阅读说明：本技术 诱导t细胞的疫苗组合物组合及其用途 (T cell-inducing vaccine composition combination and application thereof ) 是由 伯特兰·乔治 斯科特·罗伯茨 于 2019-04-04 设计创作，主要内容包括：本文提供了疫苗组合和用于在有相应需要的受试者中增强抗原特异性T细胞诱导的应答的方法。所述方法将全身性疫苗接种与第一组合物和/或第二组合物以及任选地第三组合物组合,所述第一组合物包含编码抗原或免疫原的非复制型病毒载体,所述抗原或免疫原包含一个或更多个CD8+ T细胞表位；所述第二组合物具有包含碳氟化合物连接的肽的胶束,其中每种与碳氟化合物连接的肽：i)长度为15个至75个氨基酸残基；ii)来自第一组合物的抗原或免疫原；并且iii)包含第一组合物的来自抗原或免疫原的一个或更多个CD8+ T细胞表位,以诱导抗原特异性CD8+ T细胞；所述第三组合物包含免疫调节剂组合物。(Provided herein are vaccine combinations and methods for enhancing antigen-specific T cell-induced responses in a subject in need thereof. The method combines systemic vaccination with a first composition comprising a non-replicating viral vector encoding an antigen or immunogen comprising one or more CD8+ T cell epitopes and/or a second composition and optionally a third composition; the second composition has micelles comprising fluorocarbon-linked peptides, wherein each fluorocarbon-linked peptide: i) 15 to 75 amino acid residues in length; ii) an antigen or immunogen from the first composition; and iii) one or more CD8+ T cell epitopes from an antigen or immunogen comprising the first composition to induce antigen-specific CD8+ T cells; the third composition comprises an immunomodulator composition.)

1.A vaccine combination comprising two to three compositions selected from:

a) a first composition comprising a non-replicating viral vector encoding an antigen or immunogen comprising one or more CD8+ T cell epitopes;

b) a second composition comprising micelles comprising fluorocarbon-linked peptides, wherein each peptide linked to a fluorocarbon: i) 15 to 75 amino acid residues in length; ii) an antigen or immunogen from the first composition; and iii) one or more CD8+ T cell epitopes from the antigen or immunogen comprising the first composition; and

c) a third composition comprising an immunomodulator selected from an anti-immune repressor or an immune activator,

wherein when the first composition and the second composition are selected, either the first composition or the second composition is a priming composition or a boosting composition.

2. The vaccine combination of claim 1, wherein the anti-immune repressor targets PD, PDL, CD, CTLA, B7RP, ICOS, B7RPI, B-H, BTLA, HVEM, KIR, TCR, LAG, CD137, OX40, CD40, TIM, GAL, ADORA, CD276, VTCN, IDO, KIR3DL, HAVCR, VISTA, CD244, ADAM, COX, PGE-2, iNOS, PDE, c-kit, ARG, PI3, CSF-1R, caspase-8, CCL, RON, ROS, or S100A/a.

3. The vaccine combination of claim 2, wherein the anti-immune repressor targets PD1 or PDLl.

4. The vaccine combination of claim 2, wherein the anti-immune repressor is an anti-PD 1 antibody or an anti-PDLl antibody.

5. The vaccine combination of claim 1, wherein the immune activator targets Toll-like receptor (TLR)3, TLR4, TLR5, TLR7, TLR8, TLR9, NOD1, NOD2, STING, cGAS, IFR3, IL-2 receptor, IL12 receptor, or IFN-a receptor.

6. The vaccine combination of claim 1, wherein the non-replicating viral vector is an adenoviral vector, an alphaviral vector, a herpesvirus vector, a measles virus vector, a poxvirus vector, or a vesicular stomatitis virus vector.

7. The vaccine combination of claim 6, wherein the non-replicating viral vector is an E1 and E3 deleted adenovirus vector.

8. The vaccine combination of claim 1, wherein the first, second, or third composition is formulated for parenteral administration, oral administration, ocular administration, rectal administration, nasal administration, transdermal administration, topical administration, or vaginal administration.

9. The vaccine combination of claim 1, wherein the antigen or immunogen is from a pathogen.

10. The vaccine combination of claim 9, wherein the pathogen is a virus, fungus, parasite or bacterium.

11. The vaccine combination of claim 10, wherein the virus is EBV, HPV, HTLV-1, MCPvV, KSHV, HERV, HCV, or HBV.

12. The vaccine combination of claim 1, wherein the antigen or immunogen is a cancer antigen.

13. The vaccine combination of claim 1, wherein the first composition is a priming composition and the second composition is a boosting composition.

14. The vaccine combination of claim 1, wherein the second composition is a priming composition and the first composition is a boosting composition.

15. The vaccine combination of claim 1, wherein the fluorocarbon moiety of the fluorocarbon-linked peptide is a fluorocarbon chain of 3 to 30 carbon atoms.

16. The vaccine combination of claim 15, wherein one or more fluorine moieties of the fluorocarbon chain are substituted with chlorine, bromine, iodine, hydrogen or methyl groups.

17. The vaccine combination of claim 1, wherein the fluorocarbon-linked peptide has structure C_mF_n—C_yH_x- (Sp) -R, wherein m is 3 to 30, n<2m +1, y 0 to 15, x<2y, (m + y) ═ 3-30, Sp is an optional chemical spacer moiety, and R is the peptide.

18. The vaccine combination of claim 1, wherein the fluorocarbon-linked peptide is according to structure

Wherein Sp is an optional chemical spacer moiety and R is the peptide.

19. The vaccine combination of claim 1, wherein the fluorocarbon-linked peptide of the second composition comprises at least one MHC class II binding epitope and at least one MHC class I binding epitope.

20. The vaccine combination of claim 1, wherein each of the first, second or third compositions independently further comprises one or more pharmaceutically acceptable carriers, excipients, diluents or adjuvants.

21. The vaccine combination of claim 20, wherein the adjuvant is an agonist of TLR2, TLR3, TLR7, TLR8 or TLR9, STING, cGAS, IFR3, NOD1 or NOD 2.

22. The vaccine combination of claim 1, wherein the first composition, the second composition, or the third composition is in the form of a liquid, an emulsion, a solid, an aerosol, a mist, or a gas.

23. The vaccine combination of claim 1, wherein the first composition and the second composition are selected.

24. The vaccine combination of claim 1, wherein one of the first or second compositions is selected and the third composition is selected.

25. A method of inducing an immune response in a subject in need thereof, the method comprising:

a) administering a first composition comprising a non-replicating viral vector encoding an antigen or immunogen comprising one or more CD8+ T cell epitopes; and the number of the first and second groups,

b) administering a second composition comprising micelles comprising fluorocarbon-linked peptides, wherein each peptide linked to the fluorocarbon: i) 15 to 75 amino acid residues in length; ii) an antigen or immunogen from the first composition; and iii) one or more CD8+ T cell epitopes from an antigen or immunogen of the first composition,

wherein one of the first composition or the second composition is administered as a priming dose and one of the first composition or the second composition is administered as a boosting dose, provided that both the first composition and the second composition are administered,

thereby inducing an antigen-specific CD8+ T cell response.

26. The method of claim 25, wherein the prime and boost doses are administered at least 14 days apart.

27. The method of claim 25, wherein the second composition is a priming dose and the first composition is a boosting dose.

28. The method of claim 25, wherein the first composition is a prime dose and the second composition is a boost dose.

29. The method of claim 25, wherein the subject is a mammal, bird, reptile, amphibian, or fish.

30. The method of claim 2, wherein the subject is a human, bovine, canine, feline, goat, ovine, porcine, equine, turkey, duck, or chicken.

31. The method of claim 25, further comprising administering a third composition comprising an immunomodulator selected from an anti-immune repressor or an immune activator.

32. The method of claim 31, wherein the anti-immune repressor targets PD, PDL, CD, CTLA, B7RP, ICOS, B7RPI, B-H, BTLA, HVEM, KIR, TCR, LAG, CD137, OX40, CD40, TIM, GAL, ADORA, CD276, VTCN, IDO, KIR3DL, HAVCR, VISTA, CD244, ADAM, COX, PGE-2, iNOS, PDE, c-kit, ARG, PI3, CSF-1R, caspase-8, CCL, RON, ROS, or S100A/a.

33. The method of claim 31, wherein the anti-immune repressor targets PD1 or PDLl.

34. The method of claim 31, wherein the anti-immune repressor is an anti-PD 1 antibody or an anti-PDLl antibody.

35. The method of claim 31, wherein the immune activator targets Toll-like receptor (TLR)3, TLR4, TLR5, TLR7, TLR8, TLR9, NOD1, NOD2, STING, cGAS, IFR3, IL-2 receptor, IL12 receptor, or IFN- α receptor.

36. The method of claim 31, wherein the step of administering the third composition is performed after administering the first composition.

37. The method of claim 25, wherein the antigen or immunogen is from a pathogen.

38. The method of claim 37, wherein the pathogen is a virus, fungus, parasite, or bacterium.

39. The method of claim 38, wherein the virus is EBV, HPV, HTLV-1, MCPvV, KSHV, HERV, HCV, or HBV.

40. The method of claim 25, wherein the antigen or immunogen is a cancer antigen.

41. The method of claim 25, wherein the induced immune response is a therapeutic or prophylactic treatment for: non-small cell lung cancer, breast cancer, liver cancer, brain cancer, stomach cancer, pancreatic cancer, kidney cancer, ovarian cancer, myeloma cancer, acute myelogenous leukemia, chronic myelogenous leukemia, head and neck cancer, colorectal cancer, kidney cancer, esophageal cancer, melanoma skin cancer, and/or prostate cancer.

42. The method of claim 25, wherein the induced immune response is a therapeutic or prophylactic treatment against a subject having a chronic infection or at risk of exposure to a pathogen that causes a chronic infection, wherein the pathogen is selected from Human Immunodeficiency Virus (HIV), hepatitis b virus, hepatitis d virus, herpes virus, epstein barr virus, cytomegalovirus, human papilloma virus, or human T-lymphotropic virus type III.

43. The method according to claim 26, wherein the route of administration of the first composition and/or the second composition is selected from parenteral, subcutaneous, oral, epidermal, intradermal, intramuscular, intraarterial, intraperitoneal, intravenous, nasal, intratracheal, enteral, sublingual, rectal or vaginal.

44. The method of claim 25, wherein the non-replicating viral vector is an adenoviral vector, an alphaviral vector, a herpesvirus vector, a measles virus vector, a poxvirus vector, or a vesicular stomatitis virus vector.

45. The method of claim 44, wherein the non-replicating viral vector is an E1 and/or E3 deleted adenovirus vector.

46. The method of claim 25, wherein the fluorocarbon moiety of the fluorocarbon-linked peptide is a fluorocarbon chain of 3 to 30 carbon atoms.

47. The method of claim 46, wherein one or more fluorine moieties of the fluorocarbon chain are substituted with chlorine, bromine, iodine, hydrogen or methyl groups.

48. The method of claim 25, wherein the fluorocarbon-linked peptide has structure C_mF_n—C_yH_x- (Sp) -R, wherein m is 3 to 30, n<2m +1, y 0 to 15, x<2y, (m + y) ═ 3-30, Sp is an optional chemical spacer moiety, and R is the peptide.

49. The method of claim 25, wherein the fluorocarbon-linked peptide is according to structure

Wherein Sp is an optional chemical spacer moiety and R is the peptide.

50. The method of claim 25, wherein the fluorocarbon-linked peptide of the second composition comprises at least one MHC class II binding epitope and at least one MHC class I binding epitope.

51. A method of inducing an immune response in a subject in need thereof, the method comprising:

a) administering a first composition comprising a non-replicating viral vector encoding an antigen or immunogen or fragment thereof comprising one or more CD8+ T cell epitopes; alternatively, the first and second electrodes may be,

b) administering a second composition comprising micelles comprising fluorocarbon-linked peptides, wherein each peptide linked to the fluorocarbon:

i) 15 to 75 amino acid residues in length;

ii) from an antigen or immunogen; and the number of the first and second electrodes,

iii) comprises one or more CD8+ T cell epitopes; and the number of the first and second groups,

c) separately administering a third composition comprising an immunomodulator selected from an anti-immune repressor or an immune activator,

thereby inducing an antigen-specific CD8+ T cell response.

52. The method of claim 51, wherein the subject is a mammal, bird, reptile, amphibian, or fish.

53. The method of claim 51, wherein the subject is a human, bovine, canine, feline, goat, ovine, porcine, equine, turkey, duck, or chicken.

54. The method of claim 51, wherein the anti-immune repressor targets PD, PDL, CD, CTLA, B7RP, ICOS, B7RPI, B-H, BTLA, HVEM, KIR, TCR, LAG, CD137, OX40, CD40, TIM, GAL, ADORA, CD276, VTCN, IDO, KIR3DL, HAVCR, VISTA, CD244, ADAM, COX, PGE-2, iNOS, PDE, c-kit, ARG, PI3, CSF-1R, caspase-8, CCL, RON, ROS, or S100A/A.

55. The method of claim 51, wherein the anti-immune repressor targets PD1 or PDLl.

56. The method of claim 51, wherein the anti-immune repressor is an anti-PD 1 antibody or an anti-PDLl antibody.

57. The method of claim 51, wherein the immune activator targets Toll-like receptor (TLR)3, TLR4, TLR5, TLR7, TLR8, TLR9, NOD1, NOD2, STING, cGAS, IFR3, IL-2 receptor, IL12 receptor, or IFN- α receptor.

58. The method of claim 51, wherein the step of administering the third composition is performed after administering the first composition or the second composition.

59. The method of claim 51, wherein the antigen or immunogen is from a pathogen.

60. The method of claim 59, wherein the pathogen is a virus, fungus, parasite or bacterium.

61. The method of claim 60, wherein the virus is EBV, HPV, HTLV-1, MCPvV, KSHV, HERV, HCV or HBV.

62. The method of claim 51, wherein the antigen or immunogen is a cancer antigen.

63. The method of claim 51, wherein the induced immune response is a therapeutic or prophylactic treatment for: non-small cell lung cancer, breast cancer, liver cancer, brain cancer, stomach cancer, pancreatic cancer, kidney cancer, ovarian cancer, myeloma cancer, acute myelogenous leukemia, chronic myelogenous leukemia, head and neck cancer, colorectal cancer, kidney cancer, esophageal cancer, melanoma skin cancer, and/or prostate cancer.

64. The method of claim 51, wherein the induced immune response is a therapeutic or prophylactic treatment against a subject having a chronic infection or at risk of exposure to a pathogen that causes a chronic infection, wherein the pathogen is selected from Human Immunodeficiency Virus (HIV), hepatitis B virus, hepatitis D virus, herpes virus, Epstein-Barr virus, cytomegalovirus, human papilloma virus, or human T-lymphotropic virus type III.

65. The method of claim 51, wherein the route of administration of the first composition or the second composition and the third composition is independently selected from parenteral, subcutaneous, oral, epidermal, intradermal, intramuscular, intraarterial, intraperitoneal, intravenous, nasal, intratracheal, enteral, sublingual, rectal, or vaginal.

66. The method of claim 51, wherein the non-replicating viral vector is an adenoviral vector, an alphaviral vector, a herpesvirus vector, a measles virus vector, a poxvirus vector, or a vesicular stomatitis virus vector.

67. The method of claim 66, wherein the non-replicating viral vector is an E1 and/or E3 deleted adenovirus vector.

68. The method of claim 51, wherein the fluorocarbon moiety of the fluorocarbon-linked peptide is a fluorocarbon chain of 3 to 30 carbon atoms.

69. The method of claim 68, wherein one or more fluorine moieties of the fluorocarbon chain are substituted with chlorine, bromine, iodine, hydrogen or methyl groups.

70. The method of claim 51, wherein the fluorocarbon-linked peptide has structure C_mF_n—C_yH_x- (Sp) -R, wherein m is 3 to 30, n<2m +1, y 0 to 15, x<2y, (m + y) ═ 3-30, Sp is an optional chemical spacer moiety, and R is the peptide.

71. The method of claim 51, wherein the fluorocarbon-linked peptide is according to structure

Wherein Sp is an optional chemical spacer moiety and R is the peptide.

72. The method of claim 51, wherein the fluorocarbon-linked peptide of the second composition comprises at least one MHC class II binding epitope and at least one MHC class I binding epitope.

Technical Field

The present application relates generally to vaccine combinations comprising a priming dose and/or a heterologous booster dose, and optionally an immunosuppressant dose, and methods for inducing an antigen-specific CD8+ T cell response.

Background

Vaccines are limited in their ability to promote robust CD4+ and/or CD8+ T cell responses, which are known to play an important role against diseases induced by intracellular pathogens and cancer. The only exception is that BCG vaccines against tuberculosis appear to be protected primarily by induction of T cells.

Much effort has been made to generate T cell-inducing vaccines designed to induce CD4+ T cells and/or CD8+ T cells of sufficient magnitude and effector function to directly promote clearance of infected or tumor cells. Vaccines using synthetic peptides or DNA in combination with a delivery system or recombinant viral vector are of particular interest for inducing cell-mediated immunity by providing the ability to deliver or express antigens intracellularly. However, homologous priming boost regimens, in which the priming and boosting doses of antigen/immunogen/peptide are presented to the immune system via the same delivery vehicle (carrier) and/or vector (vector), have not been able to demonstrate significant clinical efficacy due to limited ability to generate robust and durable antiviral and antitumor T cell immunity.

Heterologous prime boost vaccination using different delivery vehicles and/or vectors represents a promising strategy in inducing T cell immunity compared to homologous prime boost, due to: i) attenuated anti-viral vector antibody responses (anti-viral vector antibodies are known to interfere with immunity to the target antigen by clearing the vaccine through the vaccine-antibody immune complex); and ii) the potential of different vaccine technologies to stimulate immune responses differently and work synergistically. Heterologous prime boost approaches have been investigated for vaccines of various compositions, but have recently focused mainly on the clinical development of DNA/viral vector combinations or viral vector/viral vector combinations. Heterologous prime/boost vaccine strategies are of particular interest for acute and chronic viral infections, cancer, allergy and autoimmunity due to the prospect of stimulating strong and persistent T cell immunity.

Vaccines against cancer or chronic viral infections represent an attractive approach to offer high specificity, a good safety profile, ready applicability and a life-long anti-tumor immune prospect compared to other therapies. Although T cell vaccines have been greatly improved, they still fail to provide any clinical benefit as monotherapy in patients with advanced cancer or chronic viral infection in general.

In the case of cancer and chronic viral infections, immunosuppressive mechanisms prevent or reduce the effector activity of antigen-specific T cells produced by vaccine immunotherapy. These immunosuppressive mechanisms may act directly or indirectly through inhibitory myeloid cells, tumor-associated macrophages, regulatory T cells and/or inhibitory receptors expressed on T cells. Thus, anti-immune repressors (anti-immune repressors), such as checkpoint blockade inhibitors, inhibitory myeloid inhibitors or compounds involved in inhibitory myeloid cell reprogramming or repolarization, represent a class of therapeutic agents with the potential to improve the induction and antiviral or anti-tumor function of antigen-specific T cells generated by vaccine immunotherapy. In addition, immune activators such as agents targeting co-stimulatory receptors expressed by T cells, cytokines, or immune stimulators may also be used to improve the induction of antigen-specific T cells and antiviral or anti-tumor function resulting from vaccine immunotherapy.

Therefore, there is a need for more robust vaccine compositions that induce cell-mediated immune responses, particularly in the case of cancer and chronic infections.

Summary of The Invention

Some embodiments provided herein include vaccine combinations and methods for administering a heterologous prime boost dosing regimen, wherein the prime dose is different from the booster dose of the vaccine composition, provided that each dose comprises one or more of the same CD8+ T cell epitopes. In other words, the vaccine composition comprises polypeptides having at least one common CD8+ T cell epitope. The vaccine composition is a T cell-inducing vaccine composition; and CD4 only⁺T cell helper B cell production protective antibody response compared to vaccines designed to induce CD4 of sufficient magnitude and necessary phenotype or effector function⁺T cells and/or CD8⁺T cells, said CD4⁺T cells and/or CD8⁺T cells directly promote pathogen or tumor clearance via cell-mediated effector mechanisms.

In certain embodiments, there is provided a vaccine combination comprising: a) a first composition comprising a non-replicating viral vector encoding an antigen or immunogen comprising one or more CD8+ T cell epitopes; and b) a second composition comprising micelles comprising fluorocarbon-linked peptides, wherein each fluorocarbon-linked peptide: i) 15 to 75 amino acid residues in length; ii) an antigen or immunogen from the first composition; and iii) one or more CD8+ T cell epitopes from the antigen or immunogen comprising the first composition. See example 1. Either of the first composition or the second composition may be a priming composition or a boosting composition. In certain embodiments, the vaccine combination may further comprise an immunomodulator comprising an anti-immune repressor or an immune activator.

In embodiments, the non-replicating viral vector is an adenoviral vector, an alphaviral vector, a herpesvirus vector, a measles virus vector, a poxvirus vector, or a vesicular stomatitis virus vector. In certain embodiments, the non-replicating viral vector is an E1 and E3 deleted adenovirus vector.

In exemplary embodiments, the vaccine combination is used (prophylactically or therapeutically) as a cancer vaccine or a chronic infection vaccine in a method of inducing an immune response. For cancer vaccines, the challenge of finding patient-specific antigens is not too great (although this is still important), but the challenge lies in developing a more immunogenic approach to inducing the desired immune response, which requires breaking immune tolerance to self-antigens. Herein, the applicant provides a highly immunogenic dosing regimen, wherein the heterologous dosing regimen (heterologous dosing regein) is a synergistic agent compared to the homologous dosing in the murine (murine) tumor model tested herein. See example 3 and figures 1-2.

Accordingly, provided herein are methods for inducing an immune response (e.g., an antigen CD8+ T cell response) in a subject in need thereof, the method comprising administering a first composition comprising a non-replicating viral vector encoding an antigen or immunogen comprising one or more CD8+ T cell epitopes; and administering a second composition comprising micelles comprising fluorocarbon-linked peptides, wherein each fluorocarbon-linked peptide: i) 15 to 75 amino acid residues in length; ii) an antigen or immunogen from the first composition; and iii) one or more CD8+ T cell epitopes from the antigen or immunogen comprising the first composition. One of the first or second compositions is administered as a priming dose and the other of the first or second compositions is administered as a boosting dose, provided that both the first and second compositions are administered.

Further embodiments provided herein include vaccine combinations and methods for administering those compositions of vaccine combinations, wherein the combination comprises induced T cellsAnd compositions comprising an immunosuppressive agent selected from an immune checkpoint inhibitor or a Myeloid Derived Suppressor Cell (MDSC) inhibitor. And CD4 only⁺T cell helper B cell production protective antibody response in contrast to a vaccine composition that induces T cells is CD4 designed to induce sufficient magnitude and necessary phenotypic or effector function⁺T cells and/or CD8⁺Vaccine for T cells, said CD4⁺T cells and/or CD8⁺T cells directly promote pathogen or tumor clearance via cell-mediated effector mechanisms. In embodiments, the vaccine composition comprises a polypeptide having at least one CD8+ T cell epitope. In certain embodiments, the T cell-inducing vaccine compositions of the present invention comprise a non-replicating viral vector or a fluorocarbon-linked peptide.

In certain embodiments, there is provided a vaccine combination comprising: a first composition comprising a non-replicating viral vector encoding an antigen or immunogen comprising one or more CD8+ T cell epitopes; or b) a second composition comprising micelles comprising fluorocarbon-linked peptides, wherein each fluorocarbon-linked peptide: i) 15 to 75 amino acid residues in length; ii) from an antigen or immunogen; and iii) one or more CD8+ T cell epitopes comprising an antigen or immunogen; and c) a third composition comprising an immunomodulator selected from an anti-immune repressor or an immune activator.

In embodiments, the anti-immune repressor targets PD1, PDL1, PDL2, CD28, CD80, CD86, CTLA4, B7RP1, ICOS, B7RPI, B7-H3, B7-H4, BTLA, HVEM, KIR, TCR, LAG3, CD137L, OX40, OX40L, CD27, CD70, CD40, CD40L, TIM3, GAL1, GAL3, GAL9, ADORA, CD276, VTCN1, IDO1, KIR3DL1, HAVCR 1, VISTA, CD244, ADAM1, COX 1, PGE-2, iipde 1, PDE 1, c-kit, ARG1, PI 1, CSF-1R, CCL-8-CCL 1, caspase 1, COX 1, ROS 1, or ROS 1 a. In embodiments, the anti-immune repressor is Pembrolizumab (KEYTRUDA), nivolumab (OPDIVO), Cemiplimumab (LIBTAYO), Attributumab (TECENTREQ), Avermectimab (BAVENTIO), Devolumab (IMFINZI), Ipilimumab (Iilimumab, YERVOY), REGN2810, BMS-936558, SHR1210, KN035, IBI308, PDR001, BGB-A317, BCD-100, or JS 001.

In certain other embodiments, the anti-immune repressor is an MDSC inhibitor that targets PGE-2, COX2, NOS2, ARG1, PI3K, CSF-1R, caspase-8, CCL2, RON, ROSS100A8/A9, or the liver X nuclear receptor. In embodiments, the MDSC inhibitor is PF-5480090, INCB7839, nitroaspirin, SC58236, celecoxib, IPI-549, PLX3397, BLZ945, GW2580, RG7155, IMC-CS4, AMG-820, ARRY-382, sildenafil, tadalafil, vardenafil, N-hydroxy-nor-L-Arg, imatinib, z-IETD-FMK, trabectedin, Emricasan, anti-CCL 2 antibody (carlumab or ABN912), taconimod, ASLAN002, IMC-RON8, or GW 3965.

In certain embodiments, the immune activator targets Toll-like receptor (TLR)3, TLR4, TLR5, TLR7, TLR8, TLR9, NOD1, NOD2, STING, cGAS, IFR3, IL-2 receptor, IL12 receptor, or IFN- α receptor. In certain embodiments, the immune activator is IMO-2125, SD-101, DV281, ADZ1419, PF-3512676(AGATOLIMOD), CMP-001, Lefitolimod, IC31, MEDI9197, RO6864018, RO7020531, GS-9620, AZD8848, LFX453, CV8102, Motolimod (VTX-2337), BDB001, HILTONOL, KIN131A, MK-4621(RGT100), Inarigivir (SB9200), 39815 (ADU-S100), MK-1454, BMS-986301, SB 11285, IL-2, IL-12, or IFN- α.

In embodiments, the non-replicating viral vector is an adenoviral vector, an alphaviral vector, a herpesvirus vector, a measles virus vector, a poxvirus vector, or a vesicular stomatitis virus vector. In certain embodiments, the non-replicating viral vector is an E1 and E3 deleted adenovirus vector.

In certain embodiments, provided herein are vaccine combinations comprising a first composition comprising a non-replicating viral vector encoding a peptide of an antigen or immunogen comprising one or more CD8+ T cell epitopes; and a third composition comprising an immunomodulator selected from an anti-immune repressor or an immune activator. In certain other embodiments, provided herein are vaccine compositions comprising a second composition comprising micelles comprising fluorocarbon-linked peptides, wherein each fluorocarbon-linked peptide: i) 15 to 75 amino acid residues in length; ii) from an antigen or immunogen; and iii) one or more CD8+ T cell epitopes comprising an antigen or immunogen; and a third composition comprising an immunomodulator selected from an anti-immune repressor or an immune activator.

In exemplary embodiments, the vaccine combination is used (prophylactically or therapeutically) as a cancer vaccine or a chronic infection vaccine in a method of inducing an immune response. Herein, the applicant provides a dosing regimen that is highly immunogenic in the murine tumor models tested herein compared to an immunomodulatory agent alone or a T cell inducing vaccine composition, wherein the T cell inducing vaccine is a synergist of immunomodulatory agents selected from immune checkpoint inhibitors or Myeloid Derived Suppressor Cell (MDSC) inhibitors. See example 5 and example 7.

Accordingly, provided herein are methods for inducing an immune response (e.g., an antigen CD8+ T cell response) in a subject in need thereof, the methods comprising administering a composition comprising a non-replicating viral vector encoding a peptide of an antigen or immunogen comprising one or more CD8+ T cell epitopes, or administering a composition comprising micelles comprising fluorocarbon-linked peptides, wherein each fluorocarbon-linked peptide: i) 15 to 75 amino acid residues in length; ii) from an antigen or immunogen; and iii) one or more CD8+ T cell epitopes comprising an antigen or immunogen; and, separately administering a composition comprising an immunomodulator selected from an anti-immune repressor or an immune activator.

Brief Description of Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments of the disclosure and, together with the detailed description and examples, serve to explain the principles and implementations of the disclosure.

Figure 1 shows the cumulative IFN- γ ELISpot response to the four peptides expressed as the number of IFN- γ producing cells (spot forming cells, SFC) per million splenocytes calculated for each group on day 24 after administration of the priming dose. Group 5 (AdGP70 as priming dose and PepGP70 as booster dose) and group 6 (PepGP70 as priming dose and AdGP70 as booster dose) are heterologous prime boost dosing regimens showing a synergistic effect compared to the homologous prime boost groups (groups 1 to 4). See example 1 and example 3.

Figure 2 shows the percentage of CD8+ T cells staining positive for dextramer at day 24 after administration of the priming dose. Animals in groups 1 to 4 were subjected to a homologous prime boost dosing regimen (AdGP70 or PepGP70) and animals in groups 4 and 5 were subjected to a heterologous prime boost dosing regimen; AdGP70 as the priming dose and PepGP70 as the booster dose (group 5), or PepGP70 as the priming dose and AdGP70 as the booster dose (group 6). See example 3.

Figure 3 shows that the vaccine compositions PepGP70 and AdGP70 tested alone or as a prime boost combination had a synergistic effect with anti-PD 1 treatment in terms of their ability to promote anti-tumor immune responses, with antigen-specific CD8+ T cells measured at day 6 (D6) and day 20 (D20). Animals: group 1 was administered PepGP70 (as prime and boost doses) + anti-PD 1; group 2 was administered AdGP70 (as a prime and booster dose) + anti-PD 1; group 3 was administered AdGP70 as a priming dose and PepGP70+ anti-PD 1 as a booster dose; group 4 was administered PepGP70 as a priming dose and AdGP70+ anti-PD 1 as a booster dose; group 5 was administered anti-PD 1 only (control group); and group 6 was not administered treatment (control). The AH1 peptide is a T cell epitope SPSYVYHQF (SEQ ID NO 72) which is present in GP70-472 and GP 70-CM. See example 1, example 2 and example 5.

Fig. 4A and 4B show the IFN- γ ELISpot response to the FP-OVA vaccine compared to vehicle group (fig. 4A), and the anti-tumor activity of the FP-OVA vaccine against the e.g. 7-OVA tumor model in terms of overall survival compared to vehicle (fig. 4B). The animals in fig. 4B were induced with e.g. g7-OVA cells at day 24, where the previously administered vaccine composition provided protection from death due to tumor growth. See example 6.

Fig. 5A and 5B show the antitumor activity of the FP-OVA vaccine (fig. 5B) against the e.g. 7-OVA tumor model in preventing tumor growth compared to vehicle (fig. 5A). The animals in fig. 5B were induced with e.g. g7-OVA cells at day 24, where the vaccine compositions previously administered at day 0 and day 14 provided protection from tumor growth. See example 6.

Figure 6 shows the anti-tumor activity of FP-OVA vaccines (group 1 and group 2) against e.g7-OVA tumor models in terms of overall survival compared to vehicle (group 3), where the therapeutic effect of the vaccine composition is demonstrated upon administration of the vaccine composition to animals following induction of the animals with e.g7-OVA cells.

Figure 7 shows that the vaccine compositions PepGP70 or AdGP70 tested alone or in combination with anti-PD 1 had a synergistic effect with anti-PD 1 treatment in terms of their ability to promote anti-tumor immune responses, where tumor size was measured over a period of days (over a period of days). Group 2 (AdGP70+ anti-PD 1) provided 79% tumor-free animals compared to group 1 (AdGP70) which was only 43% tumor-free animals; group 4 (PepGP70+ anti-PD 1) provided 62% tumor-free animals compared to only 15% tumor-free animals in group 3 (PepGP 70). anti-PD 1 alone provided only 29% tumor-free animals. See example 7.

Fig. 8A and 8B show the overall survival rate for each group; adenoviral vectors GP70 (fig. 8A) and fluorocarbon-linked GP70 peptide (fig. 8B) with and without immune checkpoint inhibitor anti-PD 1. See example 7.

Detailed Description

Introduction to the design reside in

The present invention provides compositions and methods for inducing enhanced T cell responses. In particular, the vaccine combinations of the present invention induce T cell mediated immunity for prophylactic or therapeutic control of persistent viral infections and cancer. The methods and compositions provided include administering a priming dose and a boosting dose of a heterologous vaccine that produces induction of a T cell response, where "heterologous" means that the priming dose is different from the boosting dose, provided both comprise at least one or more identical T cell epitopes. In certain embodiments, this means priming and boosting doses where the antigen/immunogen/peptide is presented to the immune system via different delivery vehicles and/or carriers. Applicants have discovered that administration of different T cell-inducing vaccines (e.g., viral vectors encoding antigens and micelles comprising peptides comprising T cell epitopes in common with the antigen expressed by the viral vector) synergistically potentiate the induction of antigen-specific T cell responses. See example 3. In embodiments, the priming dose and the booster dose comprise one or more of the same CD8+ T cell epitopes. In certain embodiments, the prime and/or boost dose of polypeptide further comprises one or more CD4+ T cell epitopes.

The sequence of the antigen/immunogen may not be identical, but there must be at least one or more T cell epitope overlaps in the priming and booster doses (e.g., one may be full-length and the other a short peptide derived from the full-length antigen, including chimeric peptides). These T cell epitopes can be identified in antigens or immunogens using techniques well known in the art, including from published references, including databases or websites containing peptide sequences known to bind MHC class I and/or MHC class II molecules compiled from published reports (e.g., SYFPEITHI website), or algorithms such as Artificial Neural Networks (ANN) or Stabilization Matrix Methods (SMM) (e.g., using the Immune Epitope Database and Analysis Resource website). See example 1.In embodiments, the priming dose and the booster dose comprise one or more CD8+ T cell epitopes in common.

In certain embodiments, provided herein are vaccine combinations comprising: a first composition comprising a non-replicating viral vector encoding an antigen or immunogen comprising one or more CD8+ T cell epitopes; and a second composition comprising micelles comprising fluorocarbon-linked peptides, wherein each fluorocarbon-linked peptide: i) 15 to 75 amino acid residues in length; ii) an antigen or immunogen from the first composition; and iii) one or more CD8+ T cell epitopes from the antigen or immunogen comprising the first composition. In certain embodiments, the first composition is a priming dose and the second composition is a boosting dose. In certain other embodiments, the first composition is a booster dose and the second composition is a prime dose.

As used herein, "heterologous dosing regimen" means priming and boosting doses in which the antigen/immunogen/peptide is presented to the immune system via different delivery vehicles and/or carriers. For example, in the present invention, the first composition (as a priming dose or booster dose) comprises a viral vector antigen or immunogen and the second composition (as a priming dose or booster dose) comprises a fluorocarbon-linked peptide, wherein the peptide comprises one or more T cell epitopes in common with the viral vector antigen or immunogen. In other words, in certain embodiments, the vaccine combination of the invention is two different T cell inducing vaccine compositions, wherein each composition induces antigen specific CD8+ T cells against the same antigen.

Applicants have discovered that a heterologous dosing regimen with adenoviral vector antigens and fluorocarbon-linked peptides, wherein at least one priming dose and at least one boosting dose are administered, provides a synergistic effect in inducing an immune response compared to a homologous dosing regimen with adenoviral vector antigens or fluorocarbon-linked peptides. See example 3 and figures 1 and 2. Groups 5 and 6 of figures 1 and 2 represent animals administered a heterologous dosing regimen, as compared to groups 1 to 4 which represent animals administered a homologous dosing regimen. As shown in fig. 1 and 2, administration of heterologous prime and boost doses induced antigen-specific CD8+ T cell responses.

In certain embodiments, the vaccine combinations of the present invention are used as therapeutic agents for the treatment of tumors and/or as prophylactic agents against cancer. The synergistic effect of the vaccine combination of the present invention may be used to treat certain tumors. See example 4. In certain other embodiments, the vaccine combinations of the present invention are used as therapeutic agents for the treatment of chronic infections.

In certain embodiments, the vaccine combination of the invention further comprises a third composition comprising an immunomodulator composition selected from the group consisting of an anti-immune repressor and an immune activator. Certain immune checkpoint proteins, such as PD1 on T cells and PD-L1 on tumor cells, assist in maintaining the immune response to these tumors silent, wherein this interaction inhibits T cells from killing these tumor cells when PD1 is bound by PD-L1. Thus, blocking the interaction of certain immune checkpoint proteins with anti-immune repressors allows T cells to kill tumor cells. The present invention combines a heterologous dosing regimen, such as a cancer or viral antigen, that induces antigen-specific CD8+ T cells specific for a tumor or cancer type (e.g., melanoma, lymphoma, lung cancer, etc.) with a synergistic effect against an immune repressor, such as an immune checkpoint inhibitor that non-specifically signals T cells that kill cancer cells.

In embodiments, the anti-immune repressor is an MDSC inhibitor. Myeloid-derived suppressor cells (MDSCs) are heterogeneous populations of immune cells derived from the myeloid lineage (serogroups), and they are amplified under certain pathological conditions, such as chronic infections and cancer; they may also play a role in certain autoimmune diseases. MDSCs have potent immunosuppressive, rather than immunostimulatory, properties, in which MDSCs interact with T cells and certain Antigen Presenting Cells (APCs) to modulate their function. MDSCs are known inhibitors of T cells (both CD8+ T and CD4+ T cells) and mediate induction of tolerance in autoimmune diseases and cancer. Thus, inhibitors of MDSC activity may assist in the disruption of tolerance in certain disease states. The present invention combines the synergistic effect of a heterologous dosing regimen that induces antigen-specific CD8+ T cells specific for a tumor or cancer type, such as a cancer or viral antigen, with myeloid-derived suppressor cell (MDSC) inhibitors that aid in the disruption of tolerance by inhibiting tolerant MSDCs and allow for a more robust immune response of the induced CD8+ T cells.

The applicants have found that the heterologous dosing regimen of the invention is synergistic in combination with the immune checkpoint inhibitor anti-PD 1 when the second composition (fluorocarbon linked peptide) is administered as a priming dose and the first composition (non-replicating viral vector antigen or immunogen) is administered as a boosting dose. See example 5 and figure 3. In certain embodiments, micelles comprising fluorocarbon-linked peptides (e.g., PepGP70) are administered as a priming dose, wherein each fluorocarbon-linked peptide: i) 15 to 75 amino acid residues in length; ii) an antigen or immunogen from a booster dose; and iii) comprises a booster dose of one or more CD8+ T cell epitopes; and a non-replicating viral vector (e.g., AdGP70) encoding an antigen or immunogen comprising one or more CD8+ T cell epitopes is administered as a booster dose in combination with administration of an immune checkpoint inhibitor anti-PD 1. Fluorocarbon-linked peptides are amphiphilic and spontaneously form micelles in certain solvents, where upon administration to an animal or human, these micelles are preferentially taken up by Antigen Presenting Cells (APCs) for processing. See U.S. patent nos. 7,687,455 and 9,119,811; the disclosure of each is incorporated herein by reference.

The applicant has found that administration of the first vaccine composition or the second vaccine composition in combination with a third composition (immune checkpoint inhibitor) provides a synergistic effect of the induced immune response compared to administration of those compositions alone. See example 7 and fig. 7, 8A and 8B. Groups 2 and 4 in figure 7 represent animals administered the vaccine combination, compared to groups 1 and 3 which represent animals administered either of the two separate T cell inducing vaccine compositions. As shown in fig. 8A and 8B, administration of the vaccine combination also increased the overall survival rate of the animals compared to animals administered the monotherapy.

In certain embodiments, provided herein are vaccine combinations comprising a first composition comprising a non-replicating viral vector encoding an antigen or immunogen comprising one or more CD8+ T cell epitopes; and a third composition comprising an immunomodulator selected from an anti-immune repressor or an immune activator. In certain other embodiments, provided herein are vaccine combinations comprising a second composition comprising micelles comprising fluorocarbon-linked peptides, wherein each fluorocarbon-linked peptide: i) 15 to 75 amino acid residues in length; ii) from an antigen or immunogen; and iii) one or more CD8+ T cell epitopes comprising an antigen or immunogen; and a third composition comprising an immunomodulator selected from an anti-immune repressor or an immune activator.

In embodiments, the first composition comprises a non-replicating viral vector, such as an adenoviral vector. Among all available replication-defective viral vectors, adenovirus is most effective in eliciting a T cell response to recombinant antigens. In embodiments, the second composition comprises a fluorocarbon-linked peptide.

Definition of

As used herein, the terms "a" or "an" are used as is conventional in patent documents to include one or more than one, independent of any other instances or usages of "at least one" or "one or more.

As used herein, unless otherwise indicated, the term "or" is used to refer to a non-exclusive or, such that "a or B" includes "a but not B," B but not a, "and" a and B.

As used herein, the term "about" is used to refer to an amount that is approximately, near, nearly, or nearly equal to or equal to the recited amount, e.g., plus/minus about 5%, about 4%, about 3%, about 2%, or about 1% of the recited amount.

As used herein, "adjuvant" refers to a substance that enhances the immune response of the body to an antigen, in the present disclosure, the adjuvant enhances the cell-mediated immune response induced by the combination of the first, second, and/or third compositions. As used herein, an adjuvant is combined into any or all of the first, second, and/or third compositions. The adjuvant is different from the third composition of the present disclosure. Examples of adjuvants that may be used to enhance a cell-mediated immune response include, but are not limited to, Toll-like receptor (TLR) agonists: agonists such as TLR2, TLR3, TLR7, TLR8 or TLR 9; or an agonist of STING, cGAS, NOD1, NOD2 or IRF 3.

By "administering" is meant introducing a vaccine composition or vaccine combination of the present disclosure into a subject; "administering" may also refer to the act of providing the composition of the present disclosure to the subject (e.g., by prescribing). The term "therapeutically effective amount" as used herein refers to the amount of compound administered that will induce a cell-mediated immune response. The term also refers to the amount of a composition or combination of the invention that will alleviate or prevent to some extent one or more of the symptoms of the condition to be treated. Referring to conditions/diseases that can be directly treated with the compositions of the present disclosure, a therapeutically effective amount refers to an amount that has the following effects: preventing the occurrence of the condition/disease in an animal that may be predisposed to the disease but has not yet experienced or exhibited symptoms of the condition/disease (prophylactic treatment), alleviation of symptoms of the condition/disease, diminishment of extent of the condition/disease, stabilization (e.g., not worsening) of the condition/disease, preventing spread of the condition/disease, delaying or slowing of the condition/disease progression, amelioration or palliation of the condition/disease state, and combinations thereof. The term "effective amount" refers to the amount of compound administered that will produce a different reaction than would occur in the absence of the compound. Reference to an embodiment of the present disclosure that includes an immunotherapeutic compound of the present disclosure, an "effective amount" is an amount that increases the immune response in a recipient relative to the response expected in the absence of administration of the compound.

The term "animal" refers to mammalian subjects, including humans, horses, dogs, cats, pigs, livestock and any other mammal, as well as birds. The term "animal" as referred to herein also includes individual animals at all stages of development, including neonatal, embryonic and fetal stages.

The term "host" or "organism" as used herein includes humans, mammals (e.g., cats, dogs, horses, etc.), insects, living cells, and other living organisms. For example, a living organism may be as simple as a single eukaryotic cell, or as complex as a mammal. Typical hosts to which embodiments of the present disclosure relate will include mammals, particularly primates, and especially humans. For veterinary applications, a wide variety of subjects will be suitable, for example livestock such as cattle (cattle), sheep, goats, cattle (cow), pigs, and the like; poultry, such as chickens, ducks, geese, turkeys, and the like; and domestic animals, particularly pets, such as dogs and cats. For research applications, a wide variety of mammals will be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and pigs such as inbred pigs, among others. Furthermore, for in vitro applications, such as in vitro research applications, body fluids and cell samples of the above subjects will be suitable for use, such as blood, urine or tissue samples of mammals (in particular primates, such as humans), or to mention blood, urine or tissue samples of animals for veterinary applications. A "predisposed" to a condition may be defined as a host that does not exhibit overt symptoms of one or more of these conditions, but is genetically, physiologically, or otherwise at risk of developing one or more of these conditions.

The terms "protein," "polypeptide," and "peptide" may be referred to interchangeably herein. However, these terms can be distinguished as follows. "protein" generally refers to the final product of transcription, translation, and post-translational modification in a cell. As used herein, "polypeptide" may refer to a "protein" or a "peptide". In contrast to "proteins," peptides "are typically short polymers of amino acids, typically 100 amino acids or less in length.

The compositions, formulations, and methods of the invention can comprise, consist essentially of, or consist of: the components and ingredients of the present invention, as well as other ingredients described herein. As used herein, "consisting essentially of means that the compositions, formulations, and methods may include additional steps, components, or ingredients, provided that the additional steps, components, or ingredients do not materially alter the basic and novel characteristics of the claimed compositions, formulations, and methods.

It should also be noted that, as used in this specification and the appended claims, the term "configured" describes a system, apparatus, or other structure that is constructed or arranged to perform a particular task or take a particular configuration. The term "configured" may be used interchangeably with other similar terms, such as arranged and configured, constructed and arranged, adapted and configured, adapted, constructed, prepared and arranged, and the like.

As used herein, the term "human adenovirus" is intended to include all human adenoviruses of the family Adenoviridae (adefoviraceae), including members of the mammalian genus adenoviruses (mattadenoviridus). To date, over 51 adenovirus human serotypes have been identified (see, e.g., Fields et al, Virology2, chapter 67 (3 rd edition, Lippincott-Raven Publishers)). The adenovirus may be serogroup A, B, C, D, E or F. The human adenovirus may be of serotype 1(Ad 1), serotype 2(Ad2), serotype 3(Ad3), serotype 4(Ad4), serotype 5(Ad5), serotype 6(Ad6), serotype 7(Ad7), serotype 8(Ad8), serotype 9(Ad9), serotype 10(Ad10), serotype 11(Ad11), serotype 12(Ad 11), serotype 13(Ad 11), serotype 14(Ad 11), serotype 15(Ad 11), serotype 16(Ad 11), serotype 17(Ad 11), serotype 18(Ad 11), serotype 19a (Ad19 11), serotype 19p (Ad19 11), serotype 20(Ad 11), serotype 21(Ad 11), serotype 22(Ad 11), serotype 23(Ad 11), serotype 24(Ad 11), serotype 25(Ad 11), serotype 26(Ad 11), serotype 27(Ad 11), serotype 3628 (Ad11), serotype 3629 (Ad11), serotype 11), Serotype 30(Ad30), serotype 31(Ad31), serotype 32(Ad32), serotype 33(Ad33), serotype 34(Ad34), serotype 35(Ad35), serotype 36(Ad36), serotype 37(Ad37), serotype 38(Ad38), serotype 39(Ad39), serotype 40(Ad40), serotype 41(Ad41), serotype 42(Ad42), serotype 43(Ad43), serotype 44(Ad44), serotype 45(Ad45), serotype 46(Ad46), serotype 47(Ad47), serotype 48(Ad48), serotype 49(Ad49), serotype 50(Ad50), serotype 51(Ad51), or a combination thereof, but is not limited to these examples. In certain embodiments, the adenovirus is serotype 5(Ad 5).

As used herein, "immunomodulator" refers to a series of treatments intended to utilize the immune system of a patient to achieve immune control, stabilization and potentially elimination of a disease. For example, an immunomodulator may be a substance used to break tolerance (such as may be present in chronic infections or certain autoimmune diseases); or for inactivating immunosuppressive cells to induce or enhance a host cell-mediated immune response against a foreign or self (e.g., cancer) antigen; or also for inducing or enhancing host cell-mediated immune responses against foreign or self (e.g., cancer) antigens. In embodiments, the immunomodulator comprises a substance that modulates a T cell pathway, and has the potential to reassume an anti-tumor or anti-viral immune response.

"pharmaceutical composition" refers to a mixture of one or more vaccine compounds, derivatives thereof, or pharmaceutically acceptable salts thereof described herein with other chemical components such as pharmaceutically acceptable carriers and excipients. One purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

As used herein, "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered vaccine composition.

The terms "treatment", "treating" and "treatment" are methods for obtaining a beneficial or desired clinical result. In particular, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (e.g., not worsening) disease, delay or slowing of disease progression, substantial prevention of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Furthermore, "treatment" (therapy), "treatment" and "treatment" may also mean an extended survival rate compared to the expected survival rate if not treated and/or may be therapeutic in terms of a partial or complete cure of the disease and/or side effects caused by the disease. As used herein, the term "prophylactic treatment" or "prophylactic treatment" refers to the complete, substantial, or partial prevention of a disease/condition or one or more symptoms thereof in a host. Likewise, "delaying the onset of a condition" may also be included in "prophylactic treatment" and refers to an act of increasing the time before the actual onset of a condition in a patient susceptible to the condition.

As referred to herein, a "vaccine" may include an antigen or carrier, along with other components of the vaccine formulation, including, for example, adjuvants, slow release compounds, solvents, and the like. Although vaccines are traditionally used for the prevention or treatment of infectious diseases, vaccines can also alter the function of metabolites by binding to signalling peptides or proteins or their receptors and by blocking antigens that are characteristic of certain abnormal cell types, such as for example tumours. Accordingly, embodiments of the present invention provide vaccines that improve the immune response to any antigen, regardless of the antigen source or its function, including antigens that alter physiological functions (required for improved health, such as immunity against cancer).

As referred to herein, a "vector" carries the genetic code of an antigen or a portion thereof, but the vector is not the antigen itself. In exemplary aspects, the vector may comprise a viral vector or a bacterial vector. By "antigen" as referred to herein is meant a substance that induces a specific immune response in a subject, including humans and/or animals. Antigens may include whole organisms, killed, attenuated or live; a subunit or a portion of an organism; a recombinant vector comprising an insert having immunogenic properties; fragments (pieces) or fragments of DNA capable of inducing an immune response upon presentation to a host animal; a polypeptide, an epitope, a hapten, or any combination thereof. In various aspects, the antigen is a virus, a bacterium, a subunit of an organism, an autoantigen, or a cancer antigen.

Vaccine combinations

Provided herein are vaccine combinations comprising a first composition and a second composition, wherein the compositions are used as heterologous prime and boost doses for inducing T cell mediated immunity. The first and second compositions comprise one or more of the same CD8+ T cell epitopes. In certain embodiments, the first composition or the second composition may comprise a full-length antigen or immunogen or a fragment thereof, each of which is also referred to herein as a "polypeptide. In certain embodiments, the priming dose (as the first or second composition) comprises the full-length antigen or immunogen, while the boosting dose (as the first or second composition) comprises a fragment of the antigen or immunogen, such as a peptide, including a chimeric peptide. In certain other embodiments, the priming dose (as the first or second composition) comprises a fragment of an antigen or immunogen, such as a peptide, including a chimeric peptide, and the boosting dose (as the first or second composition) comprises a full-length antigen or immunogen. In certain other embodiments, the priming dose (as the first or second composition) comprises a fragment of an antigen or immunogen, such as a peptide, including a chimeric peptide, and the boosting dose (as the first or second composition) comprises a fragment of an antigen or immunogen, such as a peptide, including a chimeric peptide. Heterologous as used herein does not refer to an antigen or immunogen but rather to a delivery vehicle or carrier linked to an antigen or immunogen.

In embodiments, the first and second compositions comprise a delivery vector or vehicle, which may include a peptide, a lipophilic chain, or an expression vector, including a non-replicating viral vector. In certain embodiments, the carrier may be a hydrocarbon chain, optionally substituted with one or more halogen atoms. In certain embodiments, the carrier may be a fluorocarbon chain. In certain embodiments, the delivery vector may be a non-replicating viral vector, such as those selected from an adenoviral vector, an alphaviral vector, a herpesvirus vector, a measles virus vector, a poxvirus vector, or a vesicular stomatitis virus vector. In certain embodiments, the non-replicating viral vector is an E1 and/or E3 deleted adenovirus vector. In certain embodiments, the non-replicative adenovirus vector is a human adenovirus vector.

Provided herein are vaccine combinations comprising a first composition and a second composition, wherein the first composition comprises a non-replicating viral vector encoding an antigen or immunogen comprising one or more CD8+ T cell epitopes; and the second composition comprises micelles comprising fluorocarbon-linked peptides, wherein each fluorocarbon-linked peptide: i) 15 to 75 amino acid residues in length; ii) an antigen or immunogen from the first composition; and iii) one or more CD8+ T cell epitopes from the antigen or immunogen comprising the first composition. The first composition or the second composition may be a prime dose or a boost dose. To provide a heterologous dosing regimen, each of the first and second compositions must be administered to an animal in need thereof, one as a priming dose and the other as a booster dose.

Further embodiments provided herein are vaccine combinations comprising a first composition or a second composition and a third composition, which combination enhances an antigen-specific T cell response, wherein the first composition or the second composition induces T cell immunity and the third composition is an immunomodulator selected from an anti-immune repressor or an immune activator. The first and second compositions comprise one or more CD8+ T cell epitopes. In certain embodiments, the first composition or the second composition may comprise a full-length antigen or immunogen or a fragment thereof, each of which is also referred to herein as a "polypeptide. In certain embodiments, the first composition comprises a full-length antigen or immunogen. In certain other embodiments, the first composition comprises a fragment of an antigen, such as a peptide, including a chimeric peptide. In certain embodiments, the second composition comprises a full-length antigen or immunogen. In certain other embodiments, the second composition comprises a fragment of an antigen or immunogen, such as a peptide, including a chimeric peptide.

Provided herein are vaccine combinations comprising a first composition or a second composition and a third composition, wherein the first composition comprises a non-replicating viral vector encoding an antigen or immunogen comprising one or more CD8+ T cell epitopes; the second composition comprises micelles comprising fluorocarbon-linked peptides, wherein each fluorocarbon-linked peptide: i) 15 to 75 amino acid residues in length; ii) an antigen or immunogen from the first composition; and iii) comprises one or more CD8+ T cell epitopes from an antigen or immunogen; and the third composition comprises an immunomodulator selected from an anti-immune repressor or an immune activator.

Antigens and immunogens

The choice of antigen or immunogen (which may be used interchangeably herein) depends on whether the T cell response to the antigen has been found to be protective and/or therapeutic or is expected to be protective and/or therapeutic in a human or animal model. The degree of polymorphism that exists between different antigen isolates, as well as highly conserved antigens, polymorphisms of HLA molecules, or highly conserved epitopes within an antigen, are also factors in selecting antigens. For T cell vaccine compositions based on highly conserved antigenic T cell epitopes and HLA molecule polymorphisms across different populations and/or ethnic groups, see U.S. patent No. 8,642,531 and U.S. patent publication No. 2016/0106830, the contents of which are incorporated herein by reference. See also, U.S. patent publication No. 2016/0199469, paragraphs [0121] to [0134], the contents of which are incorporated herein by reference. In certain embodiments, the antigen may also be selected to be an antigen that is a combination of conserved regions or conserved epitopes from one or more antigens to produce a synthetic vaccine antigen [ Goodman AL, et AL, New cancer vaccines and antigen blood plasma bacteria and potential viral vectors expressing an optimized anti-bacterial substrate protein 1.Infection and Immunity 2010; 78(11) 4601-12; letourneau S, et al Design and pre-clinical evaluation of a univeral HIV-1vaccine. PLoS ONE 2007; 2, (10) e 984. In certain embodiments, the combination may be in the form of a pool (pool) of peptides or polypeptides in the first vaccine composition or the second vaccine composition, or as a chimeric peptide or polypeptide in the first vaccine composition or the second vaccine composition. In certain embodiments, the first vaccine composition and the second vaccine composition comprise more than one antigen to reduce the likelihood of immune evasion. For T cell tumor vaccines comprising two or more antigens, see U.S. patent publication No. 2016/0199469, the contents of which are incorporated herein by reference.

T cell epitopes in antigens can be identified by bioinformatic predictions and experimental validation, or empirically using peptide libraries spanning the entire antigen sequence. See example 1 for more disclosure on the identification of T cell epitopes (both CD8+ and CD4 +). However, while knowledge of epitopes presented by common HLA types may be helpful in detailed phenotypic studies of T cell responses in clinical studies and vaccine development, complete knowledge of all possible epitopes contained in an antigen is not necessary. The vaccine composition, the first composition and/or the second composition of the invention comprises at least one CD8+ T cell epitope. In certain embodiments, the first and second compositions comprise at least two, three, four, five, or at least six CD8+ T cell epitopes. In certain other embodiments, the first vaccine composition and/or the second vaccine composition further comprises one or more CD4+ T cell epitopes. It will be appreciated that the antigen in the form of a polypeptide in the vaccine composition may comprise one or more T cell epitopes which have not been identified using tools available for identification.

In embodiments, the antigen/immunogen may correspond to a smaller portion or polypeptide derived from a full-length antigen sequence, which may be selected based on sequence conservation and/or the presence of CD8+ T cell epitopes and CD4+ T cell epitopes. CD4+ T cell epitopes and/or CD8+ T cell epitopes can be identified using bioinformatic tools to identify HLA class I and/or HLA class II binding sequences, using in vitro assays of human PBMC samples or in vitro HLA class I and/or HLA class II binding assays. Immunogens can be produced by artificial concatenation of smaller sequence portions or polypeptides derived from single or multiple antigenic sequences of the same pathogen. The tandem sequences may be used to generate synthetic peptide immunogens or the corresponding DNA sequences may be incorporated into adenoviral vectors.

In certain embodiments, the antigen or immunogen is from a pathogen, a cancer antigen, or an autoantigen. In certain embodiments, the antigen or immunogen is a neoantigen. As used herein, a "neoantigen" refers to a known host protein having one or more amino acid changes as compared to the wild type that allows the host immune system to recognize as foreign. The neoantigen may be unique to each patient and, therefore, as used herein, may be referred to as a protein variant, including clinical variants identified from individual patients. Human clinical variants can be identified using well-known databases that provide published reported archives of relationships between human variants and phenotypes. Landrum MJ, et al ClinVar, public area of intervention of clinical relevance of nucleic Acids Res.2016Jan 4; 44(D1): D862-8. CD8+ T cell epitope mapping of these neo-antigens or clinical variants then allows the development of the vaccine combinations of the present invention. The disclosure of the use of well-known tools for T cell epitope mapping of antigens or immunogens is provided in example 1.

In certain embodiments, the antigen or immunogen is from a pathogen, including pathogens known to cause cancer development (and thus may be expressed on the surface of cancer cells). In embodiments, the pathogen is a virus, fungus, parasite, or bacterium. In certain embodiments, the antigen or immunogen is from a virus selected from EBV, HPV, HTLV-1, MCPvV, KSHV or HERV. Each of these viruses is known to induce cancer or tumor growth. In certain embodiments, the antigen or immunogen is from the virus HCV (hepatitis c virus) or HBV (hepatitis b virus).

In certain embodiments, the antigen or immunogen may be any one or more of the following: EBV (EBNA1, LMP1, LMP2 and BARF1), HPV (E1 to E7), HTLV-1(tax), MCPYV (big T, small T), KSHV (Orf73, Orf57, K10.5 and K12), CMV (glycoprotein B and phosphoprotein 65), BKV ((big T, small T), JCV (big T, small T), SV40 (big T, small T), HERV (Env, Gag, Pol), HMTV (Env, Gag, Pol), HIV-1(Env, Gag, Pol, Nef, Tat, Vif), HCV (Core, NS 38, NS4, NS5), HBV (Core, Pol, Env and X), influenza (HA, NP, NA, M1, PB1, PB 8, PA 1-4, NS4, 2A-3992), HBV (Core, Pol, Pol and X), Mylcobacter (Mylcobacter, Mylcobacter-P638, Mycobacterium tuberculosis, RTS-1, RTS-10, RTS, RTV, Mycobacterium).

In embodiments, the antigen induces an immune response against a pathogen, including, for example, a virus. Exemplary viruses include orthomyxoviruses (orthomyxoviruses), paramyxoviruses (paramyxoviruses), rhinoviruses (rhinoviruses), coronaviruses, influenza viruses, Respiratory Syncytial Viruses (RSV), common cold viruses (common cold viruses), or measles viruses, herpes viruses, rabies viruses, varicella, Human Papilloma Viruses (HPV), hepatitis viruses, or other known viral pathogens.

In embodiments, the antigen induces an immune response against a bacterial pathogen. Exemplary bacteria include Bacillus (Bacillus), Mycobacterium (Mycobacterium), Staphylococcus (Staphylococcus), Streptococcus (Streptococcus), Pseudomonas (Pseudomonas), Klebsiella (Klebsiella), Haemophilus (Haemophilus), Mycoplasma (Mycoplasma) and/or Bacillus anthracis (Bacillus anthracensis). In certain embodiments, the antigen induces an immune response against a fungal pathogen. In embodiments, the fungi include Aspergillus (Aspergillus), Candida (Candida), Cryptococcus (Cryptococcus), Histoplasma (Histoplasma), Pneumocystis (Pneumocystis), and/or Stachybotrys (Stachybotrys).

In embodiments, the antigen may comprise an allergen or a tumor associated antigen. In still further aspects, the antigen can include a polypeptide, peptide, or group thereof (panels) comprising one or more epitopes of a protein associated with a disease. For example, a suitable polypeptide, peptide, or group thereof can comprise one or more epitopes of a pathogen-associated protein. Suitable polypeptides may comprise the full-length amino acid sequence of the corresponding protein of the pathogen, or a fragment thereof.

First composition of vaccine combination

In embodiments, the first composition comprises a non-replicating viral vector encoding an antigen or immunogen comprising one or more CD8+ T cell epitopes. In certain embodiments, the non-replicating viral vector is an adenoviral vector, an alphaviral vector, a herpes viral vector, a measles viral vector, a poxvirus vector, or a vesicular stomatitis viral vector. In an exemplary embodiment, the non-replicating viral vector is an adenoviral vector. Deletion or disruption of the E1 and/or E3 sequences produces a non-replicating adenoviral vector.

In embodiments, the compositions and formulations comprise a vector, i.e., a viral vector. As referred to herein, a "viral vector" is an engineered virus that incorporates an antigen gene and expresses an antigen. Viral vectors may be non-replicating and safe for the host and environment. It will be appreciated that any viral vector may be incorporated into the compositions, formulations and methods of the invention to achieve delivery into a cell.

Exemplary viral vectors include, inter alia, adenovirus, retrovirus, lentivirus, herpes virus, poxvirus, alphavirus, adeno-associated virus. Many such viral vectors are available in the art. The vectors described herein can be constructed using standard recombinant techniques widely available to those skilled in the art. Such techniques can be found in common Molecular biology references such as Molecular Cloning, A Laboratory Manual (Sambrook, et al, 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol.185, D.Goeddel, eds., 1991.Academic Press, San Diego, Calif.) and PCR Protocols, A Guide Methods and Applications (Innis, et al, 1990.Academic Press, San Diego, Calif.).

In certain embodiments, the non-replicating viral vector is an adenovirus, including, for example, where the adenovirus is a bovine adenovirus, a canine adenovirus, a non-human primate adenovirus, a chicken adenovirus, or a porcine (porcine/swine) adenovirus. In certain embodiments, the non-replicating viral vector is a human adenovirus.

In embodiments, the non-replicating adenoviral vector is particularly useful for gene transfer into eukaryotic cells and vaccine development, as well as in animal models.

In embodiments, any adenoviral vector (Ad-vector) known to those of skill in the art and prepared for administration to a mammal that can contain and express an antigen or immunogen can be used in the compositions and with the methods of the present application. Such Ad-vectors include any one of the following: U.S. patent No. 6,706,693; 6,716,823 No; 6,348,450 No; or U.S. patent publication No. 2003/0045492; 2004/0009936 No; 2005/0271689 No; 2007/0178115 No; 2012/0276138 (incorporated herein by reference in its entirety).

In certain embodiments, the recombinant adenoviral vector can be non-replicative or replication-deficient, requiring complementary E1 activity for replication. In embodiments, the recombinant adenoviral vector can comprise an E1-deficient (defective), E3-deficient, and/or E4-deficient adenoviral vector, or a "viral gene free" (gut) adenoviral vector in which viral genes are deleted. The E1 mutation improves the safety margin of the vector because the E1 deficient adenovirus mutant is unable to replicate in non-permissive cells. The E3 mutation enhances the immunogenicity of the antigen by disrupting the mechanism by which the adenovirus down-regulates MHC class I molecules. The E4 mutation reduces the immunogenicity of the adenoviral vector by inhibiting late gene expression, thus allowing repeated re-vaccination with the same vector. In embodiments, the recombinant adenoviral vector is an E1 and/or E3 deficient vector.

Replication of "virus-free" adenoviral vectors requires helper virus and a specific human 293 cell line expressing both E1a and Cre, which is not present in the natural environment; the vector does not contain viral genes, and thus the vector is non-immunogenic as a vaccine vehicle and can be vaccinated multiple times for re-vaccination. "Virus-gene-free" adenoviral vectors also contain a 36kb space for accommodating the transgene, thus allowing co-delivery of large amounts of antigenic genes into the cell. Specific sequence motifs such as the RGD motif can be inserted into the H-I loop of an adenoviral vector to enhance its infectivity. Adenovirus recombinants can be constructed by cloning a particular transgene or segment of a transgene into any adenoviral vector, such as those described below. Adenoviral recombinant vectors are used as immunizing agents for non-invasively transducing vertebrate epidermal cells. Adenovirus vectors may also be used in invasive methods of administration, such as intravenous, intramuscular, or subcutaneous injection.

In embodiments, a first composition comprising a non-replicating viral vector expressing an antigen or immunogen of a vaccine combination of interest can be formulated for administration to a mammal. With regard to the dosage, route of administration, formulation, adjuvant and use of the recombinant virus and the expression product therefrom, the composition of the invention may be used for parenteral or mucosal administration, preferably by the intradermal, subcutaneous, intranasal or intramuscular route. When mucosal administration is used, it is possible to use the oral, ocular or nasal route.

Formulations which may contain the adenoviral vector of interest may be prepared according to standard techniques well known to those skilled in the pharmaceutical or veterinary arts. Such formulations may be administered in dosages and techniques well known to those skilled in the clinical art, taking into account factors such as age, sex, weight and route of administration. The formulations may be administered alone, or may be co-administered or sequentially administered with a composition, e.g., with an "other" immunological composition, or an attenuated, inactivated, recombinant vaccine or therapeutic composition, to provide multivalent or "mixed" or combination compositions of the invention and methods of using the same. In embodiments, the formulation may comprise sucrose as a cryoprotectant and polysorbate-80 as a non-ionic surfactant. In certain embodiments, the formulation further comprises free radical oxidation inhibitors ethanol and histidine, a metal ion chelator, ethylenediaminetetraacetic acid (EDTA), or other agents with comparable activity (e.g., to block or prevent metal ions from catalyzing free radical oxidation).

The formulations may be presented in liquid preparations for mucosal administration, e.g. oral, nasal, ocular, etc., formulations such as suspensions, and preparations for parenteral, subcutaneous, intradermal, intramuscular, intravenous (e.g. injectable administration) such as sterile suspensions or emulsions. In such formulations, the adenoviral vector can be mixed with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, and the like. The formulation may also be lyophilized or frozen. Depending on the route of administration and the desired preparation, the formulation may contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, adjuvants, preservatives and the like. The formulation may comprise at least one adjuvant compound.

Reference may be made to standard textbooks, such as "REMINGTON' S PHARMACEUTICAL SCIENCE, 17 th edition, 1985, incorporated herein by reference, to prepare suitable articles without undue experimentation.

Second composition of vaccine combination

In embodiments, the second composition comprises micelles comprising fluorocarbon-linked peptides, wherein each peptide linked to the fluorocarbon: i) 15 to 75 amino acid residues in length; ii) an antigen or immunogen from the first composition; and iii) one or more CD8+ T cell epitopes comprising the antigen or immunogen.

In embodiments, the peptide has a length of 20 to 60 amino acids, such as 25 to 50 amino acids, 30 to 40 amino acids, e.g., 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acids. In certain embodiments, the peptide may comprise an additional short amino acid sequence. The additional sequences may facilitate the preparation or formulation of the peptide, or enhance the stability of the peptide. For example, the peptide may comprise one or more additional amino acids, typically at the N-terminus and/or C-terminus, to enhance the net positive charge of the peptide and/or reduce the hydrophobicity of the peptide. The net positive charge can be increased such that the peptide has an isoelectric point greater than or equal to 7.

In certain embodiments, one or more, such as two or three positively charged amino acids (arginine and/or lysine) are added to the N-terminus and/or C-terminus of one or more peptides in the composition. For example, three lysine residues (KKK) may be added to the N-terminus and/or C-terminus of one or more peptides. See table 2. Typically, a positive amino acid is added at the end of a peptide having more than 65% total hydrophobicity, a net charge of less than zero, and/or comprising a hydrophobic amino acid cluster.

In embodiments where the peptide is linked to a fluorocarbon, the end of the peptide, such as the end not conjugated to the fluorocarbon or other attachment, can be altered, for example, to promote solubility of the fluorocarbon-peptide construct via micelle formation. To facilitate large scale synthesis of the constructs, the N-terminal or C-terminal amino acid residues of the peptides may be modified. When the desired peptide is particularly sensitive to cleavage by peptidases, the normal peptide bond can be replaced by a non-cleavable peptidomimetic. Such linkages and synthetic methods are well known in the art.

The peptide may be a native peptide. The native peptide may have free or modified termini. The peptide may be modified to increase the life of the peptide in vivo (such as half-life or persistence at the site of administration) or to direct the peptide to antigen presenting cells. For example, an immunogenic peptide can comprise one or more non-naturally occurring amino acids and/or non-naturally occurring covalent bonds for covalently linking adjacent amino acids. In certain embodiments, non-standard, non-naturally occurring amino acids may also be incorporated into immunogenic peptides, provided that they do not interfere with the ability of the peptide to interact with HLA molecules, and retain cross-reactivity with T cells that recognize the native sequence. Unnatural amino acids can be used to improve the resistance or chemical stability of peptides to proteases. Examples of unnatural amino acids include D-amino acid and cysteine modifications.

In embodiments, the second composition may comprise a plurality of peptides linked to fluorocarbon chains. Accordingly, the composition may comprise at least two, such as at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more peptides. In an exemplary embodiment, the second composition comprises four peptides, wherein each peptide is linked to a fluorocarbon chain. See example 1.

Whether a peptide is capable of inducing a peptide-specific response in T cells of a cancer patient, a patient with a chronic infection, or a healthy subject can be determined by any suitable means, typically by testing a sample of Peripheral Blood Mononuclear Cells (PBMCs) collected from the patient or subject in a suitable assay. Thus, the T cell response of the sample is tested in vitro. Suitable assays can measure or detect the activation of T cells after incubation with the test peptide. Activation of T cells can generally be indicated by secretion of cytokines such as IFN- γ, which can be detected in any suitable assay, typically an immunoassay, such as ELISA or ELISpot. After incubation with the test peptide, the magnitude of the T cell response of the patient or subject can be determined in the same assay, e.g., by quantifying the amount of cytokine released in the sample as a whole or by specific cells in the sample. Suitable assays are further described below and in the examples.

In certain embodiments, the second composition of the invention comprises one or more peptides that induce a specific CD8+ T cell response in at least 20% of healthy subjects and/or cancer patients. Immunoassays for measuring peptide-specific T cell responses in human Peripheral Blood Mononuclear Cells (PBMCs) from healthy subjects or cancer patients can be performed by means of cytokine ELISpot, such as IFN- γ ELISpot assays or intracellular cytokine staining using flow cytometry. Assays can be performed from fresh or frozen PBMCs. The assay can be performed ex vivo or after a short period of in vitro culture of PBMCs incubated with a single peptide or a composition comprising several antigenic peptides. The amount of peptide in the short term in vitro culture can vary from 0.001. mu.g/peptide to 100. mu.g/peptide. The incubation time for the short term in vitro culture may be between 5 days and 15 days, such as between 7 days and 13 days or between 9 days and 11 days. The short term in vitro culture may be carried out in the presence of cytokines, such as one or more of IL-2, IL-15 and IL-7, preferably IL-2 and IL-15. Short term in vitro culture can be performed after depletion of regulatory T cells and/or NK cells. Such depletion of cells may be particularly desirable when the PBMCs are from a cancer patient. Short in vitro cultures can be performed in the presence of IL-10 neutralizing antibodies, anti-PD 1 antibodies, anti-CTLA-4 antibodies, anti-OX-40 antibodies, anti-GITR antibodies, denileukin (diftitox), kinase inhibitors and/or toll receptor agonists including agonists for TLR2, TLR3, TLR7, TLR8 and TLR 9.

In certain embodiments, the carrier is a hydrocarbon chain substituted with one or more halogen atoms. In embodiments, the carrier is a hydrocarbon chain substituted with one or more fluorine atoms, referred to herein as a "fluorocarbon chain".

The fluorocarbon may comprise one or more chains derived from perfluorocarbon or mixed fluorocarbon/hydrocarbon groups, and may be saturated or unsaturated, each chain having from 3 to 30 carbon atoms. Thus, the chains in the fluorocarbon attachments are typically saturated or unsaturated, preferably saturated. The chain in the fluorocarbon attachment may be linear or branched, but is preferably linear. Each chain typically has from 3 to 30 carbon atoms, from 5 to 25 carbon atoms, or from 8 to 20 carbon atoms. To covalently link the fluorocarbon support to the peptide, the support contains reactive groups or ligands, such as-CO-, -NH-, S, O or any other suitable group. The use of such ligands for achieving covalent attachment is well known in the art. The reactive group may be located at any position on the fluorocarbon support.

Coupling of the fluorocarbon carrier to the peptide may be through functional groups naturally occurring or introduced at any site of the peptide, such as-OH, -SH, -COOH and-NH₂To be implemented. Examples of such linkages include amide, hydrazone, disulfide, thioether, and oxime linkages.

Optionally, spacer elements (peptidic or non-peptidic) may be incorporated to allow cleavage of the peptide from the fluorocarbon element for processing within the antigen presenting cell and to optimize spatial presentation of the peptide. Spacers may also be incorporated to aid in the synthesis of the molecule and to improve its stability and/or solubility. Examples of spacers include polyethylene glycol (PEG) or amino acids such as lysine or arginine which can be cleaved by proteolytic enzymes.

In certain embodiments, the fluorocarbon linked peptide may have chemical structure C_mF_n—C_yH_x(Sp) -R or a derivative thereof, wherein m ≦ 2m +1, y ≦ 0 to 15, x ≦ 2y, (m + y) ≦ 3 to 30, and Sp is an optional chemical spacer moiety, and R is an immunogenic peptide. Typically, m and n satisfy the relationship 2m-1 ≦ n ≦ 2m +1, and preferably n ≦ 2m + 1. Typically x and y satisfy the relationship 2y-2 ≦ x ≦ 2y, and preferably x ≦ 2 y. Preferably, C_mF_n-C_yH_xThe sections are linear.

In embodiments, m is 5 to 15, more preferably 8 to 12. In other embodiments, y is 0 to 8, more preferably 0 to 6 or 0 to 4. In an embodiment, C_mF_n—C_yH_xThe moieties are saturated (i.e., n-2 m +1 and x-2 y) and linear, and m-8 to 12 and y-0 to 6 or 0 to 4.

In certain embodiments, the fluorocarbon support is derived from 2H, 3H-perfluoroundecanoic acid of the formula:

in embodiments, the fluorocarbon attachment is a linear saturated moiety C₈F₁₇(CH₂)₂It is derived from C₈F₁₇(CH₂)₂COOH. In certain embodiments, the fluorocarbon attachment has the formula: c₆F₁₃(CH₂)₂—、C₇F₁₅(CH₂)₂—、C₉F₁₉(CH₂)₂—、C₁₀F₂₁(CH₂)₂—、C₅F₁₁(CH₂)₃—、C₆F₁₃(CH₂)₃—、C₇F₁₅(CH₂)₃—、C₈F₁₇(CH₂)₃-and C₉F₁₉(CH₂)₃Each of them is derived from C₆F₁₃(CH₂)₂COOH、C₇F₁₅(CH₂)₂COOH、C₉F₁₉(CH₂)₂COOH、C₁₀F₂₁(CH₂)₂COOH、C₅F₁₁(CH₂)₃COOH、C₆F₁₃(CH₂)₃COOH、C₇F₁₅(CH₂)₃COOH、C₈F₁₇(CH₂)₃COOH and C₉F₁₉(CH₂)₃COOH。

In embodiments, an example of a suitable structure for a fluorocarbon vector-antigen construct has the formula:

wherein Sp and R are as defined above. In certain embodiments, Sp is derived from a lysine residue and has the formula-CONH- (CH)₂)₄—CH(NH₂) -CO-. In embodiments, R is a peptide that: 15 to 75 amino acid residues in length; ii) an antigen or immunogen from the first composition; and iii) one or more CD8+ T cell epitopes from the antigen or immunogen comprising the first composition.

In certain embodiments, the fluorocarbon attachments may be modified such that the resulting compounds are still capable of delivering the peptides to antigen presenting cells. Thus, for example, many fluorine atoms may be replaced by other halogen atoms such as chlorine, bromine or iodine. Furthermore, it is possible to replace many of the fluorine atoms with methyl groups or hydrogens and still retain the properties of the molecules described herein.

In embodiments, the peptide may be linked to the fluorocarbon support via a spacer moiety. In one embodiment, the spacer moiety is a lysine residue. The spacer residue may be present in addition to any terminal lysine residue as described above, such that the peptide may have, for example, a total of four N-terminal lysine residues. Accordingly, in certain embodiments, the second composition of the present invention may comprise a fluorocarbon-linked peptide, wherein the peptide has a C-terminal or N-terminal lysine residue, preferably an N-terminal lysine residue. In embodiments, the terminal lysine in the peptide has formula C₈F₁₇(CH₂)₂Fluorocarbon linkage of COOH. In embodiments, the fluorocarbon is coupled to the epsilon chain of the N-terminal lysine residue.

In embodiments, the second composition comprises at least 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 or more immunogenic peptides linked to its own fluorocarbon carrier.

Third composition of vaccine combination

In embodiments, the third composition comprises an immunomodulatory agent. Immunomodulators include a range of treatments (e.g., substances or compounds) intended to take advantage of the patient's immune system to achieve immune control, stabilization and potential disease elimination.

In embodiments, the immune modulator comprises an immune checkpoint blockade antibody that modulates a T cell pathway and has the potential to recruit an anti-tumor or anti-viral immune response. In certain embodiments, immune checkpoint inhibitors include, but are not limited to, ipilimumab (ipilimumab is the first FDA approved immune checkpoint antibody approved for the treatment of metastatic melanoma, blocking the checkpoint molecule known as cytotoxic T lymphocyte antigen 4 (CTLA-4)) and other compounds (e.g., antibodies) that target co-inhibitory receptors such as CTLA-4, PD-1, Lag-3, Tim-3, TIGIT, and/or Vista.

In embodiments, the immunomodulator comprises agents that target co-stimulatory receptors expressed by T cells, such as those selected from Tumor Necrosis Factor Receptors (TNFR), including but not limited to glucocorticoid-induced TNFR (GITR; CD357), CD27, OX40(CD134), ICOS (CD278), or 4-1BB (CD137), wherein linkage of these glycoproteins to agonist antibodies actively transport activation signals to lymphocytes.

In certain embodiments, the immunomodulator comprises an inhibitor of inhibitory myeloid cells, such as, for example, PDL1, PDL2, VISTA, B7-1, CD47, CD200, GLA1, GAL3, CLECG4, or SIRPa. In embodiments, the immune modulators include compounds that target a range of Toll-like receptors (TLRs) and NOD-like receptors (NLRs), which represent a class of agonist molecules as well as targets for antagonist molecules. In additional embodiments, the immunomodulator comprises a cytokine known to modulate a T cell response, such as granulocyte colony stimulating factor (G-CSF), interferon, IL-2, IL-7 or IL-12.

In embodiments, the third composition comprises an immune checkpoint inhibitor targeting: PD1, PDL1, PDL2, CD28, CD80, CD86, CTLA4, B7RP1, ICOS, B7RPI, B7-H3, B7-H4, BTLA, HVEM, KIR, TCR, LAG3, CD137L, OX40, OX40L, CD27, CD70, CD40, CD40L, TIM3, GAL9, ADORA, CD276, VTCN1, IDO1, KIR3DL1, HAVCR2, VISTA or CD 244. See, e.g., example 5, which provides a method of targeting PD 1.

In embodiments, the third composition comprises an immune checkpoint inhibitor selected from the group consisting of: ipilimumab, nivolumab, pembrolizumab, alemtuzumab, avilluzumab, Devolumab, Cemiplimab, REGN2810, BMS-936558, SHR1210, KN035, IBI308, PDR001, BGB-A317, BCD-100, or JS 001.

In other embodiments, the third composition comprises an MDSC inhibitor that targets: ADAM17, PEG-2, PDE5, COX2, iNOS2, PDE5, c-kit, ARG1, PI3K, CSF-1R, caspase-8, CCL2, RON, ROS, S100A8/A9, or liver-X nuclear receptor.

In embodiments, the MDSC inhibitor is PF-5480090, INCB7839, nitroaspirin, SC58236, celecoxib, IPI-549, PLX3397, BLZ945, GW2580, RG7155, IMC-CS4, AMG-820, ARRY-382, sildenafil, tadalafil, vardenafil, N-hydroxy-nor-L-Arg, imatinib, z-IETD-FMK, trabectedin, Emricasan, anti-CCL 2 antibody (carlumab or ABN912), taconimod, ASLAN002, IMC-RON8, or GW 3965. See, e.g., Fleming et al, "Targeting Myeloid-Derived supressor Cells to Bypass Tumor-Induced immunity" Front immunity.2018; 9:398.

In embodiments, the immunosuppressive agent is formulated in a suitable aqueous solution for administration, wherein the third composition is administered as a separate composition from either the first vaccine composition or the second vaccine composition. In embodiments, the immunosuppressant composition is administered at a different time and date to the first vaccine composition and/or the second vaccine composition.

Application method

In embodiments, provided herein are methods for inducing an antigen CD8+ T cell response via administration of heterologous prime and boost doses of a vaccine composition. In certain embodiments, these methods comprise administering a first composition comprising a non-replicating viral vector encoding an antigen or immunogen comprising one or more CD8+ T cell epitopes; and administering a second composition comprising micelles comprising fluorocarbon-linked peptides, wherein each fluorocarbon-linked peptide: i) 15 to 75 amino acid residues in length; ii) an antigen or immunogen from the first composition; and iii) one or more CD8+ T cell epitopes from an antigen or immunogen comprising a first composition, wherein one of the first or second compositions is administered as a priming dose and the other of the first or second compositions is administered as a boosting dose, provided that both the first and second compositions are administered.

In embodiments, the priming dose and the booster dose are administered at least 7 days apart, at least 14 days apart, or longer. In embodiments, the priming dose and the booster dose are administered about 7 days apart, about 14 days apart, about 20 days apart, about 25 days apart, about 30 days apart, about 35 days apart, about 40 days apart, about 45 days apart, about 50 days apart, about 55 days apart, about 60 days apart, or about 65 days apart. Advantageously, the doses are administered at about 40 days apart, at about 41 days apart, at about 42 days apart, at about 43 days apart, at about 44 days apart, at about 45 days apart, at about 46 days apart, at about 47 days apart, at about 48 days apart, at about 49 days apart, or at about 50 days apart. In certain embodiments, the prime and boost doses are administered at about 1 week apart, about 2 weeks apart, about 3 weeks apart, about 4 weeks apart, about 5 weeks apart, about 6 weeks apart, about 7 weeks apart, about 8 weeks apart, about 9 weeks apart, about 10 weeks apart, about 11 weeks apart, or about 12 weeks apart. In certain other embodiments, the prime and boost doses are administered at about 1 month apart, about 2 months apart, about 3 months apart, about 4 months apart, about 5 months apart, about 6 months apart, about 7 months apart, about 8 months apart, about 9 months apart, about 10 months apart, about 11 months apart, or about 12 months apart.

In certain embodiments, the methods comprise administering a first composition comprising a non-replicating viral vector encoding an antigen or immunogen comprising one or more CD8+ T cell epitopes; or administering a second composition comprising micelles comprising fluorocarbon-linked peptides, wherein each fluorocarbon-linked peptide: i) 15 to 75 amino acid residues in length; ii) an antigen or immunogen from the first composition; and iii) one or more CD8+ T cell epitopes comprising an antigen or immunogen; and separately administering a third composition comprising an immunomodulator. In embodiments, the immune modulator is selected from an anti-immune repressor or an immune activator. The immunomodulator enhances a cell-mediated immune response induced by the first composition and/or the second composition.

In embodiments, the first vaccine composition or the second vaccine composition is administered as a prime dose and a booster dose, separated by at least 7 days, separated by at least 14 days, or longer. In embodiments, the priming dose and the booster dose are administered about 7 days apart, about 14 days apart, about 20 days apart, about 25 days apart, about 30 days apart, about 35 days apart, about 40 days apart, about 45 days apart, about 50 days apart, about 55 days apart, about 60 days apart, or about 65 days apart. Advantageously, the doses are administered at about 40 days apart, at about 41 days apart, at about 42 days apart, at about 43 days apart, at about 44 days apart, at about 45 days apart, at about 46 days apart, at about 47 days apart, at about 48 days apart, at about 49 days apart, or at about 50 days apart. In certain embodiments, the prime and boost doses are administered at about 1 week apart, about 2 weeks apart, about 3 weeks apart, about 4 weeks apart, about 5 weeks apart, about 6 weeks apart, about 7 weeks apart, about 8 weeks apart, about 9 weeks apart, about 10 weeks apart, about 11 weeks apart, or about 12 weeks apart. In certain other embodiments, the prime and boost doses are administered at about 1 month apart, about 2 months apart, about 3 months apart, about 4 months apart, about 5 months apart, about 6 months apart, about 7 months apart, about 8 months apart, about 9 months apart, about 10 months apart, about 11 months apart, or about 12 months apart.

In embodiments, the third composition (immunomodulator) is administered on the same date as the first vaccine composition or the second vaccine composition. In embodiments, the third composition is administered on multiple days, i.e., at least two days, at least three days, at least four days, at least five days, or at least six days. In embodiments, the third composition is administered at least once between the priming dose and the booster dose of the first vaccine composition or the second vaccine composition, and at least once after the booster dose of the first vaccine composition or the second vaccine composition. In exemplary embodiments, the third composition is administered on days 4, 7, 11, 15, 18, and 22 after the priming dose of the first vaccine composition or the second vaccine composition.

In embodiments, the vaccine composition is administered to a subject in need thereof. In embodiments, the subject in need thereof is a vertebrate, such as a mammal, bird, reptile, amphibian, or fish. In certain embodiments, the subject is a human, companion or domestic or food-or feed-producing or domestic or game or racing or sports animal, such as a cow, dog, cat, goat, sheep or pig or horse, or even poultry such as turkey, duck or chicken. In certain embodiments, the vertebrate is a human.

As used herein, an immunologically effective amount is the amount or concentration of a composition of a vaccine combination that, when administered to a subject in need thereof, generates an immune response against the delivered antigen. In certain embodiments, the immunologically effective amount of the vaccine combination or vaccine composition produces an antigen-specific CD8+ T cell response.

The method of the present invention may suitably be used for the prevention of disease as a prophylactic vaccination or for the treatment of disease as a therapeutic vaccination. The vaccine combinations of the present invention can be administered to a subject in need thereof as a priming dose and a booster dose alone or in combination with an immunosuppressant composition. Furthermore, the vaccine combination of the present invention may be administered as a priming dose or a booster dose in combination with an immunosuppressant composition to a subject in need thereof. The immunomodulator composition is a different third composition of the vaccine combination and may be administered on the same date and time as the first vaccine composition and/or the second vaccine composition, or on a different date. In exemplary embodiments, the immunomodulator composition is administered after the first vaccine composition.

In certain embodiments, the vaccine combination is administered to a subject in need thereof for use in the treatment or prevention of cancer. In embodiments, the vaccine combination is used as a therapeutic or prophylactic agent for: non-small cell lung cancer, breast cancer, liver cancer, brain cancer, stomach cancer, pancreatic cancer, kidney cancer, ovarian cancer, myeloma cancer, acute myelogenous leukemia, chronic myelogenous leukemia, head and neck cancer, colorectal cancer, kidney cancer, esophageal cancer, melanoma skin cancer, and/or prostate cancer. These cancer cells may express a neoantigen or viral antigen and the vaccine composition will comprise a suitable polypeptide comprising one or more CD8+ T cell epitopes, depending on the biology of the particular cancer/tumor.

In certain embodiments, the vaccine combination is administered to a subject in need thereof for use in treating or preventing chronic infection. In embodiments, the vaccine combination is used as a therapeutic or prophylactic agent for individuals having a chronic infection or at risk of exposure to a pathogen causing the chronic infection. Such pathogens include, but are not limited to, HIV, hepatitis b and delta viruses, herpes viruses, EB viruses, cytomegalovirus and human T-lymphotropic virus type III.

The vaccine combination can be administered in vivo to a human or animal subject using a variety of known routes and techniques. For example, the composition of the vaccine combination may be provided as an injectable solution, suspension or emulsion and administered via parenteral injection, subcutaneous injection, oral injection, epidermal injection, intradermal injection, intramuscular injection, intraarterial injection, intraperitoneal injection, intravenous injection using conventional needles and syringes or using liquid jet injection systems. The composition of the vaccine combination may be administered topically (topically) to the skin or mucosal tissue, such as nasally, intratracheally, enterally, sublingually, rectally or vaginally, or provided as a finely divided (fine powdered) spray, such as a mist (mist), suitable for respiratory or pulmonary administration. In certain embodiments, the vaccine composition is administered intramuscularly.

The composition of the vaccine combination may be administered to a subject in an amount compatible with the dosage composition that will be prophylactically and/or therapeutically effective. Administration of the compositions of the invention may be for "prophylactic" or "therapeutic" purposes. As used herein, the term "therapeutic" or "treatment" includes any one or more of the following: preventing infection; treatment of chronic infections; prevention of tumorigenesis/canceration; reduction or elimination of symptoms; and/or reduce or eliminate tumors or cancers altogether.

In embodiments, the vaccine combination comprises a cancer antigen or a viral antigen associated with cancer, wherein the treatment can be prophylactic (before the diagnosis of cancer) or therapeutic (after the diagnosis of cancer). Therapeutic treatment may be given to stage I, II, III or IV cancer before or after surgical intervention. The treatment may be a post-operative maintenance treatment or a long-term treatment to improve progression-free survival or overall survival and/or disease clearance.

The choice of carrier will generally vary with the route of delivery of the composition, if desired. In the present invention, the compositions may be formulated for any suitable route and mode of administration. Pharmaceutically acceptable carriers or diluents include those used in compositions suitable for oral, ocular, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, transdermal) administration.

The composition may be administered in any suitable form, for example as a liquid, solid or aerosol. For example, oral formulations may take the form of emulsions, syrups or solutions or tablets or capsules, which may be enterically coated to protect the active ingredient from degradation in the stomach. Nasal formulations may be sprays, mists or solutions. Transdermal formulations may be adapted for their particular delivery system and may include patches. Formulations for injection may be solutions or suspensions in distilled water or another pharmaceutically acceptable solvent or suspending agent.

The appropriate dose of prophylactic or therapeutic vaccine to be administered to a patient will be determined clinically. Multiple doses in addition to the prime and boost doses may be required in order to achieve an immunological or clinical effect, and are usually administered between 1 and 12 weeks apart if required. If it is desired to boost the immune response over a longer period of time, repeat doses may be administered at intervals of 1 month to 5 years.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the embodiments provided herein are used, and are not intended to limit the scope of the disclosure nor are they intended to represent that the examples below are all or only experiments performed. Although efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), some experimental errors and deviations should be accounted for. Parts are parts by volume and temperatures are degrees celsius unless otherwise indicated. It should be appreciated that variations to the methods described may be made without altering the basic aspects of the embodiments that are intended to be illustrative.

Example 1: preparation of vaccine combinations comprising heterologous prime and boost dose combinations: a first composition (a non-replicating viral vector composition); and a second composition (fluorocarbon-linked peptide composition)

Non-replicating viral vector compositions

AdGP70 was designed as a recombinant adenovirus serotype 5 vector, conferring replication defects by deletion of E1 and E3 (Δ E1E3), and allowing GP70 (envelope (Env) protein of murine leukemia virus (MuLV), GenBank accession No. ABC94931.1) to be expressed under the Cytomegalovirus (CMV) promoter. First, the shuttle plasmid was obtained after cloning the chemically synthesized, codon optimized GP70 gene for expression in mammalian cells into the HindIII/XbaI restriction site of the pAdHigh γ shuttle vector (Altimmune Inc) at Genscript (Piscataway, NJ). Recombination between the transgenic pAdHigh γ shuttle vector and pAdEasy-1 adenovirus 5 backbone plasmid was performed in escherichia coli (e. coli) strain BJ5183 under kanamycin selection toThe genomic plasmid pAdGP70 was generated. The pAdEasy-1 plasmid contains all of the Ad5 sequences except nucleotides 1-3,533 (containing the E1 gene) and nucleotides 28,130-30,820 (containing E3). By colony isolation, the selected genomic plasmid, pAdGP70, was retransformed into e.coli strain DH10B cells under kanamycin selection. Selected colonies of pAdGP70 were amplified and purified. AdGP70 recombinant adenovirus vector seeds were generated by transfection of the PacI linearized purified pAdGP70 genomic plasmid into the adenovirus packaging cell line HEK 293. The AdGP70 vector was propagated on HEK293 cells and purified by cesium chloride gradient ultracentrifugation. Purified Ad5 vector was sterilized by 0.22- μm-pore size filtration and stored at-80 ℃ in A195 adenovirus storage buffer. AdGP70 titers (4X 10) were determined on HEK293 cells by using the Adeno-X Rapid Titer kit (Clontech, Mountain View, Calif.)¹¹ifu/ml). AdGP70 was further diluted to 4X 10 in PBS prior to in vivo administration¹⁰ifu/ml。

SEQ ID NO.1 murine leukemia virus GP70 protein sequence-accession ABC94931.1

T cell epitopes present in antigens can be identified using a variety of methods including published epitopes, Artificial Neural Networks (ANN) or Stable Matrix Methods (SMM) (using the Immune Epitope Database and Analysis Resource website) or the SYFPEITHI website, the SYFPEITHI website is a Database containing more than 7000 peptide sequences known to bind MHC class I and MHC class II molecules compiled from published reports. T cell epitopes from GP70 were identified by using an Artificial Neural Network (ANN) and a Stabilization Matrix Method (SMM) using a predicted IC50 cut-off value of <50nM, and SYFPEITHI (http:// www.syfpeithi.de /) using a threshold of >20 to predict high affinity binding peptides to murine MHC class I and MHC class II molecules (H-2Kd, H-2Dd, H-2Ld, IAd, and IEd). The T cell epitopes of the GP70 protein sequence identified using a combination of these methods are provided in table 1.

Table 1: t cell epitope of GP70

N Casares；J J Lasarte；A L de Cerio；P Sarobe；M Ruiz；I Melero；J Prieto；F Borrás-Cuesta.Immunization with a tumor-associated CTL epitope plus a tumor-related or unrelated Th1 helper peptide elicits protective CTL immunity.2001.Eur J Immunol.31:1780-9。

Fluorocarbon linked peptide compositions

The fluorocarbon-linked peptide composition (PepGP70) comprises four fluorocarbon-modified peptides derived from GP70 (GP70-142, GP70-196, GP70-472, GP70-CM), GP70 is the envelope (Env) protein of murine leukemia virus (MuLV). The sequences of GP70-142, GP70-196, GP70-472, GP70-CM are presented in Table 1 and Table 2. These four peptides are predicted based on the use of Artificial Neural Networks (ANNs) and usage<Stable Matrix Method (SMM) with IC50 cutoff of 50nM (www.iedb.org /) and use>A threshold of SYFPEITHI (www.syfpeithi.de /) of 20 and published information was selected for prediction of high affinity binding peptides for murine MHC class I and MHC class II molecules (H-2Kd, H-2Dd, H-2Ld, IAd and IEd). GP70-142, GP70-196, GP70-472, GP70-CM were obtained by Solid Phase Peptide Synthesis (SPPS). All peptides were synthesized by the American Peptide Company (Sunnyvale, Calif.) using standard 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry to construct the Peptide on a resin. Reacting 2H,2H,3H, 3H-perfluoroundecanoic acid fluorocarbon chain (C)₈F₁₇(CH₂)₂COOH) to the optionally deprotected C-terminal or N-terminal epsilon-chain of a further lysine to obtain a fluorocarbon modified peptide. After cleavage and removal of the side chain protecting groups, the crude peptide was precipitated from cold ether and collected by filtration. Purity was assessed by RP-HPLC and all peptides were better than 90% pure. The lyophilized fluorocarbon-linked peptide was stored at-20 ℃. PepGP70 was obtained after peptides GP70-142, GP70-196, GP70-472, GP70-CM were dissolved, mixed, added with mannitol/water solution, filtered using a 0.22 μm filter, and freeze dried to give separate vials containing 1400 μ g/peptide. The freeze-dried PepGP70 was stored at-20 ℃. Prior to in vivo administration, lyophilized PepGP70 vials were reconstituted with 1.4ml of ODN1585(TLR 9 agonist; Invivogen, Toulose, France) containing 28mM L-histidine to yield intensities of 100. mu.g/ODN 1585/ml and 1000. mu.g/peptide/ml.

Table 2: four peptides of the PepGP70 composition

FA₁＝C₈F₁₇(CH₂)₂-CO-

The above peptide derived from the GP70 protein sequence was underlined in the full length sequence of the protein above (SEQ ID NO:1) and the identified T cell epitopes are in Table 1. GP70-472 and GP70-CM comprise the Cytotoxic T Lymphocyte (CTL) epitope SPSYVYHQF (SEQ ID NO:72) (also known as AH1 in dextramer). KKK is a linker and is not present in the full length GP70 protein sequence. GP70-CM is a chimeric peptide comprising a CTL epitope (CD8+ epitope), a KKK linker and a T helper lymphocyte (HTL) epitope.

Accordingly, provided herein is a vaccine combination for use in a heterologous prime boost dosing regimen, wherein the combination comprises a first composition comprising a non-replicating viral vector encoding an antigen or immunogen comprising one or more CD8+ T cell epitopes; and a second composition comprising micelles comprising fluorocarbon-linked peptides, wherein each fluorocarbon-linked peptide: i) 15 to 75 amino acid residues in length; ii) an antigen or immunogen from the first composition; and iii) one or more CD8+ T cell epitopes comprising the antigen or immunogen of the first composition; wherein either of the first composition or the second composition is a priming composition or a boosting composition.

Example 2: preparation of a vaccine combination comprising a combination of a T cell vaccine composition and an immunomodulator composition: a first composition (a non-replicating viral vector composition); or a second composition (fluorocarbon-linked peptide composition); and a third composition (immunomodulator composition)

The non-replicating viral vector composition (first composition) and the fluorocarbon-linked peptide composition (second composition) were prepared as disclosed in example 1.

Immune modulator composition

The immunomodulator composition can be any immunomodulator composition disclosed herein, and is provided in a suitable aqueous solution. In this example, anti-PD-1 was provided in PBS and administered separately as disclosed below, and at a different time point than the first vaccine composition or the second vaccine composition disclosed above.

Accordingly, provided herein is a vaccine combination for use in inducing a CD8+ T cell response and reducing tumor size, wherein the combination comprises a first composition comprising a non-replicating viral vector encoding an antigen or immunogen comprising one or more CD8+ T cell epitopes;orA second composition comprising micelles comprising fluorocarbon-linked peptides, wherein each fluorocarbon-linked peptide: i) 15 to 75 amino acid residues in length; ii) an antigen or immunogen from the first composition; and iii) one or more CD8+ T cell epitopes comprising an antigen or immunogen; and a third composition comprising an immunomodulator selected from an anti-immune repressor or an immune activator. The first composition or the second composition is administered separately from the third composition.

In certain embodiments, provided herein are vaccine combinations comprising two to three compositions selected from the group consisting of: a) a first composition comprising a non-replicating viral vector encoding an antigen or immunogen comprising one or more CD8+ T cell epitopes; b) a second composition comprising micelles comprising fluorocarbon-linked peptides, wherein each fluorocarbon-linked peptide: i) 15 to 75 amino acid residues in length; ii) an antigen or immunogen from the first composition; and iii) one or more CD8+ T cell epitopes from an antigen or immunogen comprising the first composition; and c) a third composition comprising an immunomodulator selected from an anti-immune repressor or an immune activator, wherein when the first and second compositions are selected, either the first or second composition is a priming or boosting composition.

Example 3: inducing an antigen-specific CD8+ T cell response using a non-replicating viral vector composition and a fluorocarbon-linked peptide composition (heterologous vaccine combination);

the preparation of AdGP70 (non-viral vector composition) and PepGP70 (fluorocarbon-linked peptide composition) is disclosed in example 1, where AdGP70 is a replication-defective adenovirus vector expressing GP70(SEQ ID NO:1) and PepGP70 is a combination of four peptides (SEQ ID NO:68 to 71) attached separately to fluorocarbon chains and present in micelles.

A comparison of the homologous prime/boost dose and the heterologous prime/boost dose of the AdGP70 and PepGP70 compositions was made, wherein the immunogenicity of the AdGP70 and PepGP70 compositions was compared to the immunogenicity of the heterologous prime/boost combination of AdGP70 and PepGP70 after one or two administrations in BALB/c mice. Mice were immunized using the subcutaneous route with a 14 day interval between the administration of the prime dose and the administration of the boost dose. Group 1 (n-8) received 50 μ l of PepGP70(50 ug/peptide) subcutaneously on day 0. Group 2 (n-8) received 50 μ l of AdGP70(2 × 10) subcutaneously on day 0⁹ifu). Group 3 (n-8) received 50 μ l of PepGP subcutaneously on days 0 and 1470(50 ug/peptide). Group 4 (n-8) received 50 μ l of PepGP70(50 ug/peptide) subcutaneously on days 0 and 14. Group 5 (n-8) received 50 μ l of AdGP70(2 × 10) subcutaneously on days 0 and 14, respectively⁹ifu) and PepGP70(50 ug/peptide). Group 6 (n-8) received 50 μ l of PepGP70(50 ug/peptide) and AdGP70(2 × 10) subcutaneously on days 1 and 14, respectively⁹ifu). 10 days after the final administration (measured on day 24), splenocytes were isolated from each animal and processed by two types of immunoassays: (1) an in vitro IFN- γ ELISpot assay to assess the frequency of GP 70-specific T cells in individual animals (fig. 1), and (2) a dextramer staining assay to measure the frequency of CD8+ T cells specific for the H-2 Ld-restricted epitope derived from GP70 (AH1 peptide, sequence SPSYVYHQF (SEQ ID NO:72)) by flow cytometry. See fig. 1 and 2.

Clinical trials have demonstrated the efficacy of T cell-inducing vaccines against a number of diseases, and while a number of methods can be employed to assess protective T cell responses, ELISpot assays have become the most suitable method of determining vaccine immunogenicity as is well established. Slota m, et al, ELISpot for measuring human immune responses to Vaccines. expert Rev Vaccines 2011; 10(3):299-306. For IFN-. gamma.ELISpot assays, ELISpot plates (MSIPS4510 Merck Millipore) were pre-coated with 100. mu.l/well of 5. mu.g/ml capture IFN-. gamma.antibody (mouse IFNg ELISPOT pair, BD, ref 551881) diluted in PBS under sterile conditions and incubated overnight at 4 ℃. The coated antibody was removed and the plates were washed and then incubated at room temperature for 2hr with 200 μ L/well of complete medium containing RPMI Glutamax 1640(GIBCO, ref 11540) supplemented with 10% fetal bovine serum (GIBCO, ref10270-098) and 1% penicillin-streptomycin solution (GIBCO, ref 11548876). After removal of the medium, the spleen cell suspension in complete medium was incubated at 5X 10⁵The concentration of individual cells/well was added to a pre-coated ELISpot plate in a volume of 200. mu.l per well, where 10ug/ml of each individual peptide (GP70-142, GP70-196, GP70-472, GP70-CM) or a mixture of four peptides, concanavalin A (eBiosciences, ref 00-4978-03) was present as a positive control. Medium alone was used as a negative control. Each test condition was performed in duplicate. The board is moistenedIncubate at 37 ℃ for 18 hours with 5% CO2 in ambient. After two washing steps to completely remove cells with deionized water (DI), plates were washed with ELISpot wash buffer (1 × Dulbecco's PBS, Gibco, Fisher Cat. No. 11540486, 0.05%Fisher catalog No. 10113103). The ELISpot plates were then incubated with 100 μ l/well of 2ug/ml anti-IFN-g detection antibody (mouse IFNg ELISpot pair, BD, ref 551881) diluted in PBS supplemented with 10% fetal bovine serum for 2hrs at room temperature. The detection antibody solution was discarded, and the plate was washed with ELISpot wash buffer, then diluted with 100. mu.L/well of streptavidin-HRP (BD)^TMELISPOT Streptavidin-HRP, catalog No. 557630) were incubated together at room temperature for 1 hr. The streptavidin-HRP solution was removed and the plate was washed with ELISpot wash buffer. 100 μ L of final substrate solution (AEC) was added to each well. Spot development was monitored from 15-20min and substrate reaction was stopped by washing the wells with DI water. The plates were then air dried overnight at room temperature in the dark. Using an ELISPOT analyzer:the ELISpot plate reader (CTL-Europe GmbH, Germany) performed spot counting. Spot-forming cells, SFC/well, were counted to quantify the number of cells producing IFN- γ in response to a particular stimulus.

Referring to fig. 1, the graph shows the cumulative IFN- γ ELISpot response to the four peptides, expressed as the number of IFN- γ producing cells (spot forming cells, SFC) per million splenocytes calculated for each group at day 24 after administration of the priming dose. Groups 5 and 6 are heterologous prime boost groups and show a synergistic effect compared to the homologous prime boost group.

For the dextramer assay, murine splenocytes were stained with H-2Ld dextramer prepared from AH1 peptide (CD8+ T cell epitope from GP70, sequence: SPSYVYHQF (SEQ ID NO:73)) or an irrelevant control H-2Ld dextramer prepared from NP118-126 peptide (CD8+ T cell epitope from nucleoprotein LCMV, sequence: RPQASGVYM (SEQ ID NO:74)),both were labeled with Phycoerythrin (PE). The Dextramer reagent consists of a dextran polymer scaffold carrying an optimized number of MHC-peptide complexes and a fluorescent dye, providing the ability to detect antigen-specific T cells, where the T cell receptor specifically recognizes the MHC/peptide complexes carried by the dextran polymer. The cell staining method for flow cytometry analysis is described below. Individual spleen cells (1 × 10) from group 1 to group 6 animals⁶Individual cells) were cultured in 200 μ l of complete medium containing RPMI Glutamax 1640(GIBCO, ref 11548876) supplemented with 10% fetal bovine serum (GIBCO, ref10270-098) and 1% penicillin-streptomycin solution (GIBCO, ref 11548876) in 96-well plates. The plates were incubated overnight at 37 ℃ in a humid environment with 5% CO 2. After incubation, splenocytes from individual mice were pooled by group and washed with staining buffer (DPBS 1 x supplemented with 5% fetal bovine serum, 2mM EDTA, and 1% penicillin-streptomycin solution) by centrifugation at 1300rpm for 6min at 4 ℃. Fc receptor blockade was achieved by incubating splenocytes with 25ul of cold staining buffer containing anti-mouse CD16/32 antibody diluted to 1:200 at 4 ℃ for 10 minutes. Cells were then stained with either the relevant AH-1/H-2Ld dextramer (Immundex, ref JG3294-OPT) or the irrelevant NP118-126/H-2Ld dextramer (Immundex, ref JG 2750-OPT). 25ul of dextramer suitably pre-diluted 1:2.5 in staining buffer was added to the Fc receptor blocked cells, followed by incubation at 4 ℃ for 30 min. After incubation, a 2 x antibody mixture comprising: anti-mouse CD4 Pe-Cy7 (zyme, ref BLE100422), anti-mouse CD8a BV510 (zyme, ref BLE100752), anti-mouse CD44BV650 (zyme, ref BLE103049), anti-mouse CD62L BV711 (zyme, ref BLE104445), anti-mouse CD45 APC (zyme, ref BLE103112), anti-mouse PD1PERCP-Cy5.5 (zyme, ref BLE109119), anti-mouse CD69 APC-eFluor780(eBioscience ref 47-0691-80) and viability dye eFluor^TM520(eBioscience, ref 65-0867-14). 50ul of the antibody mixture was added to the dextramer stained cells, followed by incubation at 4 ℃ for 20 minutes. After the staining step, the cells were washed twice with staining buffer by centrifugation at 1300rpm for 6min at 4 ℃ and resuspended in 200ul staining buffer, thenFollowed by flow cytometry collection and analysis. Live CD45+ CD8 cell events were gated. Figure 2 shows the percentage of CD8+ T cells staining positive for dextramer.

Referring to fig. 2, the graph shows the percentage of CD8+ T cells that were positive for dextramer staining on day 24 after administration of the priming dose. Groups 5 and 6 are heterologous prime boost groups and show a synergistic effect compared to the homologous prime boost group.

Results from ELISpot assays demonstrated an increase in the number of IFN- γ producing splenocytes compared to single and double administration of PepGP70 or AdGP 70. Surprisingly, heterologous prime boost dose administration (AdGP70 followed by PepGP70 or vice versa) induced a stronger peripheral immune response than homologous prime boost dose administration with AgGP70 or PepGP 70. See fig. 1 and 2. The data reveal an unexpected synergy between the two different types of immunogens in their ability to promote more robust T cell responses, including CD8+ T cell responses, when combined in heterologous prime boost doses. The results also indicate that the heterologous prime boost regimen using the first administration of PepGP70 and then the administration of AdGP70 represents a more favorable condition for induction of antigen-CD 8+ T cells, as revealed by the dextramer immunoassay. See fig. 2.

Accordingly, provided herein is a method of inducing an immune response in a subject in need thereof using a heterologous dosage regimen, wherein the method comprises administering a first composition comprising a non-replicating viral vector encoding an antigen or immunogen comprising one or more CD8+ T cell epitopes; and administering a second composition comprising micelles comprising fluorocarbon-linked peptides, wherein each fluorocarbon-linked peptide: i) 15 to 75 amino acid residues in length; ii) an antigen or immunogen from the first composition; and iii) one or more CD8+ T cell epitopes in an antigen or immunogen comprising the viral vector, wherein an antigen-specific CD8+ T cell response is induced. The first composition or the second composition is administered as a priming dose and one of the first composition or the second composition is administered as a boosting dose, provided that both the first composition and the second composition are administered.

Example 4: use of non-replicating viral vector compositions and fluorocarbon-linked peptide compositions in heterologous dosing regimens

Synergy between AdGP70 and PepGP70 was examined by assessing antitumor activity in BALB/c mice challenged with CT26 tumor cells. Experiments were performed using female BALB/c mice 6-8 weeks old. On day 0, 100ul of 2X 10 in PBS was added⁴Individual CT26 cells were injected subcutaneously in the flank of the mouse. Vaccine compositions of AdGP70 and PepGP70 were prepared according to example 1, wherein the formulated vaccine was prepared as 50ul of injectable solution. The compositions were administered subcutaneously according to a prime/boost regimen with 14 days between the administration of the prime and boost doses.

Group 1 received 50. mu.l of PepGP70 (50. mu.g/peptide) subcutaneously on days 1 and 14, respectively. Group 2 received 50. mu.l of AdGP70 (2X 10) subcutaneously on days 7 and 14, respectively⁹ifu). Group 3 received only 50 μ l of AdGP70 (2X 10) subcutaneously on days 7 and 14, respectively⁹ifu) and PepGP70(50 μ g/peptide). Group 4 received 50. mu.l of PepGP70(50 ug/peptide) and 50. mu.l of AdGP70 (2X 10) subcutaneously on days 1 and 14, respectively⁹ifu). Group 5 received no treatment.

Both vaccines PepGP70 and AdGP70 tested alone or as a combination of prime boost promoted anti-tumor responses. The vaccine regimen consisting of the prime/boost combination promoted a better anti-tumor response compared to PepGP70 and AdGP70 tested alone.

Example 5: use of non-replicating viral vector compositions and fluorocarbon-linked peptide compositions in a heterologous dosing regimen in combination with an immune checkpoint inhibitor (anti-PD 1)

Preparation of AdGP70 (non-viral vector composition) and PepGP70 (fluorocarbon-linked peptide composition) is disclosed in example 1, where AdGP70 is a replication-defective adenovirus vector expressing GP70(SEQ ID NO:1) and PepGP70 is a combination of four peptides (SEQ ID NO:68 to 71) attached separately to fluorocarbon chains and present in micelles; also, the preparation of immune checkpoint inhibitor compositions is disclosed in example 2.

Synergy between AdGP70 and PepGP70 and anti-PD 1 therapeutic combinations was examined by assessing the immune response induced by the vaccine in BALB/c mice challenged with CT26 tumor cells. Experiments were performed using 6-8 week old female BALB/c mice (n ═ 12 per group). Animals were anesthetized with a mixture of ketamine-domitor (0.7mg/80mg/kg) to allow shaving of the animals at the tumor cell inoculation site and awakening was induced by injection of anti dean (2 mg/kg). On day 0, 100ul of 2X 10 in PBS was added⁴Individual CT26 cells were injected subcutaneously in the flank of the mouse.

Vaccine compositions of AdGP70 and PepGP70 were prepared according to example 1, wherein the formulated vaccine was prepared as 50ul of injectable solution. The compositions were administered subcutaneously according to a prime/boost regimen with 7 days between the administration of the prime and boost doses. In this experimental model of tumors, booster doses were administered 7 days later, rather than the typical 14 days later, to ensure that a robust immune response was generated to test the effect of the immune response against the tumor. Six injections of the immune checkpoint inhibitor composition anti-PD 1 (anti-CD 279-clone RMP1-14, InVivoplus, Euromedex, ref BP0146-100 mg; GoInVivo, Ozyme, ref BLE114115) were administered by intraperitoneal route.

Group 1 (n-12) received 50 μ l of PepGP70(50 μ g/peptide) subcutaneously on days 7 and 14, respectively, and 100 μ l of 200 μ g anti-mouse PD1 intraperitoneally on days 7, 11, 14, 18, 22, and 25. Group 2 (n-12) received 50 μ l of AdGP70(2 × 10) subcutaneously on days 7 and 14, respectively⁹ifu) and received 100 μ l of 200 μ g anti-mouse PD1 intraperitoneally on days 7, 11, 14, 18, 22, and 25. Group 3 (n-12) received only 50 μ l of AdGP70(2 × 10) subcutaneously on days 7 and 14, respectively⁹ifu) and PepGP70(50 μ g/peptide) and received 100 μ l of 200 μ g anti-mouse PD1 intraperitoneally on days 7, 11, 14, 18, 22 and 25. Group 4 (n-12) received 50 μ l of PepGP70(50 ug/peptide) and 50 μ l of AdGP70(2 × 10) subcutaneously on days 7 and 14, respectively⁹ifu) and at day 7, day 11, day 14, day 18, day 2Day 2 and day 25 received 100 μ l of 200 μ g anti-mouse PD1 intraperitoneally. Group 5 received 100 μ l of 200 μ g anti-mouse PD1 intraperitoneally on days 7, 11, 14, 18, 22, and 25. Group 6 (n-12) received no treatment.

For the dextramer assay, PBMCs collected at day 6 (D6) or day 20 (D20) were stained with Phycoerythrin (PE) -labeled H-2Ld dextramer prepared from AH1 peptide (CD8+ T cell epitope from GP70, sequence: SPSYVYHQF (SEQ ID NO: 73)). The cell staining method for flow cytometry analysis is described below. 200 μ l of whole blood samples from group 1 to 6 animals were collected via retro-orbital bleeding technique into EDTA-coated tubes and inverted several times to prevent coagulation. Erythrocytes were lysed using multi-species RBC lysis buffer (eBiosciences, ref 00-4300-54) according to the manufacturer's recommendations. Individual PBMC samples from group 1 to group 6 animals were cultured in 200 μ l of complete medium comprising RPMI Glutamax 1640(GIBCO, ref 11548876) supplemented with 10% fetal bovine serum (GIBCO, ref 10270-. The plates were incubated overnight at 37 ℃ in a humid environment with 5% CO 2. After completion of erythrocyte lysis, incubation, PBMCs from individual mice were washed with staining buffer (DPBS 1 x supplemented with 5% fetal bovine serum, 2mM EDTA, and 1% penicillin-streptomycin solution) by centrifugation at 1300rpm for 6min at room temperature. Fc receptor blockade was achieved by incubating splenocytes with 25ul of cold staining buffer containing anti-mouse CD16/32 antibody diluted to 1:200 at 4 ℃ for 10 minutes. Cells were then stained with either the relevant AH-1/H-2Ld dextramer (Immundex, ref JG3294-OPT) or the irrelevant NP118-126/H-2Ld dextramer (Immundex, ref JG 2750-OPT). Mu.l of dextramer suitably pre-diluted 1:2.5 in staining buffer was added to Fc receptor-blocked cells, followed by incubation at 4 ℃ for 30 min. After incubation, a 2 x antibody mixture comprising: anti-mouse CD4 Pe-Cy7(Ozyme, ref BLE100422), anti-mouse CD8a BV510(Ozyme, ref BLE100752), anti-mouse CD44 VioBlue anti-mouse (Miltenyi, ref 130-ce, ref 61-0251-82), CD11b Alexa 700(eBioscience, ref 56-0112-82) and the viability dye eFluor^TM520(eBioscience, ref 65-0867-14). Add 50. mu.l of antibody mix to the dextramer stained cells and incubate at 4 ℃ for 20 min. After the staining step, the cells were washed twice with staining buffer by centrifugation at 1300rpm for 6min at 4 ℃. The surface stained cells were resuspended in 200. mu.l of a Fixation/Permeabilization working solution (Fixation/Permeabilization and Permeabilization buffer set kit), eBioscience, ref 00-5523-00, prepared according to the manufacturer's recommendations, and incubated at 4 ℃ for 25 minutes. The cells were then washed with 1 Xpermeabilization buffer by centrifugation at 1300rpm for 6min at 4 ℃ and then stained with anti-mouse Perforin APC (eBioscience, ref 17-9392-80). Add 50. mu.l of prediluted Perforin APC to the dextramer stained cells and incubate for 20min at 4 ℃. Cells were washed twice with 1 x permeabilization buffer by centrifugation at 1300rpm for 6min at 4 ℃ and then resuspended in staining buffer before flow cytometry collection and analysis. Live Dextramer + CD8 cell events were gated. Figure 3 shows the percentage of CD8+ T cells staining positive for dextramer on days 6 (D6) and 20 (D20), expressed as median for each group.

As presented in figure 3, both vaccine compositions PepGP70 and AdGP70 tested alone or as a prime boost combination had a synergistic effect with anti-PD 1 treatment in terms of their ability to promote anti-tumor immune responses, with antigen-specific CD8+ T cells measured at day 6 (D6) and day 20 (D20). The vaccine combination consisting of prime with PepGP70 and boost with AdGP70 with anti-PD 1 treatment promoted a much higher response compared to other vaccine regimens. All vaccine dosing regimens (group 1 to group 4) were synergistic with anti-PD 1, but the dosing regimen with PepGP70 as the priming dose and AdGP70 as the booster dose (group 4) showed synergy compared to the homologous dosing regimen vaccine (groups 1 and 2).

Example 6: use of fluorocarbon-linked peptide compositions in a homologous dosing regimen to induce antigen-specific CD4+ and CD8+ T cell responses and anti-tumor activity

Fluorocarbon-linked peptide (FP-OVA) compositions were prepared according to the disclosure in example 1, except that the OVA peptide was used instead of the GP70 peptide. FP-OVA comprises an ovalbumin-derived peptide (sequence ISQAVHAAHAEINEAGRESIINFEKLTEWT (SEQ ID NO:75)) comprising a CD4+ T cell epitope (OVA 323-339, sequence ISQAVHAAHAEINEAGR (SEQ ID NO:76)) and a CD8+ T cell epitope (amino acid position 257-264, sequence SIINFEKL (SEQ ID NO: 77)).

In this experiment, female C57BL/6 mice (n-15/group) were vaccinated subcutaneously with 100ul of 200ug FP-OVA vaccine (group 1) or 100ul of vehicle solution (group 2) on days 0 and 14. On day 24, 5 mice were sacrificed in each group and the immune response was measured by ELISpot assay. The immune response to the vaccine composition was measured as follows: in the IFN γ ELISpot assay, spleens were harvested and cells were stimulated with 1ng/mL OVA-CTL epitope (OVA 257-264, sequence SIINFEKL (SEQ ID NO:77) (CD8+ T cell epitope)) or 10 μ g/mL OVA-HTL (OVA 323-339, sequence ISQAVHAAHAEINEAGR (SEQ ID NO:76) (CD4+ T cell epitope)). The number of IFN γ spot-forming cells (SFC) was counted. FIG. 4A shows the number of IFN γ spot-forming cells (SFC) produced by individual mice in response to OVA-CTL or OVA-HTL/10 after subtraction of control well values⁶Individual splenocytes (line represents median response). Data were defined as parameters using the Kolmogorov-Smirnov normality test and statistically analyzed using student's t test, where P<0.001＝***。

On day 24, the remaining 10 mice in each group received 2 × 10⁶Subcutaneous injection of individual e.g. g7-OVA cells; mouse thymoma EL4 cells were stably transfected with complementary DNA to chicken Ovalbumin (OVA) and thus expressed OVA epitopes as unique antigens. When the tumor size reaches 150mm²At that time, the challenged mice were sacrificed (hill). Fig. 4B shows the overall survival measured over time in two different groups. Fig. 5 shows tumor growth measured over time in the vehicle group (fig. 5A) and the vaccine group (fig. 5B). Figures 4 and 5 demonstrate the effectiveness of fluorocarbon-linked peptides as prophylactic agents against cancer, where the peptides are unique cancer antigens.

In a second experiment, female C57BL/6 mice (n ═ 10/group) received 2 × 10 on one flank on day 0⁶S. G7-OVA cells, and received 2X 10 flank of the opposite flank in surviving animals on day 50⁶Second administration of individual E.G7-OVA cells. Group 1 then received 100ul of 200ug FP-OVA vaccine subcutaneously on days 1 and 8. Group 2 received 100ul of 200ug of FP-OVA vaccine subcutaneously on day 3 (corresponding to the day on which palpable tumors were detected in at least one animal) and day 10. Group 3 received 100ul of vehicle subcutaneously on days 1 and 8. When the tumor size reaches 150mm²At that time, the challenged mice were sacrificed. Figure 6 shows the overall survival measured over time in different groups, where each of group 1 and group 2 has at least 60% survival, demonstrating the effectiveness of fluorocarbon-linked peptides as cancer therapeutics, where the peptides are unique cancer antigens.

Example 7: use of non-replicating viral vector compositions or fluorocarbon-linked peptide compositions in combination with immune checkpoint inhibitors (anti-PD 1)

Preparation of AdGP70 (non-viral vector composition) and PepGP70 (fluorocarbon-linked peptide composition) is disclosed in example 1, where AdGP70 is a replication-defective adenovirus vector expressing GP70(SEQ ID NO:1) and PepGP70 is a combination of four peptides (SEQ ID NO:68 to 71) attached separately to fluorocarbon chains and present in micelles; also, the preparation of immune checkpoint inhibitor compositions is disclosed in example 2.

Synergy between AdGP70 or PepGP70 and anti-PD 1 treatment was examined by assessing their respective antitumor activity against CT26 colon cancer tumors in BALB/c mice. Experiments were performed using 6-8 week female BALB/c mice (n ═ 12 per group). Animals were anesthetized with a mixture of ketamine-domitor (0.7mg/80mg/kg) to allow shaving of the animals at the tumor cell inoculation site and awakening was induced by injection of anti dean (2 mg/kg). On day 0, 2X 10^4 CT26 cells in 100ul PBS were injected into the flank of the mice. Five tumor palpations were performed per week to determine the date of the first positive tumor detection. After detection of solid tumors, their size is measured weeklyAbout three times. Tumor size measurements were performed in two dimensions with calipers, and the volume was determined according to the following equation: l ^ L2/2; (L ═ major axis, and L ═ minor axis). Animals were sacrificed according to the following humane endpoints: tumor volume is more than or equal to 2000mm³Tumors with necrosis or ulceration, impaired mobility including transient prone or hunched posture, interference with important physiological functions including respiration, significant abdominal distension or weight loss over a week>20％。

The formulated vaccine compositions were prepared as 50ul injectable solutions according to examples 1 and 2 and administered subcutaneously according to a prime/boost regimen with 14 days intervals between prime and boost doses. Six injections of immune checkpoint anti-PD 1 (anti-CD 279-clone RMP1-14, InVivoplus, Euromedex, ref BP0146-100 mg; GoInVivo, Ozyme, ref BLE114115) were administered by intraperitoneal route every 3 or 4 days after tumor initiation.

Group 1 (n ═ 15) received only vaccine composition preparations on days 1 and 15, respectively: 50 μ l of PepGP70(50 ug/peptide). Group 2 (n-14) received 50 μ l of PepGP70(50 ug/peptide) on days 1 and 15, respectively, and received a 100ul delivered dose of 200 μ g of anti-mouse PD1 (anti-CD 279-clone RMP1-14) on days 4, 7, 11, 15, 18 and 22. Group 3 (n-14) received only vaccine preparations on day 0 and 14, respectively: 50 μ l of AdGP70 (2X 10)⁹ifu). Group 4 (n-15) received 50 μ l of AdGP70(2 × 10) on days 0 and 14, respectively⁹ifu) and received a 100ul delivered dose of 200 μ g of anti-mouse PD1 (anti-CD 279-clone RMP1-14) on days 4, 7, 11, 15, 18 and 22. Group 5 (n-15) received 50ul of a TLR9 agonist only vaccine preparation on days 1 and 15, respectively, and received a 100ul delivered dose of 200 μ g of anti-mouse PD1 (anti-CD 279-clone RMP1-14) on days 4, 7, 11, 15, 18 and 22. Group 6 (n-12) received no treatment. The results are presented in figure 7 (tumor volume of individual animals) and figures 8A and 8B (overall survival of each group).

As presented in fig. 7 and fig. 8A and 8B, respectively, both compositions PepGP70 and AgGP70 promoted anti-tumor activity with delayed tumor growth and improved overall survival. Furthermore, both vaccine compositions benefit from combined treatment with anti-PD 1, showing superior overall survival and delayed tumor growth profile compared to animals receiving vaccine composition alone or anti-PD 1 treatment.

Example 8: use of a non-replicating viral vector composition or a fluorocarbon-linked peptide composition in combination with a myeloid-derived suppressor cell (MDSC) inhibitor composition

AdGP70 or PepGP70 in combination with MDSC inhibitors was examined by assessing anti-tumor activity in BALB/c mice challenged with CT26 tumor cells. Experiments were performed using female BALB/c mice 6-8 weeks old. On day 0, 100. mu.l of 2X 10 in PBS⁴Individual CT26 cells were injected subcutaneously in the flank of the mouse. Vaccine compositions of AdGP70 and PepGP70 were prepared according to example 1, wherein the formulated vaccine was prepared as 50ul of injectable solution. The compositions are administered subcutaneously according to a prime/boost regimen wherein individual animals receiving either an AdGP70 dose or a PepGP70 dose are administered 14 days apart between the prime and boost doses.

Group 1 was administered 50 μ l of PepGP70(50 μ g/peptide) subcutaneously on days 1 and 14, and the MDSC inhibitor was administered intraperitoneally daily. Group 2 subcutaneous administration of 50 μ l of AdGP70 (2X 10) on days 1 and 14⁹ifu). Group 3 was administered daily intraperitoneally with MDSC inhibitors. Group 4 was not administered treatment.

Sequence listing

<110> Elite Immunity

Portland georgette

Scott Roberts

<120> T cell-inducing vaccine composition combination and use thereof

<130> IPF08-9.PCT

<150> 62/652,478

<151> 2018-04-04

<150> 62/652,484

<151> 2018-04-04

<160> 77

<170> PatentIn version 3.5

<210> 1

<211> 677

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 1

Met Asp Thr Arg Arg Pro Arg Gln Gly Ser Asp His Thr Pro Asp Lys

1 5 10 15

Thr Ile Met Glu Ser Thr Thr Leu Ser Lys Pro Phe Lys Asn Gln Val

20 25 30

Asn Pro Trp Gly Pro Leu Ile Val Leu Leu Ile Leu Gly Gly Val Asn

35 40 45

Pro Val Ala Leu Gly Asn Ser Pro His Gln Val Phe Asn Leu Ser Trp

50 55 60

Glu Val Thr Asn Gly Asp Arg Glu Thr Val Trp Ala Ile Thr Gly Asn

65 70 75 80

His Pro Leu Trp Thr Trp Trp Pro Asp Leu Thr Pro Asp Leu Cys Met

85 90 95

Leu Ala Leu His Gly Pro Phe Ser Pro Pro Pro Gly Pro Pro Cys Cys

100 105 110

Ser Gly Ser Ser Asp Ser Thr Pro Gly Cys Ser Arg Asp Cys Glu Glu

115 120 125

Pro Leu Thr Ser Tyr Thr Pro Arg Cys Asn Thr Ala Trp Asn Arg Leu

130 135 140

Lys Leu Ser Lys Val Thr His Ala His Asn Glu Gly Phe Tyr Val Cys

145 150 155 160

Pro Gly Pro His Arg Pro Arg Trp Ala Arg Ser Cys Gly Gly Pro Glu

165 170 175

Ser Phe Tyr Cys Ala Ser Trp Gly Cys Glu Thr Thr Gly Arg Ala Ser

180 185 190

Trp Lys Pro Ser Ser Ser Trp Asp Tyr Ile Thr Val Ser Asn Asn Leu

195 200 205

Thr Ser Asp Gln Ala Thr Pro Val Cys Lys Gly Asn Glu Trp Cys Asn

210 215 220

Ser Leu Thr Ile Arg Phe Thr Ser Phe Gly Lys Gln Ala Thr Ser Trp

225 230 235 240

Val Thr Gly His Trp Trp Gly Leu Arg Leu Tyr Val Ser Gly His Asp

245 250 255

Pro Gly Leu Ile Phe Gly Ile Arg Leu Lys Ile Thr Asp Ser Gly Pro

260 265 270

Arg Val Pro Ile Gly Pro Asn Pro Val Leu Ser Asp Arg Arg Pro Pro

275 280 285

Ser Arg Pro Arg Pro Thr Arg Ser Pro Pro Pro Ser Asn Ser Thr Pro

290 295 300

Thr Glu Thr Pro Leu Thr Leu Pro Glu Pro Pro Pro Ala Gly Val Glu

305 310 315 320

Asn Arg Leu Leu Asn Leu Val Lys Gly Ala Tyr Gln Ala Leu Asn Leu

325 330 335

Thr Ser Pro Asp Lys Thr Gln Glu Cys Trp Leu Cys Leu Val Ser Gly

340 345 350

Pro Pro Tyr Tyr Glu Gly Val Ala Val Leu Gly Thr Tyr Ser Asn His

355 360 365

Thr Ser Ala Pro Ala Asn Cys Ser Val Ala Ser Gln His Lys Leu Thr

370 375 380

Leu Ser Glu Val Thr Gly Gln Gly Leu Cys Ile Gly Ala Val Pro Lys

385 390 395 400

Thr His Gln Val Leu Cys Asn Thr Thr Gln Lys Thr Ser Asp Gly Ser

405 410 415

Tyr Tyr Leu Ala Ala Pro Thr Gly Thr Thr Trp Ala Cys Ser Thr Gly

420 425 430

Leu Thr Pro Cys Ile Ser Thr Thr Ile Leu Asp Leu Thr Thr Asp Tyr

435 440 445

Cys Val Leu Val Glu Leu Trp Pro Arg Val Thr Tyr His Ser Pro Ser

450 455 460

Tyr Val Tyr His Gln Phe Glu Arg Arg Ala Lys Tyr Lys Arg Glu Pro

465 470 475 480

Val Ser Leu Thr Leu Ala Leu Leu Leu Gly Gly Leu Thr Met Gly Gly

485 490 495

Ile Ala Ala Gly Val Gly Thr Gly Thr Thr Ala Leu Val Ala Thr Gln

500 505 510

Gln Phe Gln Gln Leu Gln Ala Ala Met His Asp Asp Leu Lys Glu Val

515 520 525

Glu Lys Ser Ile Thr Asn Leu Glu Lys Ser Leu Thr Ser Leu Ser Glu

530 535 540

Val Val Leu Gln Asn Arg Arg Gly Leu Asp Leu Leu Phe Leu Lys Glu

545 550 555 560

Gly Gly Leu Cys Ala Ala Leu Lys Glu Glu Cys Cys Phe Tyr Ala Asp

565 570 575

His Thr Gly Leu Val Arg Asp Ser Met Ala Lys Leu Arg Glu Arg Leu

580 585 590

Ser Gln Arg Gln Lys Leu Phe Glu Ser Gln Gln Gly Trp Phe Glu Gly

595 600 605

Leu Phe Asn Lys Ser Pro Trp Phe Thr Thr Leu Ile Ser Thr Ile Met

610 615 620

Gly Pro Leu Ile Ile Leu Leu Leu Ile Leu Leu Phe Gly Pro Cys Ile

625 630 635 640

Leu Asn Arg Leu Val Gln Phe Ile Lys Asp Arg Ile Ser Val Val Gln

645 650 655

Ala Leu Val Leu Thr Gln Gln Tyr His Gln Leu Lys Thr Ile Gly Asp

660 665 670

Cys Lys Ser Arg Glu

675

<210> 2

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 2

Val Asn Pro Trp Gly Pro Leu Ile Val Leu Leu Ile Leu Gly Gly

1 5 10 15

<210> 3

<211> 9

<212> PRT

<213> murine leukemia virus (murine leukemia virus) GP70

<400> 3

Asn Pro Trp Gly Pro Leu Ile Val Leu

1 5

<210> 4

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 4

Pro Trp Gly Pro Leu Ile Val Leu Leu Ile Leu Gly Gly Val Asn

1 5 10 15

<210> 5

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 5

Gly Pro Leu Ile Val Leu Leu Ile Leu

1 5

<210> 6

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 6

Gly Pro Leu Ile Val Leu Leu Ile Leu Gly Gly Val Asn Pro Val

1 5 10 15

<210> 7

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 7

Leu Gly Gly Val Asn Pro Val Ala Leu Gly Asn Ser Pro His Gln

1 5 10 15

<210> 8

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 8

Asn Ser Pro His Gln Val Phe Asn Leu

1 5

<210> 9

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 9

Asn Leu Ser Trp Glu Val Thr Asn Gly Asp Arg Glu Thr Val Trp

1 5 10 15

<210> 10

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 10

Asn Gly Asp Arg Glu Thr Val Trp Ala Ile Thr Gly Asn His Pro

1 5 10 15

<210> 11

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 11

Thr Pro Asp Leu Cys Met Leu Ala Leu

1 5

<210> 12

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 12

Ser Tyr Trp Gly Leu Glu Tyr Arg Ala

1 5

<210> 13

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 13

Ser Tyr Thr Pro Arg Cys Asn Thr Ala

1 5

<210> 14

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 14

Arg Cys Asn Thr Ala Trp Asn Arg Leu Lys Leu Ser Lys Val Thr

1 5 10 15

<210> 15

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 15

Asn Thr Ala Trp Asn Arg Leu Lys Leu Ser Lys Val Thr His Ala

1 5 10 15

<210> 16

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 16

Trp Asn Arg Leu Lys Leu Ser Lys Val Thr His Ala His Asn Glu

1 5 10 15

<210> 17

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 17

Asn Glu Gly Phe Tyr Val Cys Pro Gly Pro His Arg Pro Arg Trp

1 5 10 15

<210> 18

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 18

Arg Ser Cys Gly Gly Pro Glu Ser Phe

1 5

<210> 19

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 19

Cys Glu Thr Thr Gly Arg Ala Ser Trp Lys Pro Ser Ser Ser Trp

1 5 10 15

<210> 20

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 20

Lys Pro Ser Ser Ser Trp Asp Tyr Ile

1 5

<210> 21

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 21

Asp Tyr Ile Thr Val Ser Asn Asn Leu

1 5

<210> 22

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 22

Ile Thr Val Ser Asn Asn Leu Thr Ser Asp Gln Ala Thr Pro Val

1 5 10 15

<210> 23

<211> 15

<212> PRT

<213> murine leukemia virus (murine leukemia virus) GP

<400> 23

Thr Gly His Trp Trp Gly Leu Arg Leu Tyr Val Ser Gly His Asp

1 5 10 15

<210> 24

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 24

Leu Tyr Val Ser Gly His Asp Pro Gly

1 5

<210> 25

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 25

Val Pro Ile Gly Pro Asn Pro Val Leu

1 5

<210> 26

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 26

Asn Pro Val Leu Ser Asp Arg Arg Pro Pro Ser Arg Pro Arg Pro

1 5 10 15

<210> 27

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 27

Asn Ser Thr Pro Thr Glu Thr Pro Leu

1 5

<210> 28

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 28

Thr Pro Thr Glu Thr Pro Leu Thr Leu

1 5

<210> 29

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 29

Pro Pro Ala Gly Val Glu Asn Arg Leu

1 5

<210> 30

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 30

Lys Thr Gln Glu Cys Trp Leu Cys Leu Val Ser Gly Pro Pro Tyr

1 5 10 15

<210> 31

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 31

Pro Pro Tyr Tyr Glu Gly Val Ala Val Leu Gly Thr Tyr Ser Asn

1 5 10 15

<210> 32

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 32

Pro Tyr Tyr Glu Gly Val Ala Val Leu

1 5

<210> 33

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 33

Tyr Ser Asn His Thr Ser Ala Pro Ala Asn Cys Ser Val Ala Ser

1 5 10 15

<210> 34

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 34

Asn His Thr Ser Ala Pro Ala Asn Cys Ser Val Ala Ser Gln His

1 5 10 15

<210> 35

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 35

Cys Ser Val Ala Ser Gln His Lys Leu

1 5

<210> 36

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 36

Val Ala Ser Gln His Lys Leu Thr Leu Ser Glu Val Thr Gly Gln

1 5 10 15

<210> 37

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 37

Ser Asp Gly Ser Tyr Tyr Leu Ala Ala Pro Thr Gly Thr Thr Trp

1 5 10 15

<210> 38

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 38

Trp Ala Cys Ser Thr Gly Leu Thr Pro Cys Ile Ser Thr Thr Ile

1 5 10 15

<210> 39

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 39

Thr Pro Cys Ile Ser Thr Thr Ile Leu

1 5

<210> 40

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 40

Ser Pro Ser Tyr Val Tyr His Gln Phe

1 5

<210> 41

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 41

Ser Pro Ser Tyr Val Tyr His Gln Phe Glu Arg Arg Ala Lys Tyr

1 5 10 15

<210> 42

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 42

Tyr His Gln Phe Glu Arg Arg Ala Lys Tyr Lys Arg Glu Pro Val

1 5 10 15

<210> 43

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 43

Lys Tyr Lys Arg Glu Pro Val Ser Leu

1 5

<210> 44

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 44

Lys Tyr Lys Arg Glu Pro Val Ser Leu Thr Leu Ala Leu Leu Leu

1 5 10 15

<210> 45

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 45

Glu Pro Val Ser Leu Thr Leu Ala Leu

1 5

<210> 46

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 46

Glu Pro Val Ser Leu Thr Leu Ala Leu Leu Leu Gly Gly Leu Thr

1 5 10 15

<210> 47

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 47

Pro Val Ser Leu Thr Leu Ala Leu Leu Leu Gly Gly Leu Thr Met

1 5 10 15

<210> 48

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 48

Val Ser Leu Thr Leu Ala Leu Leu Leu Gly Gly Leu Thr Met Gly

1 5 10 15

<210> 49

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 49

Gly Leu Thr Met Gly Gly Ile Ala Ala Gly Val Gly Thr Gly Thr

1 5 10 15

<210> 50

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 50

Gly Thr Gly Thr Thr Ala Leu Val Ala Thr Gln Gln Phe Gln Gln

1 5 10 15

<210> 51

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 51

Thr Asn Leu Glu Lys Ser Leu Thr Ser

1 5

<210> 52

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 52

Arg Arg Gly Leu Asp Leu Leu Phe Leu Lys Glu Gly Gly Leu Cys

1 5 10 15

<210> 53

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 53

Leu Lys Glu Glu Cys Cys Leu Tyr Ala Asp His Thr Gly Leu Val

1 5 10 15

<210> 54

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 54

Cys Cys Leu Tyr Ala Asp His Thr Gly Leu Val Arg Asp Ser Met

1 5 10 15

<210> 55

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 55

Leu Tyr Ala Asp His Thr Gly Leu Val

1 5

<210> 56

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 56

Ala Asp His Thr Gly Leu Val Arg Asp Ser Met Ala Lys Leu Arg

1 5 10 15

<210> 57

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 57

Arg Asp Ser Met Ala Lys Leu Arg Glu Arg Leu Ser Gln Arg Gln

1 5 10 15

<210> 58

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 58

Met Ala Lys Leu Arg Glu Arg Leu Ser Gln Arg Gln Lys Leu Phe

1 5 10 15

<210> 59

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 59

Glu Arg Leu Ser Gln Arg Gln Lys Leu Phe Glu Ser Gln Gln Gly

1 5 10 15

<210> 60

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 60

Trp Phe Thr Thr Leu Ile Ser Thr Ile

1 5

<210> 61

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 61

Met Gly Pro Leu Ile Ile Leu Leu Leu Ile Leu Leu Phe Gly Pro

1 5 10 15

<210> 62

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 62

Pro Leu Ile Ile Leu Leu Leu Ile Leu Leu Phe Gly Pro Cys Ile

1 5 10 15

<210> 63

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 63

Ile Leu Leu Phe Gly Pro Cys Ile Leu Asn Arg Leu Val Gln Phe

1 5 10 15

<210> 64

<211> 14

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 64

Leu Val Gln Phe Ile Lys Asp Arg Ile Ser Val Val Gln Ala

1 5 10

<210> 65

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 65

Gln Phe Ile Lys Asp Arg Ile Ser Val Val Gln Ala Leu Val Leu

1 5 10 15

<210> 66

<211> 15

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 66

Lys Asp Arg Ile Ser Val Val Gln Ala Leu Val Leu Thr Gln Gln

1 5 10 15

<210> 67

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 67

Ile Ser Val Val Gln Ala Leu Val Leu

1 5

<210> 68

<211> 27

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 68

Ser Tyr Thr Pro Arg Cys Asn Thr Ala Trp Asn Arg Leu Lys Leu Ser

1 5 10 15

Lys Val Thr His Ala His Asn Glu Lys Phe Ala

20 25

<210> 69

<211> 37

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 69

Phe Ala Lys Glu Thr Thr Gly Arg Ala Ser Trp Lys Pro Ser Ser Ser

1 5 10 15

Trp Asp Tyr Ile Thr Val Ser Asn Asn Leu Thr Ser Asp Gln Ala Thr

20 25 30

Pro Val Lys Lys Lys

35

<210> 70

<211> 40

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 70

Ser Pro Ser Tyr Val Tyr His Gln Phe Glu Arg Arg Ala Lys Tyr Lys

1 5 10 15

Arg Glu Pro Val Ser Leu Thr Leu Ala Leu Leu Leu Gly Gly Leu Thr

20 25 30

Met Gly Lys Lys Lys Lys Phe Ala

35 40

<210> 71

<211> 29

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 71

Ser Pro Ser Tyr Val Tyr His Gln Phe Lys Lys Lys Leu Val Gln Phe

1 5 10 15

Ile Lys Asp Arg Ile Ser Val Val Gln Ala Lys Phe Ala

20 25

<210> 72

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 72

Ser Pro Ser Tyr Val Tyr His Gln Phe

1 5

<210> 73

<211> 9

<212> PRT

<213> murine leukemia virus GP70 (murine leukemia virus GP70)

<400> 73

Ser Pro Ser Tyr Val Tyr His Gln Phe

1 5

<210> 74

<211> 9

<212> PRT

<213> Artificial peptides (artifiacial peptides)

<220>

<223> peptide

<400> 74

Arg Pro Gln Ala Ser Gly Val Tyr Met

1 5

<210> 75

<211> 30

<212> PRT

<213> egg white albumin (chicken ovalbumin)

<400> 75

Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly

1 5 10 15

Arg Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Trp Thr

20 25 30

<210> 76

<211> 17

<212> PRT

<213> egg white albumin (chicken ovalbumin)

<400> 76

Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn Glu Ala Gly

1 5 10 15

Arg

<210> 77

<211> 8

<212> PRT

<213> egg white albumin (chicken ovalbumin)

<400> 77

Ser Ile Ile Asn Phe Glu Lys Leu

1 5