA class I genes driven by one or more promoters. In some embodiments, the modified human cancer cells further comprise at least one recombinant polynucleotide encoding one or more HLA class II genes selected from any one of class II genes listed in Table 1, codon optimized versions, mutants, or fragments thereof (SEQ ID NOs: 14-21, 30-35). For example, the modified human cancer cells may comprise recombinant polynucleotides encoding one, two, three, four, or more HLA class II genes driven by one or more promoters.

[0109] In some embodiments, the modified human cancer cells comprise at least two recombinant polynucleotides each encoding an HLA class I gene selected from any one of the class I genes, codon-optimized versions, variants, or fragments thereof set forth in Table 1 (SEQ ID NOs: 1-13, 22-29). For example, the modified human cancer cells can comprise a first recombinant polynucleotide encoding a first HLA class I gene and a second recombinant polynucleotide encoding a second HLA class I gene. In some embodiments, the modified human cancer cells comprise at least two recombinant polynucleotides each encoding an HLA class II gene selected from any of the class II genes, codon-optimized versions, variants, or fragments thereof set forth in Table 1 (SEQ ID NOs: 14-21, 30-35). For example, the modified human cancer cells can comprise a recombinant polynucleotide encoding a first HLA class II gene and a second recombinant polynucleotide encoding a second HLA class II gene.

[0110] Table 1: List of SEQ ID NOs. TIFF2025507939000002.tif254115

[0111] In some embodiments, the modified human cancer cells comprise one or more recombinant polynucleotides encoding at least a combination of a cytokine, a costimulatory molecule, a heterologous antigen (e.g., an antigen of a pathogen, a tumor-associated antigen, a neoantigen, an allergen, an antigen that is a target of an immune response), a variant thereof, or a fragment thereof. The cytokine can be a chemokine, an interferon, an interleukin, or a tumor necrosis factor. The cytokine can be an early T-cell activation antigen-1 (ETA-1), lymphocyte-activating factor (LAF), interleukin-1 family members (IL-1α, IL-β, IL-1Ra, IL-18, IL-33, IL-36Ra, IL-36α, IL-36β, IL-36γ, IL-37, IL-38), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-13), interleukin-13 (IL-14), interleukin-14 (IL-15), interleukin-15 (IL-16), interleukin-16 (IL-17), interleukin-17 (IL-18), interleukin-18 (IL-19), interleukin-19 (IL-20), interleukin-19 (IL-19), interleukin-19 (IL-19), interleukin-20 (IL-21), interleukin-20 (IL-22), interleukin-20 (IL-23), interleukin Interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interleukin-17 (IL-17), interleukin-18 (IL-19), IL-18), interleukin-21 (IL-21), interleukin-23 (IL-23), interleukin-25 (IL-25), interleukin-33 (IL-33), interferon alpha (IFN-α), interferon lambda 1 (IFNλ1 (IL-29)), interferon lambda 2 (IFNλ2 (IL-28A)), interferon lambda 3 (IFNλ3 (IL-28B)), interferon lambda 4 (IFNλ5 (IL-28C)), interferon lambda 5 (IFNλ6 (IL-28D)), interferon lambda 6 (IFNλ7 (IL-28E)), interferon lambda 7 (IFNλ8 (IL-28F)), interferon lambda 8 (IFNλ9 (IL-29F)), interferon lambda 9 (IFNλ1 (IL-29F)), interferon lambda 10 (IFNλ1 (IL-29F)), interferon lambda 11 (IFNλ1 (IL-29F)), interferon lambda 12 (IFNλ2 (IL-28A)), interferon lambda 13 (IFNλ3 (IL-28B)), interferon lambda 14 (IFNλ1 (IL-29F)), interferon lambda 15 (IFNλ2 (IL-28F)), interferon lambda 16 (IFNλ1 (IL-29F)), interferon lambda 17 (IFNλ1 (IL-29F)), interferon lambda 18 (IFNλ1 (IL-29F)), interferon lambda 19 (IFNλ1 (IL-29F)), interferon lambda 19 (IFNλ1 (IL-29F)), interferon lambda 19 (IFN The antibody can be selected from interferon lambda 4 (IFNλ4), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage CSF (CSF-1), macrophage migration inhibitory factor (MIF), CD40L molecule (CD40L), RANTES molecule (RANTES), monocyte chemotactic protein (MCP-1), monocyte inflammatory protein (MIP-1α, MIP-1β), lymphotactin, or fractalkine.The costimulatory molecule can be selected from at least one of a CD86 molecule (CD86), a CD80 molecule (CD80), a 4-1BB ligand molecule (also known as 4-1BBL, TNFSF9 or CD137L), an ICOS ligand molecule (ICOS-L), a CD70 molecule (CD70, also known as CD27L), a CD40 molecule (CD40), an OX40 ligand molecule (OX40L), a GITR ligand molecule (GITRL), a TIM-4 molecule (TIM-4), a LIGHT molecule (LIGHT), an ICAM1 molecule (ICAM1), an LFA3 molecule (LFA3), a CD30 molecule (CD30), and combinations thereof.

[0112] In some embodiments, the expression of the HLA alleles, xenoantigens, cytokines (e.g., GM-CSF and / or IFN-α2), and costimulatory molecules is under the control of two or more different promoters. In some cases, the expression of each of the HLA alleles, xenoantigens, cytokines (e.g., GM-CSF and / or IFN-α2), and costimulatory molecules is under the control of a separate promoter. In some embodiments, the expression of the HLA alleles, xenoantigens, cytokines (e.g., GM-CSF and / or IFN-α2), and costimulatory molecules is under the control of a single promoter. In some cases, the HLA alleles, xenoantigens, cytokines (e.g., GM-CSF and / or IFN-α2), and costimulatory molecules are expressed as polycistronic mRNA in a multicistronic vector. In certain examples, one or more cistrons are separated by an internal ribosome entry site. In other examples, one or more cistrons are separated by a self-cleaving peptide (e.g., T2A, P2A, E2A, F2A). One or more recombinant polynucleotides may be introduced into an expression vector to synthesize the corresponding HLA alleles. Construction of exemplary vectors is discussed in detail in Example 1 and Example 5 of Section B.

[0113] In some embodiments, the modified human cancer cells comprise: (a) one or more vectors each comprising a recombinant polynucleotide encoding at least one gene selected from CSF2, IFN-α2, CD86, IL-12, CD80, HLA-DRA, IL-7, and / or 4-1BBL (e.g., four vectors each comprising a recombinant polynucleotide encoding two different genes); (b) one or more vectors each comprising a recombinant polynucleotide encoding at least one gene selected from HLA-A*01:01, HLA-A*68:01, HLA-A*02:01, HLA-A*11:01, HLA-A*03:01, HLA-A*23:01, HLA -A*24:02, and / or HLA-A*33:03; and (c) one or more vectors comprising a recombinant polynucleotide encoding at least one gene selected from HLA-DRB3*02:02, HLA-DRB5*01:01, HLA-DRB4*01:01, HLA-DRB3*01:01, HLA-DRB3*03:01, HLA-DRB5*01:02, and / or HLA-DRB5*02:02.

[0114] In some embodiments, the modified human cancer cells comprise: (a) one or more vectors each comprising a recombinant polynucleotide encoding at least one gene selected from CSF2, IFN-α2, CD86, IL-12, CD80, HLA-DRA, IL-7, and / or 4-1BBL (e.g., four vectors each comprising a recombinant polynucleotide encoding two different genes); (b) one or more vectors each comprising a recombinant polynucleotide encoding at least one gene selected from CSF2, IFN-α2, CD86, IL-12, CD80, HLA-DRA, IL-7, and / or 4-1BBL (e.g., four vectors each comprising a recombinant polynucleotide encoding two different genes); and (c) a vector comprising a recombinant polynucleotide encoding the HLA-DRB3*02:02 allele and the HLA-DRB5*01:01 allele, or the HLA-DRB4*01:01 allele and the HLA-DRB3*01:01 allele, or the HLA-DRB3*03:01 allele and the HLA-DRB5*01:02 allele, or the HLA-DRB5*02:02 allele and the HLA-DRB3*01:01 allele.

[0115] In various embodiments, each vector comprises a recombinant polynucleotide encoding one or more 2A splicing peptides selected from T2A, P2A, or E2A located between two of the genes, two of the HLA alleles, and / or polynucleotides encoding the genes and the HLA alleles.

[0116] In some embodiments, the modified human cancer cells comprise one or more HLA-A and / or HLA-DR alleles endogenous to the cells that are inactivated. In some embodiments, the human cancer cells further comprise an inactivated HLA-DRB3 allele, an HLA-DRB4 allele, and / or an HLA-DRB5 allele. In some embodiments, the one or more HLA alleles that are inactivated have a sequence at least 85% identical to any one of SEQ ID NOs: 1-36 (Table 1), variants thereof, or fragments thereof. In some embodiments, the modified human cancer cells comprise an inactivated HLA-A*24:02 allele and / or an HLA-A*11:01 allele, variants thereof, or fragments thereof. In some embodiments, the modified human cancer cells further comprise an inactivated HLA-DRB3*01:01 allele and / or an HLA-DRB3*02:02 allele, variants thereof, or fragments thereof. As a non-limiting example, the modified human cancer cells may comprise at least one inactivated HLA-A allele and at least one inactivated HLA-DRB3 / 3 / 5 allele. In some embodiments, the modified human cancer cells comprise an inactivated HLA-A*24:02 allele, an HLA-A*11:01 allele, a DRB3*01:01 allele, an HLA-DRB3*02:02 allele, or a combination thereof. In some embodiments, the modified human cancer cells further comprise an inactivated CD47 molecule, a mutant thereof, or a fragment thereof, such that cell surface expression of CD47 is reduced or eliminated. In certain examples, the modified cancer cells described herein that lack expression of CD47 have increased immunogenicity. In some embodiments, the modified human cancer cells further comprise an inactivated Ii (CD74) molecule, a mutant thereof, or a fragment thereof. In some aspects, the modified cancer cells described herein (e.g., the Bria-OTS cell line) comprise an inactivated Ii(CD74), mutant, or fragment thereof such that cell surface expression of Ii(CD74) is reduced or eliminated.In certain instances, modified cancer cells described herein (e.g., Bria-OTS cell line) that are deficient in the expression of Ii (CD74) have increased immunogenicity. In some embodiments, modified cancer cells described herein (e.g., Bria-OTS cell line) that overexpress HLA-DRA overcome the inhibitory effect of Ii on tumor cells. In some embodiments, overexpression of HLA-DRA and deficient in Ii (CD74) expression in modified cancer cells described herein (e.g., Bria-OTS cell line) have a synergistic effect on the induction of anti-tumor immunity. As a non-limiting example, modified human cancer cells may include an inactivated HLA-A*24:02 allele, an HLA-A*11:01 allele, a DRB3*01:01 allele, an HLA-DRB3*02:02 allele, or a combination thereof, and an inactivated CD47 molecule, a mutant thereof, or a fragment thereof. As another non-limiting example, the modified human cancer cells may comprise an inactivated HLA-A*24:02 allele, an HLA-A*11:01 allele, a DRB3*01:01 allele, an HLA-DRB3*02:02 allele, or a combination thereof, and an inactivated Ii (CD74) molecule, a variant thereof, or a fragment thereof. As yet another non-limiting example, the modified human cancer cells may comprise an inactivated HLA-A*24:02 allele, an HLA-A*11:01 allele, a DRB3*01:01 allele, an HLA-DRB3*02:02 allele, or a combination thereof, an inactivated CD47 molecule, a variant thereof, or a fragment thereof, and an inactivated Ii (CD74) molecule, a variant thereof, or a fragment thereof.

[0117] In some aspects, the modified human cancer cells are derived from a human cancer cell line. For example, any number of human cancer cells or cancer cell lines, including clonal or non-clonal human cancer cells or cancer cell lines, are suitable for use in the compositions and methods described herein. Non-limiting examples of human cancer cell lines include the following cell lines and subclones thereof: SV-BR-1, SVCT, MDA-MB-231, MDA-MB-157, ZR-75-30, ZR-75-1, Hs 578T, MCF7, T47D, MTSV1-7 CE1, 1-7HB2, VP303, VP267, and VP229 breast cancer cell lines; PC-3, LNCaP (e.g., clone FGC), Shmac 5, P4E6, and VCaP prostate cancer cell lines; NCI-H2228, SHP-77, COR-L23 / R, COR-L23 / 5010, MOR / 0.2R, NCI-H69 / LX20, ChaGo-K-1, and Meta 7 lung cancer cell lines; SK-MEL-24 melanoma cell line; UM-UC-3, T24 / 83, ECV304, RT4, and HT 1197 bladder cancer cell lines; MDST8, C170, GP5d, GP2d, and LS 123 colon cancer cell lines; MFE-280 and MFE-296 endometrial cancer cell lines; CAKI 2, A.704, G-402, ACHN, G-401, UM-RC-7, and RCC4plusVHL renal cancer cell lines; SK-HEP-1, Hep 3B, PLC / PRF / 5, Hep G2, and Huh-7D12 hepatoma cell lines; HL60, Eos-HL-60, JVM-13, Sci-1, and Ri-1 leukemia cell lines; BHL-89, COR-L24, U937(CD59+), My-La CD8+, and HGC-27 lymphoma cell lines; A375-C6, GR-M, VA-ES-BJ, MEWO, and COLO 818 skin cancer cell lines; AsPC-1, HuP-T4, HuP-T3, BxPC-3, and CFPAC-1 pancreatic cancer cell lines; 8505C, 8305C, FTC-238, TT, RO82-W-1, and K1 thyroid cancer cell lines;HeLa DH, HR5-CL11, HtTA-1, HR5, X1 / 5, HeLa, C-4I, C-4 II, HeLa S3, Ca Ski, HeLa229, Hep2 (HeLa derivatives), HeLa B, Bu25 TK- HeLa Ohio, and HeLa (AC-free) cervical cancer cell lines; NB69, BE(2)-C, BE(2)-M17, SK-N-BE(2), and SK-N-DZ brain cancer cell lines; OV7, OV17R, OV58, OV56, A2780ADR, A2780, COLO 720 E, SW 626, SK-OV-3, PA-1, 59M, OAW28, TO14, PEO23, and COV362 ovarian cancer cell lines; IMR 32 abdominal cancer cell line; SW 13 adrenocortical carcinoma cell line;TR146 buccal mucosa carcinoma cell line;SK-GT-4 esophageal carcinoma cell line;TE 671 embryonal carcinoma cell line;FLYRD18 fibrosarcoma cell line;1411H germ cell tumor cell line;MFM-223 breast adenocarcinoma cell line;H-EMC-SS muscle carcinoma cell line;Detroit 562 pharyngeal carcinoma cell line;BeWo placental carcinoma cell line;Mero-95 pleural cavity carcinoma cell line,SW 837, SW 1463, CMT 93, HRT-18, and HRA-19 rectal carcinoma cell lines;Y79, WERI, and RB247C retinal carcinoma cell lines;CHP-100 spinal carcinoma cell line;KARPAS 1718 splenic lymphoma cell line;AGS and KATO-III gastric carcinoma cell lines;NTERA-2 clone D1 testicular carcinoma cell line;SCC-9, H357, H103, BICR 56, and PE / CA-PJ49 tongue cancer cell lines; MES-SA / Dx-5, MES-SA, COLO 685, and COLO 684 uterine cancer cell lines; and HMVII vaginal cancer cell line. In certain embodiments, the human cancer cell line is a breast cancer (e.g., SV-BR-1), prostate cancer (e.g., PC-3, LNCaP), melanoma (e.g., SK-MEL-24), or lung cancer (e.g., NCI-H2228) cell line. The cell lines and others described herein are available, for example, from Sigma-Aldrich (www.sigmaaldrich.com). In other embodiments, the modified human cancer cells are derived from tumor biopsies, e.g., cancer cells obtained from a subject to be treated for cancer prior to modification of the cancer cells;

[0118] A. Selected HLA alleles In one aspect, the modified human cancer cells described herein (e.g., Bria-OTS cell line) can express a combination of HLA genes having the sequence of any one of SEQ ID NOs:1-35 (Table 1), variants thereof, or fragments thereof. Illustratively, the modified human cancer cells described herein (e.g., Bria-OTS cell line) can express a combination of HLA-A*01:01, HLA-A*68:01, HLA-DRB3*02:02, and / or HLA-DRB4*01:01 alleles; HLA-A*02:01, HLA-A*11:01, HLA-DRB4*01:01, and / or HLA-DRB3*03: combination of HLA-A*03:01, HLA-A*23:01, HLA-DRB3*01:01 and / or HLA-DRB5*01:02 alleles; or combination of HLA-A*33:03, HLA-DRB5*01:01 and / or HLA-DRB5*02:02 alleles, and optionally an HLA-A*24:02 allele. As another illustration, modified human cancer cells described herein (e.g., Bria-OTS cell line) can comprise a combination of HLA-A*01:01, HLA-A*68:01, HLA-DRB3*02:02 and / or HLA-DRB5*01:01 alleles; a combination of HLA-A*02:01, HLA-A*11:01, HLA-DRB4*01:01 and / or HLA-DRB3*01:01 alleles; a combination of HLA-A*03:01, HLA-A*23:01, HLA-DRB3*03:01 and / or HLA-DRB5*01:02 alleles; or a combination of HLA-A*24:02, HLA-A*33:03, HLA-DRB5*02:02 and / or HLA-DRB3*01:01 alleles.

[0119] In another aspect, the modified human cancer cells described herein (e.g., Bria-OTS cell line) can express: (a) at least one HLA gene having the sequence of any one of SEQ ID NOs: 1-21 (Table 1), variants thereof, or fragments thereof; (b) at least one codon-optimized HLA gene (e.g., a polynucleotide terminating in an HLA class I allele and / or an HLA class II allele) having the sequence of any one of SEQ ID NOs: 22-35 (Table 1), variants thereof, or fragments thereof; and / or (c) at least one codon-optimized costimulatory molecule having the sequence of any one of SEQ ID NOs: 36-44 (Table 1), variants thereof, or fragments thereof. For example, the modified human cancer cells described herein (e.g., the Bria-OTS cell line) may comprise an HLA-A*68:01 allele, a variant thereof, or a fragment thereof; a codon-optimized HLA-A*01:01 allele, a variant thereof, or a fragment thereof; and a costimulatory molecule, a variant thereof, or a fragment thereof.

[0120] In some aspects, any one of the HLA genes, variants thereof, or fragments thereof may have a polynucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identity or similarity with its corresponding wild type sequence. In some embodiments, any one of the HLA genes, variants thereof, or fragments thereof may have a polynucleotide sequence with up to 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity or similarity with its corresponding wild type sequence. In some embodiments, the polynucleotide encoding the modified or codon-optimized HLA allele, variants thereof, or fragments thereof has at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identity or similarity with its corresponding wild type sequence. In some embodiments, a polynucleotide encoding a modified or codon-optimized HLA allele, variants thereof, or fragments thereof has at most 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity or similarity to its corresponding wild-type sequence. In some embodiments, a polynucleotide encoding a modified or codon-optimized HLA allele, variants thereof, or fragments thereof has about 10%-99%, about 30%-80%, about 40%-95%, about 60%-85% identity or similarity to its corresponding wild-type sequence.

[0121] B. Expression vectors for selected HLA alleles In some embodiments, the modified human cancer cells described herein contain one or more expression vectors to express recombinant HLA alleles. A wide variety of expression vectors can be used, such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, plasmids or transposons. The viral vectors that can be used include, for example, vectors based on HIV, SV40, EBV, HSV or BPV. The expression vector can be replication-deficient by design, so that the viral vector is deficient in one or more functions essential for the replication or synthesis of viral genome and the assembly of viral particles. Many of the currently existing replication-deficient viruses can carry large therapeutic genes, effectively transduce various types of cells, and provide long-term stable expression of the gene of interest.

[0122] Lentiviruses are a subset of retroviruses commonly used in research. Lentiviruses can transduce both dividing and non-dividing cells without significant immune response. These viruses also stably integrate into the host genome, allowing long-term transgene expression. A common lentivirus is the human immunodeficiency virus (HIV), which uses the envelope glycoproteins of other viruses to target a wide range of cell types.

[0123] One safety feature of lentivirus is that the components required for producing infectious viral particles (virions) are generally divided into multiple plasmids.For example, infectious viral particles may contain the plasmids that code for viral capsid and envelope components (typically called packaging and envelope plasmids), and the plasmids that code for viral genome (typically called transfer plasmids). Common lentiviral packaging and envelope plasmids that can be used herein include, but are not limited to, pRSV-Rev, pMDLg / pRRE, psPAX2, pCMVdeltaR8.2, pMD2.G, pCMV-VSV-G, pCMV-dR8.2 dvpr, pCI-VSVG, pCPRDEnv, pLTR-RD114A, pLTR-G, pCD / NL-BH*DDD, psPAX2-D64V, pCEP4-tat, pHEF-VSVG, pNHP, pCAG-Eco, and pCAG-VSVG. Common lentiviral transfer plasmids that can be used herein include, but are not limited to, pLKO.1 puro, pLKO.1-TRC cloning plasmid, pLKO.3G, Tet-pLKO-puro, pSico, pLJM1-EGFP, FUGW, pLVTHM, pLVUT-tTR-KRAB, pLL3.7, pLB, pWPXL, pWPI, EF.CMV.RFP, pLenti CMV Puro DEST, pLenti-puro, pLOVE, pULTRA, pLX301, pLinducer20, pHIV-EGFP, Tet-pLKO-neo, pLV-mCherry, pCW57.1, pLionII, pSLIK-Hygro, and pInducer10-mir-RUP-PheS.

[0124] There are several approaches to producing lentiviral vectors. (See Logan et al. "Factors influencing the titer and infectivity of lentiviral vectors." Hum Gene Ther. 2004 Oct;15(10):976-88. doi: 10.1089 / hum.2004.15.976. PMID: 15585113; Dull, T et al. "A third-generation lentivirus vector with a conditional packaging system." Journal of virology vol. 72,11 (1998): 8463-71. doi:10.1128 / JVI.72.11.8463-8471.1998.) Alternatively, lentiviral vectors may be purchased from commercial suppliers. Generally, lentiviral vector production involves multiple steps including plasmid generation and production, cell expansion, plasmid transfection, viral vector production, purification, packing and finishing (see, e.g., www.addgene.org / viral-vectors / lentivirus / ; www.thermofisher.com / us / en / home / clinical / cell-gene-therapy / gene-therapy / lv-production-workflow.html).

[0125] Lentiviral vectors may be designed to simultaneously express one or more genes of interest. A variety of molecular strategies are available, including the use of multiple promoters, splicing signals, gene fusions, cleavage elements, and multicistronic vectors. (See, for example, the review by Shaimardanova et al., "Production and application of multicistronic constructs for various human disease therapies." Pharmaceutics 2019,11,580).

[0126] Multicistronic vectors generally contain an internal ribosome entry site (IRES) and a sequence encoding the nucleotide sequence of a self-cleaving 2A peptide. The use of an IRES and a self-cleaving 2A peptide allows for the simultaneous expression of two or more separate proteins from the same mRNA.

[0127] Self-cleaving 2A peptides are used for the production of multicistronic vectors thanks to their small size and ability to self-cleave. 2A peptides consist of 16-20 amino acids and originate from viral RNA. Common 2A peptides used to produce multicistronic vectors are F2A (2A peptide from foot and mouth disease virus), E2A (2A peptide from equine rhinitis virus), P2A (2A peptide from porcine teschovirus-1), and T2A (2A peptide from Thosea asigna virus).

[0128] In constructs with the sequence of 2A peptides, translation is initiated once and synthesis along the mRNA occurs continuously. During translation, the first peptide is cleaved from the second peptide in the 2A region. The 2A peptide cleavage site is located between glycine and proline. The cleavage process occurs inside the ribosome during protein synthesis. The formation of normal peptide bonds between amino acids is only inhibited at the cleavage site, therefore, cleavage does not affect the translation of subsequent proteins, and synthesis continues without dissociation of the ribosome. Thus, the translation of multiple genes is interdependent. The use of different 2A peptides can affect the expression level of downstream proteins. The combination of the order of the 2A peptide sequences can prevent a gradual decrease in gene expression from the beginning to the end of the multicistronic construct. For example, the combination of the following order of 2A peptide sequences, namely T2A, P2A, and E2A, is optimal when creating a multicistronic vector containing four genes.

[0129] In some aspects, the modified human cancer cells described herein are expressed using non-viral approaches. Exemplary methods include, but are not limited to, cationic lipids such as liposomes and lipoplexes, polymers or polyplexes and dendrimers, naked plasmids for direct delivery, electroporation, ultrasound and microbubbles, magnetofection, inorganic molecules.

[0130] In one aspect, the present disclosure provides an expression vector for the modified human cancer cells described herein. The vector may comprise one or more recombinant polynucleotides encoding at least one HLA-A class I allele and at least one HLA-A class II allele (e.g., at least one HLA-DRB3 allele, at least one HLA-DRB4 allele, and / or at least one HLA-DRB5 allele) selected from any one of SEQ ID NOs: 1-35 (Table 1), variants thereof, or fragments thereof. The recombinant polynucleotide may further comprise at least one co-stimulatory molecule and / or at least one HLA allele selected from any one of SEQ ID NOs: 36-44 (Table 1). In some embodiments, the HLA allele is a codon-optimized HLA allele (e.g., HLA-DRA). In some embodiments, the recombinant polynucleotide further comprises a sequence encoding one or more cytokines (eg, GM-CSF, IFN-α2, including IFN-α2a and IFN-α2b). In some embodiments, recombinant polynucleotides encoding HLA-A class I and II alleles (e.g., HLA-DRB3, HLA-DRB4, and / or HLA-DRB5 alleles), costimulatory molecules, xenoantigens, and / or cytokines each have sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical or similar to their corresponding wild-type sequences; each have sequences that are up to 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar to their corresponding wild-type sequences; or each have sequences that are about 10%-99%, about 30%-80%, about 40%-95%, about 60%-85% identical or similar to their corresponding wild-type sequences. In various embodiments, the expression vector comprises one or more recombinant polynucleotides encoding one or more promoters to drive expression of a selected HLA allele, a codon-optimized HLA allele, a costimulatory molecule, or an adjuvant.The expression vector may comprise a recombinant polynucleotide that encodes the MNDU3 promoter, the EF1α promoter, or both. For example, the expression vector may comprise a first recombinant polynucleotide that encodes the MNDU3 promoter and a second recombinant polynucleotide that encodes the EF1α promoter. In certain embodiments, the expression vector contains a recombinant polynucleotide that encodes the MNDU3 promoter and the EF1α promoter.

[0131] Table 2 shows the construction of an exemplary expression vector using multicistronic lentiviral vector. Each lentiviral vector carries two cytokine genes and four HLA alleles. GM-CSF is encoded by CSF2 gene, while IFN-α2a and IFN-α2b are variants encoded by IFNA2.

[0132] Table 2. Constructs for the generation of exemplary engineered human cancer cell lines TIFF2025507939000003.tif58160# Self-splicing 2A peptide

[0133] In some illustrative examples, Figures 1-4 show components of multicistronic lentiviral vectors for expressing selected HLA alleles in human modified cancer cells. Construction of the expression vectors is described in Example 1.

[0134] Referring to Figure 1, from 5' to 3', recombinant polynucleotides encoding EF1α promoter, GM-CSF (encoded by CSF2 gene), T2A splicing peptide, IFN-α2b (encoded by IFNA2 gene), MNDU3 promoter, HLA-A*01:01 allele, T2A splicing peptide, HLA-A*68:01 allele, P2A splicing peptide, HLA-DRB3*02:02, E2A splicing peptide, and HLA-DRB4*01:01 allele are inserted into the pCCLc backbone. As shown in Figure 1, the EF1α promoter is located at the 5' end of the sequence encoding GM-CSF and IFN-α (e.g., IFN-α2a, or IFN-α2b). Thus, the synthesis of GM-CSF and IFN-α is driven by the EF1α promoter. The MNDU3 promoter is located at the 3' end of the sequence encoding GM-CSF and IFN-α and at the 5' end of the sequence encoding four HLA alleles and three 2A splicing peptides. Each of the three 2A splicing peptides is located between the polynucleotide sequences encoding two HLA alleles. In particular, the three 2A splicing peptides are in the order of T2A, P2A, and E2A to maximize the expression of all four HLA genes.

[0135] Referring to Figure 2, from 5' to 3', recombinant polynucleotides encoding the EF1α promoter, GM-CSF (encoded by the CSF2 gene), T2A splicing peptide, IFN-α (such as IFN-α2a or IFN-α2b encoded by the IFNA2 gene), MNDU3 promoter, HLA-A02:01 allele, T2A splicing peptide, HLA-A*11:01 allele, P2A splicing peptide, HLA-DRB4*01:01 allele, E2A peptide, and HLA-DRB3*03:01 allele are inserted into the pCCLc backbone. As shown in Figure 2, the EF1α promoter is located at the 5' end of the sequence encoding GM-CSF, T2A splicing peptide, and IFN-α. Thus, the synthesis of both GM-CSF and IFN-α is driven by the EF1α promoter. The MNDU3 promoter is located at the 3' end of the sequence encoding GM-CSF and IFN-α and at the 5' end of the sequence encoding four HLA alleles and three 2A splicing peptides. Each of the three 2A splicing peptides is located between the polynucleotide sequences encoding two HLA alleles. In particular, the three 2A splicing peptides are in the order of T2A, P2A, and E2A to maximize the expression of all four HLA genes.

[0136] Figures 3 and 4 show similar constructions of the expression vector in Figure 2. In particular, the expression vector in Figure 3 is designed to express HLA alleles (5' to 3'): HLA-A*03:01, HLA-A*23:01, HLA-DRB3*01:01, and HLA-DRB5*01:02. The expression vector in Figure 4 is designed to express HLA alleles (5' to 3'): HLA-A*24:02, HLA-A*33:03, HLA-DRB5*01:01, and HLA-DRB5*02:02. As shown in Figures 3 and 4, 2A splicing peptides (in the order of T2A, P2A, and E2A) are located between the polynucleotide sequences encoding two HLA alleles, two promoters, or HLA alleles and promoters, respectively.

[0137] In another embodiment, the expression vector is a multicistronic lentiviral vector designed to express any one combination of cytokines or HLA genes under the control of the EF1α promoter and / or the MNDU3 promoter. Exemplary constructs of multicistronic lentiviral vectors are shown in Table 3. Illustratively, the expression vector comprises one or more recombinant polynucleotides each encoding at least one gene selected from (a) CSF2, IFNA2, CD86, IL-12, CD80, HLA-DRA, IL-7, and / or 4-1BBL (also known as TNFSF9 or CD137L); (b) a gene encoding at least one gene selected from HLA-A*01:01, HLA-A*68:01, HLA-A*02:01, HLA-A*11:01, HLA-A*03:01, HLA-A*23:01, HLA-A*24:02, and / or HLA-A*33:03; or (c) one or more recombinant polynucleotides each encoding at least one gene selected from HLA-DRB3*02:02 allele, HLA-DRB5*01:01 allele, HLA-DRB3*03:01 allele, HLA-DRB4*01:01 allele, HLA-DRB3*01:01 allele, HLA-DRB5*01:02 allele, and / or HLA-DRB5*02:02 allele. As an example, the expression vector may comprise a recombinant polypeptide encoding a CD86 gene driven under the control of an EF1α promoter and an IL-12 gene driven under the control of an MDNU3 promoter. As another example, the expression vector may comprise a recombinant polypeptide encoding a CD86 gene driven under the control of an MDNU3 promoter and an IL-12 gene driven under the control of an EF1α promoter.

[0138] Table 3. Lentiviral constructs for the generation of exemplary modified human cell lines, including costimulatory molecules and HLA alleles. TIFF2025507939000004.tif102160

[0139] In some embodiments, the expression vector comprises one or more recombinant polynucleotides encoding at least one immunomodulatory molecule, costimulatory molecule, or cytokine (e.g., two or more immunomodulatory molecules, costimulatory molecules, or cytokines) including, but not limited to, CSF2, IFNA2, CD86, IL-12, CD80, HLA-DRA, IL-7, and / or 4-1BBL (also known as TNFSF9 or CD137L). In some embodiments, the expression vector comprises one or more recombinant polynucleotides encoding at least one immunomodulatory molecule, costimulatory molecule, or cytokine (e.g., two or more immunomodulatory molecules, costimulatory molecules, or cytokines) selected from CSF2 and IFNA2; CSF2; CD86 and IL-12; CD80 and HLA-DRA alleles; or IL-7 and 4-1BBL.

[0140] In some embodiments, the expression vector comprises one or more recombinant polynucleotides each encoding at least one HLA class I allele (e.g., two or more HLA class I alleles), including but not limited to an HLA-A*01:01 allele, an HLA-A*68:01 allele, an HLA-A*02:01 allele, an HLA-A*11:01 allele, an HLA-A*03:01 allele, an HLA-A*23:01 allele, an HLA-A*24:02 allele, and / or an HLA-A*33:03 allele. In some embodiments, the expression vector comprises one or more recombinant polynucleotides each encoding at least one HLA class I allele (e.g., two or more HLA-A alleles) selected from the HLA-A*01:01 and HLA-A*68:01 alleles, the HLA-A*02:01 and HLA-A*11:01 alleles, the HLA-A*03:01 and HLA-A*23:01 alleles, and / or the HLA-A*24:02 and HLA-A*33:03 alleles.

[0141] In some embodiments, the expression vector comprises one or more recombinant polynucleotides each encoding at least one HLA class II allele (e.g., two or more HLA class II alleles), including but not limited to, an HLA-DRB3*02:02 allele, an HLA-DRB5*01:01 allele, an HLA-DRB4*01:01 allele, an HLA-DRB3*01:01 allele, an HLA-DRB3*03:01 allele, an HLA-DRB5*01:02 allele, and / or an HLA-DRB5*02:02 allele. In some embodiments, the expression vector comprises one or more recombinant polynucleotides encoding at least one HLA class II allele (e.g., two or more HLA-DRB alleles) selected from the HLA-DRB3*02:02 allele and the HLA-DRB5*01:01 allele, the HLA-DRB4*01:01 allele and the HLA-DRB3*01:01 allele, the HLA-DRB3*03:01 allele and the HLA-DRB5*01:02 allele, and / or the HLA-DRB5*02:02 allele and the HLA-DRB3*01:01 allele, respectively.

[0142] In some embodiments, the expression vector is capable of expressing at least one immunomodulatory molecule, costimulatory molecule, or cytokine, at least one HLA class I allele (e.g., HLA-A allele), and / or at least one HLA class II allele (e.g., HLA-DRB allele) in the cancer cell line (e.g., modified human cancer cell line). In some embodiments, the expression vector is capable of expressing one or more (e.g., at least two) immunomodulatory molecules, costimulatory molecules, or cytokines in the cancer cell line (e.g., modified human cancer cell line); or the expression vector is capable of expressing one or more (e.g., at least two) HLA class I alleles in the cancer cell line (e.g., modified human cancer cell line); or the expression vector is capable of expressing one or more (e.g., at least two) HLA class II alleles in the cancer cell line (e.g., modified human cancer cell line). In various embodiments, one or more endogenous HLA-A and / or HLA-DR3 alleles in the cancer cell line are inactivated. In some embodiments, at least one of the endogenous HLA-A*24:02 allele, HLA-A*11:01 allele, HLA-DRB3*01:01 allele, and / or HLA-DRB3*02:02 allele in the cancer cell line is inactivated.

[0143] In various embodiments, each of the one or more recombinant polynucleotides comprising a gene encoding an immune modulating molecule, costimulatory molecule, or cytokine selected from CSF2, IFNA2, CD86, IL-12, CD80, HLA-DRA, IL-7, and / or 4-1BBL, or any one of the HLA-A alleles, or any one of the HLA-DRB alleles (e.g., the HLA-DRB3 allele, the HLA-DRB4 allele, and the HLA-DRB5 allele), further comprises a 2A splicing peptide located between any of the two genes, any of the two HLA alleles, or the gene and the HLA allele.

[0144] In some embodiments, the expression vector comprises a recombinant polynucleotide encoding a cytokine, a chemokine, an interferon, an interleukin, and / or a tumor necrosis factor. In some embodiments, the recombinant polynucleotide encodes an early T-cell activation antigen-1 (ETA-1), lymphocyte-activating factor (LAF), an interleukin-1 family member (IL-1α, IL-β, IL-1Ra, IL-18, IL-33, IL-36Ra, IL-36α, IL-36β, IL-36γ, IL-37, IL-38), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), , interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interleukin-17 (IL-17), interleukin-18 (IL-18) , interleukin-21 (IL-21), interleukin-23 (IL-23), interleukin-25 (IL-25), interleukin-33 (IL-33), interferon alpha (IFN-α), interferon lambda 1 (IFNλ1 (IL-29)), interferon lambda 2 (IFNλ2 (IL-28A)), interferon lambda 3 (IFNλ3 (IL-28B)), interferon lambda 4 (IFNλ4 (IL-28C)), interferon lambda 5 (IFNλ6 (IL-28D)), interferon lambda 6 (IFNλ7 (IL-28E)), interferon lambda 7 (IFNλ8 (IL-28F)), interferon lambda 8 (IFNλ9 (IL-28G)), interferon lambda 9 (IFNλ10 (IL-28H)), interferon lambda 10 (IFNλ110 (IL-28H)), interferon lambda 11 (IFNλ120 (IL-28H)), interferon lambda 12 (IFNλ130 (IL-28H)), interferon lambda 13 (IFNλ140 (IL-28H)), interferon lambda 14 (IFNλ150 (IL-28H)), interferon lambda 15 (IFNλ160 (IL-28H)), interferon lambda 16 (IFNλ170 (IL-28H)), interferon lambda 17 (IFNλ180 (IL-28H)), interferon lambda 18 (IFNλ190 (IL-28H)), interferon lambda 19 (IFNλ110 (IL-28H)), interferon lambda 19 (IFNλ190 ( ), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage CSF (CSF-1), macrophage migration inhibitory factor (MIF), CD40L molecule (CD40L), RANTES molecule (RANTES), monocyte chemotactic protein (MCP-1), monocyte inflammatory proteins (MIP-1α, MIP-1β), lymphotactin, and / or fractalkine. In some embodiments, the cytokine comprises GM-CSF. In some embodiments, the cytokine comprises IFN-α, e.g., IFN-α2a or IFN-α2b.

[0145] In some embodiments, the expression vector further comprises a recombinant polynucleotide encoding a costimulatory molecule selected from at least one of a CD86 molecule (CD86), a CD80 molecule (CD80), a 4-1BB ligand molecule (4-1BBL, also known as CD137L), an ICOS ligand molecule (ICOS-L), a CD70 molecule (CD70, also known as CD27L), a CD40 molecule (CD40), an OX40 ligand molecule (OX40L), a GITR ligand molecule (GITRL), a TIM-4 molecule (TIM-4), a LIGHT molecule (LIGHT), an ICAM1 molecule (ICAM1), an LFA3 molecule (LFA3), a CD30 molecule (CD30), and / or a combination thereof.

[0146] In some aspects, the expression vector further comprises a recombinant polynucleotide encoding a heterologous antigen (e.g., an antigen of a pathogen, a tumor-associated antigen, a neoantigen, an allergen, an antigen that is the target of an autoimmune response, or a fragment thereof).

[0147] In one aspect, the disclosure provides an expression vector comprising a recombinant polynucleotide comprising a sequence encoding at least one allele of an HLA class I gene having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 22-29 (Table 1), and optionally at least one heterologous polynucleotide sequence. In some embodiments, the disclosure provides an expression vector comprising a recombinant polynucleotide comprising a sequence encoding at least one allele of an HLA class I gene having a polynucleotide of any one of SEQ ID NOs: 22-29 (Table 1), and optionally at least one heterologous polynucleotide sequence. In some embodiments, the recombinant polynucleotide further comprises a sequence encoding a second allele of an HLA class I gene or an allele of a second HLA class I gene. In some embodiments, the recombinant polynucleotide further comprises a sequence encoding an allele of an HLA class II gene. The allele of the HLA class II gene has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 30-35 (Table 1). In some embodiments, the allele of the HLA class II gene has a polynucleotide of any one of SEQ ID NOs: 30-35 (Table 1).

[0148] In various embodiments, the heterologous polynucleotide sequence encodes the amino acid sequence of a cytokine, chemokine, interferon, interleukin, or tumor necrosis factor. In some embodiments, the heterologous polynucleotide sequence encodes an amino acid sequence of an early T cell activation antigen-1 (ETA-1), lymphocyte activating factor (LAF), interleukin-1 family members (IL-1α, IL-β, IL-1Ra, IL-18, IL-33, IL-36Ra, IL-36α, IL-36β, IL-36γ, IL-37, IL-38), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), or a combination thereof. ), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interleukin-17 (IL-17), interleukin-18 (IL-18 ), interleukin-21 (IL-21), interleukin-23 (IL-23), interleukin-25 (IL-25), interleukin-33 (IL-33), interferon alpha (IFN-α), interferon lambda 1 (IFNλ1 (IL-29)), interferon lambda 2 (IFNλ2 (IL-28A)), interferon lambda 3 (IFNλ3 (IL-28B)), interferon lambda 4 (IFNλ4 ), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage CSF (CSF-1), macrophage migration inhibitory factor (MIF), CD40L molecule (CD40L), RANTES molecule (RANTES), monocyte chemotactic protein (MCP-1), monocyte inflammatory proteins (MIP-1α, MIP-1β), lymphotactin, and / or fractalkine. In some embodiments, the cytokine comprises GM-CSF. In some embodiments, the cytokine comprises IFN-α, preferably IFN-α2a or IFN-α2b.

[0149] In some embodiments, the heterologous polynucleotide sequence encodes one of the costimulatory molecules selected from at least one of a CD86 molecule (CD86), a CD80 molecule (CD80), a 4-1BB ligand molecule (4-1BBL, also known as CD137L), an ICOS ligand molecule (ICOS-L), a CD70 molecule (CD70, also known as CD27L), a CD40 molecule (CD40), an OX40 ligand molecule (OX40L), a GITR ligand molecule (GITRL), a TIM-4 molecule (TIM-4), a LIGHT molecule (LIGHT), an ICAM1 molecule (ICAM1), an LFA3 molecule (LFA3), a CD30 molecule (CD30), and / or a combination thereof.

[0150] In various embodiments, the heterologous polynucleotide sequence is located between the sequences encoding two HLA class I genes, for example, the heterologous polynucleotide sequence is located at the 3' end of the sequence encoding a first HLA class I gene and at the 5' end of the sequence encoding a second HLA class I gene.Similarly, the heterologous polynucleotide sequence can be located between the sequences encoding two cytokines, two costimulatory molecules, or two heterologous antigens.In some embodiments, the heterologous polynucleotide sequence can be located between the sequences encoding HLA alleles, cytokines, costimulatory molecules, and / or heterologous antigens.In various embodiments, the heterologous polynucleotide sequence encodes one of the 2A splicing peptides selected from T2A, P2A, or E2A.

[0151] C. Composition In one aspect, the disclosure provides a composition comprising a modified human cancer cell as described herein, wherein the modified human cancer comprises (a) one or more recombinant polynucleotides encoding respective alleles of a human leukocyte antigen (HLA) class I gene; and / or (b) one or more recombinant polynucleotides encoding respective alleles of an HLA class II gene, wherein one or more HLA alleles endogenous to the cell are inactivated. In some embodiments, the HLA class I gene and the HLA class II gene are selected from any of SEQ ID NOs: 1-35 set forth in Table 1.

[0152] In one aspect, the disclosure provides a composition comprising a modified human cancer cell as described herein, the modified human cancer cell comprising one or more vectors each comprising a recombinant polynucleotide encoding at least one gene selected from (A) CSF2, IFN-α2, CD86, IL-12, CD80, HLA-DRA, IL-7, and / or 4-1BBL (also known as TNFSF9 or CD137L); (B) (c) one or more vectors each comprising a recombinant polynucleotide encoding at least one gene selected from (a) HLA-A*01:01, HLA-A*68:01, HLA-A*02:01, or (b) one or more vectors each comprising a recombinant polynucleotide encoding at least one gene selected from HLA-A*11:01, HLA-A*03:01, HLA-A*23:01, HLA-A*24:02, and / or HLA-A*33:03; or (c) one or more vectors each comprising a recombinant polynucleotide encoding at least one gene selected from HLA-A*01:01 allele and HLA-A*68:01 allele, or HLA-A*02:01 allele and HLA-A*11:01 allele, or HLA-A*03:01 allele and HLA-A*33:03. (C) a vector comprising a recombinant polynucleotide encoding at least one gene selected from (a) HLA-DRB3*02:02, HLA-DRB5*01:01, HLA-DRB4*01:01, HLA-DRB3*01:01, HLA-DRB3*03:01, HLA-DRB5*01:02, and / or HLA-DRB5*02:02; or (b) a vector comprising a recombinant polynucleotide encoding an HLA-DRB3*02:02 allele and an HLA-DRB5*01:01 allele, or an HLA-DRB4*01:01 allele and an HLA-DRB3*01:01 allele, or an HLA-DRB3*03:01 allele and an HLA-DRB5*01:02 allele, or an HLA-DRB5*02:02 allele and an HLA-DRB3*01:01 allele.In some embodiments, one or more HLA-A and / or HLA-DR3 (e.g., HLA-DRB3) alleles endogenous to the human cancer cell are inactivated. In some embodiments, at least one of the endogenous HLA-A*24:02 alleles, HLA-A*11:01 alleles, HLA-DRB3*01:01 alleles, and / or HLA-DRB3*02:02 alleles endogenous to the human cancer cell are inactivated.

[0153] In some embodiments, the composition comprises at least 10,000, at least 100,000, at least 1,000,000, at least 1,250,000, at least 1,500,000, at least 2,000,000, at least 2,500,000, at least 3,000,000, at least 3,500,000, at least 4,000,000, at least 4,500,000, at least The composition may comprise 5,000,000, at least 10,000,000, at least 12,500,000, at least 15,000,000, at least 20,000,000, at least 25,000,000, at least 30,000,000, at least 35,000,000, at least 40,000,000, at least 45,000,000, or at least 50,000,000 cells. In some embodiments, the composition comprises at least 1,000,000 cells. In some embodiments, the composition comprises at least 20,000,000 cells.

[0154] In some embodiments, the composition comprises up to 10,000, up to 100,000, up to 1,000,000, up to 1,250,000, up to 1,500,000, up to 2,000,000, up to 2,500,000, up to 3,000,000, up to 3,500,000, up to 4,000,000, up to 4,500,000, up to 5,0 In some embodiments, the composition comprises up to 20 million cells. In some embodiments, the composition comprises up to 40 million cells. In some embodiments, the composition comprises up to 50 million cells.

[0155] In some embodiments, the composition comprises about 1,000,000 to about 50,000,000 cells, about 5,000,000 to about 35,000,000 cells, about 10,000,000 to about 25,000,000 cells, about 15,000,000 to about 20,000,000 cells, or about 35,000,000 to about 40,000,000 cells. In some embodiments, the composition comprises about 1,000,000 cells. In some embodiments, the composition comprises about 20,000,000 cells. In some embodiments, the composition comprises about 40,000,000 cells.

[0156] In another aspect, the disclosure provides a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises any of the compositions described herein and a pharma- ceutically acceptable carrier. For example, the pharmaceutical composition may comprise a modified human cancer cell or cell line comprising at least one HLA class I allele having SEQ ID NO: 1-13, 22-29 in Table 1, and at least one HLA class II allele having SEQ ID NO: 14-21, 30-35 in Table 1, and at least one, two, three, four, five, or more recombinant polynucleotides encoding one or more endogenous HLA alleles that are inactivated. Additionally, the CD47 and / or Ii (CD74) molecules endogenous to the modified human cancer cell or cell line may be inactivated. Furthermore, the modified human cancer cell or cell line may comprise one or more co-stimulatory molecules, xenoantigens, and / or cytokines described herein (e.g., Table 3, Figure 5). At least one, two, three, four, five, or more recombinant polynucleotides may include heterologous sequences encoding, for example, costimulatory molecules, heterologous antigens, cytokines, or 2A splicing peptides. Thus, the recombinant polynucleotides encoding one or more HLA alleles, costimulatory molecules, heterologous antigens, and / or cytokines may be separated by sequences encoding 2A splicing peptides (e.g., T2A, P2A, E2A). In general, at least one, two, three, four, five, or more recombinant polynucleotides are cloned into expression vectors (e.g., replication-defective lentiviral vectors) and introduced into modified human cancer cells or cell lines for the synthesis of HLA alleles, costimulatory molecules, heterologous antigens, and / or cytokines. Thus, the modified human cancer cells or cell lines provided in the pharmaceutical compositions may have at least one, two, three, four, five, or more expression vectors comprising at least one, two, three, four, five, or more recombinant polynucleotides encoding HLA alleles, costimulatory molecules, xenoantigens, and / or cytokines, respectively.

[0157] In some embodiments, the pharmaceutical composition further comprises a cryoprotectant, interferon alpha (e.g., IFN-α2a or IFN-α2b), and / or an interferon lambda family member (e.g., interferon lambda 1 (IFNλ1(IL-29)), interferon lambda 2 (IFNλ2(IL-28A)), interferon lambda 3 (IFNλ1(IL-28B)), interferon lambda 4 (IFNλ4)). In some embodiments, the interferon alpha (e.g., IFN-α2a or IFN-α2b) is expressed by a vector comprising a polynucleotide sequence of an IFNA2 gene in the modified cancer cells described herein. In some embodiments, the interferon alpha is exogenously provided pegylated IFN-α2a. In some embodiments, the pharmaceutical composition further comprises one or more excipients. In some embodiments, the pharmaceutical composition further comprises CryoStor CS10, CryoStor CS2, or CryoStor CS5 cryopreservation medium. In certain embodiments, the pharmaceutical composition comprises cells cryopreserved in CryoStor CS10, CryoStor CS2, or CryoStor CS5 cryopreservation medium.

[0158] In some embodiments, the pharmaceutical composition is formulated into a dosage form that includes the total number of modified cancer cells per dose for administration to a subject in need thereof. In some embodiments, the pharmaceutical composition is formulated as an "off-the-shelf" product for self-administration to a subject in need thereof. In some embodiments, the pharmaceutical composition is formulated into a dosage form that includes the total number of modified cancer cells per dose for administration to a subject in need thereof. In some embodiments, the pharmaceutical composition is formulated into a dosage form that includes at least 10,000 cells, at least 100,000 cells, at least 1,000,000 cells, at least 1,250,000 cells, at least 1,500,000 cells, at least 2,000,000 cells, at least 2,500,000 cells, at least 3,000,000 cells, at least 3,500,000 cells, at least 4,000,000 cells, at least 4,500,000 cells, at least 5,000,000 cells, at least 6,000,000 cells, at least 7,000,000 cells, at least 8,000,000 cells, at least 9,000,000 cells, at least 10 ... The pharmaceutical composition may have at least 5,000,000 cells, at least 10,000,000 cells, at least 12,500,000 cells, at least 15,000,000 cells, at least 20,000,000 cells, at least 25,000,000 cells, at least 30,000,000 cells, at least 35,000,000 cells, at least 40,000,000 cells, at least 45,000,000 cells, or at least 50,000,000 cells. In some embodiments, the pharmaceutical composition comprises at least 1,000,000 cells. In some embodiments, the pharmaceutical composition comprises at least 20,000,000 cells.

[0159] In some embodiments, the pharmaceutical composition comprises up to 10,000 cells, up to 100,000 cells, up to 1,000,000 cells, up to 1,250,000 cells, up to 1,500,000 cells, up to 2,000,000 cells, up to 2,500,000 cells, up to 3,000,000 cells, up to 3,500,000 cells, up to 4,000,000 cells, up to 4,500,000 cells, up to In some embodiments, the pharmaceutical composition comprises up to 5,000,000 cells, up to 10,000,000 cells, up to 12,500,000 cells, up to 15,000,000 cells, up to 20,000,000 cells, up to 25,000,000 cells, up to 30,000,000 cells, up to 35,000,000 cells, up to 40,000,000 cells, up to 45,000,000 cells, or up to 50,000,000 cells. In some embodiments, the pharmaceutical composition comprises up to 20,000,000 cells. In some embodiments, the pharmaceutical composition comprises up to 40,000,000 cells.

[0160] In some embodiments, the pharmaceutical composition comprises about 1,000,000 to about 50,000,000 cells, about 5,000,000 to about 35,000,000 cells, about 10,000,000 to about 25,000,000 cells, about 15,000,000 to about 20,000,000 cells, or about 35,000,000 to about 40,000,000 cells. In some embodiments, the pharmaceutical composition comprises about 1,000,000 cells. In some embodiments, the pharmaceutical composition comprises about 20,000,000 cells. In some embodiments, the pharmaceutical composition comprises about 40,000,000 cells.

[0161] In some embodiments, the pharmaceutical composition is formulated in the form of a suspension.The formulation of pharmaceutical compositions is generally known in the art (see, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18TH ED., Mack Publishing Co., Easton, PA (1990)).Prevention of microbial contamination can be achieved by adding one or more of various antibacterial and antifungal agents.In certain embodiments, the pharmaceutical composition is a liquid formulation that includes cells resuspended in lactated Ringer's solution.

[0162] Pharmaceutical dosage forms suitable for administration include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.Typical carriers include, for example, aqueous buffer solutions (i.e., biocompatible buffers, non-limiting examples of which include lactated Ringer's solution and CryoStor cryopreservation medium (e.g., CS2, CS5, and CS10, each containing 2%, 5%, and 10% DMSO; available from BioLife Solutions, Bothell, WA)), ethanol, glycerol, polyol such as propylene glycol, polyethylene glycol, or suitable mixtures thereof, surfactants, or vegetable oils, as solvents or dispersion media.

[0163] Sterilization can be accomplished by art-recognized techniques, including, but not limited to, the addition of antibacterial and antifungal agents, such as parabens, chlorobutanol, sorbic acid or thimerosal. Additionally, isotonic agents, such as sugars or sodium chloride, may be incorporated into the subject compositions.

[0164] Sterile injectable solutions and / or other compositions containing modified cancer cells of the present disclosure can be produced by incorporating the required amount of the compound in an appropriate solvent with the various ingredients described above, as appropriate, followed by sterilization. To obtain a sterile powder, the sterile solution can be vacuum dried or freeze-dried as necessary.

[0165] In some embodiments, the modified cancer cells and / or other compositions provided herein are formulated in unit dosage form for administration, e.g., intradermal injection, intralymphatic injection, oral, nasal, topical, or parenteral administration, to facilitate administration and uniformity of dosage. Unit dosage form as used herein refers to a physically separate unit suitable as a unit dosage for a subject, e.g., a human or other mammal to be treated, each unit containing a predetermined amount of active agent calculated to produce a desired therapeutic effect, together with a necessary pharmaceutical carrier. In some cases, a more concentrated dosage form may be prepared, from which a more diluted unit dosage form may then be produced. Thus, a more concentrated dosage form contains substantially more, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more, of the amount of modified cancer cells and / or other compositions.

[0166] In some embodiments, modified cancer cells and / or other compositions provided herein are formulated to be administered, for example, one or more doses over a period of time.In some embodiments, modified cancer cells and / or other compositions are formulated to be administered every week, every two weeks, every three weeks, every four weeks, every five weeks, or every six weeks.In some embodiments, modified cancer cells and / or other compositions are formulated to be administered every month, every two months, every three months, every four months, every five months, every six months, every twelve months, every eighteen months, or every twenty-four months.

[0167] The dosage is, for example, about 50,000 to 50,000,000 (e.g., about 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 250,000, 300, 000, 350,000, 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000, 900,000, 950,000, 1,000,000, 1,500,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000 , 4,000,000, 4,500,000, 5,000,000, 5,500,000, 6,000,000, 6,500,000, 7,000,000, 7,500,000, 8,000,000, 8,500,000, 9,000,000, 9,500,000, 10,000,000, 11,000,000, 12,000,000, 13,000,000, 14 In some embodiments, the dose may contain about 1 million modified human cancer cells. In some embodiments, the dose may contain about 5 million modified human cancer cells. In some embodiments, the dose may contain about 10 million modified human cancer cells. In some embodiments, a dose may contain about 20 million modified human cancer cells.

[0168] Doses may also be, for example, at least about 5,000,000 to 100,000,000 (e.g., about 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000, 10,000,000, 15,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, 40,000, In some embodiments, the dose may comprise at least about 1 million modified human cancer cells. In some embodiments, the dose may comprise at least about 5 million modified human cancer cells. In some embodiments, the dose may comprise at least about 10 million modified human cancer cells. In some embodiments, the dose may comprise at least about 20,000,000 modified human cancer cells.

[0169] Alternatively, the dose may be, for example, at least about 100,000,000 to 1,000,000,000 (e.g., about 100,000,000, 150,000,000, 200,000,000, 250,000,000, 300,000,000, 350,000,000, 400,000,000, 450,000,000, 500,000, In some embodiments, the modified human cancer cell may comprise at least 100,000,000, 550,000,000, 600,000,000, 650,000,000, 700,000,000, 750,000,000, 800,000,000, 850,000,000, 900,000,000, 950,000,000, 1,000,000,000 or more modified human cancer cells.

[0170] In some embodiments, the modified human cancer cells are irradiated. The radiation dose can be, for example, between about 2 and 2,000 Gy (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 800, 810, 820, 830, 840, The modified human cancer cells may be irradiated at a dose of about 40, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, or 2,000 Gy). In some embodiments, the modified human cancer cells are irradiated at a dose of about 200 Gy. In some embodiments, the modified human cancer cells are irradiated at a dose of about 100 Gy.

[0171] Methods for preparing such dosage forms are known to those skilled in the art (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra). Dosage forms typically contain a conventional pharmaceutical carrier or excipient and may additionally contain other medicinal agents, carriers, adjuvants, diluents, tissue permeation enhancers, solubilizers, and the like. Appropriate excipients can be tailored to the particular dosage form and route of administration by methods well known to those skilled in the art (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra).

[0172] Examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, and polyacrylic acid such as Carbopol, e.g., Carbopol 941, Carbopol 980, Carbopol 981. The dosage form can additionally include lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying agents; suspending agents; preservatives such as methyl, ethyl, and propyl hydroxybenzoates (i.e., parabens); pH adjusting agents such as inorganic and organic acids and bases; sweeteners; and flavoring agents. The dosage form can also include biodegradable polymer beads, dextran, and cyclodextrin inclusion complexes.

[0173] In some aspects, the pharmaceutical composition for administration can be an oral delivery vehicle such as capsules, cachets or tablets, each of which contains a predetermined amount of the composition to provide the patient with the correct incremental dose.The oral delivery vehicle can be useful, for example, to avoid contact between the composition and the mouth and upper digestive tract.For oral administration, the therapeutically effective dose can be in the form of tablets, capsules, emulsions, suspensions, solutions, syrups, sprays, lozenges, powders, and sustained release formulations.Excipients suitable for oral administration include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, etc.

[0174] In some embodiments, the therapeutically effective dose takes the form of a pill, tablet, or capsule, and thus the dosage form may contain any of the following, along with the modified cancer cells, and / or other compositions described herein: diluents such as lactose, sucrose, dicalcium phosphate, and the like; disintegrants such as starch or derivatives thereof; lubricants such as magnesium stearate; and binders such as starch, gum arabic, polyvinylpyrrolidone, gelatin, cellulose and its derivatives.

[0175] In some embodiments, a suitable carrier conceals the composition, e.g., modified cancer cells and / or other compositions, from the mouth and upper gastrointestinal (GI) tract, reducing or preventing local itching / swelling reactions in these areas upon administration. For example, the carrier may contain one or more lipid, polysaccharide, or protein components. In some cases, the carrier is a food product.

[0176] For topical administration, the therapeutically effective dose can be in the form of emulsion, lotion, gel, foam, cream, jelly, solution, suspension, ointment, and transdermal patch.For administration by inhalation, the modified cancer cells and / or other compositions described herein can be delivered as dry powder or in liquid form via nebulizer.Aerosol formulations can be placed in an acceptable pressurized propellant such as dichlorodifluoromethane.For parenteral administration, the therapeutically effective dose can be in the form of sterile injection solutions and packaged sterile powders.Preferably, the injection solutions are formulated at pH of about 4.5 to about 7.5.

[0177] The therapeutically effective dose can also be provided in lyophilized form. Such dosage forms may contain a buffer, such as bicarbonate, for reconstitution before administration, or a buffer may be included in the lyophilized dosage form for reconstitution, for example, with water. The lyophilized dosage form may further contain a suitable vasoconstrictor, such as epinephrine. The lyophilized dosage form can be provided in a syringe packaged with a buffer solution for reconstitution, optionally, so that the reconstituted dosage form can be immediately administered to an individual.

[0178] In some embodiments, the therapeutically effective dose may further include other ingredients, such as anti-allergy drugs, such as antihistamines, steroids, bronchodilators, leukotriene stabilizers, and mast cell stabilizers. Suitable anti-allergy drugs are well known in the art.

[0179] D. Methods for Treating Cancer In another aspect, the present disclosure provides a method for treating cancer in a subject. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure (e.g., a pharmaceutical composition comprising a modified cancer cell of the present disclosure) described herein.

[0180] In some embodiments, the method includes administering an effective amount of the pharmaceutical composition intradermally to the upper back or thigh of the subject. Without being bound by any theory, the upper back and thigh are selected for patient tolerance because the skin of these areas has fewer nerves and is therefore less sensitive. Additionally, nearby draining lymph nodes may transport antigens from breast tumors to the upper and lower torso, which are common sites of breast cancer metastasis. The method further includes administering the pharmaceutical composition to the subject at weekly, biweekly, 3weekly, 4weekly, 5weekly, or 6weekly intervals. In some embodiments, the method includes administering the pharmaceutical composition to the subject at monthly, bimonthly, 3monthly, 4monthly, 5monthly, 6monthly, 12monthly, 18monthly, or 24monthly intervals. In some embodiments, the method includes administering the pharmaceutical composition to the subject for at least 6 weeks, 12 weeks, 24 weeks, 36 weeks, 48 ​​weeks, 52 weeks, or longer. In some embodiments, the method comprises administering to the subject the pharmaceutical composition for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, 12 months, or longer. In some embodiments, the method comprises administering to the subject the pharmaceutical composition for no more than 6 weeks, 12 weeks, 24 weeks, 36 weeks, 48 ​​weeks, or 52 weeks. In some embodiments, the method comprises administering to the subject the pharmaceutical composition for no more than 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, or 12 months.

[0181] In some embodiments, the method comprises administering to the subject an effective amount of the pharmaceutical composition by oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intratumoral, intradermal, intralymphatic, intrathecal, intranasal, or subcutaneous administration. In some embodiments, the administration of the effective amount of the pharmaceutical composition is performed by parenteral administration (e.g., intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial) or transmucosal administration (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). In some embodiments, the method comprises the use of a liposomal formulation, intravenous infusion, or a transdermal patch.

[0182] In some embodiments, the method further comprises administering one or more doses of cyclophosphamide intravenously to the subject at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, or more prior to administering to the subject a pharmaceutical composition described herein. In some embodiments, the cyclophosphamide is administered at least about 2-3 days prior to administering to the subject a pharmaceutical composition described herein. In some embodiments, the cyclophosphamide is administered at about 100, 150, 200, 250, 300, or 450 mg / m 2 A low dose of cyclophosphamide is administered to the subject.

[0183] In some embodiments, the method further comprises administering one or more doses of interferon-alpha-2b (IFN-α2b), IFN-α2a, or pegylated IFN-α2a intradermally to the subject at the site of inoculation of the pharmaceutical composition described herein. In some embodiments, the method further comprises administering one or more doses of IFN-α2b, IFN-α2a, or pegylated IFN-α2a intradermally to the subject within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 60, 72, or 84 hours after administering to the subject a pharmaceutical composition described herein. In some embodiments, the method further comprises administering to the subject one or more doses of IFN-α2b, IFN-α2a, or pegylated IFN-α2a intradermally about 1-4 hours, about 2-6 hours, about 8-12 hours, about 10-24 hours, about 20-48 hours, or about 60-72 hours after administering to the subject a pharmaceutical composition described herein. In some embodiments, the method further comprises administering to the subject one or more doses of IFN-α2b, IFN-α2a, or pegylated IFN-α2a intradermally about 5, 10, 15, 20, 25, 30, 45, 50, 60, 72, or 84 hours after administering to the subject a pharmaceutical composition. In some embodiments, the method further comprises administering intradermally to the subject one or more doses of IFN-α2bIFN-α2b, IFN-α2a, or pegylated IFN-α2a within 1, 2, 3, 4, 5, or 6 days after administering the pharmaceutical composition to the subject. In some embodiments, the method further comprises administering intradermally to the subject one or more doses of IFN-α2b, IFN-α2a, or pegylated IFN-α2a within about 1-6 days, 2-3 days, or 3-5 days after administering the pharmaceutical composition to the subject. In some embodiments, the method further comprises administering intradermally to the subject a first dose of IFN-α2b, IFN-α2a, or pegylated IFN-α2a 1-4 hours after administering the pharmaceutical composition to the subject, and a second dose of IFN-α2b, IFN-α2a, or pegylated IFN-α2a 1-3 days after administering the pharmaceutical composition to the subject. In some embodiments, the IFN-α2b administered is a low dose of about 1-20,000 IU, 100-15,000 IU, 5000-12,000 IU, or 9,000-11,000 IU.In some embodiments, the IFN-α2b administered is at a dose of about 10,000 IU. In some embodiments, the IFN-α2a or pegylated IFN-α2a administered is at a low dose of about 0.01-0.1 micrograms (mcg), 0.05-0.15 mcg, 0.06-0.12 mcg, or 0.09-0.11 mcg. In some embodiments, the IFN-α2b administered is at a dose of about 0.1 mcg.

[0184] In some embodiments, the method further comprises administering one or more additional therapeutic methods to the subject.Suitable additional types include, but are not limited to, chemotherapy, immunotherapy, radiation therapy, hormone therapy, differentiation inducer, and small molecule drug.Those skilled in the art can easily select suitable additional therapeutic methods.

[0185] Chemotherapeutic agents that can be used in the present invention include alkylating agents (e.g., nitrogen mustards (e.g., mechlorethamine, chlorambucil, cyclophosphamide, ifosfamide, melphalan), nitrosoureas (e.g., streptozocin, carmustine (BCNU), lomustine), alkyl sulfonates (e.g., busulfan), triazines (e.g., dacarbazine (DTIC), temozlomide), ethylenimines (e.g., thiotepa, altretamine (hexamethylmelamine))), platinum drugs (e.g., cisplatin, carboplatin, oxalaplatin), antimetabolites (e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxacin, These include, but are not limited to, uridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed), anthracycline antitumor antibiotics (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin), non-anthracycline antitumor antibiotics (e.g., actinomycin D, bleomycin, mitomycin C, mitoxantrone), mitotic inhibitors (e.g., taxanes (e.g., paclitaxel, docetaxel), epothilones (e.g., ixabepilone), vinca alkaloids (e.g., vinblastine, vincristine, vinorelbine), estramustine), corticosteroids (e.g., prednisone, methylprednisolone, dexamethasone), L-asparaginase, bortezomib, and topoisomerase inhibitors. Combinations of chemotherapeutic agents can be used.

[0186] Topoisomerase inhibitors are compounds that inhibit the activity of topoisomerase, an enzyme that promotes changes in DNA structure by catalyzing the breakage and rejoining of phosphodiester bonds in the backbone of the DNA strand. Such changes in DNA structure are necessary for DNA replication during normal cell cycle. Topoisomerase inhibitors inhibit DNA ligation during the cell cycle and increase the number of single- and double-strand breaks, leading to decreased genomic stability. Such decreased genomic stability leads to apoptosis and cell death.

[0187] Topoisomerases are often divided into type I and type II topoisomerases. Type I topoisomerases are essential for the relaxation of DNA supercoils during DNA replication and transcription. Type I topoisomerases also generate single-strand breaks in DNA and religate the breaks to reestablish intact double-stranded DNA molecules. Examples of inhibitors of type I topoisomerases include irinotecan, topotecan, camptothecin, and lamellarin D, all of which target type IB topoisomerases.

[0188] Type II topoisomerase inhibitors are broadly classified as topoisomerase toxins and topoisomerase inhibitors. Topoisomerase toxins target the topoisomerase-DNA complex, while topoisomerase inhibitors disrupt the catalytic turnover of the enzyme. Examples of type II topoisomerase inhibitors include amsacrine, etoposide, etoposide phosphate, teniposide, doxorubicin, and fluoroquinolones.

[0189] In some embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. In some cases, the topoisomerase inhibitor is a topoisomerase I inhibitor, a topoisomerase II inhibitor, or a combination thereof. In certain embodiments, the topoisomerase inhibitor is selected from the group consisting of doxorubicin, etoposide, teniposide, daunorubicin, mitoxantrone, amsacrine, ellipticine, aurintricarboxylic acid, HU-331, irinotecan, topotecan, camptothecin, lamellarin D, resveratrol, genistein, quercetin, epigallocatechin gallate (EGCG), and combinations thereof. EGCG is an example of a plant-derived natural phenol that serves as a suitable topoisomerase inhibitor. In some cases, the topoisomerase inhibitor is doxorubicin.

[0190] Immunotherapy refers to any treatment that utilizes a subject's immune system to fight disease (e.g., cancer). Immunotherapy can be directed to either enhance or suppress immune function. In the context of cancer therapy, immunotherapy is typically directed to enhance or activate immune function. In some cases, immunotherapeutic agents include monoclonal antibodies that target specific types or parts of cancer cells. In some cases, the antibodies are conjugated to moieties such as drug molecules or radioactive substances. The antibodies can be of murine, chimeric, or humanized origin, as non-limiting examples. Non-limiting examples of therapeutic monoclonal antibodies include alemtuzumab, bevacizumab, cetuximab, daratumumab, ipilimumab (MDX-101), nivolumab, ofatumumab, panitumumab, pembrolizumab, retifanlimab, rituximab, tositumomab, and trastuzumab.

[0191] Immunotherapeutic agents can also include immune checkpoint inhibitors that modulate the immune system's ability to distinguish between normal and "foreign" cells. Programmed cell death protein 1 (PD-1) and protein cell death ligand 1 (PD-L1) are common targets of immune checkpoint inhibitors, as disruption of the interaction between PD1 and PD-L1 enhances the activity of immune cells against foreign cells, such as cancer cells. Examples of PD-1 inhibitors include pembrolizumab, retifanlimab, and nivolumab. An example of a PD-L1 inhibitor is atezolizumab.

[0192] Another immune checkpoint target for the treatment of cancer is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), a receptor that downregulates immune cell responses. Therefore, drugs that inhibit CTLA-4 can promote immune function. An example of such a drug is ipilimumab, a monoclonal antibody that binds and inhibits CTLA-4.

[0193] The term "radiotherapy" refers to the delivery of high-energy radiation to a subject for the treatment of disease (e.g., cancer). Radiation therapy can include the delivery of X-rays, gamma rays, and / or charged particles. Radiation therapy can be delivered locally (e.g., to the site or region of a tumor) or systemically (e.g., a radioactive substance such as radioactive iodine is administered systemically and travels to the tumor site).

[0194] The term "hormonal therapy" can refer to inhibitors of hormone synthesis, hormone receptor antagonists, or hormone supplements. Inhibitors of hormone synthesis include, but are not limited to, aromatase inhibitors and gonadotropin releasing hormone (GnRH) analogs. Hormone receptor antagonists include, but are not limited to, selective receptor antagonists and antiandrogens. Hormone supplements include, but are not limited to, progestogens, androgens, estrogens, and somatostatin analogs. Aromatase inhibitors are used, for example, to treat breast cancer. Non-limiting examples include letrozole, anastrozole, and aminoglutethimide. GnRH analogs can be used, for example, to induce chemical castration. Selective estrogen receptor antagonists commonly used for the treatment of breast cancer include tamoxifen, raloxifene, toremifene, and fulvestrant. Antiandrogens that bind to and inhibit the androgen receptor are commonly used to inhibit the growth and survival effects of testosterone on prostate cancer. Non-limiting examples include flutamide, apalutamide, and bicalutamide.

[0195] The term "differentiation agent" refers to any substance that promotes cell differentiation that can encourage malignant cells to assume a less stem cell-like state in the context of cancer. A non-limiting example of an anti-cancer differentiation agent is retinoic acid.

[0196] Small molecule drugs are generally pharmacological agents with low molecular weight (i.e., less than about 900 daltons). Non-limiting examples of small molecule drugs used to treat cancer include bortezomib (proteasome inhibitor), imatinib (tyrosine kinase inhibitor), and seliciclib (cyclin-dependent kinase inhibitor), and epacadostat (indoleamine 2,3-dioxygenase (IDO1) inhibitor).

[0197] In some embodiments, the method of treating cancer of the present disclosure further comprises selecting a whole cell cancer vaccine for the subject according to the method of the present disclosure described herein.In certain embodiments, the subject has stage I, stage II, stage III, and / or stage IV cancer.In other embodiments, the cancer is in transition between stages.In some embodiments, the subject has precancerous lesions.In some embodiments, the subject does not have cancer.

[0198] In some embodiments, treating the subject includes inhibiting cancer cell growth, inhibiting cancer cell proliferation, inhibiting cancer cell migration, inhibiting cancer cell invasion, ameliorating or eliminating symptoms of cancer, reducing cancer tumor size (e.g., volume), reducing cancer tumor number, reducing cancer cell number, inducing cancer cell necrosis, pyroptosis, oncosis, apoptosis, autophagy, or other cell death, or enhancing the therapeutic effect of the composition or pharmaceutical composition. In some embodiments, treating the subject results in increased survival. In some cases, overall survival is increased. In other cases, disease-free survival is increased. In some cases, progression-free survival is increased. In certain embodiments, treating the subject results in reduced tumor volume and / or increased survival.

[0199] In certain embodiments, treating the subject enhances the therapeutic effect of an anti-cancer therapy, such as a chemotherapeutic agent, an immunotherapeutic agent, radiation therapy, hormone therapy, a differentiation agent, and / or a small molecule drug.

[0200] Therapeutic methods, such as modified cancer cells, compositions, and pharmaceutical compositions of the present disclosure, can be administered using routes, dosages, and protocols that are readily known to those skilled in the art. Administration can be once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once a week. Therapeutic methods can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more times a week. In some cases, modified cancer cells, compositions, and / or pharmaceutical compositions of the present disclosure are administered as a single dose, co-administered (e.g., administered in separate doses or by different routes, but administered close in time), or administered separately (e.g., administered in different doses, including by the same or different routes, but administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours or more apart). If multiple doses are administered on the same day, or if a single dose contains one or more components (e.g., modified cancer cells and IFNa are administered separately), then administration can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more times per day.

[0201] In some cases, therapeutic administration can be performed about once a week, about every two weeks, about every three weeks, or about once a month. In other cases, therapeutic administration can be performed about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 times or more per month. Treatment can be continued for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or more; about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more; or longer. At any time during treatment, the therapeutic regimen can be adjusted as needed. For example, depending on the response to the modified cancer cells, compositions, or pharmaceutical compositions of the present disclosure, a different vaccine may be selected, one or more additional therapeutic agents or drugs may be chosen, or any aspect of the treatment regimen may be discontinued. One of skill in the art can readily make such determinations, informed, for example, by the results of comparing allelic profiles, changes in immune cell activity and / or numbers, and / or changes in the presence or level of one or more biomarkers.

[0202] The modified cancer cells, compositions, and pharmaceutical compositions of the present disclosure can be administered by any suitable route, including those described herein. In some embodiments, administration is by intradermal or intralymphatic injection. In some embodiments, the whole cell cancer vaccine (e.g., comprising the modified cancer cells of the present disclosure) is given separately from interferon alpha (IFNa). In some cases, IFNa is injected locally. IFNa can be given before and / or after the vaccine is administered. The timing of the separate injections can be any suitable interval, including those described herein.

[0203] One of skill in the art can easily administer the appropriate number of modified cancer cells to include in a particular dose. One dose may be, for example, about 50,000 to 50,000,000 cells (e.g., about 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 250,000, 30 0,000, 350,000, 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000, 900,000, 950,000, 1,000,000, 1,500,000, 2,000,000, 2,500,000, 3,000,000, 3,500,0 00, 4,000,000, 4,500,000, 5,000,000, 5,500,000, 6,000,000, 6,500,000, 7,000,000, 7,500,000, 8,000,000, 8,500,000, 9,000,000, 9,500,000, 10,000,000, 11,000,000, 12,000,000, 13,000,000, In some embodiments, a dose may comprise about 1 million modified cancer cells. In some embodiments, a dose may comprise about 5 million modified cancer cells. In some embodiments, a dose may comprise about 10 million modified cancer cells. In some embodiments, one dose may comprise about 20,000,000 modified cancer cells.

[0204] One dose may contain, for example, at least about 5,000,000 to 100,000,000 (e.g., about 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000, 10,000,000, 15,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, 40,000, The modified cancer cells may also include 00,000, 45,000,000, 50,000,000, 55,000,000, 60,000,000, 65,000,000, 70,000,000, 75,000,000, 80,000,000, 85,000,000, 90,000,000, 95,000,000, 100,000,000 or more modified cancer cells.

[0205] Alternatively, one dose may contain, for example, at least about 100,000,000 to 1,000,000,000 (e.g., about 100,000,000, 150,000,000, 200,000,000, 250,000,000, 300,000,000, 350,000,000, 400,000,000, 450,000,000, 500,000, In one embodiment, the modified cancer cell may comprise at least 100,000, 550,000,000, 600,000,000, 650,000,000, 700,000,000, 750,000,000, 800,000,000, 850,000,000, 900,000,000, 950,000,000, 1,000,000,000 or more modified cancer cells.

[0206] In some embodiments, the modified cancer cells are irradiated. The radiation dose can be, for example, between about 2 and 2,000 Gy (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 800, 810, 820, 830, 840, 8 In certain embodiments, the modified cancer cells are irradiated with a dose of about 200 Gy.

[0207] In some embodiments, subject treatment leads to the reduction of the presence or level of one or more xenoantigens measured or detected in the sample obtained from subject.In some embodiments, subject treatment leads to the increase of the presence or level of one or more biomarkers measured or detected in the sample obtained from subject.In certain embodiments, subject treatment does not lead to the change of the presence or level of one or more biomarkers.

[0208] In some embodiments, treating a subject results in an increase in the activity and / or number of one or more immune cells. In some cases, the increase occurs in one cell type. In other cases, the increase occurs in multiple cell types. In some embodiments, the cells whose activity and / or number levels are increased are selected from the group consisting of peripheral blood mononuclear cells (PBMCs), lymphocytes (e.g., T lymphocytes, B lymphocytes, NK cells), monocytes, dendritic cells, macrophages, myeloid-derived suppressor cells (MDSCs), and combinations thereof. In certain embodiments, the activity and / or number levels of immune cells are measured using the methods of the present disclosure described herein.

[0209] In some embodiments, the increase in the activity and / or number of immune cells indicates that the subject should be administered one or more additional doses of pharmaceutical composition (e.g., comprising modified cancer cells of the present disclosure).In some cases, different vaccines are administered.Those skilled in the art recognize that the increase in the activity and / or number of immune cells may occur after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more doses of vaccine are administered in some cases.

[0210] In some embodiments, the sample is obtained from a subject. In other embodiments, the sample is obtained from different subjects or subject populations. The sample can be used to select an appropriate cancer vaccine of the present disclosure, to monitor the response to a vaccine therapy, and / or to predict how a subject will respond to a vaccine therapy. Samples obtained from different subjects and / or subject populations can be used, for example, to establish a reference range to facilitate the comparison that is part of the method of the present disclosure. Samples can be obtained at any time, including before and / or after administration of the modified cancer cells, pharmaceutical compositions, and / or other compositions of the present disclosure. In some embodiments, the sample includes whole blood, plasma, serum, cerebrospinal fluid, tissue, saliva, oral cells, tumor tissue, urine, liquid obtained from pleural effusion, hair, skin, or combinations thereof. In general, the sample can include any biological fluid. For HLA typing, any type of cell, tissue, or biological fluid is suitable, so long as it contains a sufficient amount of DNA or RNA to allow typing. In some cases, the sample includes circulating tumor cells (CTCs). The sample can also be composed of a combination of normal cells and cancer cells. In certain embodiments, the sample comprises circulating tumor cells (CTCs).The sample can be obtained, for example, from biopsy, from surgical resection, and / or as fine needle aspirate (FNA).The sample can be used to determine, measure, or detect HLA alleles, immune cell activity and / or number, and / or biomarkers as described herein.

[0211] In some embodiments, the results of HLA typing (e.g., the alleles present in the allele profile, the results of comparing allele profiles), measuring the activity and / or number of immune cells, and / or determining the presence or level of biomarkers are recorded on tangible media.For example, the results of assay (e.g., the alleles present in the allele profile, the results of comparing allele profiles, the activity level and / or number of immune cells, the presence or level (e.g., expression) of one or more biomarkers) and / or prognosis or diagnosis (e.g., whether cancer exists, whether the subject responds to a vaccine, or prediction of whether the subject responds to a vaccine) can be recorded, for example, on paper or electronic media (e.g., audiotape, computer disk, CD, flash drive, etc.).

[0212] In other embodiments, the method further comprises providing the patient (ie, subject) with the assay, prognostic, and / or diagnostic results and / or treatment results.

[0213] E. Kit In another aspect, the present disclosure provides a kit for treating a subject with cancer.In some embodiments, the kit comprises the modified cancer cell line, composition, and / or pharmaceutical composition of the present disclosure described herein.The kit is useful for treating any cancer, some non-limiting examples of which include breast cancer, ovarian cancer, cervical cancer, prostate cancer, pancreatic cancer, colorectal cancer, gastric cancer, lung cancer, skin cancer, liver cancer, brain cancer, eye cancer, soft tissue cancer, renal cancer, bladder cancer, head and neck cancer, mesothelioma, acute leukemia, chronic leukemia, medulloblastoma, multiple myeloma, sarcoma, and any other cancer described herein, including combinations thereof.

[0214] Materials and reagents for carrying out the various methods of the present disclosure can be provided in kits to facilitate carrying out the methods. As used herein, the term "kit" includes a combination of items that facilitate a step, assay, analysis, or operation. In particular, the kits of the present disclosure are useful for a wide range of applications, including, for example, diagnostics, prognostics, therapeutics, and the like.

[0215] The kit may include chemical reagents and other components. In addition, the kit of the present disclosure may include, but is not limited to, instructions for the user of the kit, devices and reagents for sample collection and / or purification, devices and reagents for product collection and / or purification, devices and reagents for administering the modified cancer cells or other compositions of the present disclosure, devices and reagents for determining the level of biomarkers and / or the activity and / or number of immune cells, devices and reagents for detecting HLA alleles, sample tubes, holders, trays, racks, dishes, plates, solutions, buffers or other chemical reagents, samples suitable for use for standardization, normalization and / or control samples. The kit of the present disclosure may also be packaged, for example, in a box with a lid, for convenient storage and safe transportation. For example, the kit may be stored and transported at room temperature, on wet ice or with a cooling pack, or frozen in the vapor phase of liquid nitrogen or in dry ice.

[0216] In some embodiments, the kit also includes negative and positive control samples for detection of HLA alleles, activity and / or number of immune cells, and / or presence or level of biomarkers. In some embodiments, the negative control sample is a non-cancerous cell, tissue, or biological fluid obtained from a subject to be treated or a subject already undergoing treatment. In other embodiments, the negative control sample is obtained from an individual or group of individuals without cancer. In other embodiments, the positive control sample is obtained from a subject with cancer or from another individual or group of individuals. In some embodiments, the kit includes samples for preparing a titration curve of one or more biomarkers in the sample to help determine the quantified level of activity and / or number of one or more immune cells and / or biomarkers in the biological sample. EXAMPLES

[0217] IV. Working Examples The present disclosure will be described in more detail by specific examples. The following examples are provided for illustrative purposes only and are not intended to limit the present invention in any way. Those skilled in the art will easily recognize various non-critical parameters that can be changed or modified to produce essentially the same results.

[0218] Example 1: Generation of Bria-OTS breast cancer immunotherapy cell line This example illustrates one embodiment of generating the cell lines described herein.

[0219] Bria-OTS is an experimental HER-2 / neu positive allogeneic whole cell breast cancer (BC) cell line designed to secrete granulocyte-macrophage colony stimulating factor (GM-CSF) in situ and enhance dendritic cell activity. In addition, Bria-OTS cell lines have been engineered to express multiple human leukocyte antigen (HLA) alleles to match >99% of the breast cancer patient population. Bria-OTS consists of four separate BC cell lines: BC1, BC2, BC3, and BC4, as detailed below. Each of the four Bria-OTS cell lines expresses the CSF2 gene, which encodes GM-CSF.

[0220] Parental Cell Line. The Bria-OTS cell line was derived from a breast cancer parental cell line, SV-BR-1, which expresses multiple cancer-associated antigens and immune stimulatory factors, including class II HLA molecules that directly activate CD4+ T cells to enhance the immune response. The generation of the original SV-BR-1 cell line is described in WO 2017 / 147600, which is incorporated herein in its entirety.

[0221] The parental cell line, SV-BR-1, contains the expressed endogenous HLA alleles HLA-A*24:02, HLA-B*35:08, HLA-B*55:01, HLA-C*01:02, HLA-C*04-01, and HLA-DRB3*01:01, as well as HLA-DRB3*02:02, HLA-DRB1*11:04, and HLA-DRB1*13:03, which are expressed at lower levels but enhanced by exposure to interferon-gamma (IFNγ); HLA-A*11:01 is undetectable. In the Bria-OTS cell line, expression of HLA-A*24:02 and DRB3*01:01 has been functionally knocked out using CRISPR techniques as described below. Instead, the Bria-OTS cell line expresses the following combinations of HLA genes: BC1.68.3.4 (BC1): HLA-A*01:01, HLA-A*68:01, HLA-DRB3*02:02, HLA-DRB4*01:01; BC2.11.4.3 (BC2): HLA-A*02:01, HLA-A*11:01, HLA-D RB4*01:01, HLA-DRB3*03:01;BC3.23.3.5(BC3):HLA-A*03:01, HLA-A*23:01, HLA-DRB3*01: 01, HLA-DRB5*01:02; and BC33.5.5 (BC4): HLA-A*33:03, HLA-DRB5*01:01, HLA-DRB5*02:02.

[0222] To prepare the therapeutic compositions described herein, vials from a Master Cell Bank (MCB) can be thawed, grown in culture, harvested, and cryopreserved.

[0223] Inactivation of endogenous HLA antigen expression in cell lines. SV-BR-1 endogenous HLA alleles include HLA-A*24:02, HLA-A*11:01, HLA-B*35:08, HLA-B*55:01, HLA-DRB3*01:01, and HLA-DRB3*02:02, with all but HLA-A*11:01 being expressed in standard culture conditions. To eliminate the possibility of competition between endogenous SV-BR-1 HLA alleles and exogenous HLA alleles introduced as part of the Bria-OTS-1 construct, Bria-KO (SV-BR-1-KO) was engineered by inactivating the endogenous HLA-A and HLA-DRB3 alleles. Using clustered regularly interspaced short interdigitated repeats / CRISPR associated protein-9 (CRISPR / Cas9) technology, synthetic guide RNAs (sgRNAs) were designed to introduce frameshift mutations into endogenous HLA alleles, rendering the HLA proteins nonfunctional as described below. Bria-SV-BR-1 cells were transfected with sgRNA guide RNAs using Lipofectamine CRISPRMAX Cas9 transfection reagent. Knockout (KO) cells were assessed by flow cytometry using anti-HLA-A or anti-HLA-DR antibodies. For example, HLA-A null cells were sorted via negative isolation using Dynabeads (Invitrogen) and HLA-A24 antibodies. Two rounds of negative selection were performed to enrich for HLA-A*24:02 negative cells. Further rounds of negative selection were similarly performed using Dynabeads coupled with anti-HLA-DR antibodies (which recognize HLA-DRβ3, encoded by the HLA-DRB3 gene and complexed with HLA-DRα) to obtain clonal derivatives. Subsequently, genes for immunostimulants (e.g., GM-CSF) and other exogenous HLA alleles were introduced either by transfection or transduction. Clone 17 was confirmed by flow cytometry assay to contain HLA-A*24:02 and HLA-DRB3*01:01 frameshift mutations that result in mutant HLA antigens not recognized by the antibodies.Thus, two of the four targeted HLA alleles in the parental cell line Bria-SV-BR-1 were successfully inactivated, resulting in the generation of clone 17.

[0224] Clone 17 was then transduced separately with four lentiviral vectors (V1, V2, V3, and V4, respectively) to induce the expression of GM-CSF, interferon-alpha (IFN-α), and four exogenous HLA genes—two HLA-A genes and two HLA-DRB3 or HLA-DRB4 or HLA-DRB5 genes. The lentiviral constructs used to generate the corresponding Bria-OTS cell lines are shown in Table 2.

[0225] As shown in Table 2 and Figures 1-4, the results were that all four cell lines expressed GM-CSF and IFN-α, BC1, BC2, and BC3 each expressed four unique HLA genes—two class I HLA genes and two class II HLA genes—and BC4 expressed three unique HLA genes—two class I HLA genes and one class II HLA gene. These cell lines were examined using flow cytometry on the cell lines generated after lentiviral transduction and by measuring GM-CSF in the culture supernatants.

[0226] CRISPR inactivation of endogenous HLA-A and HLA-DRB3 expression. CRISPR / Cas9 technology was used to remove HLA-A (*11:01 and *24:02) and HLA-DRB3 (*01:01 and *02:02). Synthetic sgRNAs with chemical modifications were purchased from Synthego (HLA-A: TIFF2025507939000005.tif5128 and HLA-DBR3: TIFF2025507939000006.tif4131). SV-BR-1 cells were stored in LN2 and cultured in supplemented RPMI medium at 37°C, 5% CO2, and 95% humidity. Cells were cryopreserved. SV-BR-1 cells were returned to culture and continued to grow before seeding. Cells were cultured at 0.5 × 10 6 The cells were seeded at a concentration of 1000 cells / well. The next day, cells were transfected using Lipofectamine CRISPRMAX reagent. sgRNA (HLA-A) was reconstituted as per manufacturer's recommendations and diluted to 30uM. RNP complexes were then created by mixing 21ul of 30um sgRNA + 3.5ul of synthego with 20uM Cas9 nuclease + 5ul of Lipofectamine Cas9 Plus Reagent + 95.5ul of Optimem and incubating at room temperature for 10 minutes. After the RNP complexes formed, they were mixed with pre-incubated Lipofectamine CRISPRMAX (5ul of Lipofectamine + 120ul of Optimem) and incubated for 5 minutes. Finally, 350ul of the mixture was added to the wells containing the cells and incubated for 2-3 days. Cells were expanded for 10 days and KO was verified by flow cytometry using anti-HLA-A24 antibody on March 1, 2018 (20% HLA-A24 negative cells). HLA-A null cells were sorted via negative isolation using Dynabeads (Invitrogen) and HLA-A24 antibody. After bead sorting, 40% of cells were HLA-A24 negative. Cells were expanded and subjected to a second round of negative isolation using HLA-A24 antibody and Dynabeads. After bead sorting, 100% of SV-BR-1 cells were HLA-24 negative as shown by flow cytometry. Cells were expanded and cryopreserved.

[0227] 0.15 x 10 in a 24-well plate 6SV-BR-1 HLA-A knockout cells (KO) were returned to culture before seeding at 1000 / well. The next day, cells were transfected using Lipofectamine CRISPRMAX reagent. sgRNA (HLA-DRB3) was reconstituted and diluted to 30uM according to the manufacturer's recommendations. RNP complexes were then created by mixing 21ul of sgRNA 30um + 3.5ul of synthego, 20uM Cas9 nuclease + 5ul of Lipofectamine Cas9 Plus Reagent + 95.5ul of Optimen and incubating at room temperature for 10 minutes. After the RNP complexes formed, they were mixed with pre-incubated Lipofectamine CRISPRMAX (5ul of Lipofectamine + 120ul of Optimen) and incubated for 5 minutes. Finally, 350ul of the mixture was added to the wells containing the cells and incubated for 2-3 days.

[0228] Single cell cloning of SV-BR-1 KO was then initiated. Cells were seeded at 1 cell / well in 30% complete medium + 70% conditioned medium (20% FBS) in 96-well plates. Single cell clones were subsequently screened and frozen, and HLA-A / DRB3 KO clones were identified by using the Guide-IT genotyping kit (Takara). Clone 17 was selected and used for further development. Clone 17 was further validated using Topo cloning and sequencing. Genomic DNA was isolated from the clone 17 cell line and specific primers were used to amplify HLA-A (*11:01 and *24:02) and HLA-DRB3 (*01:01 and *02:02). Following PCR, the products were cloned according to the manufacturer's recommendations. Bacterial colonies were picked and sequenced using rolling circle amplification (RCA) prior to Sanger sequencing.

[0229] Preparation of plasmids for lentivirus production. The plasmid backbone pCCl-c (SEQ ID NO:47) for the construction of this plasmid was originally constructed in the Naldini laboratory (Dull et al., 1998) and further modified in the Donald B Kohn laboratory (Department of Microbiology and Immunology UCLA). For example, the final plasmid for BC4 contains the following elements as shown in Figure 4. Specifically, the ampicillin resistance gene (AMP) is located at bases 132-992; the CMV enhanced 5' the LTR is located at bases 2358-3070; the packaging signal (PSI) is located at bases 3120-3258; the Rev-responsive element (RRE) is located at bases 3745-3948; the central polypurine tract (cPPT) is located at bases 4461-4648; the hEF1a promoter is located at bases 4653-5842; the CSF2-T2A-IFN-α is located at bases 5843-6916; the MNDU3 promoter is located at bases 6917-7453; the HLA allele containing HLA-A*24:02-T2A-HLA-A*33:03-P2A-HLA-DRB5*01:01-E2A-HLA-DRB5*01:02 is located at bases 7454-11452; and the U3 Sin region 3' The LTR is located at bases 11609-11664; the R region 3' LTR is located at bases 11665-11761; and the U5 region 3' LTR is located at bases 11763-11845.

[0230] Cloning strategy. pCCLc-Ef1a-X-WPRE (SEQ ID NO:47) was created by removing the unique gene using BAMH1 and SAL1, and MCS was cloned in. The final vector was confirmed by having band sizes of 351bp, 1111bp, and 6380bp by Sac2. Additionally, the vector was confirmed by sequencing.

[0231] CSF2-T2A-IFN-α was synthesized by Genscript and the coding sequence was codon-optimized to contain BamH1 at the 5' and 3' ends, with CSF2 and IFN-α separated by the sequence of the T2A self-cleaving peptide.

[0232] HLA-A*24:02-T2A-HLA-A*33:03-P2A-HLA-DRB5*01:01-E2A-HLA-DRB5*02:02 were synthesized by Genscript with ECOR1 sites at the 5' and 3' ends, and the coding sequences were codon-optimized. HLA-A*24:02 and HLA-A*33:03 are separated by the sequence of the T2A self-cleaving peptide. HLA-A*33:03 and HLA-DRB5*01:01 are separated by the sequence of the P2A self-cleaving peptide. HLA-DRB5*01:01 and HLA-DRB5*02:02 are separated by the sequence of the E2A self-cleaving peptide. This fragment was cloned into the EcoR1 site of pCCLc-EF1a-X-WPRE. The final plasmid was confirmed by NCO1 to give band sizes 221 bp, 1519 bp, 3271 bp, 3441 bp, 4439 bp and also by sequencing.

[0233] Lentivirus production. To produce lentiviral vectors, HEK 293T cells (Clontech Lenti-X 293 cells) were cultured in D10HG medium and transfected with VSV-G envelope plasmid, packaging plasmid and vector using Mirus TRANS-IT 293 transfection reagent with DMEM medium (Figures 1-4). On day 5, conditioned medium was collected, centrifuged, and the supernatant was concentrated using Millipore Centricon Plus 70 PL-100 and then filtered using Costar 0.45 Spinx centrifugal filter tubes. Viral titers were determined using the ABM LV900 Lentivirus Titer Kit with Mastermix R. Lentiviral vector testing was performed by, for example, determining titer, sequence, RCL, bioburden, and / or endotoxin.

[0234] Lentiviral transduction with lentivirus. 0.29 × 10 6 Cells were seeded at a density of 10 cells / well and transduced the following day at an MOI of 150. The medium for transduction was 1 ml RPMI 1640 + 10% FBS + L-glutamine + 20 mg / mL protamine sulfate. After cell recovery and reattachment, cells were lifted using 4 mL TrypLE Express for 5 min. The reaction was stopped with 8 mL medium. Cells were counted and diluted to approximately 8 × 10 cells using CS10. 5 The cells were cryopreserved at 1 cell per vial.

[0235] Lentiviral transduced cells (e.g., clone 17 cells) were transferred to culture and expanded. HLA-DR+ cells were isolated using Dynabeads (Invitrogen) and HLA-DR antibody-L243 (BioLegend) according to the manufacturer's recommendations. Cells were further expanded and clone 17 cells were subjected to a second round of HLA-DR+ isolation using Dynabeads. Cells were further expanded and then single cell cloning was initiated by seeding 2 cells / well in 96-well plates. Single cell clones were transferred to 48-well plates. GM-CSF ELISA (BioLegend) was completed to identify positive clones. GM-CSF positive clones were transferred to 12-well plates. For example, V4-2 clone (BC4 cell line) was expanded and then cryopreserved using CS10 freezing medium.

[0236] Characterization of cell lines using GM-CSF and IFN-α ELISA. BC4 cell line and clone 17 cells were thawed and GM-CSF production was measured using a GM-CSF ELISA kit (R&D Systems Cat. Number DGM00) following the manufacturer's recommendations. The concentration of GM-CSF detected in BC4 was 36.9 ng / mL / 24h, whereas no GM-CSF was detected from clone 17. IFN-α production in BC4 was measured using an IFN-α ELISA kit (Invitrogen) and IFN-α concentrations were below the detection limit of the assay for both cells.

[0237] SYBR Green quantitative RT-PCR of HLA. BC4, SV-BR-1, and clone 17 cells were thawed and cultured for 7 days until the cells reached 80% confluence. Cells were detached using accutase dissociation reagent, then counted and cultured at 5 × 10 for subsequent analysis. 6 The cells were stored at -80°C.

[0238] RNA was extracted using the PureLink RNA Mini kit (ThermoFisher) according to the manufacturer's recommendations. 500 ng of RNA was used for synthesis of first strand cDNA. This cDNA (2 μL) was subsequently subjected to SYBR Green quantitative RT-PCR (Applied Biosystems) using custom designed primers (Genescript) for HLA-A*24:02, HLA-A*33:03, HLA-DRB5*01:01 and HLA-DRB5*02:02. High levels of HLA-DRB5*02:02 and HLA-A*33:03 and low levels of HLA-DRB5*01:01 were confirmed in BC4 but not in SV-BR-1 and clone 17. No cDNA detection of HLA-A*24:02 was observed. Therefore, it was assumed that HLA-A*24:02 was not expressed and the cell line was designated BC33.5.5 (BC4).

[0239] Flow cytometry of HLA-DR: BC4, SV-BR-1, and clone 17 cells were seeded in 6-well plates. The next day, cells were treated with IFNγ for 48 h. Treated cells were subjected to flow cytometry using anti-HLA-DR antibody (clone L243, Biolegend). The expression levels of HLA-DR in BC4 and SV-BR-1 were similar and higher than clone 17.

[0240] Example 2: Preparation of MCB for each cell line A master cell bank (MCB) may be created and tested for each cell line. By way of illustration, BC1, BC2, BC3 and BC4 cell lines are grown in T-25, T-75 and T-150 flasks using RPMI medium with 10% FBS. To confirm production of GM-CSF, randomly selected flasks are incubated for 72 hours in antibiotic-free RPMI + 10% FBS. Supernatants are collected and the concentration of GM-CSF is determined by ELISA. To confirm expression of specific HLA alleles, cells are subjected to flow cytometry using HLA-specific antibodies and RT-PCR using HLA-specific primers.

[0241] The expanded cells are harvested at passage 8 using 0.25% porcine trypsin + 1 mM EDTA (Gibco). The cells are resuspended in freezing medium (CS10) and placed in a freezing vial at 1.5 × 10 6 Aliquot at a viable cell concentration of 10 cells / vial. Examine cells as outlined below.

[0242] Testing MCB for each cell line. A Master Cell Bank (MCB) may be created and tested for each cell line. Briefly, cells may be tested for total cell recovery, viability, identity, purity, potency, mycoplasma, direct and indirect detection of mycoplasma, mycoplasma culture (mycoplasmastasis), endotoxin, 14-day axenic culture, specific viruses such as AAV2, EBV, HAV, HBV, HCMV, HCV, herpes virus (broad range), HHV-6, HHV-7, HHV-8, HIV (HIV-1, HIV-2), HTLV-1, HTLV-2, human parvovirus, polyoma virus (broad range), WNV, retroviral contaminants, bovine or porcine virus detection as per 9CFR, replication competent lentivirus (RCL), and / or non-endogenous / adventitious viruses. Details of the testing methods and criteria are listed in Table 4 below.

[0243] Table 4: MCB release test for BC1-BC4 cell lines TIFF2025507939000007.tif244160*Validated PCR or USP <63> Either method is used for mycoplasma detection.

[0244] Example 3: Pharmaceutical Compositions This example describes one embodiment of the composition of the pharmaceutical preparation described herein. The pharmaceutical preparation consists of a suspension of cells grown and irradiated from a single vial of MCB.

[0245] BC1-BC4 Drug Products (DPs) will each be prepared as frozen suspensions of approximately 20 million / mL total BC cells (target single viable cells 12.5 million / mL) in CryoStor CS10 filled at 1.25 mL in 2 mL cryovials. DPs will be stored in 2 mL male threaded freestanding CryoElite Natural Cap vials. Vials are made of low binding cryo-grade virgin polypropylene.

[0246] As an illustration, the BC33.5.5 (BC4) cell line is a HER-2 / neu positive allogeneic whole cell breast cancer (BC) cell line that has been engineered to express GM-CSF and the following HLA genes: HLA-A*33:03, HLA-DRB5*01:01, HLA-DRB5*02:02. CRISPR techniques were used to functionally knock out the native HLA genes. The drug substance (DS) consists of a suspension of cells that have been grown and irradiated from a single vial of MCB. The DS is immediately resuspended in cryopreservation solution and frozen to produce the final drug product.

[0247] The composition of the BC4 DP vaccine is provided in Table 5. The composition is the same for each of the BC1-BC4 DPs. The composition contains active ingredients with approximately 20 million total BC1 cells / mL, and is made up to 1 mL with an appropriate amount of CryoStor CS10 as a buffer and cryoprotectant. CryoStor CS10 is an animal component-free, defined cryopreservation medium with 10% DMSO. It is manufactured under cGMP using USP grade reagents.

[0248] (Table 5) Composition of BC4 Drugs (BC DP) TIFF2025507939000008.tif20170

[0249] Example 4: Safety and Efficacy of Modified Human Cancer Cell Lines in Cancer Patients This example illustrates one embodiment of a clinical trial testing the safety and efficacy of modified human cancer cell lines (e.g., Bria-OTS cell lines) in patients with advanced metastatic or locally recurrent breast cancer. This study provides preliminary data on safety, tolerability, and tumor response in patients with advanced (e.g., metastatic or locally recurrent) breast cancer. An overview of the clinical trial design is provided below.

[0250] This is a Phase 1 / 2a open-label clinical trial with patients assigned to treatment with breast cancer immunotherapy cell lines based on their HLA type. The primary goal of the study is to evaluate the safety of HLA-matched cellular immunotherapy in patients with advanced breast cancer. The secondary objective of the study is to evaluate the tumor response of HLA-matched cellular immunotherapy in patients with advanced breast cancer. Additionally, the study may include the objectives of evaluating progression-free survival (PFS) and overall survival (OS) in advanced breast cancer patients treated with HLA-matched cellular immunotherapy, evaluating the immune response elicited by HLA-matched cellular immunotherapy in patients with advanced breast cancer, evaluating patient and tumor characteristics that may predict response to HLA-matched cellular immunotherapy in patients with advanced breast cancer, and / or evaluating quality of life (QOL) in advanced breast cancer patients treated with HLA-matched cellular immunotherapy. The cell line being studied is the Bria OTS cell line (e.g., BC1, BC2, BC3, or BC4). Patients are treated with the cell line with which their HLA type is most closely matched, with at least one match required for the patient to be treated.

[0251] Additionally, to boost the immune response, patients are pretreated with low-dose cyclophosphamide, which down-regulates regulatory T cell mechanisms, 48-72 hours (2-3 days) prior to each vaccination. Low-dose pegylated interferon-alpha-2a (IFN-α2a) serves as an adjuvant and is given by intradermal injection of 0.1 mcg at the site of vaccination approximately 1-4 hours and approximately 24-72 hours (1-3 days) after vaccination. Biological samples are collected at regular intervals as per the protocol and stored in a repository.

[0252] By way of illustration, in the first part (Phase 1) of the study there is intrapatient dose escalation where each patient is treated with a single cell line. After the cell dose is determined in Part 1, in the second part (Phase 2a) patients may be treated with one or two cell lines with the goal of matching the patient with the cell line used in at least two HLA types, preferably one class II (HLA-DR) type and one class I (HLA-A, B, or C) type.

[0253] Patient Population. Patients will be screened to ensure they meet the enrollment criteria. Screening must occur within 30 days of initiation of treatment, and imaging studies must occur within 2 weeks of initiation of treatment. All patients will be women with histologically confirmed breast cancer with recurrent and / or metastatic disease via the study site who have failed prior treatment. Patients with any of the four breast cancer subtypes, namely, luminal A (HR+ / HER2-), triple negative (HR- / HER2-), luminal B (HR+ / HER2+), and HER2 enriched (HR- / HER2+), will be eligible if they meet the required criteria for failed prior treatment. Patients with de novo or progressive breast cancer that has metastasized to the brain will also be eligible, provided their intracranial disease is stable and not life-threatening, as described in the protocol outline.

[0254] Planned enrollment for both arms of the study is up to 48 patients, with 12 to 24 patients (at least three patients per cell line) being evaluated initially. If the cell lines are found safe in Part 1, an expansion cohort of 24 patients (at least four patients per cell line) will be enrolled in Part 2.

[0255] Test Treatment and Dose Escalation. Bria-OTS cell lines are irradiated to render them replication incompetent prior to freezing in viable freezing medium. They are shipped frozen to the clinical site and thawed on-site for inoculation. Patients are initially evaluated weekly during the dose escalation phase, including all safety assessments.

[0256] The dosage form is formulated as a suspension of irradiated cells. The patient is administered a therapeutically effective amount of the composition by intradermal injection into the upper back or thigh over a period of time. Table 6 shows the dosing regimen for phase 1 monotherapy phase.

[0257] Table 6. Dosing regimens for the monotherapy phase TIFF2025507939000009.tif36160

[0258] Patients return 2±1 days after each inoculation for safety assessments and to measure delayed-type hypersensitivity (DTH) responses midway through the monotherapy phase. After the monotherapy phase, patients are treated every 3 weeks with the highest cell dose that was safely tolerated (i.e., the maximum tolerated dose [MTD] or pharmacologically active dose). During this phase, patients also receive 300 mg / m 2–3 days prior to inoculation of each cell line. 2 of cyclophosphamide at each inoculation site, and 0.1 mcg of pegylated IFN-α2a at each inoculation site 1-4 hours and 1-3 days after inoculation of each cell line. The entire cycle is repeated every 3 weeks.

[0259] After at least three patients have been safely treated with each cell line as a single cell line, the second part of the trial (phase 2a) will begin. During part 2, all patients will be treated with one or two cell lines, with the goal of matching patients with the cell line used in at least two HLA types, preferably one class II (HLA-DR) type and one class I (HLA-A, B or C) type. 2 Treatment is preceded by cyclophosphamide. Treatment uses 20,000,000 cells (up to 4 intradermal injections in the upper back and thighs). Patients receive 0.1 mcg of pegylated IFN-α2a at each inoculation site 1-4 hours and 1-3 days after inoculation of each cell line. Patients return 2±1 days after each inoculation for safety evaluation and to measure DTH response. Treatment cycles are repeated every 3 weeks.

[0260] Evaluation. Study participants will be closely monitored for adverse events (e.g., toxicity) using the Common Terminology Criteria for Adverse Events (i.e., CTCAE v4.03) scale. Development of new or progressing tumors, or treatment-related grade III allergy / hypersensitivity will terminate further vaccinations for any particular subject.

[0261] Although the core success measures are safety and lack of toxicity, any of the following may be applied as success measures: 1) clinical response as defined by irRC RECIST 1.1 criteria in 25% of patients, 2) improved quality of life in 50% or more patients as evidenced by significant change in one or more scales on the SF-36 questionnaire (quality of life), 3) prolonged disease-free and overall survival compared to historical controls from other salvage therapy reports in the published literature, 4) evidence of development or amplification of an immune response, especially if correlated with prolonged survival. Other success measures may include induction of tumor regression.

[0262] Clinical response is primarily assessed by radiological assessment of tumor burden. This can be done by, for example, computed tomography (CT), magnetic resonance imaging (MRI), and / or positron emission tomography (PET). To assess whether objective tumor regression is particularly pronounced in patients with HLA alleles that are also found in SV-BR-1-GM cells, for example, HLA type is determined from oral or blood cells of clinical trial subjects. Several methods known to those skilled in the art are suitable for determining HLA alleles.

[0263] Example 5: Conversion of tumor cells into antigen-presenting cells for cancer immunotherapy This example shows one embodiment of converting tumor cells into antigen-presenting cells for cancer immunotherapy. The tumor cells may include engineered human cancer cell lines (e.g., Bria-OTS cell lines) and can be prepared for administration to patients with advanced metastatic or locally recurrent breast cancer.

[0264] Background. Therapeutic cancer vaccines are based on the specific stimulation of the immune system using tumor antigens to induce antitumor responses. Clinical trials are underway using the breast cancer cell line SV-BR-1-GM as a therapeutic vaccine. SV-BR-1-GM is a GM-CSF-expressing breast cancer cell line with the characteristics of an antigen-presenting cell (APC) that recognizes the expression of several immune-regulatory molecules, including MHC-I (HLA-A, B, and C) and MHC-II (HLA-DRB3 and -DRA). Initial results in patients treated with irradiated SV-BR-1-GM cells, low-dose cyclophosphamide, and topical IFN-α suggest that patients who match SV-BR-1-GM at at least one HLA allele are more likely to obtain clinical benefit. This clinical observation, together with the fact that SV-BR-1-GM cells can directly activate CD4+ T cells in an antigen-specific, HLA-restricted manner, as demonstrated by in vitro antigen-presentation assays, leads us to hypothesize that SV-BR-1 (the parent cell line) can function as an APC. To further enhance direct antigen presentation to T cells, SV-BR-1 cells have been genetically modified to express costimulatory molecules, immunomodulatory cytokines, and a broad repertoire of HLA alleles.

[0265] Methods. To generate an off-the-shelf semi-allogeneic cell therapy covering a large proportion of the population, SV-BR-1 was genetically modified to express a broad repertoire of HLA alleles. Based on population analysis, the four cell lines, each carrying two (2) HLA-A and two (2) HLA-DRB3 / 4 / 5 alleles, can generate at least one match to 99% of the population, with 92% matches at class I HLA-A alleles and 98% matches at class II HLA-DRB3 / 4 / 5 alleles. SV-BR-1 was genetically modified using CRISPR / cas9 deletion of endogenous HLA-A and HLA-DRB3 alleles and subsequent lentivirus-mediated expression of alternative HLA-A and DRB3 alleles. To generate tumor cell lines with enhanced direct antigen presentation to T cells, SV-BR-1 cells were genetically modified to express costimulatory molecules and immunomodulatory cytokines by using a lentivirus-mediated expression system.

[0266] Results: After sequential CRISPR / Cas9 editing, SV-BR-1 cells were cloned and one clone (clone 17) was selected for further manipulation. Flow cytometry and DNA sequencing were used to confirm the lack of expression of HLA-A and HLA-DRB3. Clone 17 was subsequently transduced with six lentiviral vectors expressing two genes under the control of separate promoters: CD86-IL12, CD80-HLA-DRA, 4-1BBL-IL7, GM-CSF-IFNA2, HLA-allele-1-HLA-allele-2 and HLA-DR-allele-1-HLA-DR-allele-2, respectively. Four cell lines were generated with different combinations of HLA alleles. After selection, cells were assessed by ELISA, flow cytometry and RT-PCR to confirm gene expression. Cell lines that secreted GM-CSF, IFN-α2 (e.g., IFN-α2a, IFN-α2b), IL-12, IL-7, and expressed CD80, CD86, 4-1BBL, and both class I and class II HLA alleles were then transferred to GMP manufacturing. In some cases, IFN-α, such as pegylated IFN-α2a, is exogenously provided to the modified cancer cell line.

[0267] Clinical trials. These modified breast cancer cell lines will be used in planned clinical trials to first evaluate the safety of intradermal inoculation of the irradiated cells, and then combine them with other agents to enhance the immune response. Each patient will be treated with a cell line that is matched to the patient at at least one HLA allele.

[0268] Example 6: Off-the-shelf personalized cell approaches towards immunotherapy for the treatment of advanced solid tumors Background: We are developing an off-the-shelf personalized cellular immunotherapy based on SV-BR-1-GM in a Phase I / IIa clinical trial in patients with metastatic or locally recurrent breast cancer. SV-BR-1-GM is a breast cancer cell line with antigen-presenting cell (APC) characteristics that has been stably transfected with the CSF2 gene encoding GM-CSF (SV-BR-1-GM). Favorable clinical outcomes have been reported in patient populations matched with SV-BR-1-GM at one or more HLA alleles. This clinical observation, together with the fact that SV-BR-1-GM cells can directly activate CD4+ T cells in an antigen-specific, HLA-restricted manner as demonstrated by an in vitro antigen presentation assay (Lacher MD et al, Front Immunol. 2018 May 15;9:776), has led us to hypothesize that SV-BR-1-GM can function as an APC. This example describes a therapeutic approach in which patients are treated with cell lines expressing HLA class I and II molecules that match their genotype. In addition, the parental SV-BR-1 cells were genetically modified to express costimulatory molecules and additional immunomodulatory cytokines to further enhance direct antigen presentation to T cells.

[0269] Methods: Using CRISPR / Cas9 techniques, we inactivated several endogenous HLA-A and HLA-DRB alleles present in five cancer cell lines (SV-BR-1, PC-3, LNCaP, SK-MEL-24, and NCI-H2228). Cells with inactivated HLA-A / DRB genes were transduced with lentiviral-based vectors expressing selected cytokines and costimulatory molecules (GM-CSF, IFN-α, CD80, CD86, IL-12, IL-7, HLA-DRA, and 4-1BBL). The lentiviral-based vectors were then used to transduce cells with unique combinations of HLA-A and HLA-DRB3 / 4 / 5 alleles to generate a collection of cell lines (e.g., Bria-OTS cell line) that matched with more than 99% of the patient population for at least one HLA allele. Flow cytometry and cell-based assays were used to establish the expression and functionality of stimulatory molecules and transgenic HLA alleles.

[0270] Results: Four cell lines (per tumor type) were selected that secreted GM-CSF, IFN-α, IL-12, IL-7, and expressed different combinations of CD80, CD86, 4-1BBL, and both class I and class II HLA alleles. Using cell-based assays, including a mixed lymphocyte reaction assay, we demonstrated that the generated cells stimulated naive T cells.

[0271] Conclusion: We have successfully generated "off-the-shelf" personalized cell-based therapeutic cancer vaccines that induce potent T cell responses. These modified cancer cell lines will be used in planned clinical trials to first evaluate the safety of intradermal inoculation of irradiated cells, and then combined with other agents to enhance the immune response.

[0272] Example 7: Bria-OTS cells with HLA-matched alleles for personalized treatment of advanced solid tumors This example describes the development of a novel precision-based immunotherapy cell line for the treatment of advanced solid tumors that can be HLA-matched to over 99% of the cancer patient population in the U.S. This immunotherapy platform shows promise as a safe, potent and effective treatment for solid tumors, and is the first rapid off-the-shelf therapy that does not require personalized manufacturing.

[0273] overview Cancer vaccines are promising immunotherapies thanks to their potent and long-lasting cancer-specific immune responses. Different types of cancer vaccines have been developed, including DNA / RNA-based, protein / peptide-based, dendritic cell, and whole cell-based vaccines. Although tumor cell vaccines express both tumor-specific and tumor-associated antigens, they do not induce robust responses in the clinic and there is a large variability in human leukocyte antigen (HLA)-restricted cellular responses among patients, which limits the efficacy of the vaccines. Thus, there remains an urgent and unmet need for new treatment regimens with greater impact on long-term survival.

[0274] To address the need for new immunotherapeutic approaches to treat advanced solid tumors, we are developing a novel whole-cell immunotherapeutic approach that acts via two complementary mechanisms of action: 1) cross-presentation of cancer cell antigens and 2) direct T-cell activation. This dual mechanism of action is believed to be unique and represents a significant advance from previous attempts to develop whole-cell cancer vaccines. Enhanced clinical responses are achieved through direct T-cell activation if the patient's HLA molecules match those in the therapeutic cell line. Our first generation Bria-IMT product has demonstrated substantial tumor regression in patients with metastatic breast cancer who are matched at least at one HLA allele with Bria-IMT. Bria-IMT secretes granulocyte-macrophage colony-stimulating factor, functions as an antigen-presenting cell (APC), and binds CD4 +SV-BR-1-GM is a breast cancer cell line that can directly activate T cells. We have completed molecular analysis of SV-BR-1-GM cells by microarray gene expression profiling and identified a unique 22-gene immune signature that confers APC-like activity. Based on this unique immune signature, we have selected additional cell lines from different origins (e.g., melanoma, prostate and lung cancer) in addition to breast cancer, and engineered cell lines that express a distinct set of HLA alleles as well as a set of cytokines and costimulatory molecules, which collectively have the potential to treat nearly 100% of the American population at the level of matching one HLA class I or II allele, and 90% when matching two HLA alleles. These cells (named Bria-OTS for "off the shelf") are used as a pre-manufactured, ready-to-use personalized immunotherapy for the treatment of advanced solid tumors.

[0275] Specific Goals Cellular cancer immunotherapy must be tailored to each individual patient to induce an effective immune response, which increases manufacturing complexity and associated costs. To circumvent this limitation, we are developing Bria-OTS™ as an off-the-shelf, pre-manufactured, ready-to-use personalized immunotherapy for the treatment of advanced solid tumors.

[0276] Cancer immunotherapy relies on activating a patient's immune system to eliminate cancer cells based on the presence of tumor-associated antigens (TAA). Despite significant advances, current immunotherapies are effective in only a very limited number of patients. 1,2 Cancer vaccines are promising because they often result in strong and long-lasting cancer-specific immune responses. Different types of cancer vaccines have been developed, including DNA / RNA-based, protein / peptide-based, dendritic cell (DC), and whole cell-based vaccines. Although autologous DC vaccines have been successfully administered in clinical trials, 3 ,Their use is limited by complex manufacturing logistics that impair feasibility and increase costs. 4Although tumor cell-based vaccines express both tumor-specific and tumor-associated antigens, they do not induce robust responses in the clinic and there is variability in human leukocyte antigen (HLA)-restricted cellular responses among patients. 5 Thus, there is an urgent and unmet need for new treatment regimens that have a major impact on long-term survival.

[0277] The inventors are developing their most advanced lead candidate - an off-the-shelf personalized cellular immunotherapy based on Bria-IMT™ - Bria-OTS - which is in a Phase I / IIa clinical trial (NCT03328026) in patients with metastatic or locally recurrent breast cancer, and preliminary data have shown substantial tumor regression in heavily pretreated patients matched for Bria-IMT at two HLA alleles. 6 Bria-IMT™ is a breast cancer cell line (SV-BR-1-GM) stably transfected with the CSF2 gene and therefore secretes granulocyte-macrophage colony-stimulating factor (GM-CSF). Bria-IMT™ expresses class I and II human leukocyte antigen (HLA) complex genes. SV-BR-1-GM functions as an antigen-presenting cell (APC) to secrete CD4 + Directly activating T cells 7 Preliminary clinical data of metastatic breast cancer patients with at least one HLA allele compatible with the Bria-IMT cell line 6,8 Based on the Bria-IMT technique, which has demonstrated excellent safety and tumor regression in mice, the inventors have developed engineered Bria-OTS cell lines for the treatment of solid tumors. The inventors have engineered Bria-OTS cell lines derived from breast cancer (SV-BR-1), prostate cancer (PC-3), melanoma (SK-MEL-24), or lung cancer cells (NCI-H2228). These cell lines were selected based on the expression of a distinctive 22-gene immune signature originally characterized in SV-BR-1 cells, which includes MHC class I and II components, certain cytokines and chemokines known to promote immune cell attraction. 7Bria-OTS cells are engineered to express a distinct set of HLA alleles that collectively have the potential to treat nearly 100% of the American population at a level of matching one HLA class I or II allele, and approximately 90% at a level of >= one HLA class I plus >= one HLA class II (double match). After the patient's unique HLA signature is known, the patient is assigned a complementary Bria-OTS product with at least one matching HLA allele. Treatment can begin immediately without the need for patient-specific manufacturing.

[0278] Generation of Bria-OTS breast, prostate, melanoma, and lung cancer cell lines. Using CRISPR / Cas9 technology, we inactivated several endogenous HLA-A and HLA-DRB alleles present in all four parental cell lines (SV-BR-1, PC-3, SK-MEL-24, and NCI-H2228). We used pCCLc-based lentiviral vectors to express HLA-DRA, selected cytokines (GM-CSF, IFNα, IL-12, IL-7), and costimulatory molecules (CD80, CD86, 4-1BBL), and subsequently overexpressed unique combinations of HLA-A and HLA-DRB3 / 4 / 5 alleles. As a final product, we generate four distinct Bria-OTS clonal cell lines that match more than 99% of the patient population within a particular cancer type. The final Bria-OTS cells are rigorously examined to confirm chromosomal integration (whole genome sequencing) and lack of deviation in expression of key genes from the parental cells (RNA-seq).

[0279] Demonstration of in vitro functionality of Bria-OTS cells. Mixed lymphocyte reaction (MLR) assays are used to measure T cell proliferation, secreted interferon gamma (IFNγ), interleukin-2 (IL-2), IL-4, and IL-17 levels at baseline and after 1, 2, 5, and 7 days to assess T cell activation (to cover CD8+ cytotoxic T cells, CD4+ Th1, Th2, and Th17 T cell subsets). Eight T cell receptor (TCR) transgenic Jurkat cell lines expressing selected HLA-A and HLA-DR restricted TCRs are generated to assess functionality of the transgenic HLA using T cell activation bioassays.

[0280] The clinical efficacy of Bria-OTS cells can be verified in xenograft tumor mouse models of advanced solid tumors, and safety and clinical activity can be demonstrated in first-in-human trials. The cellular immunotherapy strategy described herein is promising as a safe and efficient treatment option for solid tumors in need of effective therapy, with the advantage of personalized therapy without complex manufacturing.

[0281] significance Cancer immunotherapy for advanced solid tumors. In contrast to conventional chemotherapy or radiation therapy, which indiscriminately kill all rapidly dividing cells, cancer immunotherapy relies on the activation of the patient's immune system to eliminate cancer cells based on the presence of tumor-associated antigens (TAA). Despite great advances, current immunotherapies are effective only in a very limited number of patients. 1,2 Cancer vaccines are promising because they often result in strong and long-lasting immune responses. Development efforts have focused on peptide and protein tumor antigens or neoantigens. 9-11 , cancer antigen-pulsed dendritic cells 12-22 , and whole tumor cells 23,24 The advantage of a whole cell vaccine is that it delivers a range of TAAs. 25Sipuleucel-T (Provenge®) is currently the only whole-cell vaccination-based personalized immunotherapy approved by the US FDA for the treatment of advanced prostate cancer. However, sipuleucel-T does not offer the antigen repertoire that whole-cell vaccines do, as it is directed only against a fusion protein of prostatic acid phosphatase antigen (PAP) and GM-CSF with a mechanism of action similar to "cross-presentation". In essence, sipuleucel-T is a cellular therapy that contains antigen-presenting cells (APCs) "loaded" with PAP-GM-CSF that are recognized by a subset of patient T cells. The addition of GM-CSF to whole-cell tumor vaccines has been shown to stimulate the migration of DCs, T cells, and macrophages to the vaccination site. 26 Despite great efforts and promising results in early-phase studies, whole-cell cancer vaccines have shown poor clinical efficacy, likely due to multiple factors including, but not limited to, poor immunogenicity, lack of costimulatory molecules, down-regulation of human leukocyte antigen (HLA) expression, and / or suppression by the tumor microenvironment (TME). 27 Addressing these significant obstacles, we have developed the Bria-OTS cell line, which expresses not only TAAs, costimulatory molecules, and HLA genes that enable direct presentation of tumor antigens to T cells, but also immunostimulatory cytokines that lead to enhanced immune responses (Figure 6).

[0282] Scientific rationale: Bria-IMT (also called SV-BR-1-GM) is a HER2 / neu positive breast cancer cell line genetically engineered to overexpress GM-CSF. We performed molecular fingerprinting of SV-BR-1-GM cells by microarray gene expression profiling, quantitative reverse transcription PCR (qRT-PCR), transcript counting (nCounter), ELISA, and flow cytometry. 7We found that SV-BR-1-GM, despite its mammary epithelial origin, expresses a gene signature associated with immunostimulatory functions. In addition to HLA class I molecules, Bria-IMT expresses several HLA class II genes, namely human leukocyte antigen (HLA)-DMA, -DMB, -DRA, and -DRB3. 7 We identified a unique 22-gene immune signature that included cell surface ligands for T cell costimulatory receptors, cytokines and other soluble factors with positive T cell stimulatory functions, factors that promote maturation, survival, chemotaxis, and / or in vitro generation of DCs, and factors that promote antigen presentation. 7 We also identified 31 genes that were potential TAA candidates expressed in SV-BR-1-GM cells, including Her2 / neu, MIEN1 (migration and invasion enhancer 1), PGAP3 (post-GPI attachment to proteins phospholipase 3), and STARD3 (Star related lipid transfer domain containing 3), and several cancer / testis antigens (CTAs) were strongly expressed (PRAME, kinesin-like protein KIF2C, 55 kDa centrosomal protein-CEP55, and PDZ-binding kinase-PBK). 7,28 In vitro studies demonstrated that Bria-IMT directly stimulates T cell activation in an antigen-specific, HLA-restricted manner and thus has characteristics of antigen-presenting cells. 7 Importantly, this feature of Bria-IMT requires that the endogenous HLA class II genes expressed in the Bria-IMT cell line be "matched" with those of the co-cultured T cells. Thus, Bria-IMT cell lines possess critical characteristics of both breast cancer cells and dendritic cells. In the clinic, patients carrying HLA class II alleles that match the endogenous HLA alleles in Bria-IMT show substantial tumor regression and other signs of clinical activity. 6,8Thus, our clinical data demonstrate that if the endogenous HLA barcode unique to the Bria-IMT cell line matches that of the patient, the therapy has the potential to induce high clinical responses. These clinical data highlight direct T cell stimulation via class II HLA matching as a previously unrecognized contributor to robust immunotherapeutic activity that was not present or considered when evaluating previous whole-cell immunotherapies. Moreover, our studies collectively demonstrate a novel "dual mechanism" of Bria-IMT action in which the immune system is stimulated by both antigen cross-presentation and direct T cell stimulation (Figure 6). However, despite the demonstrated clinical potential of Bria-IMT, only 47% of the US population carries one or more HLA alleles that match the alleles in the Bria-IMT cell line, and approximately 10% of the population matches at two or more HLA alleles expressed in the Bria-IMT cell line. 29 .

[0283] To expand the patient population that can benefit from our immunotherapy, we are developing a collection of "off-the-shelf" Bria-OTS™ cell lines, four cell lines per parental tumor cell. Each cell line expresses four exogenous HLA alleles, two HLA-A and two HLA-DRB3 / 4 / 5 alleles, for a total of eight HLA-A and seven HLA-DRB alleles for the four cell lines. Collectively, the collection of Bria-OTS cell lines has the potential to treat nearly 100% of the American population with ≧1 HLA allele match and about 90% of the American population with a double match of HLA class I and II. After the patient's unique HLA signature is known, the patient is assigned one or two complementary Bria-OTS products that are well matched to the patient's HLA profile.

[0284] technological innovation The transformation techniques described herein are the development of therapeutic strategies that address the current gap in safe and reliable treatment for patients with advanced solid tumors. Because our platform works through at least two distinct but complementary mechanisms to stimulate the patient's immune system, our tumor cell-based immunotherapy significantly improves upon previous attempts to develop whole cell cancer vaccines. The Bria-OTS cell line population offers the following characteristics: - Dual mechanism of action to achieve strong immune responses and clinical activity. Bria-OTS cell line possesses characteristics of both cancer cells (expressing various TAAs) and dendritic cells (by directly presenting TAAs to T cells) to enhance immune responses. - Precision Therapy: Bria-OTS cell lines are matched to patients based on HLA antigens, covering over 99% of the US population, and induce a cellular response that recognizes the patient's cancer cells. - Improved safety compared to current chemotherapy and hormone-based therapies, which are associated with serious, potentially life-threatening adverse events. - Rapid and cost-effective treatment. Our "off-the-shelf" cell lines do not require individualized manufacturing and can be administered immediately after patient HLA genotyping.

[0285] procedure This example describes engineering Bria-OTS cell lines derived from different types of solid tumors (melanoma, breast, prostate, and lung cancer) to express HLA alleles not present in the parental tumor cell lines, as well as GM-CSF, other cytokines and costimulatory molecules. Figure 7 shows the general strategy for developing Bria-OTS cell lines. Briefly, the parental tumor cell lines are selected by gene expression profile based on the immune signature of 22 genes originally identified in SV-BR-1 breast cancer cells. 7Endogenous HLA-A and HLA-DR alleles are knocked out (KO) by CRISPR / Cas9 strategy, followed by single-cell cloning and validation resulting in Bria-KO cells. Eight predetermined cytokines and costimulatory molecules are then overexpressed by lentiviral transduction, their expression is confirmed by qRT-PCR, flow cytometry, and / or ELISA in single-cell clones, and Bria-APTCs with APC properties are recovered. Finally, a unique combination of eight HLA-A and seven HLA-DRB alleles (two HLA-A and two HLA-DRB alleles / cell line) is overexpressed to generate the final Bria-OTS product (four different cell lines per cancer cell line). These cell lines deliver precision therapy for patients with solid tumors without the need for personalized manufacturing.

[0286] Generation of Bria-OTS breast, prostate, melanoma, and lung cancer cell lines. We selected SV-BR-1 (Her2-positive breast cancer cell line), PC3 (prostate cancer cell line), NCI-H2228 (lung cancer), and SK-MEL-24 cells (melanoma) as parental cancer cell lines based on the expression of a 22-gene immune signature characterized in SV-BR-1 cells, consisting of HLA molecules, cytokines, and chemokines. 7 Following the general scheme shown in Figure 7A, a population of four Bria-OTS cell lines expressing four unique combinations of HLA-A and HLA-DRB alleles that match >99% of the patient population for each particular parental cancer cell line can be generated.

[0287] Manipulation of SV-BR-1-Bria-OTS cells.Following sequential CRISPR / Cas9 inactivation of endogenous HLA-A and HLA-DRB3 alleles present in parental SV-BR-1 cells, as demonstrated by flow cytometry and sequencing, we selected clone 17 for further manipulation. We constructed four lentiviral vectors with a pCCLc backbone encoding two genes each from two independent promoters (EF1a and MNDU3) for expression of HLA-DRA, four cytokines (GM-CSF, IFN-α, IL-7, IL-12), and three costimulatory molecules (CD80, CD86, and 4-1BBL). 30 GM-CSF stimulates DC activation and antigen presentation 31,32 IFN-α induces a type I response that reduces the occurrence of suppressor mechanisms 33,34 IL-7 is a critical cytokine for T cell homeostasis 35,36 IL-12 is typically expressed in DCs and provides a signal during the priming process. 37-39 Costimulatory molecules are typically expressed by APCs and activate naive T cells. 40 SV-BR-1-KO cells, clone 17, were transduced sequentially with all four lentiviruses to generate SV-BR-1-APTC (for antigen-presenting tumor cells, APTC). Expression of all cytokines and costimulatory molecules was confirmed by ELISA, flow cytometry, and quantitative real-time PCR (qRT-PCR) (Figure 8). Enhanced expression of IL-12, IL-7, GM-CSF, and IFN-α was demonstrated by ELISA in clone 17 transduced with all four plasmids (Figure 8A); elevated expression of cell surface receptors CD80 and CD86 was detected by flow cytometry (Figure 8B), while 4-1BBL was expressed at lower levels.

[0288] A modified mixed lymphocyte release (MLR) assay with pooled SV-BR-1-APTC was used to demonstrate the functionality of these costimulators. SV-BR-1-APTC (Figure 9A), which expresses endogenous HLA class I and class II that were not knocked out by CRISPR / Cas9, was cocultured with peripheral blood mononuclear cells (PBMCs) isolated from healthy donors at different ratios of 1:5, 1:10, and 1:20 (tumor cells:PBMCs). Supernatants were collected on days 1, 2, 3, and 7, and secreted IFNγ levels as a measure of T cell activation were determined by ELISA (data for day 1 are shown in Figure 9B). Although the levels of secreted IFNγ were significantly increased in the presence of SV-BR-1-OTS-APTC depending on the PBMC:tumor ratio; in contrast, a decrease in IFNγ was detected when PBMCs were incubated with SV-BR-1-KO cells or when tumor cells were incubated alone.

[0289] Following selection and cell cloning, SV-BR-1-APTC was then transduced with lentiviral vectors expressing two unique HLA-A alleles. We first constructed eight pCCLc-based lentiviral vectors, each containing two HLA-A or two HLA-DRB3 / 4 / 5 alleles. The rationale for allele selection was based on a study by Gragert et al., which reported the abundance of HLA allele frequencies among 16 different American populations. 29 We ranked these frequencies based on overall abundance to account for different population sizes. The 15 selected HLAs (Figure 7B) should yield at least one match in 99% of the American population, with 92% matches for class I HLA-A and 98% matches for class II HLA-DR3 / 4 / 5 alleles. Plasmid integrity was confirmed by restriction digestion and Sanger sequencing, and HLA-A expression was confirmed by qRT-PCR.

[0290] Generation of PC3-APTC cells.After inactivation of endogenous HLA-A and HLA-DRB3 alleles present in parental PC3 cells by sequential CRISPR / Cas9, demonstrated by flow cytometry and sequencing, we selected clone A4 for further manipulation. We constructed four lentiviral vectors with pCCLc backbone, encoding two genes each from two independent promoters (EF1a and MNDU3) to express HLA-DRA, three cytokines (GM-CSF, IL-7, IL-12), and three costimulatory molecules (CD80, CD86, and 4-1BBL). These genes were selected based on their immune functions. GM-CSF stimulates DC activation and antigen presentation. IL-7 is a cytokine critical for T cell homeostasis. IL-12 is typically expressed in DCs and provides signal 3 during the priming process. Costimulatory molecules are typically expressed by APCs to activate naive T cells. PC3-KO cells were transduced sequentially with all four lentiviruses to generate PC3-APTC (for antigen-presenting tumor cells). Expression of cytokines and costimulatory molecules was confirmed by flow cytometry, ELISA, and qRT-PCR, and their functionality was demonstrated by MLR assay. PC3-APTC, but not PC3-KO control, induced substantial IFNγ secretion when co-cultured with PBMCs (Figure 10).

[0291] Manipulation of PC3-Bria-OTS cells.PC3-APTC was subsequently transduced with pCCLc-based lentiviral plasmids encoding one of the following HLA-A allele combinations: A01-A68, A02-A11, A03-A23 or A24-A33 to generate PC3-Bria-OTS cells. Expression of cytokines and costimulatory molecules and HLA-A alleles was confirmed by ELISA and flow cytometry (Figure 11). Elevated expression of cell surface receptors CD80 and CD86 as well as several HLA-A alleles was detected by flow cytometry in clone A4 transduced with all four plasmids (Figures 11A-B). Enhanced expression of IL-7, GM-CSF and IL-12 was demonstrated by ELISA (Figure 11C).

[0292] In summary, we have successfully generated tumor cell lines expressing cytokines, costimulatory molecules, and diverse HLA.

[0293] Engineering of H2228-Bria-OTS (lung) and SK-MEL-24-Bria-OTS (melanoma) cells. Following the same overall strategy, we generated H2228 and SK-MEL-24 cells lacking endogenous HLA-DR alleles (Figure 12) and undergoing CRISPR / Cas9 KO of endogenous HLA-A alleles. The resulting Bria-KO clonal cell line is sequentially transduced with all four lentiviral plasmids to produce Bria-APTC by overexpression of GM-CSF, IFN-α, CD80, CD86, IL-12, IL-7, HLA-DRA, and 4-1BBL. ≥100ng / 1x10 for GM-CSF. 6 Cytokine secretion was assessed by ELISA at 100 cells / 24 h target secretion. 6 , which is sufficient to enhance dendritic cell activity 6,7Cell surface expression of costimulatory molecules is confirmed by flow cytometry. In addition, all ectopic gene expression is further verified by qRT-PCR. All analyses can be performed at least in triplicate for each cell line, and the mean ± SD can be reported. As a final step, clones can be isolated, expanded, and cryopreserved. Selected clones are transduced sequentially with two lentiviral vectors expressing two HLA-A and two HLA-DR alleles, respectively, to obtain four final clonal cells for each of H2228 and SK-MEL-24 cells.

[0294] Demonstration of in vitro functionality of Bria-OTS cells. Using the MLR assay, we measure the levels of T cell activation induced by Bria-OTS and control Bria-KO cell lines. We measure T cell proliferation and levels of secreted IFNγ, IL-2, IL-4, and IL-17 as proxies for T cell activation (to cover CD8+ cytotoxic T cells, CD4+ Th1, Th2, and Th17 T cell subsets) at baseline and after 1, 2, 3, and 7 days. We generated TCR transgenic Jurkat cell lines that recognize antigens in the context of HLA-A / DR alleles expressed on Bria-OTS cells to verify HLA functionality of the final Bria-OTS product.

[0295] Modified MLR assay to demonstrate T cell activation by Bria-OTS cells. MLR assay is performed using the above final cell lines. Bria-KO cells are used as a control. Briefly, Bria-OTS cells are co-cultured with PBMCs isolated from at least two healthy donors (one male and one female) in 96-well plates at different tumor:PBMC ratios. T cell proliferation is measured by cell count and dye dilution every 24 hours. Supernatants are collected on days 0, 1, 2, 3, and 7 to measure the levels of IFNγ, IL-2, IL-4, and IL-17 by ELISA.

[0296] Validation of HLA functionality of Bria-OTS cells using a T cell activation bioassay with TCR transgenic Jurkat cells. The T cell activation bioassay (Promega) consists of a genetically engineered Jurkat T cell line (obtained by CRISPR / Cas9-mediated knockout of TCRα / β) that lacks expression of endogenous TCRα / β subunits and also expresses an NFAT-driven luciferase reporter. This cell line is useful for measuring the potency of transgenic TCR constructs in activated T cells independent of endogenous TCR expression. We have obtained three HLA-A-restricted TCR constructs (HLA-A*02:01, HLA-A*01:01, HLA-A*24:02) from Creative Biolabs and four HLA-DR-restricted TCRs (DRB4*0101, DRB3*0101, DRB3*0202, DRB5*0101) from Benaroya Research Institute (Table 7) to generate seven different TCR-transgenic Jurkat cell lines restricted by some of the HLA-A and HLA-A / DR alleles present in Bria-OTS cells. All transgenic TCRs can be expressed using the pCDTCR1 vector (HLA-A) or the lentiviral gene expression vector pLV (VectorBuilder, HLA-DR). Co-culture of Bria-OTS cells with TCR-transgenic Jurkat cells expressing TCRs restricted by HLA-A or DR alleles results in increased luciferase activity (Figure 13). Negative controls include TCR-transgenic Jurkat cells incubated with Bria-KO / APTC and Bria-OTS incubated with Jurkat TCRα / β-KO cells lacking a TCR. The Bria-OTS cell line, but not Bria-KO / APTC, induces a strong IFNγ response in the MLR and NFAT-driven luciferase activity in an antigen-specific HLA-restricted manner in the TCR-transgenic Jurkat assay.

[0297] Table 7. Constructs for generating TCR transgenic Jurkat cells TIFF2025507939000010.tif61170

[0298] Validation of HLA functionality in PC3-Bria-OTS cells using a T cell activation bioassay (NFAT). Co-culture of PC3-Bria-OTS-4 or PC3-Bria-OTS-1 cells with HLA-A*24 or HLA-A*01 TCR transgenic Jurkat cells resulted in an increase in luciferase activity (Figures 14A and 14C). Each experiment included appropriate negative controls: TCR transgenic Jurkat cells incubated with PC3 or PC3-KO, and PC3-Bria-OTS cells incubated with Jurkat TCRα / β-KO cells (no TCR). Interestingly, activation of transgenic Jurkat cells correlates with the expression level of HLA-A (Figures 14B and 14D).

[0299] Validation of HLA functionality in SV-BR-1-Bria-OTS cells using a T cell activation bioassay (NFAT). Similarly, coculture of SV-BR-1-OTS-4 cells with HLA-A24 TCR transgenic Jurkat cells resulted in significant activation as measured by bioluminescence monitoring, whereas the same cells lacking expression of HLA-A24 (SV-BR-1-APTC and SV-BR-1-KO) did not (Figure 15).

[0300] Overall, the above results demonstrate that tumor cells expressing transgenic HLA-A alleles are capable of inducing T cell activation.

[0301] References (Example 7 only) TIFF2025507939000011.tif157160TIFF2025507939000012.tif238160TIFF2025507939000013.tif155160

[0302] V. References TIFF2025507939000014.tif67160TIFF2025507939000015.tif236160TIFF2025507939000016.tif233160 TIFF2025507939000017.tif233160TIFF2025507939000018.tif238160TIFF2025507939000019.tif103160

[0303] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the appended claims. All publications, patents, patent applications, and sequence accession numbers cited herein are incorporated by reference in their entirety for all purposes.

[0304] Informal sequence listing <h2 style=";text-align:left;direction:ltr">TIFF2025507939000020.tif231149TIFF2025507939000021.tif239152TIFF2025507939000022.tif239153TIFF2025507939000023.tif238152TIFF2025507939000024.tif239149TIFF2025507939000025.tif239152TIFF2025507939000026.tif235149TIFF2025507939000027.tif239152TIFF2 025507939000028.tif239152TIFF2025507939000029.tif239152TIFF2025507939000030.tif239152TIFF2025507939000031.tif243149TIFF2025507939000032.tif243149TIFF2025507939000033.tif243149TIFF2025507939000034.tif243149TIFF2025507939000035.tif243149TIFF202550 7939000036.tif243149TIFF2025507939000037.tif243149TIFF2025507939000038.tif243149TIFF2025507939000039.tif243149TIFF2025507939000040.tif238149TIFF2025507939000041.tif243149TIFF2025507939000042.tif243149TIFF2025507939000043.tif243149TIFF20255079390 00044.tif238149TIFF2025507939000045.tif243149TIFF2025507939000046.tif243149TIFF2025507939000047.tif243149TIFF2025507939000048.tif243149TIFF2025507939000049.tif243149TIFF2025507939000050.tif243149TIFF2025507939000051.tif243149TIFF2025507939000052.tif243149TIFF2025507939000053.tif243160TIFF2025507939000054.tif243160TIFF2025507939000055.tif243160TIFF2025507939000056.tif243160TIFF2025507939000057.tif243160TIFF2025507939000058.tif239160TIFF2025507939000059.tif243160TIFF2025507939000060.tif243160TIFF2025507939000061.tif175160.

Claims

1. A modified human cancer cell comprising: (a) one or more recombinant polynucleotides each encoding an allele of a human leukocyte antigen (HLA) class I gene; and (b) one or more recombinant polynucleotides each encoding an allele of an HLA class II gene, wherein one or more HLA alleles endogenous to the cell are inactivated.

2. 2. The modified human cancer cell of claim 1, wherein (a) comprises a first recombinant polynucleotide encoding a first allele of an HLA class I gene and a second recombinant polynucleotide encoding a second allele of an HLA class I gene.

3. 3. The modified human cancer cell of claim 1 or 2, wherein (b) comprises a first recombinant polynucleotide encoding an allele of a first HLA class II gene and a second recombinant polynucleotide encoding an allele of a second HLA class II gene.

4. 3. The modified human cancer cell of claim 1 or 2, wherein (b) comprises a first recombinant polynucleotide encoding a first allele of an HLA class II gene and a second recombinant polynucleotide encoding a second allele of an HLA class II gene.

5. 5. The modified human cancer cell of any one of claims 1 to 4, wherein the HLA class I genes comprise an HLA-A gene, an HLA-B gene, an HLA-C gene, an HLA-E gene, an HLA-F gene, an HLA-G gene, a beta-2-microglobulin (B2M) gene, or a combination thereof.

6. 6. The modified human cancer cell of claim 5, wherein the alleles of the HLA-A gene comprise an HLA-A*01:01 allele, an HLA-A*02:01 allele, an HLA-A*02:06 allele, an HLA-A*03:01 allele, an HLA-A*11:01 allele, an HLA-A*23:01 allele, an HLA-A*24:02 allele, an HLA-A*26:01 allele, an HLA-A*29:02 allele, an HLA-A*31:01 allele, an HLA-A*32:01 allele, an HLA-A*33:03 allele, an HLA-A*68:01 allele, or a combination thereof.

7. The allele of the HLA-A gene is (i) HLA-A*01:01 and HLA-A*68:01 alleles; (ii) HLA-A*02:01 allele and HLA-A*11:01 allele; (iii) the HLA-A*03:01 allele and the HLA-A*23:01 allele; or (iv) HLA-A*33:03 allele 7. The modified human cancer cell of claim 5 or 6, comprising:

8. The modified human cancer cell of claim 7, wherein (iv) further comprises an HLA-A*24:02 allele.

9. The modified human cancer cell of any one of claims 1 to 8, wherein the HLA class II genes comprise HLA class II alpha subunit genes, HLA class II beta subunit genes, or a combination thereof.

10. 9. The modified human cancer cell of any one of claims 1 to 8, wherein the HLA class II genes comprise an HLA-DP gene, an HLA-DM gene, an HLA-DO gene, an HLA-DQ gene, an HLA-DR gene, or a combination thereof.

11. The modified human cancer cell of claim 10, wherein the HLA-DM gene comprises an HLA-DMA gene, an HLA-DMB gene, or a combination thereof.

12. The modified human cancer cell of claim 10, wherein the HLA-DR gene comprises an HLA-DRA gene, an HLA-DRB1 gene, an HLA-DRB3 gene, an HLA-DRB4 gene, an HLA-DRB5 gene, or a combination thereof.

13. 13. The modified human cancer cell of claim 11 or 12, wherein the alleles of the HLA-DRB3 gene include an HLA-DRB3*01:01 allele, an HLA-DRB3*02:02 allele, an HLA-DRB3*03:01 allele, or a combination thereof.

14. 14. The modified human cancer cell of any one of claims 11 to 13, wherein the alleles of the HLA-DRB4 gene comprise an HLA-DRB4*01:01 allele, an HLA-DRB4*01:03 allele, or a combination thereof.

15. 15. The modified human cancer cell of any one of claims 11 to 14, wherein the alleles of the HLA-DRB5 gene comprise an HLA-DRB5*01:01 allele, an HLA-DRB5*01:02 allele, an HLA-DRB5*02:02 allele, or a combination thereof.

16. The allele of the HLA-DR gene is (i) the HLA-DRB3*02:02 allele and the HLA-DRB5*01:01 allele; (ii) HLA-DRB4*01:01 allele and HLA-DRB3*01:01 allele; (iii) the HLA-DRB3*03:01 allele and the HLA-DRB5*01:02 allele; or (iv) HLA-DRB5*02:02 allele and HLA-DRB3*01:01 allele The modified human cancer cell of any one of claims 10 to 15, comprising:

17. The allele of the HLA-DR gene is (i) the HLA-DRB3*02:02 allele and the HLA-DRB4*01:01 allele; (ii) HLA-DRB4*01:01 allele and HLA-DRB3*03:01 allele; (iii) the HLA-DRB3*01:01 allele and the HLA-DRB5*01:02 allele; or (iv) HLA-DRB5*01:01 allele and HLA-DRB5*02:02 allele The modified human cancer cell of any one of claims 10 to 15, comprising:

18. (a) a recombinant polynucleotide encoding at least one of an HLA-A*01:01 allele, an HLA-A*68:01 allele, an HLA-DRB3*02:02 allele, and an HLA-DRB5*01:01 allele; (b) a recombinant polynucleotide encoding at least one of the HLA-A*02:01 allele, the HLA-A*11:01 allele, the HLA-DRB4*01:01 allele, and the HLA-DRB3*01:01 allele; (c) a recombinant polynucleotide encoding at least one of the HLA-A*03:01 allele, the HLA-A*23:01 allele, the HLA-DRB3*03:01 allele, and the HLA-DRB5*01:02 allele; or (d) a recombinant polynucleotide encoding at least one of the HLA-A*24:02 allele, the HLA-A*33:03 allele, the HLA-DRB5*02:02 allele, and the HLA-DRB3*01:01 allele. wherein one or more HLA alleles endogenous to the cell are inactivated.

19. (a) a recombinant polynucleotide encoding an HLA-A*01:01 allele, an HLA-A*68:01 allele, an HLA-DRB3*02:02 allele, and an HLA-DRB5*01:01 allele; (b) a recombinant polynucleotide encoding the HLA-A*02:01 allele, the HLA-A*11:01 allele, the HLA-DRB4*01:01 allele, and the HLA-DRB3*01:01 allele; (c) a recombinant polynucleotide encoding the HLA-A*03:01 allele, the HLA-A*23:01 allele, the HLA-DRB3*03:01 allele, and the HLA-DRB5*01:02 allele; or (d) a recombinant polynucleotide encoding the HLA-A*24:02 allele, the HLA-A*33:03 allele, the HLA-DRB5*02:02 allele, and the HLA-DRB3*01:01 allele. Including, one or more HLA alleles endogenous to the cell are inactivated, 20. The modified human cancer cell of claim 18.

20. 1. A modified human cancer cell, comprising: (a) a recombinant polynucleotide encoding at least one of an HLA-A*01:01 allele, an HLA-A*68:01 allele, an HLA-DRB3*02:02 allele, and an HLA-DRB4*01:01 allele; (b) a recombinant polynucleotide encoding at least one of the HLA-A*02:01 allele, the HLA-A*11:01 allele, the HLA-DRB4*01:01 allele, and the HLA-DRB3*03:01 allele; (c) a recombinant polynucleotide encoding at least one of the HLA-A*03:01 allele, the HLA-A*23:01 allele, the HLA-DRB3*01:01 allele, and the HLA-DRB5*01:02 allele; or (d) a recombinant polynucleotide encoding at least one of the HLA-A*33:03 allele, the HLA-DRB5*01:01 allele, and the HLA-DRB5*02:02 allele. Including, one or more HLA alleles endogenous to the cell are inactivated; Engineered human cancer cells.

21. (a) a recombinant polynucleotide encoding an HLA-A*01:01 allele, an HLA-A*68:01 allele, an HLA-DRB3*02:02 allele, and an HLA-DRB4*01:01 allele; (b) a recombinant polynucleotide encoding the HLA-A*02:01 allele, the HLA-A*11:01 allele, the HLA-DRB4*01:01 allele, and the HLA-DRB3*03:01 allele; (c) a recombinant polynucleotide encoding the HLA-A*03:01 allele, the HLA-A*23:01 allele, the HLA-DRB3*01:01 allele, and the HLA-DRB5*01:02 allele; or (d) a recombinant polynucleotide encoding the HLA-A*33:03 allele, the HLA-DRB5*01:01 allele, and the HLA-DRB5*02:02 allele. Including, one or more HLA alleles endogenous to the cell are inactivated, 21. The modified human cancer cell of claim 20.

22. 22. The modified human cancer cell of claim 20 or 21, wherein (d) further comprises an HLA-A*24:02 allele.

23. 23. The modified human cancer cell of any one of claims 18 to 22, further comprising a recombinant polynucleotide encoding a cytokine.

24. 24. The modified human cancer cell of claim 23, wherein the cytokine comprises a chemokine, an interferon, an interleukin, or a tumor necrosis factor.

25. The recombinant polynucleotide encoding the cytokine is an early T cell activation antigen-1 (ATA-1). antigen-1) (ETA-1), lymphocyte-activating factor (LAF), interleukin-1 family members (IL-1α, IL-β, IL-1Ra, IL-18, IL-33, IL-36Ra, IL-36α, IL-36β, IL-36γ, IL-37, IL-38), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-2 25. The modified human cancer cell of claim 24, comprising a recombinant polynucleotide encoding interferon-1 (IL-21), interleukin-23 (IL-23), interleukin-25 (IL-25), interleukin-33 (IL-33), interferon alpha (IFN-α), interferon lambda 1 (IFNλ1(IL-29)), interferon lambda 2 (IFNλ2(IL-28A)), interferon lambda 3 (IFNλ1(IL-28B)), interferon lambda 4 (IFNλ4), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage CSF (CSF-1), macrophage migration inhibitory factor (MIF), CD40L molecule (CD40L), RANTES molecule (RANTES), monocyte chemotactic protein (MCP-1), monocyte inflammatory proteins (MIP-1α, MIP-1β), lymphotactin, or fractalkine.

26. 26. The modified human cancer cell of any one of claims 23 to 25, wherein the cytokine comprises granulocyte-macrophage colony-stimulating factor (GM-CSF).

27. 27. The modified human cancer cell of any one of claims 23-26, wherein the cytokine comprises a recombinant polynucleotide encoding interferon alpha (IFN-α), interferon lambda 1 (IFNλ1 (IL-29)), interferon lambda 2 (IFNλ2 (IL-28A)), interferon lambda 3 (IFNλ1 (IL-28B)), or interferon lambda 4 (IFNλ4).

28. 28. The modified human cancer cell of any one of claims 1 to 27, further comprising a recombinant polynucleotide encoding a costimulatory molecule.

29. 29. The modified human cancer cell of claim 28, wherein the recombinant polynucleotide encoding the costimulatory molecule comprises a recombinant polynucleotide encoding a CD86 molecule (CD86), a CD80 molecule (CD80), a 4-1BB ligand molecule (4-1BBL, also known as CD137L), an ICOS ligand molecule (ICOS-L), a CD70 molecule (CD70, also known as CD27L), a CD40 molecule (CD40), an OX40 ligand molecule (OX40L), a GITR ligand molecule (GITRL), a TIM-4 molecule (TIM-4), a LIGHT molecule (LIGHT), an ICAM1 molecule (ICAM1), an LFA3 molecule (LFA3), a CD30 molecule (CD30), or a combination thereof.

30. 30. The modified human cancer cell of any one of claims 1-29, further comprising a recombinant polynucleotide encoding an antigen of a pathogen, a tumor-associated antigen, a neoantigen, an allergen, an antigen that is the target of an autoimmune response, or a fragment thereof.

31. 31. The modified human cancer cell of any one of claims 1 to 30, wherein one or more HLA-A and / or HLA-DR alleles endogenous to the cell are inactivated.

32. 32. The modified human cancer cell of claim 31 , wherein the one or more HLA-A alleles comprise an HLA-A*24:02 allele and / or an HLA-A*11:01 allele.

33. 33. The modified human cancer cell of claim 31 or 32, wherein the one or more HLA-DR alleles comprise one or more HLA-DRB3 alleles.

34. The modified human cancer cell of claim 33, wherein the one or more HLA-DRB3 alleles include an HLA-DRB3*01:01 allele and / or an HLA-DRB3*02:02 allele.

35. 35. The modified human cancer cell of any one of Claims 1-34, wherein one or more of said recombinant polynucleotides are present in a vector within said cell.

36. The modified human cancer cell of claim 35, wherein the vector comprises two recombinant polynucleotides each encoding an allele of an HLA class I gene.

37. The modified human cancer cell of claim 35, wherein the vector comprises two recombinant polynucleotides each encoding an allele of an HLA class II gene.

38. The modified human cancer cell of claim 35, wherein the vector comprises two recombinant polynucleotides each encoding an allele of an HLA class I gene and two recombinant polynucleotides each encoding an allele of an HLA class II gene.

39. 36. The modified human cancer cell of any one of claims 1 to 35, wherein the one or more recombinant polynucleotides encoding each allele of an HLA class I gene and the one or more recombinant polynucleotides encoding each allele of an HLA class II gene are present in the same vector within the cell.

40. 36. The modified human cancer cell of any one of claims 1-35, wherein the one or more recombinant polynucleotides encoding each allele of an HLA class I gene and the one or more recombinant polynucleotides encoding each allele of an HLA class II gene are present on separate vectors within the cell.

41. 36. The modified human cancer cell of any one of claims 1-35, wherein the one or more recombinant polynucleotides encoding each of the two alleles of an HLA class I gene and the one or more recombinant polynucleotides encoding each of the two alleles of an HLA class II gene are present on separate vectors within the cell.

42. The modified human cancer cell of any one of claims 1-41, wherein one or more of said recombinant polynucleotides comprises a sequence having at least 85% identity to any one of SEQ ID NOs: 22-35.

43. The modified human cancer cell of any one of claims 1 to 42, wherein one or more of said recombinant polynucleotides comprises the sequence of any one of SEQ ID NOs: 22 to 35.

44. 44. The modified human cancer cell of any one of claims 1 to 43, wherein the human cancer cell is a human cancer cell line.

45. 45. The modified human cancer cell of claim 44, wherein the human cancer cell line is a breast cancer, prostate cancer, melanoma, or lung cancer cell line.

46. 46. ​​The modified human cancer cell of any one of claims 1 to 45, further comprising an inactivated CD47 molecule.

47. 47. The modified human cancer cell of any one of claims 1 to 46, further comprising an inactivated Ii (CD74) molecule.

48. 48. The modified human cancer cell of any one of Claims 1-47, wherein said one or more recombinant polynucleotides each comprise a heterologous polynucleotide sequence between a polynucleotide sequence encoding a first HLA allele and a polynucleotide sequence encoding a second HLA allele, wherein the heterologous polynucleotide sequence encodes one or more self-cleaving peptides.

49. 49. The modified human cancer cell of claim 48, wherein the self-cleaving peptide is positioned at the 3' end of the first HLA allele and at the 5' end of the second HLA allele.

50. 50. The modified human cancer cell of any one of claims 1-49, wherein the self-cleaving peptide comprises T2A, P2A, E2A, or a combination thereof.

51. (a) one or more vectors, each comprising a recombinant polynucleotide encoding at least one gene selected from GM-CSF, IFN-α, CD86, IL-12, CD80, HLA-DRA, IL-7, and 4-1BBL; (b) one or more vectors each comprising a recombinant polynucleotide encoding at least one HLA-A allele selected from the HLA-A*01:01 allele, the HLA-A*68:01 allele, the HLA-A*02:01 allele, the HLA-A*11:01 allele, the HLA-A*03:01 allele, the HLA-A*23:01 allele, the HLA-A*24:02, and the HLA-A*33:03 allele; and (c) one or more vectors each comprising a recombinant polynucleotide encoding at least one HLA-DR allele selected from HLA-DRB3*02:02, HLA-DRB5*01:01, HLA-DRB4*01:01, HLA-DRB3*01:01, HLA-DRB3*03:01, HLA-DRB5*01:02, and / or HLA-DRB5*02:

02. A modified human cancer cell comprising: one or more HLA alleles endogenous to the cell are inactivated; Engineered human cancer cells.

52. (b) comprises a vector comprising a recombinant polynucleotide encoding the HLA-A*01:01 allele and the HLA-A*68:01 allele, or the HLA-A*02:01 allele and the HLA-A*11:01 allele, or the HLA-A*03:01 allele and the HLA-A*23:01 allele, or the HLA-A*24:02 allele and the HLA-A*33:03 allele; and (c) comprises a vector comprising a recombinant polynucleotide encoding the HLA-DRB3*02:02 allele and the HLA-DRB5*01:01 allele, or the HLA-DRB4*01:01 allele and the HLA-DRB3*01:01 allele, or the HLA-DRB3*03:01 allele and the HLA-DRB5*01:02 allele, or the HLA-DRB5*02:02 allele and the HLA-DRB3*01:01 allele, 52. The modified human cancer cell of claim 51.

53. (a) comprises four vectors, each of which comprises a recombinant polynucleotide encoding two different genes selected from GM-CSF, IFN-α, CD86, IL-12, CD80, HLA-DRA, IL-7, and 4-1BBL; 53. The modified human cancer cell of claim 51 or 52.

54. 54. The modified human cancer cell of any one of claims 51 to 53, wherein one or more HLA-A and / or HLA-DR alleles endogenous to the cell are inactivated.

55. 55. The modified human cancer cell of claim 54, wherein the one or more inactivated endogenous HLA-A alleles comprise an HLA-A*24:02 allele and / or an HLA-A*11:01 allele.

56. 56. The modified human cancer cell of claim 54 or 55, wherein the one or more inactivated endogenous HLA-DR alleles comprise one or more HLA-DRB3 alleles.

57. 57. The modified human cancer cell of claim 56, wherein the one or more inactivated endogenous HLA-DRB3 alleles comprise an HLA-DRB3*01:01 allele and / or an HLA-DRB3*02:02 allele.

58. The modified human cancer cell of any one of claims 51-57, wherein one or more of said recombinant polynucleotides comprises a sequence having at least 85% identity to any one of SEQ ID NOs: 22-44.

59. The modified human cancer cell of any one of claims 51-58, wherein one or more of said recombinant polynucleotides comprises the sequence of any one of SEQ ID NOs: 22-44.

60. 60. The modified human cancer cell of any one of claims 51 to 59, wherein the human cancer cell is a human cancer cell line.

61. 61. The modified human cancer cell of claim 60, wherein the human cancer cell line is a breast cancer, prostate cancer, melanoma, or lung cancer cell line.

62. 62. The replication-incompetent modified human cancer cell of any one of claims 1 to 61.

63. 63. The replication-incompetent modified human cancer cell of claim 62, wherein the modified human cancer cell has been rendered replication-incompetent by irradiation, freeze-thawing, or mitomycin C treatment.

64. 64. A composition comprising the modified human cancer cell of any one of claims 1 to 63.

65. 65. The composition of claim 64, comprising at least 1,000,000 cells.

66. 66. A pharmaceutical composition comprising the composition of claim 64 or 65 and a pharma- ceutically acceptable carrier.

67. 67. The pharmaceutical composition of claim 66, further comprising a cryoprotectant.

68. 68. A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 66 or 67.

69. 69. The method of claim 68, wherein the pharmaceutical composition is administered weekly.

70. The method of any one of claims 68 to 69, wherein the pharmaceutical composition is administered in a dose of at least 1,000,000 cells.

71. The method of any one of claims 68-70, wherein the pharmaceutical composition is administered intradermally to the upper back or thigh of the subject.

72. 72. The method of any one of claims 68-71, further comprising administering to said subject a therapeutically effective amount of cyclophosphamide at least 2 days prior to said administering of said pharmaceutical composition.

73. The method of any one of claims 68 to 72, further comprising administering to said subject a therapeutically effective amount of IFN-α2b at least one hour and at least one day after said administration of said pharmaceutical composition.

74. 68. A kit for treating a subject having cancer comprising the pharmaceutical composition of claim 66 or 67.

75. 75. The kit of claim 74, further comprising a therapeutically effective amount of IFN-α2b.

76. (a) a recombinant polynucleotide encoding an HLA-A*02:01 allele, an HLA-A*11:01 allele, an HLA-DRB4*01:01 allele, and an HLA-DRB3*03:01 allele; (b) a recombinant polynucleotide encoding the HLA-A*01:01 allele, the HLA-A*68:01 allele, the HLA-DRB3*02:02 allele, and the HLA-DRB4*01:01 allele; (c) a recombinant polynucleotide encoding the HLA-A*03:01 allele, the HLA-A*23:01 allele, the HLA-DRB3*01:01 allele, and the HLA-DRB5*01:02 allele; or (d) a recombinant polynucleotide encoding the HLA-A*33:03 allele, the HLA-DRB5*01:01 allele, and the HLA-DRB5*02:02 allele. A vector comprising: Each recombinant polynucleotide in (a) to (d) further comprises a polynucleotide encoding a self-cleaving peptide between two HLA alleles; The vector.

77. 77. The vector of claim 76, wherein (d) further comprises an HLA-A*24:02 allele.

78. 78. The vector of claim 76 or 77, wherein the polynucleotide encoding the self-cleaving peptide is positioned at the 3' end of a first HLA allele and at the 5' end of a second HLA allele.

79. 79. The vector of any one of claims 76-78, wherein the self-cleaving peptide comprises T2A, P2A, E2A, or a combination thereof.

80. The vector of any one of claims 76 to 79, wherein the recombinant polynucleotide comprises at least one of the sequences having at least 85% identity to any one of SEQ ID NOs: 22 to 35.

81. The vector of any one of claims 76 to 80, wherein the recombinant polynucleotide comprises at least one of any one of SEQ ID NOs: 22 to 35.

82. The vector of any one of claims 76-81, wherein the recombinant polynucleotide comprises the MNDU3 promoter.

83. 83. The vector of any one of claims 76 to 82, further comprising said recombinant polynucleotide encoding said cytokine.

84. 84. The vector of claim 83, wherein the cytokine comprises a chemokine, an interferon, an interleukin, or a tumor necrosis factor.

85. The recombinant polynucleotide encoding the cytokine is selected from the group consisting of early T-cell activation antigen-1 (ETA-1), lymphocyte-activating factor (LAF), interleukin-1 family members (IL-1α, IL-β, IL-1Ra, IL-18, IL-33, IL-36Ra, IL-36α, IL-36β, IL-36γ, IL-37, IL-38), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interleukin-17 (IL-17), interleukin-18 (IL-19), interleukin-20 (IL-21), interleukin-22 (IL-22), interleukin-23 (IL-23), interleukin-24 (IL-25), interleukin-26 (IL-27), interleukin-28 (IL-29), interleukin-30 (IL-30), interleukin-31 (IL-31), interleukin-32 (IL-32), interleukin-33 (IL-33), interleukin-34 (IL-34), interleukin-35 (IL-35), interleukin-36 (IL-36), interleukin-37 (IL-37), interleukin-38 (IL-38), interleukin-39 (IL-39), interleukin-40 (IL-40), interleukin-41 (IL 85. The vector of claim 84, comprising a recombinant polynucleotide encoding an IL-18), interleukin-23 (IL-21), interleukin-23 (IL-23), interleukin-25 (IL-25), interleukin-33 (IL-33), interferon alpha (IFN-α), interferon lambda 1 (IFNλ1(IL-29)), interferon lambda 2 (IFNλ2(IL-28A)), interferon lambda 3 (IFNλ1(IL-28B)), interferon lambda 4 (IFNλ4), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage CSF (CSF-1), macrophage migration inhibitory factor (MIF), CD40L molecule (CD40L), RANTES molecule (RANTES), monocyte chemotactic protein (MCP-1), monocyte inflammatory proteins (MIP-1α, MIP-1β), lymphotactin, or fractalkine.

86. The vector of any one of claims 76 to 85, wherein the cytokine comprises granulocyte-macrophage colony-stimulating factor (GM-CSF).

87. 87. The vector of any one of claims 76-86, wherein the cytokine comprises interferon alpha (IFN-α), interferon lambda 1 (IFNλ1 (IL-29)), interferon lambda 2 (IFNλ2 (IL-28A)), interferon lambda 3 (IFNλ1 (IL-28B)), or interferon lambda 4 (IFNλ4).

88. The vector of claim 87, further comprising a recombinant polynucleotide encoding a costimulatory molecule.

89. 89. The vector of claim 88, wherein the costimulatory molecule comprises a CD86 molecule (CD86), a CD80 molecule (CD80), a 4-1BB ligand molecule (4-1BBL, also known as CD137L), an ICOS ligand molecule (ICOS-L), a CD70 molecule (CD70, also known as CD27L), a CD40 molecule (CD40), an OX40 ligand molecule (OX40L), a GITR ligand molecule (GITRL), a TIM-4 molecule (TIM-4), a LIGHT molecule (LIGHT), an ICAM1 molecule (ICAM1), an LFA3 molecule (LFA3), a CD30 molecule (CD30), or a combination thereof.

90. The vector of any one of claims 76-89, further comprising a recombinant polynucleotide encoding an antigen of a pathogen, a tumor-associated antigen, a neoantigen, an allergen, an antigen that is the target of an autoimmune response, or a fragment thereof.

91. A recombinant polynucleotide comprising a sequence encoding an allele of an HLA class I gene having at least 85% identity to any one of SEQ ID NOs: 22-29, and optionally at least one heterologous polynucleotide sequence.

92. The recombinant polynucleotide of claim 91, comprising the sequence of any one of SEQ ID NOs: 22-29.

93. 93. The recombinant polynucleotide of claim 91 or 92, further comprising a recombinant polynucleotide encoding a second allele of the HLA class I gene or an allele of a second HLA class I gene.

94. The recombinant polynucleotide of any one of claims 91 to 93, further comprising a recombinant polynucleotide encoding an allele of an HLA class II gene.

95. 95. The recombinant polynucleotide of claim 94, wherein said recombinant polynucleotide encoding an allele of said HLA class II gene has at least 85% identity to any one of SEQ ID NOs: 30-35.

96. The recombinant polynucleotide of claim 95, wherein said recombinant polynucleotide encoding an allele of said HLA class II gene comprises the sequence of any one of SEQ ID NOs: 30-35.

97. 97. The recombinant polynucleotide of any one of claims 91 to 96, wherein said heterologous polynucleotide sequence encodes an amino acid sequence of a cytokine.

98. 98. The recombinant polynucleotide of claim 97, wherein said cytokine comprises a chemokine, an interferon, an interleukin, or a tumor necrosis factor.

99. The heterologous polynucleotide sequence encoding the cytokine is selected from the group consisting of early T cell activation antigen-1 (ETA-1), lymphocyte activating factor (LAF), interleukin-1 family members (IL-1α, IL-β, IL-1Ra, IL-18, IL-33, IL-36Ra, IL-36α, IL-36β, IL-36γ, IL-37, IL-38), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-5), interleukin-5 (IL-6), interleukin-6 (IL-7), interleukin-7 (IL-8), interleukin-8 (IL-9), interleukin-9 (IL-10), interleukin-11 (IL-12), interleukin-12 (IL-13), interleukin-13 (IL-14), interleukin-15 (IL-15), interleukin-16 (IL-17), interleukin-17 (IL-18), interleukin-17 (IL-19), interleukin-18 (IL-20), interleukin-19 (IL-21), interleukin-19 (IL-22), interleukin-19 (IL-23), interleukin-19 (IL-24), interleukin-19 (IL-25), interleukin-19 (IL-26), interleukin-19 (IL-27), interleukin-19 (IL-28), interleukin-19 (IL-29), interleukin-20 (IL-29), interleukin-20 (IL-20), interleukin-20 (IL-21), interleukin-20 (IL-22), interleukin-20 (IL-23), interleukin-20 (IL-24), interleukin-2 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interleukin-17 (IL-17), interleukin-18 (IL- 18), interleukin-21 (IL-21), interleukin-23 (IL-23), interleukin-25 (IL-25), interleukin-33 (IL-33), interferon alpha (IFN-α), interferon lambda 1 (IFNλ1(IL-29)), interferon lambda 2 (IFNλ2(IL-28A)), interferon lambda 3 (IFNλ1(IL-28B)), interferon lambda 4 (IFNλ4), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage CSF (CSF-1), macrophage migration inhibitory factor (MIF), CD40L molecule (CD40L), RANTES molecule (RANTES), monocyte chemotactic protein (MCP-1), monocyte inflammatory proteins (MIP-1α, MIP-1β), lymphotactin, or fractalkine.

100. 100. The recombinant polynucleotide of any one of claims 97-99, wherein said cytokine comprises granulocyte-macrophage colony-stimulating factor (GM-CSF).

101. 101. The recombinant polynucleotide of any one of claims 97-100, wherein said cytokine comprises interferon alpha (IFN-α), interferon lambda 1 (IFNλ1 (IL-29)), interferon lambda 2 (IFNλ2 (IL-28A)), interferon lambda 3 (IFNλ1 (IL-28B)), interferon lambda 4 (IFNλ4).

102. The recombinant polynucleotide of any one of claims 91-101, wherein said heterologous polynucleotide sequence encodes one or more self-cleaving peptides between two HLA alleles.

103. 103. The recombinant polynucleotide of claim 102, wherein the self-cleaving peptide is located at the 3' end of a first HLA allele and at the 5' end of a second HLA allele.

104. 104. The recombinant polynucleotide of claim 102 or 103, wherein the self-cleaving peptide comprises T2A, P2A, E2A, or a combination thereof.

105. The recombinant polynucleotide of any one of claims 91 to 104, wherein the heterologous polynucleotide sequence comprises a promoter.

106. The recombinant polynucleotide of claim 105, wherein the promoter comprises the MNDU3 promoter or the EF1α promoter.

107. The recombinant polynucleotide of any one of claims 91-106, wherein said heterologous polynucleotide sequence encodes an amino acid sequence of a costimulatory molecule.

108. 108. The recombinant polynucleotide of claim 107, wherein the costimulatory molecule comprises a CD86 molecule (CD86), a CD80 molecule (CD80), a 4-1BB ligand molecule (4-1BBL, also known as CD137L), an ICOS ligand molecule (ICOS-L), a CD70 molecule (CD70, also known as CD27L), a CD40 molecule (CD40), an OX40 ligand molecule (OX40L), a GITR ligand molecule (GITRL), a TIM-4 molecule (TIM-4), a LIGHT molecule (LIGHT), an ICAM1 molecule (ICAM1), an LFA3 molecule (LFA3), a CD30 molecule (CD30), or a combination thereof.

109. The recombinant polynucleotide of any one of claims 91-108, wherein the heterologous polynucleotide sequence comprises a sequence having at least 85% identity to any one of SEQ ID NOs: 36-44.

110. 110. The recombinant polynucleotide of claim 109, wherein said heterologous polynucleotide sequence comprises any one of SEQ ID NOs: 36-44.

111. The recombinant polynucleotide of any one of claims 91 to 110, wherein said heterologous polynucleotide sequence encodes an amino acid sequence of an antigen of a pathogen, a tumor-associated antigen, a neoantigen, an allergen, an antigen that is the target of an autoimmune response, or a fragment thereof.

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