阅读说明：本技术 Hla限制性vcx/y肽和t细胞受体及其用途 (HLA-restricted VCX/Y peptides and T cell receptors and uses thereof ) 是由 余嘉诚 潘科 于 2017-12-28 设计创作，主要内容包括：本文中提供了肿瘤抗原VCX/Y特异性肽和经改造的VCX/Y特异性T细胞受体。本文中还提供了产生VCX/Y特异性免疫细胞的方法及其用于治疗癌症的用途。另外,所述VCX/Y特异性肽可用作疫苗。(Provided herein are tumor antigen VCX/Y specific peptides and engineered VCX/Y specific T cell receptors. Also provided herein are methods of producing VCX/Y specific immune cells and their use for treating cancer. In addition, the VCX/Y specific peptides can be used as vaccines.)

1. An isolated VCX/Y peptide 35 amino acids or less in length comprising a sequence that is identical to SEQ ID NO: 1. 8, 9,12, 13, or 14, wherein the peptide is capable of inducing Cytotoxic T Lymphocytes (CTL) and selectively binding to HLA-a 2.

2. The peptide of claim 1, wherein the HLA-a2 is HLA-a x 0201.

3. The peptide of claim 1, wherein the peptide comprises a sequence identical to SEQ ID NO: 1. 8, 9,12, 13, or 14, having at least 95% sequence identity.

4. The peptide of claim 1, wherein the peptide is 30 amino acids or less in length.

5. The peptide of claim 3, wherein the peptide is 25 amino acids or less in length.

6. The peptide of claim 5, wherein the peptide is 20 amino acids or less in length.

7. The peptide of claim 4, wherein the peptide is 15 amino acids or less in length.

8. The peptide of claim 1, wherein the peptide consists of SEQ ID NO: 1. 8, 9,12, 13, or 14.

9. A pharmaceutical composition comprising the isolated peptide of any one of claims 1 to 8, and a pharmaceutical carrier.

10. The composition of claim 9, wherein the pharmaceutical composition is formulated for parenteral administration, intravenous injection, intramuscular injection, inhalation, or subcutaneous injection.

11. The composition of claim 9, wherein the peptide is comprised in a liposome, a lipid-containing nanoparticle, or in a lipid-based carrier.

12. The composition of claim 9, wherein the pharmaceutical formulation is formulated for injection or inhalation as a nasal spray.

13. An isolated nucleic acid encoding the VCX/Y peptide of any one of claims 1 to 8.

14. A vector comprising a contiguous sequence consisting of the nucleic acid of claim 13.

15. A method of promoting an immune response in a subject, comprising administering to the subject an effective amount of the peptide of any one of claims 1 to 8, wherein the peptide induces VCX/Y specific T cells in the subject.

16. The method of claim 15, wherein the subject is diagnosed with cancer.

17. The method of claim 16, wherein the cancer is a thymus tumor, bladder cancer, uterine cancer, melanoma, sarcoma, cervical cancer, or head and neck cancer.

18. The method of claim 15, wherein the subject is a human.

19. The method of claim 15, further comprising administering at least a second anti-cancer therapy.

20. The method of claim 19, wherein the second anticancer therapy is selected from chemotherapy, radiation therapy, immunotherapy, or surgery.

21. The method of claim 20, wherein the immunotherapy is an immune checkpoint inhibitor.

22. The method of claim 21, wherein the immune checkpoint inhibitor is an anti-PD 1 monoclonal antibody.

23. A method of producing a VCX/Y specific T cell, comprising:

(a) obtaining a starting T cell population; and

(b) contacting the starting T cell population with the VCX/Y peptide of claim 1, thereby generating VCX/Y-specific T cells.

24. The method of claim 23, wherein contacting is further defined as co-culturing the starting T cell population with an Antigen Presenting Cell (APC), wherein the APC presents the VCX/Y peptide of claim 1 on its surface.

25. The method of claim 24, wherein the APC is a dendritic cell.

26. The method of claim 23, wherein the starting T cell population is CD8⁺T is thinCell or CD4⁺T cells.

27. The method of claim 23, wherein the T cell is a Cytotoxic T Lymphocyte (CTL).

28. The method of claim 23, wherein obtaining comprises isolating the starting T cell population from Peripheral Blood Mononuclear Cells (PBMCs).

29. A VCX/Y specific T cell produced according to any one of claims 23 to 28.

30. A pharmaceutical composition comprising the VCX/Y-specific T cells produced according to any one of claims 23 to 28.

31. An engineered T Cell Receptor (TCR) comprising the amino acid sequence of SEQ ID NO: 2 and the alpha chain CDR3 of SEQ ID NO: 3, or the beta chain CDR3 of SEQ ID NO: 19 and the α chain CDR3 of SEQ ID NO: 20 beta chain CDR 3.

32. The TCR of claim 31, wherein the engineered TCR binds HLA-a 2.

33. The TCR of claim 31, wherein the engineered TCR binds HLA-a 0201.

34. The TCR of claim 31, wherein the TCR comprises a sequence identical to SEQ ID NO: 5 or 16 and/or an alpha chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 7 or 18, or a beta strand having at least 90% identity to the amino acid sequence of said variant.

35. The TCR of claim 31, wherein the TCR comprises a sequence identical to SEQ ID NO: 5 or 16 and/or an alpha chain having at least 95% identity to the amino acid sequence of SEQ ID NO: 7 or 18 has at least 95% identity to the beta strand of the amino acid sequence.

36. The TCR of claim 31, wherein the TCR comprises a sequence identical to SEQ ID NO: 5 or 16 and/or an alpha chain having at least 99% identity to the amino acid sequence of SEQ ID NO: 7 or 18, or a beta strand having at least 99% identity to the amino acid sequence of said polypeptide.

37. The TCR of claim 31, wherein the TCR comprises SEQ ID NO: 5 or 16 and/or the alpha chain of SEQ ID NO: 7 or 18.

38. The TCR of claim 31, wherein the TCR is further defined as a soluble TCR, wherein the soluble TCR does not comprise a transmembrane domain.

39. The TCR of any one of claims 31-38, further comprising a detectable label.

40. The TCR of any one of claims 31-38, further comprising a therapeutic agent.

41. A multivalent TCR complex comprising a plurality of TCRs according to any one of claims 31 to 38.

42. The complex of claim 41, wherein the multivalent TCR comprises 2, 3, 4, or more TCRs associated with one another.

43. The complex of claim 42, wherein the multivalent TCR is present in a lipid bilayer or attached to a particle.

44. The complex of claim 42, wherein the TCRs are associated with each other by a linker molecule.

45. A polypeptide encoding a TCR as claimed in any one of claims 31 to 38.

46. A polynucleotide encoding the polypeptide of claim 45.

47. An expression vector comprising a TCR according to any one of claims 31 to 38.

48. The expression vector of claim 47, wherein the expression vector is a viral vector.

49. The expression vector of claim 48, wherein the viral vector is a retroviral vector.

50. The expression vector of claim 47, further comprising a linker domain.

51. The expression vector of claim 50, wherein the linker domain is located between the alpha and beta chains.

52. The expression vector of claim 50, wherein the linker domain comprises one or more cleavage sites.

53. The expression vector of claim 52, wherein the one or more cleavage sites are furin cleavage sites and/or P2A cleavage sites.

54. The expression vector of claim 50, wherein the one or more cleavage sites are separated by a spacer.

55. The expression vector of claim 54, wherein the spacer is SGSG or GSG.

56. A host cell engineered to express a TCR as claimed in any one of claims 31 to 38.

57. The host cell of claim 56, wherein the cell is an immune cell.

58. The host cell of claim 56, wherein the cell is an NK cell, a constant NK cell, an NKT cell, a Mesenchymal Stem Cell (MSC), or an Induced Pluripotent Stem (iPS) cell.

59. The host cell of claim 56, wherein the cell is isolated from umbilical cord.

60. The host cell of claim 56, wherein the immune cell is a T cell or a peripheral blood lymphocyte.

61. The host cell of claim 60, wherein the T cell is CD8⁺T cells, CD4+ T cells, or γ δ T cells.

62. The host cell of claim 60, wherein the T cell is a regulatory T cell (Treg).

63. The host cell of claim 56, wherein the cell is allogeneic or autologous.

64. A method for engineering the immune cell of claim 56, comprising contacting the immune cell with the TCR of claim 31 or the expression vector of claim 47.

65. The method of claim 64, wherein the immune cell is a T cell or a peripheral blood lymphocyte.

66. The method of claim 64, wherein contacting is further defined as transfecting or transducing.

67. The method of claim 66, wherein transfecting comprises electroporating RNA encoding the TCR of claim 31 into the immune cell.

68. The method of claim 66, further comprising producing a viral supernatant from the expression vector of claim 47 prior to transducing the immune cells.

69. The method of claim 67 or claim 68, wherein the immune cell is a stimulated lymphocyte.

70. The method of claim 69, wherein the stimulated lymphocytes are human lymphocytes.

71. The method of claim 69, wherein stimulating comprises OKT3 and/or IL-2.

72. The method of claim 64, further comprising sorting the immune cells to isolate TCR-engineered T cells.

73. The method of claim 72, further comprising T cell cloning by serial dilution.

74. The method of claim 73, further comprising expanding the T cell clone by a rapid expansion protocol.

75. A method of treating cancer in a subject, comprising administering to the subject an effective amount of the VCX/Y specific T cell of claim 29 or the TCR-engineered host cell of claim 56.

76. A composition comprising an effective amount of the VCX/Y specific T cell of claim 29 or the TCR engineered host cell of claim 56 for use in the treatment of cancer in a subject.

77. The method of claim 75, wherein the subject is identified as having an HLA-A0201 allele.

78. The method of claim 75, wherein the host cell is a T cell, a peripheral blood lymphocyte, an NK cell, a constant NK cell, an NKT cell, a Mesenchymal Stem Cell (MSC), or an Induced Pluripotent Stem (iPS) cell.

79. The method of claim 75, wherein the host cell is isolated from umbilical cord.

80. The method of claim 75, wherein the host cell is autologous or allogeneic.

81. The method of claim 75, wherein the T cell is CD8⁺T cell, CD4⁺T cells or γ δ T cells.

82. The method of claim 75, wherein the cancer is a thymus tumor, bladder cancer, uterine cancer, melanoma, sarcoma, cervical cancer, or head and neck cancer.

83. The method of claim 75, wherein the subject is a human.

84. The method of claim 75, wherein the VCX/Y specific T cells are autologous or allogeneic.

85. The method of claim 75, further comprising depleting lymphocytes from the subject prior to administering the VCX/Y specific T cells.

86. The method of claim 85, wherein lymphocyte depletion comprises administration of cyclophosphamide and/or fludarabine.

87. The method of claim 75, further comprising administering at least a second therapeutic agent.

88. The method of claim 87, wherein the at least second therapeutic agent comprises chemotherapy, immunotherapy, surgery, radiation therapy, or biological therapy.

89. The method of claim 87, wherein the VCX/Y specific T cells, TCR engineered immune cells and/or at least a second therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, transdermally, subcutaneously, regionally or by direct injection or infusion.

90. The method of claim 75, wherein the subject is determined to have cancer cells that express the VCX/Y family protein.

91. The method of claim 90, wherein the protein is VCX1, VCX2, VCX3A, VCX3B, or VCY.

1. Field of the invention

The present invention relates generally to the fields of immunology and medicine. More particularly, it relates to tumor antigen peptides and their use for the treatment of cancer.

Background

Brief description of the invention

In some embodiments, the disclosure provides VCX/Y (e.g., VCX1, VCX2, VCX3A, VCX3B, and VCY) peptides that are useful in adoptive T cell therapy. In some embodiments, the peptides can be used to expand VCX/Y specific T cells in vitro for administration to a mammalian subject (e.g., a human patient) to treat a disease (e.g., cancer). In further embodiments, the T cell is genetically engineered to express a T Cell Receptor (TCR) with antigenic specificity for VCX/Y. In other embodiments, the peptide may be administered to a mammalian subject to induce an immune response against the peptide or to immunize the subject against the peptide, and such an immune response may be used to treat a disease (e.g., cancer) or reduce the chance of acquiring or relapsing.

In one embodiment, the present disclosure provides an isolated VCX/Y peptide 35 amino acids or less in length comprising a sequence identical to SEQ ID NO: 1(GAATKMAAV), 8(KVAKKGKAV), 9(SEMEELPSV), 12(KVAEKGEAV), 13(KMAAVEAPEA), or 14(MAAVEAPEA), wherein the peptide selectively binds HLA-a 2. In some aspects, the peptide comprises a sequence identical to SEQ ID NO: 1. 8, 9,12, 13, or 14, or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In a particular aspect, the human HLA class I-a 2 protein is HLA-a x 0201.

In certain aspects, the peptide is 30 amino acids or less in length, e.g., 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 amino acids in length.

In another embodiment, pharmaceutical compositions are provided comprising the isolated VCX/Y peptides of some embodiments, and a pharmaceutical carrier. In some aspects, the pharmaceutical composition is formulated for parenteral administration, intravenous injection, intramuscular injection, inhalation, or subcutaneous injection. In certain aspects, the peptide is contained in a liposome, nanoparticle (e.g., a lipid-containing nanoparticle), or lipid-based carrier. In some aspects, the pharmaceutical formulation is formulated for injection or inhalation as a nasal spray.

Another embodiment provides an isolated nucleic acid encoding the VCX/Y peptide of some embodiments. Also provided herein are vectors comprising a contiguous sequence consisting of a nucleic acid encoding a VCX/Y peptide.

In another embodiment, there is provided a method of promoting an immune response in a subject, comprising administering to the subject an effective amount of the VCX/Y peptide of some embodiments, wherein the peptide induces antigen-specific T cells in the subject. In some aspects, the subject is diagnosed with cancer. In certain aspects, the cancer is pancreatic cancer, ovarian cancer, gastric cancer, or breast cancer. In a particular aspect, the subject is a human.

In other aspects, the method further comprises administering at least a second anti-cancer therapy. In some aspects, the second anticancer therapy is selected from chemotherapy, radiation therapy, immunotherapy, or surgery. In a particular aspect, the immunotherapy is an immune checkpoint inhibitor. In a particular aspect, the immune checkpoint inhibitor is an anti-PD 1 monoclonal antibody.

Another embodiment provides a method of producing a VCX/Y-specific T cell, comprising obtaining a starting T cell population, and contacting the starting T cell population with a VCX/Y peptide of some embodiments, thereby producing a VCX/Y-specific T cell. In some aspects, contacting is further defined as co-culturing the starting T cell population with Antigen Presenting Cells (APCs), wherein the APCs present some embodiments of the VCX/Y peptide on their surface. In a particular aspect, the APC is a dendritic cell. In some aspects, the starting T cell population is CD8⁺T cells. In certain aspects, the T cell is a Cytotoxic T Lymphocyte (CTL). In some aspects, obtaining comprises isolating the starting T cell population from Peripheral Blood Mononuclear Cells (PBMCs). Also provided herein are pharmaceutical compositions comprising VCX/Y-specific T cells produced by the methods herein.

Another embodiment provides an antigen receptor with antigen specificity for VCX/Y, such as a T Cell Receptor (TCR) or a Chimeric Antigen Receptor (CAR). Another embodiment provides T cells engineered to express a VCX/Y specific TCR or CAR. In certain aspects, the TCR binds to HLA-a2, e.g., HLA-a x 0201. In some aspects, the TCR comprises a sequence identical to SEQ ID NO: 2 or 19 and/or an alpha chain CDR3 having at least 95% (e.g., 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 3 or 20 has at least 95% (e.g., 96%, 97%, 98%, 99% or 100%) identity to the beta chain CDR 3. In some aspects, the TCR comprises a sequence identical to SEQ ID NO: 5 or 16 and/or an alpha chain having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 7 or 18, or a beta strand having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence of said polypeptide. In a particular aspect, the TCR comprises SEQ ID NO: 5 or 16 and/or the alpha chain of SEQ ID NO: 7 or 18.

In some aspects, the VCX/Y-specific antigen receptor comprises an intracellular signaling domain, a transmembrane domain, and/or an extracellular domain. In certain aspects, the DNA encoding the TCR or CAR is integrated into the genome of the cell. In some aspects, the extracellular domain of the TCR or CAR comprises a VCX/Y binding region. For example, the VCX/Y binding region may be F (ab ') 2, Fab', Fab, Fv or scFv. In certain aspects, the intracellular signaling domain is a T lymphocyte activation domain. For example, the intracellular signaling domain of a TCR may comprise CD3 ξ, CD3 ε, CD3 γ, CD3 δ, CD8, CD27, CD28, OX40/CD134, 4-1BB/CD137, GITR/CD357, Fc ε RI γ, ICOS/CD278, ILRB/CD122, IL-2RG/CD132, DAP molecules, CD70, cytokine receptors, CD40, or a combination thereof. In certain aspects, the transmembrane domain of the TCR comprises a CD28 transmembrane domain, an ICOS transmembrane domain, an NKG2D transmembrane domain, a DAP molecule transmembrane domain, an IgG4Fc hinge, an Fc region, a CD4 transmembrane domain, a CD3 ξ transmembrane domain, a cysteine-mutated human CD3 ξ domain, a CD16 transmembrane domain, a CD8 transmembrane domain, or an erythropoietin receptor transmembrane domain.

In some embodiments, the disclosure provides soluble TCRs, such as the VCX/Y TCRs provided herein. In some aspects, the TCR is conjugated to a detectable label or therapeutic agent. In some aspects, the soluble TCR is used to deliver a therapeutic agent (e.g., a cytotoxic compound or an immunostimulatory compound) to a cell presenting a particular antigen. In some aspects, the TCR is linked to another molecule that delivers nearby cells to the tumor. In certain aspects, the TCR delivers a toxin, cytokine, co-stimulatory ligand, or inhibitor ligand in order to direct a molecule, cell, or compound to a target cell expressing the peptide-MHC. In a particular aspect, the TCR is conjugated to anti-CD 3.

Another embodiment provides a multivalent TCR complex comprising a plurality of the VCX/Y antigen receptors of the above embodiments, such as VCX/Y TCRs. In some aspects, the multivalent TCR comprises 2, 3, 4, or more TCRs associated with each other. In certain aspects, the multivalent TCR is present in a lipid bilayer or attached to a particle.

In another embodiment, polypeptides encoding the TCRs of some embodiments are provided. Also provided are polynucleotides encoding the polypeptides and expression vectors (e.g., viral vectors, such as retroviral vectors) comprising the polynucleotides. In some aspects, the vector further comprises a linker domain. In certain aspects, the linker domain comprises one or more cleavage sites. In some aspects, the one or more cleavage sites are Furin (Furin) cleavage sites and/or P2A cleavage sites, which may be separated by a spacer (e.g., SGSG or GSG).

In another embodiment, host cells engineered to express the TCRs of some embodiments are provided. In some aspects, the cell is an immune cell. In certain aspects, the cell is an NK cell, a constant NK cell (NKcell), an NKT cell, a Mesenchymal Stem Cell (MSC), or an Induced Pluripotent Stem (iPS) cell. In some aspects, the cells are isolated from the umbilical cord. In certain aspects, the immune cell is a T cell or a peripheral blood lymphocyte. In a particular aspect, the T cell is CD8⁺T cell, CD4⁺T cells or γ δ T cells. In some aspects, the T cell is a regulatory T cell (Treg). In some aspects, the cells are allogeneic or autologous.

In another embodiment, methods for engineering an immune cell of some embodiments are provided, comprising contacting the immune cell with a TCR of some embodiments or an expression vector encoding the TCR. In some aspects, the immune cell is a T cell or a peripheral blood lymphocyte. In certain aspects, contacting is further defined as transfection or transduction. In some aspects, transfection comprises electroporating RNA encoding the TCRs of some embodiments into immune cells. In some aspects, the method further comprises producing viral supernatant from the expression vector of some embodiments prior to transducing the immune cells. In some aspects, the immune cell is a stimulated lymphocyte. In certain aspects, the stimulated lymphocytes are human lymphocytes. In some aspects, the stimulus comprises OKT3 and/or IL-2. In certain aspects, the method further comprises sorting the immune cells to isolate TCR-engineered T cells. In some aspects, the method further comprises T cell cloning by serial dilution. In certain aspects, the method further comprises expansion of the T cell clones by a rapid expansion protocol.

Another embodiment provides a method of treating cancer in a subject comprising administering to the subject an effective amount of the VCX/Y-specific T cells or TCR-engineered host cells of some embodiments. Also provided herein are compositions comprising an effective amount of the VCX/Y-specific T cells or TCR-engineered host cells of some embodiments for use in treating cancer in a subject. In some aspects, the cancer is a thymoma, bladder cancer, uterine cancer, melanoma, sarcoma, cervical cancer, or head and neck cancer. In a particular aspect, the subject is a human. In some aspects, the cells are autologous or allogeneic. In some aspects, the subject is determined to have cancer cells that express VCX/Y. In particular aspects, the subject is identified as having an HLA-a x 0201 allele.

In some aspects, the host cell is a T cell, peripheral blood lymphCells, NK cells, constant NK cells, NKT cells, Mesenchymal Stem Cells (MSC) or Induced Pluripotent Stem (iPS) cells. In certain aspects, the host cell is isolated from the umbilical cord. In some aspects, the host cell is autologous or allogeneic. In certain aspects, the T cell is CD8⁺T cell, CD4⁺T cells or γ δ T cells.

In certain aspects, the method further comprises depleting the subject of lymphocytes prior to administering the antigen-specific T cells. In some aspects, lymphocyte depletion comprises administration of cyclophosphamide and/or fludarabine.

In some aspects, the method further comprises administering at least a second therapeutic agent. In certain aspects, the at least second therapeutic agent comprises chemotherapy, immunotherapy, surgery, radiation therapy, or biological therapy. In a particular aspect, the immunotherapy is an immune checkpoint inhibitor. In a particular aspect, the immune checkpoint inhibitor is an anti-PD 1 monoclonal antibody.

In certain aspects, the VCX/Y-specific T cells and/or the at least a second therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, transdermally, subcutaneously, regionally, or by direct injection or infusion.

In some aspects, the subject is determined to have cancer cells that express a protein of the VCX/Y family. In a particular aspect, the protein is VCX1, VCX2, VCX3A, VCX3B, or VCY.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Drawings

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of some specific embodiments presented herein.

Fig. 1A to 1B: t2 cells were assayed for stable expression of HLA-A2. (A) The binding assay predicting peptides after 18 hours incubation with T2 cells showed HLA-a2 expression as fluorescent index. (B) Western blot detects the expression of VCX members (VCX1, VCX3A, and VCY) in lung cancer cell lines. H522, H2023, H1395, H82, H1355, H1755 and DFC1032 express HLA-A0201; h647 expresses HLA-A1101; PC-9 expresses HLA-A0206 and HLA-A2402.

Fig. 2A to 2B: VCX 54-specific T cell clones were generated from HLA-A0201 healthy donors. (A) CD8 and VCX54 tetramer expression of T cells after two stimulations of dendritic cells pulsed with VCX3A antigen. (B) CD8 and VCX54 tetramer expression of T cells after expansion using a Rapid Expansion Protocol (REP).

Fig. 3A to 3B: functional avidity of VCX 54-specific T cells. (A) Cytotoxic lysis of VCX54CTL clones co-cultured with T2 cells pulsed with various concentrations of VCX54 peptide at an effector to target (E: T) ratio of 20: 1. (B) Cytotoxic cleavage of VCX54CTL clone (C7) co-cultured with VCX1 positive expressing human lung cancer cell line H2023(HLA-A0201+) or primary bronchial epithelial cells NHBE (HLA-A0201+) at various E: T ratios.

FIG. 4: recognition of endogenously presented VCX54 peptide by specific T cells. Cytotoxic lysis of VCX54CTL clones co-cultured with a panel of HLA-A2+ lung cancer cell lines expressing VCX3A or VCX1 at various E: T ratios.

FIG. 5: HLA allele restriction analysis of VCX-specific T cells. Cytotoxicity of several VCX54CTL clones against HLA-a0201 negative lung cancer cell lines.

FIG. 6: specific cytotoxicity of VCX-specific T cells against lung cancer cells was demonstrated. Endogenous presented peptide specific recognition of VCX54CTL clones detected with cold target inhibition assay.

Fig. 7A to 7B: VCX54CTL clone (C7) TCR use assay. (A) The TCR α chain (TRAV) was identified using PCR. TRAV is TRAV-13.1 or TRAV-14. (B) TCR β chain (TRBV) was detected using flow cytometry. TRBV was used as TRBV-13.

FIG. 8: VCX54CTL clone (C7) TCR α chain and β chain analysis. Sequence analysis from the IMGT database showed that the VCX54CTL clone (C7) TCR was used as TRAV-14 and TRBV-13. The corresponding CDR3 amino acid sequences of TCR-. alpha.and TCR-. beta.are indicated.

FIG. 9: TCR from the VCX54CTL clone was constructed into the retroviral expression vector pMSGV 1. A linker fragment comprising a furin cleavage site, an SGSG linker and a P2A cleavage site was inserted between the TCR-. beta.chain and the TCR-. alpha.chain to ensure that both chains are expressed at equivalent levels under the MSCV promoter.

FIG. 10: the retroviral expression vector pMSGV1 containing the TCR sequences from the VCX54CTL clone and the envelope vector RD114 were co-transfected into the packaging cell line GP 2-293. CD8 and tetramer expression was shown by flow cytometry.

FIG. 11: the VCX1+/HLA-A0201 positive lung cancer cell line H2023 and HLA-A0201 positive immortalized normal human small airway epithelium HSAEC2-KT cell line were used with standards₅₁Cr Release assay (₅₁Cr release assay, CRA) 450 clones were screened. Only clones with more than 20% of cytotoxicity targeting H2023 are shown.

FIG. 12: cytotoxicity of TCR gene modified T cell clones C13 and C119 amplified using a rapid amplification protocol (REP).

Fig. 13A to 13B: tetramer staining and tetramer dissociation assay of TCR gene modified T cell clone C119. (A) Tetramer staining of parent VCX54CTL clone and TCR gene modified T cell clone C119. The tetramer staining density of C119 was compared to the parental CTL clone. (B) Tetramer dissociation detection. Half maximal binding time (T) for C119 clone_1/2) Higher than the parental VCX54CTL clone.

FIG. 14: peptide titration assay for the detection of specific response of TCR gene modified T cell clone C119.

Fig. 15A to 15B: intracellular staining assay for evaluation of specific response of TCR gene modified clone C119. The VCX54 parental CTL clone or TCR gene modified T cell clone C119 was co-cultured with the following effector to target (E: T) ratio of 10: 1: (A) HLA-A2+/VCX1+ lung cancer cell line H2023 or HLA-A0201+ immortalized normal human small airway epithelial cell line HSAEC2-KT, or (B) T2 cells pulsed with 10. mu.g/ml VCX54 peptide or control peptide M26. Intracellular TNF-alpha, CD137, IFN-gamma and IL-2 levels were detected by flow cytometry.

FIG. 16: cytotoxicity of parental CTL clone and TCR gene-modified T cell clone C119 against lung cancer cell line H2023, immortalized normal human small airway epithelial cell line HSAEC2-KT, and primary human bronchial epithelial cell NHBE.

Fig. 17A to 17C: HLA-A2 restricted peptides from the VCX/Y family. (A) HLA binding assay for peptides (HLA-A2 stabilization assay). (B) VCY-37 tetramer detection of CTL. (C) Determination of chromium release from VCY-37CTL against lung tumor or normal lung cell lines.

Detailed description of illustrative embodiments

For patients with many different cancer types, T cell-based immunotherapy represents a promising approach with demonstrated efficacy. However, due to the lack of currently known tumor associated antigens, antigen-specific T cell therapy against most cancer types is not feasible, which hinders their clinical development. The studies in this disclosure identified a novel HLA-a2 restricted peptide epitope of VCX/Y family origin found in all VCX/Y family members including VCX1, VCX2, VCX3A, VCX3B and VCY. Using the peptide epitopes, antigen-specific Cytotoxic T Lymphocytes (CTLs) are generated from patient Peripheral Blood Mononuclear Cells (PBMCs) that recognize endogenously presented antigens on HLA-matched allogeneic tumor cell lines, causing tumor cell killing. Thus, these antigen-specific CTLs can be used to target solid cancers (e.g., pancreatic cancer, ovarian cancer, gastric cancer, and breast cancer).

Thus, the present disclosure provides tumor antigen-specific peptides, e.g., against the tumor antigen VCX/Y, for use as immunotherapies for the treatment of cancer. Disclosed herein are exemplary VCX/Y peptides VCX54 (e.g., comprising SEQ ID NO: 1) whose sequences are shared with all VCX/Y family members including VCX1, VCX2, VCX3A, VCX3B, and VCY. Other VCX/Y peptides include VCX-37(SEQ ID NO: 8), VCX-178(SEQ ID NO: 9), VCY-37(SEQ ID NO: 12), VCX-58(SEQ ID NO: 13), and VCX-59(SEQ ID NO: 14). For example, the tumor antigen-specific peptide can be contacted with a population of T cells or used to stimulate a population of T cells to induce proliferation of T cells that recognize or bind the tumor antigen-specific peptide. In other embodiments, the VCX/Y specific peptides of the disclosure may be administered to a subject (e.g., a human patient) to enhance the subject's immune response against cancer.

VCX/Y specific peptides may be included in active immunotherapy (e.g., cancer vaccines) or passive immunotherapy (e.g., adoptive immunotherapy). Active immunotherapy involves the immunization of a subject with a purified tumor antigen or an immunodominant VCX/Y specific peptide (natural or modified); alternatively, antigen presenting cells pulsed with VCX/Y specific peptides (or transfected with a gene encoding a tumor antigen) may be administered to a subject. The VCX/Y specific peptide may be modified or comprise one or more mutations, such as substitution mutations. Passive immunotherapy includes adoptive immunotherapy. Adoptive immunotherapy typically involves administering cells to a subject, wherein the cells (e.g., cytotoxic T cells) have been primed in vitro against VCX/Y-specific peptides (see, e.g., US 7910109).

In particular, patient-own VCX/Y specific T cells can be generated ex vivo for effective immune-based therapy within a short period of time (e.g., 6 to 8 weeks). Autologous or allogeneic T cells (e.g., CD 4) that can be isolated from peripheral blood⁺T cell, CD8⁺T cells, γ δ T cells and tregs), for example using a tetramer-directed sorting and Rapid Expansion Protocol (REP). Next, the peptide or corresponding encoding polynucleotide can be loaded into HLA-a2 positive dendritic cells, LCLs, PBMCs, or artificial antigen presenting cells (aapcs) and then co-cultured with T cells by several rounds of stimulation to generate antigen-specific CTL cell lines or clones. In addition, the effector function and long-term persistence in vivo of these expanded antigen-specific T cells can be enhanced by manipulation of immune modulatory parameters. These autologous CTL cells can be used for adoptive immunotherapy for VCX/Y and HLA-A2 positive cancer patients. In addition, other VCX/Y specific cells that can be generated from the present disclosure includeSomatic or allogeneic NK cells, constant NK cells, NKT cells, Mesenchymal Stem Cells (MSC) and Induced Pluripotent Stem (iPS) cells. These cells can be isolated from blood or umbilical cord.

In another approach, antigen-specific cells can be generated by using a VCX54 TCR provided herein (e.g., SEQ ID NOS: 2 to 7) or a VCY37 TCR provided herein (e.g., SEQ ID NOS: 15 to 20). In this method, the TCR sequence is inserted into a vector (e.g., a retroviral or lentiviral vector) and the vector is introduced into a host cell (e.g., a T cell (e.g., CD 4)⁺T cell, CD8⁺T cells, γ δ T cells and tregs), NK cells, constant NK cells, NKT cells, Mesenchymal Stem Cells (MSCs), Induced Pluripotent Stem (iPS) cells and PBMCs) to generate antigen-specific cells, which can be used for adoptive cell therapy for cancer patients.

In addition, the present disclosure provides soluble TCRs that can be used to directly treat HLA-a2 positive cancer patients. Soluble bispecific T cell engaging molecules were generated by linking VCX54 TCR or VCY37 TCR to a CD 3-specific Fab fragment. T cells can be conjugated to TCR binding to tumor cell surface by presenting the corresponding peptide/MHC complex and Fab fragment, and then cross-linked to antigen-exposed CD8⁺TCR on the surface of T cells, leading to cell activation and elimination of target cells. Thus, this soluble bispecific TCR construct can be used to directly treat cancer patients.

Finally, soluble TCRs can be used as probes for diagnostic evaluation of peptides/MHC in tumor cells, or for directing therapeutic molecules to the tumor site. The soluble TCR molecules may also be labelled with a tracer (e.g. a fluorescent probe or a radioactive probe) and then used for diagnostic evaluation of peptide/MHC presentation in tumour cells. In addition, the soluble TCR molecules can be linked to therapeutic molecules, such as toxins, which are then directed to the tumor site to treat cancer patients.

I. Definition of

As used herein, "substantially free" with respect to a particular component is used herein to mean that none of the particular component is purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. Thus, the total amount of a particular component resulting from any unintended contamination of the composition is well below 0.05%, preferably below 0.01%. Most preferred are compositions wherein the amount of a particular component cannot be detected by standard analytical methods.

As used in this specification, a noun without a quantitative term change may mean one or more. As used in the claims, when used in combination with the word "comprising" the nouns without the numerical word can mean one or more than one.

The use of the term "or/and" in the claims is intended to mean "and/or" unless explicitly indicated to refer to alternatives only or to alternatives being mutually exclusive, but the disclosure supports definitions referring to alternatives and "and/or" only. "another" as used herein may mean at least a second or more.

Throughout this application, the term "about" is used to indicate a value that includes inherent variations in the error of the apparatus, method, or subject used to determine the value.

"treating" or "treatment" refers to administering or applying a therapeutic agent to a subject or performing some method or manner on a subject in order to obtain a therapeutic benefit of a disease or health-related disorder. For example, the treatment may comprise administering a T cell therapy.

"subject" and "patient" refer to humans or non-humans, such as primates, mammals, and vertebrates. In a particular embodiment, the subject is a human.

The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to any condition that promotes or enhances the health of a subject with respect to drug treatment of the condition. This includes, but is not limited to, reducing the frequency or severity of signs or symptoms of disease. For example, treatment of cancer may involve, for example, reducing tumor size, reducing tumor invasiveness, reducing the rate of cancer growth, or preventing metastasis. Treatment of cancer may also refer to prolonging survival of a subject having cancer.

An "anti-cancer" agent can negatively affect a cancer cell/tumor in a subject, for example, by promoting killing of the cancer cell, inducing apoptosis of the cancer cell, reducing the growth rate of the cancer cell, reducing the incidence or number of metastases, reducing the size of the tumor, inhibiting tumor growth, reducing blood supply to the tumor or cancer cell, promoting an immune response against the cancer cell or tumor, preventing or inhibiting progression of the cancer, or increasing the lifespan of the subject with the cancer.

The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.

The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal (e.g., a human), as appropriate. The preparation of pharmaceutical compositions comprising antibodies or other active ingredients will be known to those skilled in the art in light of the present disclosure. In addition, for animal (e.g., human) administration, it is understood that the formulations should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of biological standards.

As will be known to those of ordinary skill in the art, "pharmaceutically acceptable carrier" as used herein includes any and all materials such as aqueous solvents (e.g., water, alcohol/water solutions, saline solutions, parenteral carriers (e.g., sodium chloride, ringer's dextrose, etc.)), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters (e.g., ethyl oleate)), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorants, dyes, fluids, nutritional supplements, and the like, and combinations thereof. The pH and precise concentration of the various components in the pharmaceutical composition are adjusted according to well-known parameters.

The term "unit dose" or "dose" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined amount of a therapeutic composition calculated to produce the desired response discussed above in relation to its administration (i.e., the appropriate route and treatment regimen). The amount to be administered depends on the desired effect, both in terms of the amount treated and the unit dose. The actual dosage amount of the composition of the present embodiment to be administered to a patient or subject may be determined by physical and physiological factors such as the weight, age, health condition and sex of the subject, the type of disease to be treated, the degree of disease penetration, previous or concurrent therapeutic intervention, the patient's self-illness, the route of administration, and the efficacy, stability and toxicity of the particular therapeutic substance. For example, a dose may also comprise from about 1 μ g/kg/body weight to about 1000 mg/kg/body weight per administration (such ranges include intervening doses) or more, and any ranges derivable therein. In some non-limiting examples of ranges that can be inferred from the numbers listed herein, ranges of about 5 μ g/kg/body weight to about 100 mg/kg/body weight, about 5 μ g/kg/body weight to about 500 mg/kg/body weight, and the like can be administered. In any event, the practitioner responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dosage for the individual subject. In some embodiments, the dose of antigen-specific T cell infusion may comprise from about 1 to about 300 million cells, e.g., 100, 150, or 200 million cells.

The term "immune checkpoint" refers to a molecule (e.g., a protein) in the immune system that provides a signal to components of the immune system to balance the immune response. Known immune checkpoint proteins include CTLA-4, PD1 and its ligands PD-L1 and PD-L2, and additionally LAG-3, BTLA, B7H3, B7H4, TIM3, KIR. Pathways involving LAG3, BTLA, B7H3, B7H4, TIM3 and KIR are considered in the art to constitute immune checkpoint pathways similar to CTLA-4 and PD-1 dependent pathways (see e.g., pardol, 2012; Mellman et al, 2011).

By "immune checkpoint inhibitor" is meant any compound that inhibits the function of an immune checkpoint protein. Inhibition includes reduced function and complete blockade. In particular, the immune checkpoint protein is a human immune checkpoint protein. Thus, the immune checkpoint protein inhibitor is in particular a human immune checkpoint protein inhibitor.

As used herein, "protective immune response" refers to the response by the immune system of a mammalian host against cancer. The protective immune response may provide a therapeutic effect for treating cancer (e.g., reducing tumor size or increasing survival).

The term "antigen" as used herein is a molecule capable of being bound by an antibody or a T cell receptor. Antigens may generally be used to induce a humoral and/or cellular immune response, leading to the production of B and/or T lymphocytes.

The terms "tumor-associated antigen", "tumor antigen" and "cancer cell antigen" are used interchangeably herein. In each case, the term refers to a protein, glycoprotein, or carbohydrate that is specifically or preferentially expressed by the cancer cell.

The term "Chimeric Antigen Receptor (CAR)" as used herein may refer to, for example, an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor, and encompasses engineered receptors that specifically transplant an artificial onto a particular immune effector cell. The specificity of a monoclonal antibody can be conferred onto T cells using a CAR, thereby allowing the generation of a large number of specific T cells, e.g., for use in adoptive cell therapy. In particular embodiments, the CAR directs the specificity of the cell against, for example, a tumor-associated antigen. In some embodiments, the CAR comprises an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor-associated antigen binding region. In some particular aspects, the CAR comprises a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to the transmembrane and endodomain of CD3 ζ. The specificity of other CAR designs can be derived from ligands for receptors (e.g., peptides) or from pattern recognition receptors, such as dendronin (Dectin). In some cases, the spacing of the antigen recognition domains may be modified to reduce activation-induced cell death. In certain instances, the CAR comprises domains for other costimulatory signaling, e.g., CD3 ζ, FcR, CD27, CD28, CD137, DAP10, and/or OX 40. In some cases, molecules can be co-expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally ablate T cells after addition of a prodrug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

A polynucleotide or polynucleotide region (or polypeptide region) has a certain percentage (e.g., 80%, 85%, 90% or 95%) of "sequence identity" or "homology" to another sequence, meaning that the percentage of bases (or amino acids) are the same in comparing the two sequences when aligned. The alignment and percent homology or sequence identity can be determined using software programs known IN the art, such as those described IN CURRENT promoters IN MOLECULAR BIOLOGY (f.m. ausubel et al, eds., 1987) subsidiary 30, 7.7.18, table 7.7.1. Preferably, default parameters are used for alignment. The preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: the genetic code is standard; no filter; two chains; cutoff is 60; the expected value is 10; BLOSUM 62; describe 50 sequences; the ranking is according to HIGH SCORE; database-not redundant-GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR.

II.VCX/Y peptides

Certain embodiments of the present disclosure relate to tumor antigen-specific peptides, e.g., against VCX/Y tumor antigens. In particular embodiments, the tumor antigen-specific peptide has the amino acid sequence of the VCX/Y peptide (GAATKMAAV: SEQ ID NO: 1; or SEQ ID NO: 8, 9,12, 13, or 14). The tumor antigen-specific peptide may have an amino acid sequence identical to SEQ ID NO: 1. 8, 9,12, 13 or 14 has an amino acid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

The term "peptide" as used herein encompasses amino acid chains comprising: 7 to 35 amino acids, preferably 8 to 35 amino acid residues, more preferably 8 to 25 amino acids, or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 amino acids in length, or any range derivable therein. For example, in some embodiments, a VCX/Y peptide of the disclosure may comprise SEQ ID NO: 1 or the VCX54 peptide of seq id NO: 8. 9,12, 13 or 14, or consists thereof. As used herein, an "antigenic peptide" is a peptide that: which when introduced into a vertebrate can stimulate the production of antibodies (i.e., are antigenic) in the vertebrate, and wherein the antibodies can selectively recognize and/or bind to the antigenic peptide. The antigenic peptide may comprise an immunoreactive VCX/Y peptide and may comprise additional sequences. Additional sequences may be derived from a native antigen and may be heterologous, and such sequences may (but need not) be immunogenic. In some embodiments, the tumor antigen-specific peptide (e.g., VCX/Y peptide) may selectively bind to HLA-a2, particularly HLA-a 0201. In certain embodiments, the VCX/Y peptide is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids in length, or any range derivable therein. Preferably, the tumor antigen-specific peptide (e.g., VCX/Y peptide) is 8 to 35 amino acids in length. In some embodiments, the tumor antigen-specific peptide (e.g., VCX/Y peptide) is 8 to 10 amino acids in length.

As will be understood by those skilled in the art, MHC molecules can bind peptides of various sizes, but do not typically bind full-length proteins. Although MHC class I molecules have traditionally been described as binding to peptides 8 to 11 amino acids in length, it has been shown that peptides 15 amino acids in length can bind to MHC class I molecules by bulging (bucking) in the middle of the binding site or extending outside the MHC class I binding groove (Guo et al, 1992; Burrows et al, 2006; Saminio et al, 2006; Stryhn et al, 2000; Collins et al, 1994; Blanchard and Shastri, 2008). In addition, recent studies have also shown that longer peptides can be more efficiently endocytosed, processed, and presented by antigen presenting cells (Zwaveling et al, 2002; Bijker et al, 2007; Melief and van der Burg, 2008; Quintarelli et al, 2011). Peptides up to 35 amino acids in length are useful for selective binding to MHC class II and are effective, as indicated in Zwaveling et al (2002). As the skilled person will immediately understand, naturally occurring full length tumor antigens (e.g. VCX/Y) will not be available for selective binding to MHC class II, such that the antigen is endocytosed and T cell proliferation results. In general, naturally occurring full-length tumor antigen proteins do not exhibit these properties and will therefore not be useful for these immunotherapeutic purposes.

In certain embodiments, the tumor antigen-specific peptide (e.g., VCX/Y peptide) is immunogenic or antigenic. As shown in the examples below, various tumor antigen-specific peptides of the disclosure (e.g., VCX/Y peptides) can promote proliferation of T cells. Such peptides are expected to be useful in inducing a degree of protective immunity.

The tumor antigen-specific peptide (e.g., VCX/Y peptide) can be a recombinant peptide, a synthetic peptide, a purified peptide, an immobilized peptide, a detectably labeled peptide, an encapsulated peptide, or a peptide expressed via a vector (e.g., a peptide encoded by a nucleic acid in a vector comprising a heterologous promoter operably linked to the nucleic acid). In some embodiments, a synthetic tumor antigen-specific peptide (e.g., a VCX/Y peptide) can be administered to a subject (e.g., a human patient) to induce an immune response in the subject. Synthetic peptides may exhibit certain advantages, such as a reduced risk of bacterial contamination, compared to recombinantly expressed peptides. The tumor antigen-specific peptide (e.g., VCX/Y peptide) can also be included in a pharmaceutical composition (e.g., a vaccine composition) formulated for administration to a mammalian or human subject.

A. Cell penetrating peptides

In some embodiments, immunotherapy may employ a tumor antigen-specific peptide (e.g., VCX/Y peptide) of the present disclosure in association with a cell penetrating agent (e.g., a liposome or a Cell Penetrating Peptide (CPP)). Antigen presenting cells (e.g., dendritic cells) pulsed with peptides can be used to enhance anti-tumor immunity (Celluzzi et al, 1996; Young et al, 1996). Liposomes and CPPs are described in further detail below. In some embodiments, immunotherapy can employ a nucleic acid encoding a tumor antigen-specific peptide of the disclosure (e.g., a VCX/Y peptide), wherein the nucleic acid is delivered, e.g., in a viral vector or a non-viral vector.

Tumor antigen-specific peptides (e.g., VCX/Y peptides) can also be associated or covalently bound to a cell-penetrating peptide (CPP). Cell penetrating peptides which can be covalently bound to tumor antigen specific peptides (e.g., VCX/Y peptides) include, for example, HIV Tat, herpes virus VP22, Drosophila Antennapedia homeobox gene product, signal sequences, fusion sequences, or antimicrobial peptide i (protegrin i). Covalent binding of peptides to CPPs prolongs the presentation of peptides by dendritic cells, thereby enhancing anti-tumor immunity (Wang and Wang, 2002). In some embodiments, a tumor antigen-specific peptide of the present disclosure (e.g., contained within a peptide or multi-epitope string) (e.g., a VCX/Y peptide) can be covalently bound (e.g., via a peptide bond) to a CPP to produce a fusion protein. In additional embodiments, the tumor antigen-specific peptide (e.g., VCX/Y peptide) or nucleic acid encoding the tumor antigen-specific peptide can be encapsulated within or associated with a liposome (e.g., a multilamellar, vesicular, or multivesicular liposome, an extracellular vesicle, or an exosome).

As used herein, "associated" means physically associated, chemically associated, or both. For example, association may involve covalent bonds, hydrophobic interactions, encapsulation, surface adsorption, and the like.

As used herein, "cell permeabilizing agent" refers to a composition or compound that enhances the intracellular delivery of a peptide/multi-epitope string to an antigen presenting cell. For example, the cell penetrating agent may be a lipid that enhances its ability to cross the plasma membrane when associated with the peptide. Alternatively, the cell penetrating agent may be a peptide. Cell Penetrating Peptides (CPPs) are known in the art and include, for example, the Tat protein of HIV (Frankel and Pabo, 1988), the VP22 protein of HSV (Elliott and O' Hare, 1997), and fibroblast growth factor (Lin et al, 1995).

Cell penetrating peptides (or "protein transduction domains") have been identified from the third helix of the Drosophila Antennapedia homeobox gene (Antp), HIV Tat and herpes virus VP22, all of which contain positively charged domains rich in arginine and lysine residues (Schwarze et al, 2000; Schwarze et al, 1999). Furthermore, hydrophobic peptides derived from the signal sequence have also been identified as cell penetrating peptides (Rojas et al, 1996; Rojas et al, 1998; Du et al, 1998). It has been shown that coupling these peptides to a marker protein (e.g., β -galactosidase) allows the marker protein to be internalized into cells with high efficiency, and chimeric in-frame fusion proteins comprising these peptides have been used to deliver the protein to a broad spectrum of cell types both in vitro and in vivo (Drin et al, 2002). Fusion of these cell penetrating peptides with tumor antigen specific peptides according to the present disclosure (e.g., VCX/Y peptides) can enhance cellular uptake of the polypeptide.

In some embodiments, cellular uptake is facilitated by attaching a lipid (e.g., stearate or myristate) to the polypeptide. Lipidation (lipidation) has been shown to enhance peptide entry into cells. Attachment of a lipid moiety is another way of the present disclosure to increase polypeptide uptake by a cell. Cellular uptake is discussed further below.

The tumor antigen-specific peptides (e.g., VCX/Y peptides) of the present disclosure can be included in a liposome vaccine composition. For example, the liposome composition may be or comprise a proteoliposome composition (proteoliposomamal composition). Methods for producing proteoliposome compositions that can be used with the present disclosure are described, for example, in Neelapu et al (2007) and Popescu et al (2007). In some embodiments, the proteoliposome composition can be used to treat melanoma.

By enhancing uptake of tumor antigen-specific polypeptides, it may be feasible to reduce the amount of protein or peptide required for treatment. This in turn can significantly reduce treatment costs and increase the supply of therapeutic agents. Lower doses may also minimize the potential immunogenicity of the peptide and limit toxic side effects.

In some embodiments, a tumor antigen-specific peptide (e.g., a VCX/Y peptide) can be associated with a nanoparticle to form a nanoparticle-polypeptide complex. In some embodiments, the nanoparticle is a liposome or other lipid-based nanoparticle, such as a lipid-based vesicle (e.g., DOTAP: cholesterol vesicle). In a further embodiment, the nanoparticle is a superparamagnetic iron oxide-based nanoparticle. Superparamagnetic nanoparticles of about 10nm to 100nm in diameter are small enough to avoid isolation by the spleen (sequester), but large enough to avoid clearance by the liver. Particles of this size can penetrate very small capillaries and can be effectively distributed in body tissue. Superparamagnetic nanoparticle-polypeptide complexes can be used as MRI contrast agents to identify and track those cells that take up tumor antigen-specific peptides (e.g., VCX/Y peptides). In some embodiments, the nanoparticle is a semiconductor nanocrystal or a semiconductor quantum dot, both of which can be used for optical imaging. In further embodiments, the nanoparticles may be nanoshells comprising a gold layer over a silica core. One advantage of nanoshells is that standard chemistry can be used to conjugate the polypeptide to a gold layer. In further embodiments, the nanoparticle may be a fullerene or a nanotube (Gupta et al, 2005).

Peptides are rapidly removed from circulation by the kidneys and are susceptible to degradation by proteases in serum. By associating tumor antigen-specific peptides (e.g., VCX/Y peptides) with nanoparticles, the nanoparticle-polypeptide complexes of the present disclosure can provide protection against degradation and/or reduce clearance through the kidney. This may increase the serum half-life of the polypeptide, thereby reducing the dosage requirements for the polypeptide for effective treatment. Furthermore, this can reduce the cost of treatment and minimize immunological problems and toxic reactions to treatment.

B. Multi-table bit string

In some embodiments, the tumor antigen-specific peptide (e.g., VCX/Y peptide) is included or contained in a multi-epitope string. A polyepitopic bit string is a peptide or polypeptide that contains multiple epitopes from one or more antigens linked together. The multi-epitopic bit strings can be used to induce an immune response in a subject (e.g., a human subject). Multi-epitope bit strings have previously been used to target malaria and other pathogens (Baraldo et al, 2005; Moorthy et al, 2004; Baird et al, 2004). The polyepitopic bit string may refer to a nucleic acid (e.g., a nucleic acid encoding a variety of antigens including VCX/Y peptides), or a peptide or polypeptide (e.g., a nucleic acid comprising a variety of antigens including VCX/Y peptides). The multi-epitopic bit strings can be included in a cancer vaccine composition.

C. Biological functional equivalent

The tumor antigen-specific peptides (e.g., VCX/Y peptides) of the present disclosure can be modified to include amino acid substitutions, insertions, and/or deletions that do not alter their interaction with the HLA class protein (e.g., HLA-a 0101) binding region, respectively. Such biologically functional equivalents of tumor antigen-specific peptides (e.g., VCX/Y peptides) may be molecules having similar or otherwise desirable characteristics (e.g., binding of HLA-a 0201). As a non-limiting example, certain amino acids in the tumor antigen-specific peptides disclosed herein (e.g., VCX/Y peptides) can be substituted for other amino acids without appreciable loss of interaction capacity, as indicated by detectably unaltered peptides bound to HLA-a x 0201. In some embodiments, the tumor antigen-specific peptide has a substitution mutation at the anchor residence, e.g., at one, two, or all of the following positions: 1 (P1); 2 (P2); and/or 9 (P9). It is therefore contemplated that tumor antigen-specific peptides (e.g., VCX/Y peptides) disclosed herein (or nucleic acids encoding such peptides) that are modified in sequence and/or structure but not altered in biological utility or activity) remain within the scope of the compositions and methods disclosed herein.

It is also well understood by the skilled person that inherent to the definition of biologically functionally equivalent peptides is the following concept: there is a limit to the number of changes that can be made in a defined portion of a molecule while still maintaining an acceptable level of equivalent biological activity. Thus, biologically functionally equivalent peptides are defined herein as those peptides in which some (but not most or all) of the amino acids may be substituted. Of course, a variety of different peptides with different substitutions can be readily made and used in accordance with the present disclosure.

The skilled artisan also recognizes that where certain residues (e.g., residues in a particular epitope) are shown to be particularly important for biological or structural properties of the peptide, such residues may not typically be exchanged. This may be the case in the present disclosure, mutations in tumor antigen-specific peptides (e.g., VCX/Y peptides) as disclosed herein may result in a loss of species specificity and thereby reduce the utility of the resulting peptide for use in the methods of the present disclosure. Thus, peptides that are antigenic (e.g., specifically bind HLA-a x 0201) and comprise conservative amino acid substitutions are understood to be encompassed by the present disclosure. Conservative substitutions are least likely to drastically alter the activity of the protein. "conservative amino acid substitution" refers to the replacement of an amino acid with a chemically similar amino acid, i.e.: replacement of a non-polar amino acid with another non-polar amino acid; replacement of polar amino acids with other polar amino acids; replacement of acidic residues with other acidic amino acids, and the like.

Amino acid substitutions, such as those useful for modifying tumor antigen-specific peptides disclosed herein (e.g., VCX/Y peptides), are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, and the like. Analysis of the size, shape, and type of amino acid side-chain substituents revealed that arginine, lysine, and histidine were all positively charged residues; alanine, glycine and serine all have similar dimensions; and phenylalanine, tryptophan, and tyrosine all have approximately similar shapes. Accordingly, based on these considerations, arginine, lysine, and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine are defined as biofunctional equivalents. In some embodiments, the mutation can enhance TCR-pMHC interaction and/or peptide-MHC binding.

The present disclosure also contemplates isoforms (isofom) of the tumor antigen specific peptides (e.g., VCX/Y peptides) disclosed herein. The isoforms contain the same number and kind of amino acids as the peptides of the present disclosure, but the isoforms have different molecular structures. Isoforms contemplated by the present disclosure are those having the same properties as the peptides of the present disclosure described herein.

Non-standard amino acids can be incorporated into proteins by chemical modification of existing amino acids or by de novo synthesis of the peptides disclosed herein. Non-standard amino acids refer to amino acids that differ in chemical structure from the twenty standard amino acids encoded by the genetic code.

In some selected embodiments, the present disclosure contemplates chemical derivatives of the tumor antigen-specific peptides (e.g., VCX/Y peptides) disclosed herein. "chemical derivative" refers to a peptide having one or more residues chemically derivatized by reaction of a functional side group and retaining biological activity and utility. Such derivatized peptides include, for example, those in which the free amino group has been derivatized to form a particular salt or derivatized by alkylation and/or acylation to, among others, p-toluenesulfonyl, benzyloxycarbonyl, tert-butoxycarbonyl, chloroacetyl, formyl, or acetyl. The free carboxyl groups can be derivatized to form organic or inorganic salts, methyl and ethyl esters or other types of esters or hydrazides and preferably amides (primary or secondary). Chemical derivatives may include those peptides comprising one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline may be substituted with serine; and ornithine may be replaced by lysine.

It should be noted that all amino acid residue sequences are herein represented by formulas oriented in the conventional direction from the amino terminus to the carboxy terminus. Furthermore, it should be noted that a dashed line at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence having one or more amino acid residues. The amino acids described herein are preferably in the "L" isomeric form. However, residues in the "D" isomeric form may be substituted for any L-amino acid residue, as long as the protein retains the desired functional properties described herein.

Preferred tumor antigen-specific peptides (e.g. VCX/Y peptides) or analogues thereof preferably specifically or preferentially bind HLA-a 0201. Determining whether or to what extent a particular tumor antigen-specific peptide or labeled peptide, or analog thereof, binds to HLA-a0201 may be assessed using, for example, an in vitro assay as follows: enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, Radioimmunoassay (RIA), immunostaining, latex agglutination, Indirect Hemagglutination Assay (IHA), complement fixation (complement fixation), indirect immunofluorescence assay (FA), nephelometry (nephelometry), flow cytometry assay, chemiluminescence assay, lateral flow immunoassay, u-capture assay, mass spectrometry assay, particle-based assay, inhibition assay, and/or avidity assay.

D. Nucleic acids encoding tumor antigen-specific peptides

In one aspect, the present disclosure provides a nucleic acid encoding an isolated antigen-specific peptide comprising an amino acid sequence identical to SEQ ID NO: 1. 8, 9,12, 13, or 14, or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1. 8, 9,12, 13, or 14 may have 1, 2, 3, or 4 point mutations (e.g., substitution mutations) compared. As mentioned above, such tumor antigen-specific peptides may be, for example, 8 to 35 amino acids in length, or any range derivable therein. In some embodiments, the tumor antigen-specific peptide corresponds to a portion of a tumor antigen protein, e.g., VCX1, VCX2, VCX3A, VCX3B, or VCY (e.g., VCX 3A; GenBank accession No: AAI 26903.1). The term "nucleic acid" is intended to include DNA and RNA and may be double-stranded or single-stranded.

Some embodiments of the present disclosure provide recombinantly produced tumor antigen-specific peptides (e.g., VCX/Y peptides) that can specifically bind HLA-a 0201. Thus, a nucleic acid encoding a tumor antigen-specific peptide can be operably linked to an expression vector and a peptide produced in a suitable expression system using methods well known in the art of molecular biology. The nucleic acid encoding the tumor antigen-specific peptides disclosed herein can be incorporated into any expression vector that ensures good expression of the peptide. Possible expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), so long as the vector is suitable for transformation of a host cell.

By "suitable for the transformation of a host cell" of the recombinant expression vector is meant that the expression vector comprises the nucleic acid molecule of the present disclosure and operably linked to the nucleic acid molecule a regulatory sequence selected based on the host cell to be used for expression. The terms "operably linked" or "operably linked" are used interchangeably and are intended to mean that a nucleic acid is linked to a regulatory sequence in a manner that allows for expression of the nucleic acid.

Accordingly, the present disclosure provides recombinant expression vectors comprising a nucleic acid encoding a tumor antigen-specific peptide, and essential regulatory sequences for transcription and translation of the inserted protein sequence. Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal or viral genes (see, e.g., the regulatory sequences described in Goeddel (1990)).

The selection of suitable regulatory sequences generally depends on the host cell selected and can be readily accomplished by one of ordinary skill in the art. Some examples of such regulatory sequences include: transcription promoters and enhancers or RNA polymerase binding sequences; a ribosome binding sequence comprising a translation initiation signal. In addition, other sequences (e.g., origins of replication, other DNA restriction sites, enhancers, and sequences that confer transcriptional inducibility) may also be incorporated into the expression vector, depending on the host cell chosen and the vector used. It will also be appreciated that the essential regulatory sequences may be provided by the native protein and/or flanking regions thereof.

The recombinant expression vector may further comprise a selectable marker gene that facilitates selection of host cells transformed or transfected with the recombinant tumor antigen-specific peptides disclosed herein (e.g., VCX/Y peptides). Some examples of selectable marker genes are genes encoding for example the following proteins: g418 and hygromycin that confer resistance to certain drugs; beta-galactosidase; chloramphenicol acetyltransferase; or firefly luciferase. Transcription of the selectable marker gene is monitored by changes in the concentration of a selectable marker protein (e.g., β -galactosidase, chloramphenicol acetyltransferase, or firefly luciferase). If the selectable marker gene encodes a protein that confers antibiotic resistance (e.g., neomycin resistance), then G418 can be used to select for the transformant cells. Cells that have incorporated the selectable marker gene will survive, while other cells will die. This allows the expression of the recombinant expression vector to be visualized and assayed and in particular the effect of the mutation on expression and phenotype to be determined. It is understood that the selectable marker may be introduced on an isolated vector from the nucleic acid of interest.

The recombinant expression vector can be introduced into a host cell to produce a transformant host cell. The term "transformant host cell" is intended to include prokaryotic and eukaryotic cells that have been transformed or transfected with the recombinant expression vectors of the present disclosure. The terms "transformation with … …", "transfection with … …", "transformation" and "transfection" are intended to encompass the introduction of a nucleic acid (e.g., a vector) into a cell by one of many possible techniques known in the art. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the proteins of the present disclosure can be expressed in bacterial cells (e.g., e.coli), insect cells (using baculovirus), yeast cells, or mammalian cells.

The nucleic acid molecules of the present disclosure can also be chemically synthesized using standard techniques. Various methods of chemical synthesis of polydeoxyribonucleotides are known, including solid phase synthesis, which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (see, e.g., U.S. Pat. Nos. 4,598,049; 4,458,066; 4,401,796; and 4,373,071).

Antigen specific cell therapy

Certain embodiments of the present disclosure relate to obtaining antigen-specific cells (e.g., autologous or allogeneic T cells (e.g., regulatory T cells, CD 4)⁺T cell, CD8⁺T cells or γ δ T cells), NK cells, constant NK cells, NKT cells, Mesenchymal Stem Cells (MSCs) or Induced Pluripotent Stem (iPS) cells) and administered to a subject as immunotherapy against targeted cancer cells. In particular, the cells are antigen-specific T cells (e.g., VCX/Y-specific T cells). Several basic methods for the derivation, activation and expansion of functional anti-tumor effector T cells have been described in the last two decades. These include: autologous cells, such as tumor-infiltrating lymphocytes (TIL); ex vivo activated T cells using autologous DCs, lymphocytes, artificial Antigen Presenting Cells (APCs) or beads coated with T cell ligands and activated antibodies, or cells isolated by capturing the target cell membrane; allogeneic cells that naturally express a T Cell Receptor (TCR) against the host tumor; and tumor-reactive TCRs or that are genetically reprogrammed or "redirected" to express a tumor-reactive TCR exhibiting antibody-like tumor recognition capability called a "T-bodyNon-tumor specific autologous or allogeneic cells of the chimeric TCR molecule. These methods have resulted in a number of protocols for T cell preparation and immunization that can be used in the methods described herein.

A.T cell preparation

In some embodiments, the T cell is derived from blood, bone marrow, lymph, umbilical cord, or lymphoid organs. In some aspects, the cell is a human cell. The cells are typically primary cells, such as those isolated directly from the subject and/or isolated from the subject and frozen. In some embodiments, the cells comprise one or more subpopulations of T cells or other cell types, e.g., the entire T cell population, CD4⁺Cell, CD8⁺Cells, and subpopulations thereof, such as those defined by: function, activation state, maturity, differentiation potential, expansion, recycling, localization and/or persistence ability, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion characteristics, and/or degree of differentiation. The cells may be allogeneic and/or autologous with respect to the subject to be treated. In some aspects, for example for off-the-shelf technologies, the cells are pluripotent (pluripotent) and/or multipotent, such as stem cells, e.g., induced pluripotent stem cells (ipscs). In some embodiments, the methods comprise isolating cells from a subject, preparing, processing, culturing, and/or engineering them, and reintroducing them into the same patient before or after cryopreservation, as described herein.

T cells (e.g., CD 4)⁺And/or CD8⁺T cells) and the presence of incipient T (T) in subtypes and subpopulations of T cells_N) Cells, effector T cells (T)_EFF) Memory T cells, and subtypes thereof, e.g., stem cell memory T (TSC)_M) Cell, central memory T (TC)_M) Cellular, effector memory T (T)_EM) Cells or terminally differentiated effector memory T cells; tumor Infiltrating Lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (treg) cells, helper T cells (e.g., TH1 fine cells)Cell, TH2 cell, TH3 cell, TH17 cell, TH9 cell, TH22 cell, follicular helper T cell); α/β T cells and δ/γ T cells.

In some embodiments, one or more T cell populations are enriched for or depleted of cells positive for a particular marker (e.g., a surface marker), or negative for a particular marker. In some cases, such markers are those that are absent or expressed at relatively low levels on certain T cell populations (e.g., non-memory cells) but present or expressed at relatively higher levels on certain other T cell populations (e.g., memory cells).

In some embodiments, T cells are isolated from the PBMC sample by negatively selecting for a marker (e.g., CD14) expressed on non-T cells (e.g., B cells, monocytes, or other leukocytes). In some aspects, CD4⁺Or CD8⁺Selection procedure for separating CD4⁺Auxiliary sum CD8⁺Cytotoxic T cells. Such CD4 by positively or negatively selecting markers expressed or expressed to a relatively high degree on one or more naive, memory and/or effector T cell subsets⁺And CD8⁺The clusters can be further classified into subpopulations.

In some embodiments, CD8⁺T cells are further enriched for or depleted of naive cells, central memory cells, effector memory cells and/or central memory stem cells, e.g., by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, central memory T (T) is performed_CM) Enrichment of cells to increase efficacy, e.g., to improve long-term survival, expansion and/or implantation after administration, is particularly powerful in some aspects in such subpopulations. See Terakura et al, 2012; wang et al, 2012.

In some embodiments, the T cell is an autologous T cell. In this method, a tumor sample is obtained from a patient and a single cell suspension is obtained. The single cell suspension may be applied in any suitable manner, e.g.mechanically (using e.g.GentleMeC @)^TMDissociators, Miltenyi Biotec, Auburn, Calif. to dissociate tumors) or enzymatically (e.g., collagenase or DNase). Single cell suspensions of tumor enzyme digests were cultured in interleukin-2 (IL-2). Cells are cultured until confluent (e.g., about 2X 10)⁶Individual lymphocytes), for example, for about 5 to about 21 days, preferably about 10 to about 14 days.

Cultured T cells can be pooled and expanded rapidly. Rapid expansion provides at least about a 50-fold (e.g., 50, 60, 70, 80, 90, or 100-fold or greater) increase in the number of antigen-specific T cells over a period of about 10 to about 14 days. More preferably, rapid amplification provides an increase of at least about 200-fold (e.g., 200, 300, 400, 500, 600, 700, 800, 900-fold or more) over a period of about 10 to about 14 days.

Amplification may be accomplished by any of a number of methods known in the art. For example, non-specific T cell receptor stimulation can be used to rapidly expand T cells in the presence of feeder lymphocytes and interleukin-2 (IL-2) or interleukin-15 (IL-15), with IL-2 being preferred. Non-specific T cell receptor stimulation may comprise about 30ng/ml OKT3 (mouse monoclonal anti-CD 3 antibody, available fromObtained from Raritan, n.j.). Alternatively, T cells can be rapidly expanded by stimulating PBMCs in vitro with one or more cancer antigens (including antigenic portions thereof, e.g., epitopes or cells) that can optionally be expressed from a vector, such as human leukocyte antigen A2(HLA-A2) binding peptide, in the presence of T cell growth factor (e.g., 300 IU/ml IL-2 or IL-15, with IL-2 being preferred). Rapidly expanding in vitro induced T cells by restimulation with the same cancer antigen pulsed onto HLA-a2 expressing antigen presenting cells. Alternatively, T cells can be restimulated, for example, with irradiated autologous lymphocytes or with irradiated HLA-A2+ allogenic lymphocytes and IL-2.

Autologous T cells may be modified to express T cell growth factors that promote growth and activation of the autologous T cells. Suitable T cell growth factors include for example IL-2, IL-7, IL-15 and IL-12. Suitable modification methods are known in the art. See, e.g., Sambrook et al, 2001; and Ausubel et al, 1994. In particular aspects, the modified autologous T cells express T cell growth factors at high levels. T cell growth factor coding sequences, such as the coding sequence for IL-12, are readily available in the art, as are promoters, whose operative linkage to the T cell growth factor coding sequence promotes high levels of expression.

B. Genetically engineered antigen receptors

Cells of the present disclosure (e.g., autologous or allogeneic T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or γ δ T cells), NK cells, constant NK cells, NKT cells, Mesenchymal Stem Cells (MSCs), or induced pluripotent stem cells) can be genetically engineered to express antigen receptors, e.g., engineered TCRs and/or CARs. For example, host cells (e.g., autologous or allogeneic T cells) are modified to express TCRs with antigenic specificity for cancer antigens. In particular embodiments, the antigen receptor is antigen specific for VCX/Y (e.g., VCX1, VCX2, VCX3A, VCX3B, and VCY, particularly VCX54 peptides). In certain embodiments, the engineered TCR has a sequence identical to SEQ ID NO: 2 or 19, and/or an alpha chain CDR3 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to seq id NO: 3 or 20 beta chain CDR3 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, the TCR has a sequence identical to SEQ ID NO: 4. 5, 15 or 16 and/or an alpha chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to seq id NO: 6. 7, 17 or 18, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. Suitable modification methods are known in the art. See, e.g., Sambrook and Ausubel (supra). For example, cells may be transduced to express TCRs with antigenic specificity for cancer antigens using the transduction techniques described in Heemskerk et al, 2008 and Johnson et al, 2009.

In some embodiments, the cell comprises one or more nucleic acids encoding one or more antigen receptors introduced by genetic engineering, and the genetically engineered products of such nucleic acids. In some embodiments, the nucleic acid is heterologous, i.e., not normally present in a cell or sample obtained from a cell, e.g., a nucleic acid obtained from another organism or cell, e.g., which is not normally found in the cell being engineered and/or the organism from which such cell is derived. In some embodiments, the nucleic acid is not naturally occurring, such as a nucleic acid not found in nature (e.g., chimeric).

In some embodiments, the CAR comprises an extracellular antigen recognition domain that specifically binds to an antigen. In some embodiments, the antigen is a protein expressed on the surface of a cell. In some embodiments, the CAR is a TCR-like CAR and the antigen is a treated peptide antigen, e.g., a peptide antigen of an intracellular protein, which, like the TCR, is recognized on the cell surface in the context of a Major Histocompatibility Complex (MHC) molecule.

Exemplary antigen receptors, including CARs and recombinant TCRs, and methods for engineering and introducing the receptors into cells, including, for example, those described in: international patent application publication nos. WO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, WO 2013/123061; U.S. patent application publication nos. US2002131960, US2013287748, US 20130149337; U.S. Pat. nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118; and european patent application No. EP 2537416; and/or those described by: sadelain et al, 2013; davila et al, 2013; turtle et al, 2012; wu et al, 2012. In some aspects, the genetically engineered antigen receptor includes a CAR described in U.S. patent No.7,446,190, and international patent application publication No.: those described in WO/2014055668A 1.

1. Chimeric antigen receptors

In some embodiments, the engineered antigen receptor comprises a CAR, including an activating or stimulating CAR, a co-stimulating CAR (see WO2014/055668), and/or an inhibitory CAR (iCAR, see Fedorov et al, 2013). CARs typically comprise an extracellular antigen (or ligand) binding domain linked, in some aspects, to one or more intracellular signaling components through a linker and/or transmembrane domain. Such molecules typically mimic or approximate the signal through a native antigen receptor, the signal through such a receptor bound to a co-stimulatory receptor, and/or the signal through the co-stimulatory receptor alone.

In some embodiments, CARs are constructed with specificity for a particular antigen (or marker or ligand), e.g., an antigen expressed in a particular cell type to be targeted by adoptive therapy, e.g., a cancer marker; and/or antigens intended to induce an inhibitory response, such as antigens expressed on normal or non-diseased cell types. Thus, a CAR typically comprises in its extracellular portion one or more antigen binding molecules, such as one or more antigen binding fragments, domains, or portions, or one or more antibody variable domains, and/or antibody molecules. In some embodiments, the CAR comprises one or more antigen binding portions of an antibody molecule, such as a single chain antibody fragment (scFv) derived from the variable heavy chain (VH) and variable light chain (VL) of a monoclonal antibody.

In some aspects, the antigen-specific binding or recognition component is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR comprises a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some cases, the transmembrane domains are selected or modified by amino acid substitutions to avoid binding of such domains to transmembrane domains having the same or different surface membrane proteins to minimize interaction with other members of the receptor complex.

In some embodiments, the transmembrane domain is derived from a natural or synthetic source. Where the source is native, in some aspects, the domain is derived from any membrane bound or transmembrane protein. Transmembrane regions include those derived from (i.e., including at least the following transmembrane regions): the α, β or ζ chain of the T cell receptor, CD28, CD3 ζ, CD3 ∈, CD3 γ, CD3 δ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS/CD278, GITR/CD357, NKG2D and DAP molecules. Alternatively, in some embodiments the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues, such as leucine and valine. In some aspects, triplets of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain.

CARs typically comprise at least one intracellular signaling component. In some embodiments, the CAR comprises an intracellular component of the TCR complex, such as TCRCD3 that mediates T cell activation and cytotoxicity⁺Chains, such as the CD3 zeta chain. Thus, in some aspects, the antigen binding molecule is linked to one or more cell signaling modules. In some embodiments, the cell signaling module includes a CD3 transmembrane domain, a CD3 intracellular signaling domain, and/or other CD transmembrane domains. In some embodiments, the CAR further comprises a portion of one or more other molecules (e.g., Fc receptor γ, CD8, CD4, CD25, or CD 16). For example, in some aspects, the CAR comprises a chimeric molecule between CD3 ζ (CD3-Q or Fc receptor γ and CD8, CD4, CD25, or CD 16.

T Cell Receptor (TCR)

In some embodiments, the genetically engineered antigen receptor comprises a recombinant TCR and/or a TCR cloned from a naturally occurring T cell. "T cell receptor" or "TCR" refers to a molecule that comprises variable a and β chains (also known as TCR α and TCR β, respectively) or variable γ and δ chains (also known as TCR γ and TCR δ, respectively), and is capable of specifically binding to an antigenic peptide bound to an MHC receptor. In some embodiments, the TCR is in the α β form. In certain embodiments, the engineered TCR has the amino acid sequence of SEQ id no: 2 and/or the alpha chain CDR3 of SEQ ID NO: 3 beta chain CDR 3. In some embodiments, the TCR has the amino acid sequence of SEQ ID NO: 4. 5, 15 or 16 and/or the alpha chain of SEQ ID NO: 6. 7, 17 or 18.

In general, TCRs in the α β and γ δ forms are generally structurally similar, but T cells expressing them may have different structural positions or functions. The TCR may be present on the cell surface or in soluble form. Generally, TCRs are present on the surface of T cells (or T lymphocytes) that are generally responsible for recognizing antigens bound to Major Histocompatibility Complex (MHC) molecules. In some embodiments, the TCR may further comprise a constant domain, a transmembrane domain, and/or a short cytoplasmic tail (see, e.g., Janeway et al, 1997). For example, in some aspects, each chain of the TCR may have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, the TCR is associated with an invariant protein of the CD3 complex involved in mediating signal transduction. Unless otherwise indicated, the term "TCR" is understood to encompass functional TCR fragments thereof. The term also encompasses intact or full-length TCRs, including TCRs in either the α β or γ δ form.

Thus, for purposes herein, reference to a TCR includes any TCR or functional fragment, such as the antigen-binding portion of a TCR that binds to a particular antigenic peptide bound in an MHC molecule (i.e., an MHC-peptide complex). An "antigen-binding portion" or "antigen-binding fragment" of a TCR, used interchangeably, refers to a molecule that comprises a portion of the structural domain of the TCR, but binds to an antigen (e.g., an MHC-peptide complex) to which the complete TCR binds. In some cases, the antigen-binding portion comprises variable domains of a TCR, such as the variable alpha and variable beta chains of the TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex (e.g., where each chain typically comprises three complementarity determining regions).

In some embodiments, the variable domains of the TCR chains associate to form loops, or immunoglobulin-like Complementarity Determining Regions (CDRs), that confer antigen recognition and peptide specificity by forming the binding site of the TCR molecule. In general, like immunoglobulins, CDRs are separated by Framework Regions (FRs) (see, e.g., Jores et al, 1990; Chothia et al, 1988; Lefranc et al, 2003). In some embodiments, CDR3 is the primary CDR responsible for recognition of the treated antigen, although CDR1 of the α chain has also been shown to interact with the N-terminal portion of the antigenic peptide, while CDR1 of the β chain interacts with the C-terminal portion of the peptide. CDR2 is thought to recognize MHC molecules. In some embodiments, the variable region of the β -strand may comprise an additional hypervariable (HV4) region.

In some embodiments, the TCR chain comprises a constant domain. For example, similar to immunoglobulins, the extracellular portion of a TCR chain (e.g., a-chain, β -chain) may comprise two immunoglobulin domains, an N-terminal variable domain (e.g., V |)_aOr V_p(ii) a Usually amino acids 1 to 116 based on Kabat numbering, Kabat et al, "Sequences of Proteins of immunological Interest, US Dept. Health and Human Services, Public Health service National Institutes of Health, 1991, 5 th edition), and a constant domain adjacent to the cell membrane (e.g., a-chain constant domain or C.sub._aTypically amino acids 117 to 259 based on Kabat; beta chain constant domain or Cp, typically based on Kabat amino acids 117 to 259). For example, in some cases, the extracellular portion of a TCR formed by two chains comprises two membrane-proximal constant domains and two membrane-distal variable domains comprising CDRs. The constant domain of the TCR domain comprises a short linking sequence in which cysteine residues form a disulfide bond, forming a link between the two chains. In some embodiments, the TCR may have additional cysteine residues in each of the α and β chains, such that the TCR comprises two disulfide bonds in the constant domain.

In some embodiments, the TCR chains can comprise a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain comprises a cytoplasmic tail. In some cases, the structure allows the TCR to associate with other molecules (e.g., CD 3). For example, a TCR comprising a constant domain with a transmembrane region can anchor a protein to the cell membrane and associate with an invariant subunit of a CD3 signaling device or complex.

In general, CD3 is a polyprotein complex that can have three distinct chains (γ, δ, and ε) and a zeta chain in mammals. For example, in mammals, the complex may comprise a CD3 γ chain, a CD3 δ chain, two CD3 epsilon chains, and a homodimer of a CD3 zeta chain. The CD3 γ, CD3 δ, and CD3 epsilon chains are highly related cell surface proteins of the immunoglobulin superfamily that comprise a single immunoglobulin domain. The transmembrane regions of the CD3 γ, CD3 δ, and CD3 ε chains are negatively charged, a feature that allows these chains to associate with positively charged T cell receptor chains. The intracellular tails of the CD3 γ, CD3 δ, and CD3 ε chains each contain a single conserved motif called the immunoreceptor tyrosine-based activation motif, or ITAM, while there are three per CD3 ζ chain. In general, ITAMs are involved in the signaling capacity of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in transmitting signals from the TCR to the cell. The CD3 chain and the zeta chain form together with the TCR a so-called T cell receptor complex.

In some embodiments, the TCR may be a heterodimer of the two chains α and β (or optionally γ and δ), or it may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer comprising two independent chains (α and β chains or γ and δ chains) linked, for example, by one or more disulfide bonds. In some embodiments, TCRs directed against a target antigen (e.g., a cancer antigen) are identified and introduced into a cell. In some embodiments, nucleic acids encoding the TCR are available from a variety of sources, such as by Polymerase Chain Reaction (PCR) amplification of publicly available TCR DNA sequences. In some embodiments, the TCR is obtained from a biological source, e.g., from a cell, e.g., from a T cell (e.g., a cytotoxic T cell), a T cell hybridoma, or other publicly available source. In some embodiments, T cells may be obtained from cells isolated in vivo. In some embodiments, high affinity T cell clones can be isolated from a patient, and the TCR isolated. In some embodiments, the T cell may be a cultured T cell hybridoma or clone. In some embodiments, TCR clones directed against a target antigen have been generated in transgenic mice engineered with human immune system genes (e.g., human leukocyte antigen system or HLA). See, e.g., tumor antigens (see, e.g., Parkhurst et al, 2009 and Cohen et al, 2005). In some embodiments, phage display is used to isolate TCRs against a target antigen (see, e.g., Varela-Rohena et al, 2008 and Li, 2005). In some embodiments, the TCR, or antigen-binding portion thereof, can be synthetically generated based on knowledge of the TCR sequence.

3. Antigen presenting cell

Antigen presenting cells, which include macrophages, B lymphocytes and dendritic cells, are distinguished by their expression of specific MHC molecules. The APC internalizes the antigen and re-expresses a portion of the antigen along with MHC molecules on its outer cell membrane. Major Histocompatibility Complex (MHC) is a large genetic complex with multiple loci. The MHC locus encodes two major classes of MHC membrane molecules, termed MHC class I and class II. T helper lymphocytes typically recognize antigens associated with MHC class I molecules, whereas T cytotoxic lymphocytes recognize antigens associated with MHC class I molecules. In humans, the MHC is referred to as the HLA complex, and in mice as the H-2 complex.

In some cases, aapcs can be used to prepare therapeutic compositions and cell therapy products of some embodiments. For general guidance regarding the preparation and use of antigen presentation systems, see, e.g., U.S. patent nos. 6,225,042, 6,355,479, 6,362,001, and 6,790,662; U.S. patent application publication nos. 2009/0017000 and 2009/0004142; and international publication No. wo 2007/103009.

The aAPC system may comprise at least one exogenous helper molecule (assisting molecule). Any suitable number and combination of helper molecules may be used. The helper molecule may be selected from helper molecules such as co-stimulatory molecules and adhesion molecules. Some exemplary co-stimulatory molecules include CD86, CD64(Fc γ RI), 41BB ligand, and IL-21. Adhesion molecules may include: carbohydrate-binding glycoproteins, such as lectins; transmembrane binding glycoproteins, such as integrins; calcium-dependent proteins, such as cadherin; and single transmembrane immunoglobulin (Ig) superfamily proteins, such as intercellular adhesion molecules (ICAMs), which facilitate, for example, cell-to-cell or cell-to-matrix contact. Some exemplary adhesion molecules include LFA-3 and ICAMs, such as ICAM-1. Techniques, methods and reagents useful for selecting, cloning, preparing and expressing exemplary helper molecules, including co-stimulatory molecules and adhesion molecules, are exemplified in, for example, U.S. Pat. nos. 6,225,042, 6,355,479 and 6,362,001.

Soluble TCR

In some embodiments, the disclosure provides soluble TCRs, such as the VCX/Y TCRs provided herein. Soluble TCRs are useful not only for the purpose of studying specific TCR-pMHC interactions, but are potentially useful as diagnostic tools for detecting infection or for detecting autoimmune disease markers. Soluble TCRs can also be used for staining, for example, for staining cells for the presence of particular peptide antigens presented in the MHC context. Similarly, soluble TCRs can be used to deliver therapeutic agents (e.g., cytotoxic or immunostimulatory compounds) to cells presenting a particular antigen. Soluble TCRs are also useful for inhibiting T cells, e.g., those that react with autoimmune peptide antigens. In some aspects, the TCR is linked to another molecule that delivers neighboring cells to the tumor. In other aspects, the TCR delivers a toxin, cytokine, co-stimulatory ligand, or inhibitor ligand to and directs the molecule, cell, or compound into a target cell expressing the peptide-MHC.

In the context of the present application, "solubility" is defined as the concentration at 1mg/ml in Phosphate Buffered Saline (PBS) (KCl 2.7mM, KH)₂PO₄1.5mM, NaCl 137mM and Na₂PO 48 mM, pH 7.1 to 7.5. Life Technologies, Gibco BRL) was purified as monodisperse heterodimers and more than 90% of the TCR remained as monodisperse heterodimers after 1 hour of incubation at 25 ℃.

In some aspects, the present disclosure provides a soluble T cell receptor (sTCR) comprising: (i) all or part of a TCR alpha chain (e.g. SEQ ID NO: 4,5, 15 or 16), except for its transmembrane domain, and (ii) all or part of a TCR beta chain (e.g. SEQ ID NO: 6,7, 17 or 18), except for its transmembrane domain, wherein (i) and (ii) each comprise a functional variable domain and at least a portion of a constant domain of a TCR chain, and are linked by a disulfide bond between constant domain residues not present in native TCRs.

In some aspects, a soluble TCR comprises a TCR α or γ chain extracellular domain dimerized to a TCR β or δ chain extracellular domain, respectively, by a pair of C-terminal dimeric peptides (e.g., leucine zippers) (International patent publication No. WO 99/60120; U.S. Pat. No.7,666,604).

The soluble TCRs of the present disclosure (which are preferably human) can be provided in substantially pure form or as a purified or isolated preparation. For example, it may be provided in a form substantially free of other proteins.

A variety of soluble TCRs of the present disclosure can be provided in multivalent complexes. Accordingly, in one aspect, the present disclosure provides a multivalent T Cell Receptor (TCR) complex comprising a plurality of soluble T cell receptors as described herein. Each of the plurality of soluble TCRs is preferably the same.

In its simplest form, a multivalent TCR complex according to the present disclosure comprises multimers that associate (e.g., covalently or otherwise connect) with each other, preferably through two or three or four or more T cell receptor molecules of a linker molecule. Suitable linker molecules include, but are not limited to, multivalent attachment molecules such as avidin, streptavidin, neutravidin, and extravidin, each of which has four binding sites for biotin. Thus, biotinylated TCR molecules can be formed into multimers of T cell receptors with multiple TCR binding sites. The number of TCR molecules in a multimer will depend on the amount of TCR associated with the amount of linker molecules used to make the multimer, but also on the presence or absence of any other biotinylated molecule. Preferred multimers are dimeric, trimeric or tetrameric TCR complexes.

Suitable structures for use in the methods of the invention include membrane structures, such as liposomes; and solid structures, preferably particles, such as beads (e.g., latex beads). Other structures that can be coated externally with T cell receptor molecules are also suitable. Preferably, the structure is coated with a T cell receptor multimer rather than a separate T cell receptor molecule.

In the case of liposomes, the T cell receptor molecule or multimer thereof may be attached to or otherwise associated with the membrane. Techniques for this are well known to those skilled in the art.

A label or additional moiety (e.g., a toxic or therapeutic moiety) may be included in the multivalent TCR complexes of the disclosure. For example, labels or additional moieties may be included in the mixed molecular multimer. An example of such a multimeric molecule is a tetramer comprising three TCR molecules and one peroxidase molecule. This can be accomplished by mixing the TCR and enzyme in a 3: 1 molar ratio to produce a tetrameric complex, and isolating the desired complex from any complex that does not contain the correct ratio of molecules. These mixed molecules do not comprise any combination of molecules, provided that steric hindrance does not impair or does not significantly impair the desired function of the molecules. The positioning of the binding sites on the streptavidin molecules is suitable for the mixed tetramer, since steric hindrance is less likely to occur.

The TCRs (or multivalent complexes thereof) of the present disclosure may alternatively or additionally be associated with (e.g., covalently or otherwise linked to) a therapeutic agent, which may be, for example, a toxic moiety useful in cell killing, or an immunostimulatory agent (e.g., an interleukin or cytokine). Multivalent TCR complexes of the present disclosure can have enhanced binding capacity for TCR ligands compared to non-multimeric T cell receptor heterodimers. Thus, multivalent TCR complexes according to the present disclosure are particularly useful for tracking or targeting cells presenting a particular antigen in vitro or in vivo, and also as intermediates for the production of additional multivalent TCR complexes having such uses. Thus, the TCR or multivalent TCR complex can be provided in a pharmaceutically acceptable formulation for use in vivo.

The present disclosure also provides methods for delivering a therapeutic agent to a target cell, the method comprising contacting a potential target cell with a TCR or multivalent TCR complex specific for a TCR ligand and having a therapeutic agent associated therewith according to the present disclosure, while attaching the TCR or multivalent TCR complex to the target cell.

In particular, soluble TCRs or multivalent TCR complexes can be used to deliver therapeutic agents to the site of cells presenting a particular antigen. This will be useful in many cases, particularly for tumours. The therapeutic agent may be delivered so that it exerts its effect locally, but not only on the cells to which it binds. Thus, one particular strategy contemplates anti-tumor molecules linked to T cell receptors or multivalent TCR complexes specific for tumor antigens.

A number of therapeutic agents are available for this use, such as radioactive compounds, enzymes (e.g. perforins) or chemotherapeutic agents (e.g. cisplatin). To ensure that toxic effects are performed in the desired location, the toxin may be inside the liposome attached to the streptavidin, so that the compound is slowly released. This will prevent damaging effects during transport in vivo and ensure that the toxin has the greatest effect after binding of the TCR to the relevant antigen presenting cell.

Other suitable therapeutic agents include:

small molecule cytotoxic agents, i.e. compounds having the ability to kill mammalian cells having a molecular weight of less than 700 daltons. These compounds may also contain toxic metals capable of having cytotoxic effects. Furthermore, it is understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that decay or convert under physiological conditions to release the cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, rebeccin (rachelmycin), calicheamicin (calicheamicin), docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, porfimer sodium (porfimer sodium) II, temozolomide, topotecan, trimetrexate glucuronate, auristatin E vincristine, and doxorubicin;

peptide cytotoxins, i.e. proteins or fragments thereof having the ability to kill mammalian cells. Some examples include ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, DNA enzyme, and rnase;

radionuclides, i.e. labile isotopes of an element which decay with the simultaneous emission of one or more of alpha or beta particles or gamma rays. Some examples include iodine 131, rhenium 186, indium 111, yttrium 90, bismuth 210 and 213, actinium 225, and astatine 213;

prodrugs, such as antibody-directed enzyme prodrugs; and

an immunostimulant, i.e. a moiety that stimulates an immune response. Some examples include: cytokines, such as IL-2; chemokines, such as IL-8; platelet factor 4; melanoma growth stimulating protein, and the like; an antibody or fragment thereof, e.g., an anti-CD 3 antibody or fragment thereof; a complement activator; a heterologous protein domain; an allogeneic protein domain; viral/bacterial protein domains and viral/bacterial peptides.

The soluble TCRs of the present disclosure are useful for modulating T cell activation by binding to specific TCR ligands, thereby inhibiting T cell activation. Autoimmune diseases involving T cell mediated inflammation and/or tissue damage (e.g., type I diabetes) would be suitable for use in this method. For this use, knowledge of the particular peptide epitopes presented by the relevant pmhcs is required.

Also contemplated is the use of the soluble TCRs and/or multivalent TCR complexes of the present disclosure in the preparation of a composition for treating cancer or autoimmune disease.

Also provided are methods of treating cancer or autoimmune disease comprising administering to a patient in need thereof an effective amount of a soluble TCR and/or multivalent TCR complex of the disclosure.

As is common in anti-cancer and autoimmune therapies, stcrs of the present disclosure can be used in combination with other agents for the treatment of cancer and autoimmune diseases and other related conditions found in similar patient groups.

Methods of treatment

Provided herein are methods for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of an antigen-specific cell therapy, such as a VCX/Y-specific T cell therapy. Adoptive T cell therapy with genetically engineered TCR-transduced T cells conjugating the TCR with other biologically reactive proteins (e.g., anti-CD 3) is also provided herein. In additional embodiments, methods for treating cancer are provided, which include immunizing a subject with a purified tumor antigen or an immunodominant tumor antigen-specific peptide.

The VCX/Y peptides provided herein can be used to develop cancer vaccines or immunogens (e.g., peptides or modified peptide mixtures with adjuvants, encoding polynucleotides and corresponding expression products, such as inactive viral or other microbial vaccines). These peptide-specific vaccines or immunogens can be used to immunize cancer patients directly to induce an anti-tumor immune response in vivo, or to expand antigen-specific T cells in vitro stimulated with peptide-or encoded polynucleotide-loaded APCs. These large numbers of T cells can be adoptively transferred into patients to induce tumor regression.

Some examples of cancers contemplated for treatment include lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, lymphoma, pre-neoplastic lesions in the lung, colon cancer, melanoma, and bladder cancer.

In some embodiments, the T cells are autologous. However, the cells may be allogeneic. In some embodiments, the T cells are isolated from the patient themselves, and thus the cells are autologous. If the T cells are allogeneic, the T cells may be pooled from several donors. The cells are administered to the target subject in an amount sufficient to control, reduce or eliminate symptoms and signs of the disease being treated.

In some embodiments, the subject may be administered a non-myeloablative lymphocyte depleting chemotherapy (nonmyeloablative chemotherapy) prior to the T cell therapy. The non-myeloablative lymphocyte depleting chemotherapy may be any suitable such therapy: it may be administered by any suitable route. Non-myeloablative lymphocyte depletion chemotherapy may include, for example, administration of cyclophosphamide and fludarabine, which may be metastatic, particularly if the cancer is melanoma. One exemplary route of administration of cyclophosphamide and fludarabine is intravenously. Likewise, any suitable dose of cyclophosphamide and fludarabine may be administered. In a particular aspect, about 60mg/kg cyclophosphamide is administered for 2 days, after which about 25mg/m cyclophosphamide is administered²Fludarabine lasted for 5 days.

In certain embodiments, the T cell growth factor that promotes growth and activation of autologous T cells is administered to the subject simultaneously with or subsequent to the autologous T cells. The T cell growth factor may be any suitable growth factor that promotes growth and activation of autologous T cells. Some examples of suitable T cell growth factors include IL-2, IL-7, IL-15, and IL-12, which may be used alone or in various combinations (e.g., IL-2 and IL-7; IL-2 and IL-15; IL-7 and IL-15; IL-2, IL-7, and IL-15; IL-12 and IL-7; IL-12 and IL-15; or IL-12 and IL 2). IL-12 is a preferred T cell growth factor.

T cells may be administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage of T cell therapy can be determined based on the type of disease to be treated, the severity and course of the disease, the clinical status of the individual, the clinical history and response to treatment of the individual, and the judgment of the attending physician.

Intratumoral injection or injection into the tumor vasculature is specifically contemplated for discrete, solid, accessible tumors. Local, regional or systemic administration may also be suitable. For tumours of >4cm, the volume to be administered will be about 4 to 10ml (especially 10ml), whereas for tumours of < 4cm, a volume of about 1 to 3ml (especially 3ml) will be used. Multiple injections delivered as a single dose comprise a volume of about 0.1 to about 0.5 ml.

A. Pharmaceutical composition

Also provided herein are pharmaceutical compositions and formulations comprising an antigen-specific immune cell (e.g., T cell) or receptor (e.g., TCR) and a pharmaceutically acceptable carrier. A vaccine composition for pharmaceutical use in a subject can comprise a tumor antigen peptide (e.g., VCX/Y) composition disclosed herein and a pharmaceutically acceptable carrier.

Pharmaceutical compositions and formulations as described herein may be prepared by mixing an active ingredient (e.g., an antibody or polypeptide) of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's pharmaceutical sciences 22 th edition, 2012), in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited toWithout limitation: buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (for example octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens, for example methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial (injectable) drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, e.g., rHuPH20 (r: (r) () r)Baxter International, Inc.). Certain exemplary shasegps (including rHuPH20) and methods of use are described in U.S. patent publication nos. 2005/0260186 and 2006/0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases (e.g., chondroitinases).

B. Combination therapy

In certain embodiments, the compositions and methods of embodiments of the invention relate to antigen-specific immune cell populations or TCRs in combination with at least one additional therapy. The additional treatment can be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional treatment may be in the form of adjuvant or neoadjuvant therapy.

In some embodiments, the additional treatment is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional treatment is administration of a side-effect limiting agent (e.g., an agent intended to reduce the occurrence and/or severity of a side-effect of the treatment, such as an anti-nausea agent, etc.). In some embodiments, the additional treatment is radiation therapy. In some embodiments, the additional treatment is surgery. In some embodiments, the additional treatment is a combination of radiation therapy and surgery. In some embodiments, the additional treatment is gamma radiation. In some embodiments, the additional treatment is a treatment that targets the PBK/AKT/mTOR pathway, HSP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and/or chemopreventive agent. The additional treatment may be one or more chemotherapeutic agents known in the art.

The immune cell therapy can be administered before, during, after, or in various combinations relative to additional cancer therapies (e.g., immune checkpoint therapies). Administration may be carried out at intervals ranging from simultaneous to several minutes to several days to several weeks. In embodiments where immune cell therapy is provided to the patient separately from the additional therapeutic agent, it will generally be ensured that there will be no failure for a significant period of time between the time of each delivery, such that the two compounds are still able to exert a favorable combined effect on the patient. In this case, it is contemplated that the antibody treatment and the anti-cancer treatment may be provided to the patient within about 12 hours to 24 hours or 72 hours of each other, and more particularly, within about 6 to 12 hours of each other. In some cases, it may be desirable to significantly extend the time period of treatment, with intervals between each administration extending from days (2, 3, 4,5, 6, or 7) to weeks (1, 2, 3, 4,5, 6,7, or 8).

Various combinations may be used. For the following examples, the antigen-specific immune cell therapy, peptide or TCR is "a" and the anti-cancer therapy is "B":

in view of the toxicity, if any, of the agents, administration of any compound or treatment of the embodiments of the present invention to a patient will follow the general protocol for administering such compounds. Thus, in some embodiments, there is a step of monitoring toxicity attributable to the combination therapy.

1. Chemotherapy

A wide variety of chemotherapeutic agents may be used in accordance with embodiments of the present invention. The term "chemotherapy" refers to the use of drugs to treat cancer. "chemotherapeutic agent" is used to mean a compound or composition that is administered in the treatment of cancer. These agents or drugs are classified by their mode of activity within the cell (e.g., whether they affect the cell cycle and at what stage). Alternatively, agents can be characterized based on their ability to directly cross-link DNA, intercalate into DNA, or induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

Some examples of chemotherapeutic agents include: alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzotepa (benzodopa), carboquone (carboquone), metotepipa (meturedpa) and uredepa (uredpa); ethyleneimine and methylmelamine including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethlamelamine; annonaceous acetogenins (especially bullatacin and bullatacin); camptothecin (camptothecin) (including the synthetic analogue topotecan); bryostatin; caristatin (callystatin); CC-1065 (including its adozelesin (adozelesin), carvelesin (carzelesin), and bizelesin (bizelesin) synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin (dolastatin); duocarmycins (duocarmycins) (including the synthetic analogs KW-2189 and CB1-TM 1); eiscosahol (eleutherobin); coprinus atrata base (pancratistatin); sarcandra glabra alcohol (sarcodictyin); spongistatin (spongistatin); nitrogen mustards (nitrosgen mustards), such as chlorambucil (chlorambucil), chlorambucil (chlorenaphazine), cholorphamide (cholorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine hydrochloride (mechlorethamine oxydehydrochloride), melphalan, neomustard (novembichin), benzene mustard (phenyleneterenine), prednimustine (prednimustine), trofosfamide (fosfamide) and uracil mustard (uracil mustard); nitroureas such as carmustine (carmustine), chlorouretocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ranimustine (ranirnustine); antibiotics, such as enediynes (enediynes) antibiotics (e.g., calicheamicins, particularly calicheamicin γ 1I and calicheamicin ω I1; daptomycin (dynemicin), including daptomycin A; bisphosphonates, such as clodronate (clodronate), epothilones (esperamicin), and neocarcinomycin (neocarzinostatin) and related chromoprotein enediynes antibiotic chromophores, aclacinomycin (acarinomysin), actinomycin, anthranomycin (aurramycin), azaserine (azaserine), bleomycin, actinomycin C (cactinomycin), carubicin (carubicin), carzinophilin (carzinophilin), chromomycin (chloramphenicol), chlamycins (chromastatin), doxorubicin (doxorubicin), doxorubicin (5-oxo-noradriamycin), doxorubicin (5-6-noradriamycin), doxorubicin (noradriamycin), doxorubicin (5-6-noradriamycin), doxorubicin (doxorubicin, doxorubicin (noradriamycin), doxorubicin (doxorubicin, doxorubicin (noradriamycin), doxorubicin (doxorubicin, doxorubicin (noradriamycin), doxorubicin, Cyanomorpholino-doxorubicin, 2-pyrrolo-doxorubicin and deoxydoxorubicin), epirubicin, isorubicin, idarubicin (idarubicin), marijumycin (marcellomomycin), mitomycin (e.g., mitomycin C), mycophenolic acid (mycophenolic acid), nogomycin (nogalacycline), olivomycin (olivomycin), pelomycin (peplomycin), pofiomycin (potfimromycin), puromycin, doxorubicin, roxydicin, streptonigrin, streptozotocin (streptazocin), tubercidin, ubenimex, neat-stacin and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as dimethylfolic acid, pteropterin, and trimetrexate (trimetrexate); purine analogs such as fludarabine, 6-mercaptopurine, thiamine (thiamiprine), and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine (doxifluridine), enocitabine, and floxuridine; androgens such as carroterone, dromostanone propionate, epitioandrostanol (epitiostanol), meperiandrane and testolactone; anti-adrenal agents, such as mitotane (mitotane) and trostane; folic acid supplements, such as folinic acid (frilic acid); acetic acid glucurolactone; an aldehydic phosphoramide glycoside; aminolevulinic acid; eniluracil; amsacrine (amsacrine); dimoxystrobin (besrabucil); bisantrene; edatrexate (edatraxate); desphosphamide (defofamine); dimecorsine; diazaquinone (diaziqutone); eflornithine (elformithine); hydroxypyrazole acetate (ellitiniumacetate); epothilones (epothilones); etoglut (etoglucid); gallium nitrate; a hydroxyurea; lentinan (1 entinan); lonidamine (lonidainine); maytansinoids (maytansinoids), such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanol (mopidanmol); diamine nitracridine (nitrarine); pentostatin; phenamet (phenamett); pirarubicin; losoxanthraquinone; podophyllinic acid (podophyllic acid); 2-ethyl hydrazide; procarbazine; PSK polysaccharide complex); razoxane (rizoxane); root toxin (rhizoxin); sisofilan (sizofiran); germanium spiroamines (spirogyranium); alternarionic acid; triimine quinone (triaziquone); 2, 2' -trichlorotriethylamine; trichothecenes (trichothecenes), especially T-2 toxin, verrucin A (verrucatinA), tuberculin A and serpentine (anguidine); urethane (urethan); vindesine; dacarbazine; mannomustine (manomostine); dibromomannitol; dibromodulcitol; pipobromane (pipobroman); gatifloxacin (gacytosine); cytarabine ("Ara-C"); cyclophosphamide; taxanes such as paclitaxel and docetaxel (docetaxel); gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum group; etoposide (VP-16); an ifosfamide; mitoxantrone; vincristine; vinorelbine; nuantro (novantrone); (ii) teniposide; edatrexae; daunorubicin; aminopterin; (xiloda); ibandronic acid (ibandronate); irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicamycin (plicomycin), gemcitabine, noviben, farnesyl protein transferase inhibitors, antiplatin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

2. Radiation therapy

Other factors that cause DNA damage and have been widely used include those commonly referred to as gamma rays, X-rays, and/or targeted delivery of radioisotopes to tumor cells. Other forms of DNA damage factors are also contemplated, such as microwaves, proton beam irradiation (U.S. Pat. nos. 5,760,395 and 4,870,287), and UV irradiation. Most likely all of these factors cause extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The dose of X-rays ranges from a daily dose of 50 to 200 roentgens for a prolonged period of time (3 to 4 weeks) to a single dose of 2000 to 6000 roentgens. The dosage range of radioisotopes varies widely, and depends on the half-life of the isotope, the intensity and type of radiation emitted, and the uptake by neoplastic cells.

3. Immunotherapy

The skilled person will appreciate that additional immunotherapies may be combined or used in conjunction with the methods of some embodiments. In the context of cancer therapy, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. RituximabOne such example is. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may act as an effector of the treatment, or it may recruit other cells to actually affect cell killing. The antibody may also be conjugated to a drug or toxin (b)Chemotherapeutic, radionuclide, ricin a chain, cholera toxin, pertussis toxin, etc.) and act as targeting agents. Alternatively, the effector may be a surface molecule-bearing lymphocyte that interacts directly or indirectly with the tumor cell target. A variety of effector cells include cytotoxic T cells and NK cells.

A breakthrough approach to the development of antibody-drug conjugates as cancer therapeutics has emerged. Cancer is one of the causes of death in the world. Antibody-drug conjugates (ADCs) comprise a monoclonal Antibody (MAb) covalently linked to a drug that kills cells. This approach combines the high specificity of mabs for their antigen targets with highly potent cytotoxic drugs, resulting in "armed" mabs that deliver a payload (drug) to tumor cells with abundant levels of antigen. Targeted delivery of drugs also minimizes their exposure to normal tissues, resulting in reduced toxicity and an increased therapeutic index. FDA approval for two ADC drugs (2011 years)(vildagliptin-brentuximab vedotin) and 2013(trastuzumab emtansine or T-DM1)) validated the method. There are currently over 30 ADC drug candidates at various stages of clinical trials for cancer treatment (Leal et al, 2014). As antibody engineering and linker-payload optimization become more mature, the discovery and development of new ADCs is more dependent on the identification and validation of new targets suitable for this approach and targeted Mab generation. Two criteria for ADC targets are upregulation/high level expression and robust internalization in tumor cells.

In one aspect of immunotherapy, tumor cells must have some marker that can be targeted, i.e., the marker is not present on most other cells. There are many tumor markers, and any of these may be suitable for targeting in the context of embodiments of the present invention. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialylated Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B and p 155. An alternative aspect of immunotherapy is to combine an anti-cancer effect with an immunostimulating effect. Immunostimulatory molecules also exist, including: cytokines such as IL-2, IL-4, IL-12, GM-CSF, γ -IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

Some examples of immunotherapies currently under investigation or in use are immunological adjuvants, such as Mycobacterium bovis (Mycobacterium bovis), Plasmodium falciparum (Plasmodium falciparum), dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al, 1998); cytokine therapies such as interferon alpha, beta and gamma, IL-1, GM-CSF and TNF (Bukowski et al, 1998; Davidson et al, 1998; Hellstrand et al, 1998); gene therapy, such as TNF, IL-1, IL-2 and p53(Qin et al, 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, such as anti-CD 20, anti-ganglioside GM2 and anti-p 185(Hollander, 2012; Hanibuchi et al, 1998; U.S. Pat. No.5,824,311). It is contemplated that one or more anti-cancer treatments may be used with the antibody treatments described herein.

In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints up signal (e.g., co-stimulatory molecules) or down signal. Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade include: adenosine A2A receptor (A2A receptor, A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuating agents (B and Tlymphocyte attenuator, BTLA), cytotoxic T lymphocyte-associated protein 4(cytotoxic T-lymphocyte-associated protein 4, CTLA-4, also known as CD152), indoleamine 2, 3-dioxygenase (IDO), killer-cell immunoglobulin (KIR), lymphocyte activating gene-3 (lymphocyte activating gene-3, LAG3), programmed death 1(programmed death 1, PD-1), T cell immunoglobulin domain and mucin domain 3(T-cell activating gene-3, Ig-cell activating domain of T-immunoglobulin and mucin domain of TIM), VISTA). In particular, the immune checkpoint inhibitor targets the PD-1 axis and/or CTLA-4.

The immune checkpoint inhibitor may be a drug, such as a small molecule, a recombinant form of a ligand or receptor, or in particular an antibody, such as a human antibody (e.g. international patent publication WO 2015016718; pardol, Nat Rev Cancer; 12 (4): 252-64, 2012; both incorporated herein by reference). Known inhibitors of immune checkpoint proteins or analogs thereof may be used, in particular chimeric, humanized or human forms of antibodies may be used. As will be appreciated by those of skill in the art, alternative and/or equivalent names may be used for certain antibodies mentioned in the present disclosure. In the context of the present disclosure, such alternative and/or equivalent designations are interchangeable. For example, lambrolizumab is known under the alternative and equivalent names MK-3475 and pembrolizumab.

In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a particular aspect, the PD-1 ligand binding partner is PDL1 and/or PDL 2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In a particular aspect, the PDL1 binding partner is PD-1 and/or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In a particular aspect, the PDL2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesion protein, a fusion protein, or an oligopeptide. Some exemplary antibodies are described in U.S. patent nos. US8735553, US8354509, and US8008449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art, for example, as described in U.S. patent application nos. US20140294898, US2014022021, and US20110008369, all of which are incorporated herein by reference.

In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, an anti-PD-1 antibodySelected from the group consisting of nivolumab, pembrolizumab and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesion protein (e.g., an immunoadhesion protein comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558 andis an anti-PD-1 antibody described in WO 2006/121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab,And SCH-900475, which is an anti-PD-1 antibody described in WO 2009/114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO 2009/101611. AMP-224, also known as B7-DCIg, is the PDL2-Fc fusion soluble receptor described in WO2010/027827 and WO 201I/066342.

Another immune checkpoint that may be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4(CTLA-4), also known as CD 152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to T cell costimulatory protein CD28, and both molecules bind to CD80 and CD86 (also referred to as B7-1 and B7-2, respectively) on antigen presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and may be important to their function. T cell activation by T cell receptors and CD28 results in increased expression of CTLA-4, an inhibitory receptor for the B7 molecule.

In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesion protein, a fusion protein, or an oligopeptide.

Anti-human CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the methods of the invention can be produced using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies may be used. For example, anti-CTLA-4 antibodies disclosed in the following may be used in the methods disclosed herein: US8,119,129, WO 01/14424, WO 98/42752; WO 00/37504(CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Pat. No.6,207,156; hurwitz et al, (1998) Proc Natl Acad Sci USA 95 (17): 10067-10071; camacho et al, (2004) J Clin Oncology 22 (145): digest No.2505 (antibody CP-675206); and Mokyr et al, (1998) Cancer Res 58: 5301-5304. The teachings of each of the above publications are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in international patent application nos. WO2001014424, WO2000037504 and us patent No.8,017,114; all incorporated herein by reference.

Exemplary anti-CTLA-4 antibodies are ipilimumab (also referred to as 10D1, MDX-010, MDX-101, and) Or antigen-binding fragments and variants thereof (see, e.g., WO 01/14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes with the above antibody for binding to the same epitope on CTLA-4 and/or binding to the same epitope on CTLA-4. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity to the above-described antibody (e.g., at least about 90%, 95%, or 99% variable region identity to ipilimumab).

Other molecules that may be used to modulate CTLA-4 include CTLA-4 ligands and receptors such as described in U.S. Pat. Nos. US5844905, US5885796 and International patent application Nos. WO1995001994 and WO1998042752, all of which are incorporated herein by reference, and immunoadhesin such as described in U.S. Pat. No. US8329867, which is incorporated herein by reference.

Surgery

About 60% of people with cancer will undergo some type of surgery, including preventative, diagnostic or staging, curative and palliative surgery. Curative surgery includes resection in which all or a portion of cancerous tissue is physically removed, excised, and/or destroyed, and may be used in conjunction with other therapies (e.g., the therapies, chemotherapies, radiation therapies, hormonal therapies, gene therapies, immunotherapies, and/or alternative therapies of embodiments of the present invention). Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery).

After resection of a portion or all of the cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be achieved by perfusion, direct injection or local administration of an additional anti-cancer treatment to the area. Such treatment may be repeated, for example, every 1, 2, 3, 4,5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4,5, 6,7, 8, 9, 10, 11, or 12 months. These treatments may also have multiple doses.

5. Additional agents

It is contemplated that additional agents may be used in combination with certain aspects of the present embodiments to increase the therapeutic efficacy of the treatment. These additional agents include agents that affect upregulation of cell surface receptor-GAP junctions, cytostatics and differentiating agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis-inducing agents or other biological agents. Increasing intercellular signaling by increasing the number of GAP junctions will increase the anti-hyperproliferative effects of the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents may be used in combination with certain aspects of the present embodiments to increase the anti-hyperproliferative efficacy of the treatments. Cell adhesion inhibitors are contemplated to enhance the efficacy of embodiments of the present invention. Some examples of cell adhesion inhibitors are Focal Adhesion Kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis (e.g., antibody c225) may be used in combination with certain aspects of embodiments of the invention to increase the efficacy of the treatment.

Article of manufacture or kit

Also provided herein are articles of manufacture or kits comprising antigen-specific immune cells, TCRs, or antigenic peptides (e.g., VCX/Y peptides). The article of manufacture or kit can further comprise a package insert comprising instructions for using the antigen-specific immune cells to treat or delay progression of cancer in an individual or to enhance immune function of an individual having cancer. Any of the antigen-specific immune cells described herein can be contained in an article of manufacture or a kit. Suitable containers include, for example, bottles, vials, bags, and syringes. The container may be formed from a variety of materials such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or metal alloys (e.g., stainless steel or hastelloy). In some embodiments, the container contains the formulation, and a label on or associated with the container can indicate the instructions for use. The article of manufacture or kit may also contain other materials desirable from a commercial and user standpoint, including additional buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture further comprises one or more additional agents (e.g., chemotherapeutic agents and antineoplastic agents). Suitable containers for the one or more medicaments include, for example, bottles, vials, bags, and syringes.

VII. examples

The following examples are included to illustrate some preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 identification and characterization of tumor antigen-specific peptides

Epitope prediction tools including BIMAS, SYFPEITHI and NetMHC analyses were used to predict HLA-restricted peptide epitopes of the VCX3A antigen. The results of VCX3A binding and affinity prediction are shown in table 1.

Table 1: HLA-a0201 peptide binding and affinity prediction for VCX 3A.

The predicted peptide was synthesized and co-cultured with T2 cells with a series of dilution concentrations. DMSO without peptide or at the same concentration that negatively binds to HLA-a2 peptide was used as a control. After 18 hours of incubation, HLA-a2 expression was detected by flow cytometry. Fluorescence Index (FI) was calculated as follows: FI ═ average fluorescence with a given peptide-average fluorescence without peptide)/(average fluorescence without peptide). From the binding assay, the VCX54 peptide was found to show the strongest binding to the HLA-a2 allele among the 3 predicted peptides (fig. 1A). The VCX54 epitope is present in all members of the VCX/Y family including VCX1, VCX2, VCX3A, VCX3B, or VCY expressed in various lung cancer cells (fig. 1B).

Next, VCX 54-specific T cell clones were generated from HLA-a0201 healthy donors. CD8 was observed after 2 stimulations with VCX3A mRNA pulsed dendritic cells⁺And VCX54 tetramer⁺T cell population (fig. 2A). Following tetramer-directed sorting, cells are expanded using a Rapid Expansion Protocol (REP). T cell clones were generated by limiting dilution and subjected to 2 amplifications. More than 99% of the cells were observed to be CD8⁺And tetramers⁺(FIG. 2B).

The functional avidity of VCX 54-specific T cells was tested. The VCX54CTL clone was co-cultured with T2 cells pulsed with various concentrations of VCX54 peptide at an effector to target (E: T) ratio of 20: 1. Using standards₅₁The Cr Release Assay (CRA) detects cytotoxic lysis (fig. 3). VCX54CTL clone (C7) and VCX1 positive expression human lung cancer cell line H2023(HLA-A*0201⁺) Or primary bronchial epithelial cell NHBE (HLA-A0201)⁺) Co-culture at various E: T ratios. Using standards₅₁The Cr Release Assay (CRA) detects cytotoxic lysis.

To determine the recognition of endogenously presented VCX54 peptides by specific T cells, a panel of HLA-a2 positive lung cancer cell lines were tested. The VCX54CTL clones were co-cultured with the HLA-A2+ lung cancer cell line groups expressing VCY, VCX3A or VCX1 at various E: T ratios. HLA-A2 positive primary bronchial epithelial NHBE cells were used to test the cytotoxicity of CTL clones against normal lung tissue. Using standards₅₁The Cr Release Assay (CRA) detects specific cytotoxic lysis. High levels of cytotoxicity of VCX54CTL clone were observed in lung cancer cells but not in primary bronchial epithelial NHBE cells (fig. 4). Thus, the VCX54CTL clone was selectively cytotoxic to cancer cells expressing the VCX antigen.

Next, HLA allele restriction analysis of VCX/Y specific T cells was performed. Several HLA-A0201 negative lung cancer cell lines were analyzed using the CRA assay to detect HLA restriction of VCX54CTL clones. The PC-9 cell line is positive for HLA-A0206/A2402, and the H650 cell line is positive for HLA-A2402. Forced expression of the HLA-a0201 allele in both cell lines significantly enhanced the level of cytotoxic lysis of CTL clones (fig. 5). H1573 is an HLA-A0201 and HLA-A2402 negative cell line. Forced expression of the HLA-A0201 allele enhanced cytotoxic lysis, but not at high levels. Co-transfection of the HLA-A0201 allele with the VCX3A gene was found to significantly enhance the level of cytotoxic lysis of CTL clones.

In addition, specific cytotoxicity of VCX-specific T cells against lung cancer cells was demonstrated. Endogenous presented peptide specific recognition of VCX54CTL clones was detected using a cold target inhibition assay. The hot target was lung cancer cells (positive or forced expression of HLA-A2). Cold targets were T2 cells pulsed with VCX54 peptide (10. mu.g/ml). T2 cells without any peptide or pulsed with M26 peptide (10. mu.g/ml) were used as negative controls. The ratio of E to T is 10: 1. The ratio of cold target to hot target is 10: 1 or 20: 1. Significant inhibition was observed when T2 cells were pulsed with VCX54 peptide at a ratio of cold to hot targets of 20: 1 (fig. 6).