Targeted neoepitope vectors and methods thereof

文档序号：1602461 发布日期：2020-01-07 浏览：26次中文

阅读说明：本技术 靶向性新表位载体及其方法 (Targeted neoepitope vectors and methods thereof ) 是由卡伊万·尼亚兹于 2018-04-23 设计创作，主要内容包括：提出了允许选择肿瘤新表位的系统和方法,然后将这些系统和方法用于生成编码一个或多个多表位的重组核酸,将这些多表位针对适当的运输和加工进行了优化。在优选的方法中,这些多表位在质粒和/或病毒表达系统中被编码,以用作治疗剂。(Systems and methods are presented that allow for the selection of tumor neoepitopes, which are then used to generate recombinant nucleic acids encoding one or more polyepitopes that are optimized for proper transport and processing. In a preferred method, these polyepitopes are encoded in a plasmid and/or viral expression system for use as a therapeutic agent.)

1. A method of generating a recombinant expression construct for use in immunotherapy of a mammal, the method comprising:

generating a first recombinant nucleic acid having a sequence encoding a polyepitope, wherein the polyepitope comprises a plurality of filtered neo-epitope sequences;

wherein the polyepitope further comprises a trafficking element that directs the polyepitope to a subcellular location selected from the group consisting of: circulating the endocytosis body, sorting the endocytosis body and the lysosome;

wherein the first recombinant nucleic acid comprises a first promoter operably linked to a sequence encoding the polyepitope to drive expression of the polyepitope in a mammal; and

generating a second recombinant nucleic acid having a sequence encoding the polyepitope, wherein the second recombinant nucleic acid comprises a second promoter operably linked to the sequence encoding the polyepitope to drive expression of the polyepitope in the non-mammalian cell.

2. The method of claim 1, wherein the first promoter is a constitutive promoter, or wherein the first promoter is inducible by hypoxia, IFN- γ or IL-8.

3. The method of any one of the preceding claims, wherein the transport element is selected from the group consisting of: CD1b leader sequence, CD1a tail, CD1c tail, and LAMP 1-transmembrane sequence.

4. The method of any one of the preceding claims, wherein the filtered new epitope sequence is filtered by comparing the tumor of the same patient to a matched normal control.

5. The method of any one of the preceding claims, wherein the filtered neoepitope sequence is filtered to have a binding affinity to MHC complexes equal to or less than 200 nM.

6. The method of any one of claims 1-5, wherein the filtered neoepitope sequence binds to MHC-I, and wherein the trafficking element directs the polyepitope to the circulating endocytosis, sorting endocytosis, or lysosome.

7. The method of any one of claims 1-5, wherein the filtered neoepitope sequence binds to MHC-II, and wherein the trafficking element directs the polyepitope to the circulating endocytosis, sorting endocytosis, or lysosome.

8. The method of any one of the preceding claims, wherein the first recombinant nucleic acid further comprises an additional sequence encoding a second polyepitope, wherein the second polyepitope comprises a second trafficking element that directs the second polyepitope to a different subcellular location, and wherein the second polyepitope comprises a plurality of second filtered neo-epitope sequences.

9. The method of claim 8, wherein at least one of the filtered new sequence of table bits and at least one of the second filtered new sequence of table bits are the same.

10. The method of any one of the preceding claims, wherein the first recombinant nucleic acid further comprises a sequence encoding at least one of a co-stimulatory molecule, an immunostimulatory cytokine, and a protein that interferes with or down-regulates checkpoint inhibition.

11. The method of claim 10, wherein the co-stimulatory molecule is selected from the group consisting of: CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1, and LFA 3.

12. The method of claim 10, wherein the immunostimulatory cytokine is selected from the group consisting of: IL-2, IL-12, IL-15 superagonists (ALT803), IL-21, IPS1 and LMP 1.

13. The method of claim 10, wherein the interfering protein is an antibody or antagonist to CTLA-4, PD-1, TIM1 receptor, 2B4, or CD 160.

14. The method of any one of the preceding claims, wherein the first recombinant nucleic acid replicates in a bacterial cell or a yeast cell.

15. The method of any one of the preceding claims, wherein the first recombinant nucleic acid is a shuttle vector for the production of a recombinant virus.

16. The method of claim 15, wherein the recombinant virus is an adenovirus that optionally lacks at least one of the E1 and E2b genes.

17. The method of any one of the preceding claims, further comprising the step of formulating the first recombinant nucleic acid into an injectable pharmaceutical formulation.

18. The method of any one of the preceding claims, wherein the second promoter is a constitutive bacterial or yeast promoter.

19. The method of any one of the preceding claims, wherein the non-mammalian cell is an E.

20. The method of any one of the preceding claims, further comprising the steps of:

transfecting the second recombinant nucleic acid into a bacterial cell or a yeast cell;

expressing the polyepitope in the bacterial cell or yeast cell; and

the bacterial cells or yeast cells are formulated into a pharmaceutical formulation for injection.

21. The method of claim 1, wherein the transport element is selected from the group consisting of: CD1b leader sequence, CD1a tail, CD1c tail, and LAMP 1-transmembrane sequence.

22. The method of claim 1, wherein the filtered new epitope sequence is filtered by comparing tumors of the same patient to a matching normal control.

23. The method of claim 1, wherein the filtered neoepitope sequence is filtered to have a binding affinity to MHC complexes equal to or less than 200 nM.

24. The method of claim 1, wherein the filtered neoepitope sequence binds to MHC-I, and wherein the trafficking element directs the polyepitope to the circulating endocytosis, sorting endocytosis, or lysosome.

25. The method of claim 1, wherein the filtered neoepitope sequence binds to MHC-II, and wherein the trafficking element directs the polyepitope to the circulating endocytosis, sorting endocytosis, or lysosome.

26. The method of claim 1, wherein the first recombinant nucleic acid further comprises an additional sequence encoding a second polyepitope, wherein the second polyepitope comprises a second trafficking element that directs the second polyepitope to a different subcellular location, and wherein the second polyepitope comprises a plurality of second filtered neo-epitope sequences.

27. The method of claim 26, wherein at least one of the filtered new sequence of table bits and at least one of the second filtered new sequence of table bits are the same.

28. The method of claim 1, wherein the first recombinant nucleic acid further comprises a sequence encoding at least one of a co-stimulatory molecule, an immunostimulatory cytokine, and a protein that interferes with or down-regulates checkpoint inhibition.

29. The method of claim 28, wherein the co-stimulatory molecule is selected from the group consisting of: CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1, and LFA 3.

30. The method of claim 28, wherein the immunostimulatory cytokine is selected from the group consisting of: IL-2, IL-12, IL-15 superagonists (ALT803), IL-21, IPS1 and LMP 1.

31. The method of claim 28, wherein the interfering protein is an antibody or antagonist to CTLA-4, PD-1, TIM1 receptor, 2B4, or CD 160.

32. The method of claim 1, wherein the first recombinant nucleic acid replicates in a bacterial cell or a yeast cell.

33. The method of claim 1, wherein the first recombinant nucleic acid is a shuttle vector for generating a recombinant virus.

34. The method of claim 33, wherein the recombinant virus is an adenovirus that optionally lacks at least one of the E1 and E2b genes.

35. The method of claim 1, further comprising the step of formulating the first recombinant nucleic acid into an injectable pharmaceutical formulation.

36. The method of claim 1, wherein the second promoter is a constitutive bacterial or yeast promoter.

37. The method of claim 1, wherein the non-mammalian cell is an E.

38. The method of claim 1, further comprising the steps of:

transfecting the second recombinant nucleic acid into a bacterial cell or a yeast cell;

expressing the polyepitope in the bacterial cell or yeast cell; and

the bacterial cells or yeast cells are formulated into a pharmaceutical formulation for injection.

39. A recombinant bacterial or yeast expression vector for immunotherapy of a mammal, the recombinant bacterial or yeast expression vector comprising:

a recombinant nucleic acid having a sequence encoding a polyepitope operably linked to a bacterial or yeast promoter to drive expression of the polyepitope;

wherein the polyepitope comprises a trafficking element that directs the polyepitope to a subcellular location of a mammalian immune competent cell, the subcellular location selected from the group consisting of: circulating the endocytosis body, sorting the endocytosis body and the lysosome; and is

Wherein the polyepitope comprises a plurality of filtered new epitope sequences.

40. The vector of claim 39, wherein the promoter is a constitutive promoter.

41. The vector of any one of claims 39-40 wherein the transport element is selected from the group consisting of: CD1b leader sequence, CD1a tail, CD1c tail, and LAMP 1-transmembrane sequence.

42. The vector of any one of claims 39-41, wherein the filtered new epitope sequence is filtered by comparing tumors of the same patient to a matched normal control.

43. The vector of any one of claims 39-42, wherein the filtered neoepitope sequence binds to MHC-I, and wherein the trafficking element directs the polyepitope to the circulating endocytosis, sorting endocytosis, or lysosome.

44. The vector of any one of claims 39-42, wherein the filtered neoepitope sequence binds to MHC-II, and wherein the trafficking element directs the polyepitope to the circulating endocytosis, sorting endocytosis, or lysosome.

45. The vector of any one of claims 39-45, wherein the recombinant nucleic acid further comprises an additional sequence encoding a second polyepitope, wherein the second polyepitope comprises a second trafficking element that directs the second polyepitope to a different subcellular location, and wherein the second polyepitope comprises a plurality of second filtered neo-epitope sequences.

46. The carrier of claim 45, wherein at least one of the filtered new sequence of epitopes and at least one of the second filtered new sequence of epitopes are the same.

47. The vector of any one of claims 39-46, wherein the expression vector is a bacterial expression vector.

48. The vector of any one of claims 39-46, wherein the expression vector is a yeast expression vector.

49. The carrier of claim 39, wherein the transport element is selected from the group consisting of: CD1b leader sequence, CD1a tail, CD1c tail, and LAMP 1-transmembrane sequence.

50. The vector of claim 39, wherein the filtered new epitope sequence is filtered by comparing tumors of the same patient to a matched normal control.

51. The vector of claim 39, wherein the filtered neo-epitope sequence binds MHC-I, and wherein the trafficking element directs the polyepitope to the circulating endocytosis, sorting endocytosis, or lysosome.

52. The vector of claim 39, wherein the filtered neo-epitope sequence binds to MHC-II, and wherein the trafficking element directs the polyepitope to the circulating endocytosis, sorting endocytosis, or lysosome.

53. The vector of claim 39, wherein the recombinant nucleic acid further comprises an additional sequence encoding a second polyepitope, wherein the second polyepitope comprises a second trafficking element that directs the second polyepitope to a different subcellular location, and wherein the second polyepitope comprises a plurality of second filtered neo-epitope sequences.

54. The carrier of claim 53, wherein at least one of the filtered new sequence of epitopes and at least one of the second filtered new sequence of epitopes are the same.

55. The vector of claim 39, wherein the expression vector is a bacterial expression vector.

56. The vector of claim 39, wherein the expression vector is a yeast expression vector.

57. A recombinant yeast cell transfected with the vector of any one of claims 40-48.

58. A recombinant yeast cell transfected with the vector of any one of claims 49-56.

59. A recombinant bacterial cell transfected with the vector of any one of claims 40-48.

60. A recombinant bacterial cell transfected with the vector of any one of claims 49-56.

61. A pharmaceutical composition comprising the recombinant yeast cell of claim 57.

62. A pharmaceutical composition comprising the recombinant yeast cell of claim 58.

63. A pharmaceutical composition comprising the recombinant bacterial cell of claim 59.

64. A pharmaceutical composition comprising the recombinant bacterial cell of claim 60.

65. (DNA/cell-based) A method of preparing first and second treatment compositions for an individual having a tumor, the method comprising:

identifying a plurality of expressed neoepitope sequences from omics data of the tumor, wherein each expressed neoepitope sequence has a calculated binding affinity to at least one of MHC-I and MHC-II of the individual that is equal to or less than 500 nM;

generating a first recombinant nucleic acid having a sequence encoding a polyepitope, wherein the polyepitope comprises the plurality of expressed neo-epitope sequences;

wherein the first recombinant nucleic acid comprises a first promoter operably linked to a sequence encoding the polyepitope to drive expression of the polyepitope in cells of the individual;

formulating the first recombinant nucleic acid into a DNA vaccine formulation to obtain the first treatment composition;

generating a second recombinant nucleic acid comprising a sequence encoding the polyepitope, wherein the second recombinant nucleic acid comprises a second promoter operably linked to the sequence encoding the polyepitope to drive expression of the polyepitope in a bacterial cell or a yeast cell;

transfecting the bacterial cell or yeast cell with the second recombinant nucleic acid and expressing the polyepitope in the bacterial cell or yeast cell; and

formulating the transfected bacterial or yeast cells into a cell-based vaccine formulation to obtain the second treatment composition.

66. The method of claim 65, wherein the plurality of expressed novel epitope sequences are identified using an incremental simultaneous alignment of omics data from a tumor and omics data from a non-tumor sample from the same individual.

67. The method of any one of claims 65-66, wherein the first recombinant nucleic acid is an expression vector.

68. The method of any one of claims 65-67, wherein the transport element is selected from the group consisting of: CD1b leader sequence, CD1a tail, CD1c tail, and LAMP 1-transmembrane sequence.

69. The method of any one of claims 65-68, wherein the second promoter is a constitutive bacterial or yeast promoter.

70. The method of any one of claims 65-69, wherein the bacterial cell or yeast cell is an E.coli cell or a s.cerevisiae cell.

71. The method of any one of claims 65-70, wherein the cell-based vaccine formulation is formulated for injection.

72. The method of any one of claims 65-71, further comprising the step of generating a third recombinant nucleic acid that is a viral expression vector comprising a sequence encoding the polyepitope, and wherein the third recombinant nucleic acid comprises a third promoter operably linked to the sequence encoding the polyepitope to drive expression of the polyepitope in the cells of the individual.

73. The method of claim 72, wherein the third promoter is a constitutive promoter, or wherein the third promoter is inducible by hypoxia, IFN- γ or IL-8.

74. The method of claim 65, wherein the first recombinant nucleic acid is an expression vector.

75. The method of claim 65, wherein the transport element is selected from the group consisting of: CD1b leader sequence, CD1a tail, CD1c tail, and LAMP 1-transmembrane sequence.

76. The method of claim 65, wherein the second promoter is a constitutive bacterial or yeast promoter.

77. The method of claim 65, wherein the bacterial cell or yeast cell is an E.coli cell or a Saccharomyces cerevisiae cell.

78. The method of claim 65, wherein the cell-based vaccine formulation is formulated for injection.

79. The method of claim 65, further comprising the step of generating a third recombinant nucleic acid that is a viral expression vector comprising a sequence encoding the polyepitope, and wherein the third recombinant nucleic acid comprises a third promoter operably linked to the sequence encoding the polyepitope to drive expression of the polyepitope in the cells of the individual.

80. The method of claim 79, wherein the third promoter is a constitutive promoter, or wherein the third promoter is inducible by hypoxia, IFN- γ or IL-8.

A method of preparing (AdV/cell-based) first and second treatment compositions for an individual having a tumor, the method comprising:

identifying a plurality of expressed neoepitope sequences from omics data of the tumor, wherein the expressed neoepitope sequences have a calculated binding affinity to at least one of MHC-I and MHC-II of the individual that is equal to or less than 500 nM;

generating a first recombinant nucleic acid having a sequence encoding a polyepitope, wherein the polyepitope comprises the plurality of expressed neo-epitope sequences, wherein the first recombinant nucleic acid is a viral expression vector;

wherein the first recombinant nucleic acid comprises a first promoter operably linked to a sequence encoding the polyepitope to drive expression of the polyepitope in cells of the individual:

forming viral particles from the viral expression vector and formulating the viral particles into a viral vaccine formulation to obtain the first treatment composition;

transfecting the bacterial cell or yeast cell with the second recombinant nucleic acid and expressing the polyepitope in the bacterial cell or yeast cell; and

formulating the transfected bacterial or yeast cells into a cell-based vaccine formulation to obtain the second treatment composition.

82. The method of claim 81, wherein the plurality of expressed novel epitope sequences are identified using an incremental simultaneous alignment of omics data from a tumor and omics data from a non-tumor sample from the same individual.

83. The method of any one of claims 81-82, wherein the transport element is selected from the group consisting of: CD1b leader sequence, CD1a tail, CD1c tail, and LAMP 1-transmembrane sequence.

84. The method of any one of claims 81-83, wherein the first promoter is a constitutive promoter, or wherein the first promoter is inducible by hypoxia, IFN- γ, or IL-8.

85. The method of any one of claims 81-84, wherein the viral expression vector is an adenoviral expression vector optionally deleted of the E1 and E2b genes.

86. The method of any one of claims 81-85, wherein the second promoter is a constitutive bacterial or yeast promoter.

87. The method of any one of claims 81-86, wherein the non-mammalian cell or yeast cell is an E.

88. The method of any one of claims 81-87, wherein the viral vaccine formulation and cell-based vaccine formulation are formulated for injection.

89. The method of claim 81, wherein the transport element is selected from the group consisting of: CD1b leader sequence, CD1a tail, CD1c tail, and LAMP 1-transmembrane sequence.

90. The method of claim 81, wherein the first promoter is a constitutive promoter, or wherein the first promoter is inducible by hypoxia, IFN- γ or IL-8.

91. The method of claim 81, wherein the viral expression vector is an adenoviral expression vector optionally deleted of the E1 and E2b genes.

92. The method of claim 81, wherein the second promoter is a constitutive bacterial or yeast promoter.

93. The method of claim 81, wherein the non-mammalian cell or yeast cell is an E.

94. The method of claim 81, wherein the viral vaccine formulation and cell-based vaccine formulation are formulated for injection.

Technical Field

Background

The background description includes information that may be useful in understanding the present invention. There is no admission that any information provided herein is prior art or relevant to the presently claimed invention, nor that any publication specifically or implicitly referenced is prior art.

All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. If a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

Cancer immunotherapy targeting certain antigens common to specific cancers has elicited significant responses in some patients. Unfortunately, despite the dominant expression of the same antigen, many patients fail to respond to this immunotherapy. One possible reason for the failure of this response is that various effector cells of the immune system may not be present in sufficient numbers, or may have been depleted. Furthermore, intracellular antigen processing and HLA variability in patients may have resulted in insufficient processing of antigen and/or antigen display, resulting in ineffective treatment or lack of response.

In order to increase the selection of targets for immunotherapy, random mutations have recently been considered, as some random mutations in tumor cells may give rise to unique tumor-specific antigens (neo-epitopes). As such, and at least conceptually, the new epitopes can provide unique, precise targets for immunotherapy. Additionally, it has been shown that cytolytic T cell responses can be triggered by very small amounts of peptide (e.g., Sykulev et al, Immunity, Vol.4, No. 6, p.565-571, 1/6/1996). Moreover, the number of possible targets is relatively high due to the relatively high number of mutations in many cancers. In view of these findings, the identification of cancer neoepitopes as therapeutic targets has attracted considerable attention. Unfortunately, current data appears to indicate that all or almost all cancer neoepitopes are unique to both the patient and the particular tumor, and fails to provide any specific indication as to which neoepitope is useful for a therapeutically effective immunotherapeutic.

To overcome at least some of the problems associated with the large number of possible targets for immunotherapy, the mutation type of neoepitope (e.g., to determine missense or nonsense mutations), the transcription level (to confirm transcription of the mutant gene, and confirm protein expression) may be filtered. Moreover, the neo-epitopes thus filtered can be further analyzed for specific binding to the patient's HLA system, as described in WO 2016/172722. Although such systems advantageously reduce the relatively large number of potential neoepitopes, the importance of these neoepitopes with respect to the therapeutic outcome remains uncertain. Still further, and especially where multiple peptides are to be expressed in antigen presenting cells (e.g., dendritic cells), the processing of precursor proteins to generate neoepitopes is not well understood and thus leads to a lack of predictability of therapeutic success.

Immunotherapy can be performed using at least two conceptually different methods, where the first method is based on DNA vaccination and the second method is based on the use of a recombinant virus encoding one or more antigens expressed in cells infected with the virus. For example, clinical trials have shown that plasmid DNA vaccines are safe and immunologically effective in humans at doses of 300mcg of plasmid DNA encoding HIV virion protein expression regulatory (rev) protein and envelope (env) protein when administered intramuscularly. This DNA vaccination is induced in HIV-seronegative patientsAntigen-specific IFN γ -secreting T cell responses (j.infect.dis. [ journal of infectious disease)](2000)181: 476-83). In addition, the results of clinical trials targeting PSMA (prostate specific membrane antigen) in patients with prostate cancer using intradermal injection of plasmid DNA and adenovirus have been reported (see eur. urol. [ european urology)](2000),38: 208217). Here, 26 patients were immunized with PSMA-expressing adenoviral vectors followed by PSMA-encoding plasmid DNA, or plasmid DNA alone, in a prime/boost strategy, and no significant toxicity was observed. However, the therapeutic efficacy of such vaccination (particularly in the treatment of cancer) has not been demonstrated using this approach. In still other examples, adenoviral expression of cancer neoepitopes has been reported, as described in US 2017/0312351. Although such methods are highly specific to the patient and the patient's tumor, it is time consuming to generate sufficient numbers of viral particles encoding one or more neo-epitopes. For example, a single dose of 10 is generated¹¹The virus yield of individual virus particles will typically take 6-8 weeks, and in some cases even longer, and multiple administrations are often required to elicit a therapeutic effect. Depending on the type of cancer and the growth rate, such a production time period may be prohibitive. In addition, immune stimulation with only virally expressed proteins is often less effective and often requires additional therapeutic modalities (e.g., cytokines) to elicit the desired therapeutic effect.

Thus, while various methods of identifying and delivering neoepitopes to multiple cells are known in the art, all or almost all of these methods have various drawbacks, particularly in terms of efficacy and time requirements. Accordingly, it would be desirable to have improved systems and methods for selecting and generating novel epitopes to increase the likelihood of a therapeutic response in immunotherapy in a convenient manner.

Disclosure of Invention

The subject matter of the present invention relates to various immunotherapeutic compositions and methods, and in particular recombinant expression systems, in which a plurality of novel epitopes are combined to form rationally designed polypeptides with trafficking signals to increase antigen processing and presentation and thereby enhance therapeutic efficacy. Additionally, the systems and methods contemplated herein utilize multiple and different vaccination patterns that will provide significantly reduced time to first vaccination and different and complementary immune stimulation patterns.

For example, where a first vaccination mode comprises DNA vaccination encoding a polyepitope (typically comprising multiple neo-epitopes and/or TAAs), the vaccine may be prepared within a few days and will provide TLR stimulation (e.g., TLR9 stimulation), while a second vaccination mode may comprise a recombinant bacterial or yeast vaccine encoding a polyepitope (typically the same polyepitope as in the first vaccination mode) and will therefore provide different TRL stimulation (e.g., TRL1, TLR2, TLR5, etc.). In yet another example, the first vaccination mode may comprise a bacterial or yeast vaccine encoding a polyepitope (polytope) and the second vaccination mode may comprise a recombinant virus encoding a polyepitope, which two vaccination modes will again provide different (and typically complementary or even synergistic) innate immune stimulation.

Obviously, this multi-modal strategy would greatly reduce the time to generate the first vaccination, since DNA, bacterial and yeast vaccines can be prepared within a few days, rather than within a few weeks as most viral vaccines. Moreover, due to the different delivery modalities, contemplated vaccine compositions and methods will also take advantage of the different immunostimulatory effects provided by the different modalities, and will be so particularly useful in prime/boost regimens. Additionally, it should be recognized that contemplated systems and methods utilize substantially the same polyepitope in different vaccine formats. In other words, once the antigens that have potential therapeutic effects on a patient are identified, recombinant nucleic acids encoding these antigens can be assembled into a multi-epitope cassette that is then used across multiple vaccine platforms.

In one aspect of the inventive subject matter, the inventors contemplate a method of generating a recombinant expression construct for use in immunotherapy of a mammal. Such methods will typically include the step of generating a first recombinant nucleic acid having a sequence encoding a polyepitope, wherein the polyepitope comprises a plurality of filtered neoepitope sequences, wherein the polyepitope further comprises a trafficking element that directs the polyepitope to a subcellular location selected from the group consisting of: circulating the endocytosis, sorting the endocytosis and the lysosome, and wherein the first recombinant nucleic acid comprises a first promoter operably linked to a sequence encoding a polyepitope to drive expression of the polyepitope in the mammal. In another step, a second recombinant nucleic acid having the same sequence encoding the polyepitope is generated, wherein the second recombinant nucleic acid comprises a second promoter operably linked to the sequence encoding the polyepitope to drive expression of the polyepitope in the non-mammalian cell.

For example, in exemplary embodiments, the first promoter can be a constitutive promoter, or a promoter that is inducible by hypoxia, IFN- γ, or IL-8. Alternatively, the transport element may be a CD1b leader sequence, a CD1a tail, a CD1c tail, or a LAMP 1-transmembrane sequence. Most typically, the filtered neoepitope sequences are filtered by comparing tumors of the same patient to a matched normal control, and/or filtered to have a binding affinity to the MHC complex of equal to or less than 200 nM. While in some aspects the filtered neoepitope sequence is calculated to bind to MHC-I and the trafficking element directs the polyepitope to circulating endocytosis, sorting endocytosis, or lysosomes, in other aspects the filtered neoepitope sequence is calculated to bind to MHC-II and the trafficking element directs the polyepitope to circulating endocytosis, sorting endocytosis, or lysosomes.

Where desired, the first recombinant nucleic acid can further comprise additional sequences encoding a second polyepitope, wherein the second polyepitope comprises a second trafficking element that directs the second polyepitope to a different subcellular location, and wherein the second polyepitope comprises a plurality of second filtered neo-epitope sequences. In some embodiments, at least one of the filtered new sequence of table bits and at least one of the second filtered new sequence of table bits may be the same.

It is still further contemplated that the first recombinant nucleic acid further comprises a sequence encoding at least one of a co-stimulatory molecule, an immunostimulatory cytokine, and a protein that interferes with or down-regulates checkpoint inhibition. For example, suitable co-stimulatory molecules include CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1 and/or LFA3, while suitable immunostimulatory cytokines include IL-2, IL-12, IL-15 superagonists (ALT803), IL-21, IPS1 and/or LMP1, and/or suitable interfering proteins include antibodies or antagonists against CTLA-4, PD-1, 1 receptors, 2B4 and/or CD 160.

Although not limiting to the subject matter of the invention, the first recombinant nucleic acid can replicate in a bacterial cell or a yeast cell, and/or the first recombinant nucleic acid can be an expression vector or shuttle vector for generating a recombinant virus (e.g., an adenovirus that optionally lacks at least one of the E1 and E2b genes). It is also contemplated that such methods may further comprise the step of formulating the first recombinant nucleic acid into an injectable pharmaceutical formulation.

Most typically, the second promoter is a constitutive bacterial or yeast promoter. Thus, suitable non-mammalian cells include E.coli cells and Saccharomyces cerevisiae. In such cases, the method may comprise the further step of transfecting the second recombinant nucleic acid into a bacterial cell or a yeast cell; expressing the polyepitope in a bacterial cell or a yeast cell; and formulating the bacterial cells or yeast cells into a pharmaceutical formulation for injection.

Thus, the inventors also contemplate recombinant bacterial or yeast expression vectors for immunotherapy of mammals. Most preferably, such vectors will comprise a recombinant nucleic acid having a sequence encoding a polyepitope operably linked to a bacterial or yeast promoter to drive expression of the polyepitope, wherein the polyepitope comprises a trafficking element that directs the polyepitope to a subcellular location of a mammalian immune competent cell selected from the group consisting of: circulating the endocytosis body, sorting the endocytosis body and the lysosome; and wherein the polyepitope comprises a plurality of filtered new epitope sequences.

Preferably but not necessarily, the promoter is a constitutive promoter and the transport element is selected from the group consisting of: CD1b leader sequence, CD1a tail, CD1c tail, and LAMP 1-transmembrane sequence. As previously described, the filtered neoepitope sequences can be filtered by comparing tumors of the same patient to matched normative controls, and the filtered neoepitope sequences are bound to MHC-I and/or MHC-II, and the trafficking elements direct the polyepitope to circulating endocytosis, sorting endocytosis, or lysosomes. It is still further contemplated that the recombinant nucleic acid can further comprise additional sequences encoding a second polyepitope, wherein the second polyepitope comprises a second trafficking element that directs the second polyepitope to a different subcellular location, and wherein the second polyepitope comprises a plurality of second filtered neo-epitope sequences. As previously described, at least one of the filtered new sequence of table bits and at least one of the second filtered new sequence of table bits may be the same.

In still further contemplated aspects, the expression vector is a bacterial expression vector or a yeast expression vector. Thus, transfection of recombinant yeast cells and bacterial cells with the vectors considered above is specifically contemplated. These cells can then be formulated into pharmaceutical compositions comprising recombinant yeast cells or bacterial cells.

In further aspects related to the inventive subject matter, the inventors also contemplate methods of preparing first and second treatment compositions for an individual having a tumor. Such methods will typically include the step of identifying a plurality of expressed novel epitope sequences from omics data of the tumor, wherein each expressed novel epitope sequence has a calculated binding affinity to at least one of MHC-I and MHC-II of the individual that is equal to or less than 500 nM; and the additional step of generating a first recombinant nucleic acid having a sequence encoding a polyepitope, wherein the polyepitope comprises a plurality of expressed novel epitope sequences. Preferably, the polyepitope further comprises a trafficking element that directs the polyepitope to a subcellular location selected from the group consisting of: circulating the endocytosis, sorting the endocytosis and lysosome, and the first recombinant nucleic acid comprises a first promoter operably linked to a sequence encoding the polyepitope to drive expression of the polyepitope in the cells of the individual. In another step, the first recombinant nucleic acid is formulated into a DNA vaccine formulation, thereby obtaining a first treatment composition. In yet another step, a second recombinant nucleic acid comprising a sequence encoding a polyepitope is generated, wherein the second recombinant nucleic acid comprises a second promoter operably linked to the sequence encoding the polyepitope to drive expression of the polyepitope in the bacterial cell or the yeast cell, and in a further step, the bacterial cell or the yeast cell is transfected with the second recombinant nucleic acid and the polyepitope is expressed in the bacterial cell or the yeast cell. In yet another step, the transfected bacterial or yeast cells are formulated into a cell-based vaccine formulation, thereby obtaining a second treatment composition.

Most typically, incremental simultaneous alignments of omics data from tumors and omics data from non-tumor samples of the same individual are used to identify expressed novel epitope sequences. It is generally further preferred that the first recombinant nucleic acid is an expression vector and/or the trafficking element is a CD1b leader, CD1a trailer, CD1c trailer or LAMP 1-transmembrane sequence. The second promoter is preferably a constitutive bacterial or yeast promoter and the bacterial or yeast cell is preferably an E.coli cell or a s.cerevisiae cell. Most typically, cell-based vaccine formulations are formulated for injection. In addition, where desired, such methods can further include the step of generating a third recombinant nucleic acid that is a viral expression vector that includes a sequence encoding the polyepitope, wherein the third recombinant nucleic acid comprises a third promoter operably linked to the sequence encoding the polyepitope to drive expression of the polyepitope in the cells of the individual.

In yet another aspect related to the inventive subject matter, the inventors also contemplate a method of preparing first and second treatment compositions for an individual having a tumor, the method comprising the step of identifying a plurality of expressed neoepitope sequences from omics data of the tumor, wherein the expressed neoepitope sequences have a calculated binding affinity to at least one of MHC-I and MHC-II of the individual of equal to or less than 500 nM; and a step of generating a first recombinant nucleic acid having a sequence encoding a polyepitope, wherein the polyepitope comprises a plurality of expressed neo-epitope sequences, wherein the first recombinant nucleic acid is a viral expression vector. Preferably, the polyepitope further comprises a trafficking element that directs the polyepitope to a subcellular location selected from the group consisting of: circulating the endocytosis, sorting the endocytosis and lysosome, and the first recombinant nucleic acid comprises a first promoter operably linked to a sequence encoding the polyepitope to drive expression of the polyepitope in the cells of the individual. In a further step, viral particles are generated from the viral expression vector and formulated into a viral vaccine formulation, thereby obtaining a first treatment composition. Such methods will further include the step of generating a second recombinant nucleic acid having a sequence encoding the polyepitope, wherein the second recombinant nucleic acid comprises a second promoter operably linked to the sequence encoding the polyepitope to drive expression of the polyepitope in the non-mammalian cell; and the further step of transfecting the bacterial cell or yeast cell with the second recombinant nucleic acid and expressing the polyepitope in the bacterial cell or yeast cell. The bacterial or yeast cells so transfected are then formulated into a cell-based vaccine formulation to obtain a second treatment composition.

Most typically, incremental simultaneous alignments of omics data from tumors and non-tumor samples from the same individual are used to identify the new epitope sequences expressed, and/or the trafficking elements are the CD1b leader, CD1a tail, CD1c tail, or LAMP 1-transmembrane sequences. Also, preferably, the first promoter is a constitutive promoter, or the first promoter is inducible by hypoxia, IFN-. gamma.or IL-8, and/or the second promoter is a constitutive bacterial or yeast promoter. In further contemplated embodiments, the viral expression vector is an adenoviral expression vector optionally deleted for the E1 and E2b genes. Although not limiting to the subject matter of the invention, it is generally preferred that the non-mammalian cells or yeast cells are e.coli cells or saccharomyces cerevisiae cells, and/or that both viral and cell-based vaccine formulations are formulated for injection.

Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawings in which like numerals represent like components.

Drawings

FIG. 1 is a schematic illustration of various new bit arrangements.

FIG. 2 is an exemplary and partially schematic illustration of a preferred arrangement for selecting neo-epitopes.

Detailed Description

The inventors have now found that aspects in tumor antigen-based and/or neoepitope-based immunotherapy can be further improved not only by targeting antigens or neoepitopes to specific processing and cell surface presentation pathways, but also by using different vaccine modalities that preferably trigger different immunostimulatory pathways.

Thus, in addition to recombinant virus-based viral cancer vaccines that trigger expression of tumor-associated or tumor-specific antigens in host cells, the same (and/or additional) antigens may be expressed from DNA vaccines, and/or provided in yeast and/or bacterial cells genetically engineered to express these antigens. For example, in the case of plasmids used in DNA vaccines, innate immune response mechanisms against episomal DNA (e.g., TLR 9-based or STING) can be triggered, as well as adaptive immune responses based on episomal DNA expression in vivo. In another example, where the viral expression vector is used as part of a viral vaccine in which the virus infects patient cells, such infection will typically trigger different innate immune response mechanisms (typically TLR2, TLR4, TLR 7, TLR 8, TLR 9). In yet another example, where a bacterial or yeast vaccine is used, wherein the bacteria or yeast already express one or more antigens, such vaccination will again trigger a different innate immune response mechanism (typically TLR1-3 for bacteria and TLR1-4 for yeast). As will be readily appreciated, the triggering of various different innate immune response mechanisms may provide complementary or even synergistic enhancement of vaccine compositions.

Thus, it will be understood that the compositions and methods presented herein will preferably include the use of at least two different vaccine modes. For example, the first mode may be a mode selected from the group consisting of: DNA vaccines, protein vaccines, bacterial vaccines, yeast vaccines and viral vaccines, while the second/subsequent mode may be another different mode selected from the same group. Most preferably, the antigens present in any pattern will overlap or be identical to exert a prime/boost effect upon repeated antigen challenge.

In addition, it will be appreciated that in addition to the benefits of triggering multiple different innate immune pathways, contemplated compositions and methods also allow for rapid initiation of treatment if a patient's tumor-associated antigens are identified in a timely manner. Indeed, it will be appreciated that a DNA vaccine may be prepared based on the relevant antigen within a few days (typically within less than a week, and even less than 4 days or even less than 48 hours). Furthermore, bacterial or yeast vaccines can also be prepared using (the same) antigen within a few days, typically less than 2 weeks, and more typically less than 1 week. Meanwhile, a viral vaccine may be prepared when the patient has received a first vaccine (e.g., a DNA vaccine, a bacterial vaccine, and/or a yeast vaccine).

From a different perspective, it is understood that the compositions and methods presented herein will include one or more of tumor-associated antigens, tumor-specific antigens, and/or neo-epitopes that are specific to the patient and the tumor in the patient to allow for targeted therapy. Moreover, such treatments may be advantageously tailored to achieve one or more specific immune responses (including the innate immune response, CD 4)⁺Biased immune response, CD8⁺A biased immune response, an antibody biased immune response, and/or a stimulated immune response (e.g., reducing checkpoint inhibition and/or activating immune competent cells by using cytokines)). Most typically, such effects are achieved in the context of neo-epitopes derived from recombinant nucleic acids that can be administered in one or more different formats (e.g., as recombinant plasmids and as recombinant viruses) via one or more routes.

Antigens

With respect to suitable therapeutic antigens, it is contemplated that a variety of antigens are deemed suitable for use herein. However, particularly preferred antigens include tumor-associated antigens (e.g., CEA, MUC1, brachyury), tumor-specific antigens (e.g., HER2, PSA, PSMA, etc.), and in particular tumor and patient-specific antigens (i.e., neoepitopes). Neoepitopes can be characterized as random mutations expressed in tumor cells that produce unique antigens and tumor-specific antigens. Thus, from a different perspective, neoepitopes can be identified by considering the type of mutation (e.g., deletion, insertion, transversion, transition, translocation) and the impact of the mutation (e.g., nonsense, missense, frameshift, etc.), and thus can be used as a content filter through which silent mutations and other unrelated (e.g., unexpressed) mutations can be eliminated. It should also be understood that the new sequence of table bits may be defined as having a relatively short lengthA sequence extension of length (e.g., an 8-12mer or a 14-20mer), wherein such extension will include one or more changes in amino acid sequence. Most typically, but not necessarily, the altered amino acid will be at or near the central amino acid position. For example, a typical new bit may have a₄-N-A₄Or A₃-N-A₅Or A₂-N-A₇Or A₅-N-A₃Or A₇-N-A₂Wherein a is a proprotein wild-type or normal (i.e., from the corresponding healthy tissue of the same patient) amino acid, and N is an altered amino acid (relative to wild-type or relative to matched normal). Thus, the novel epitope sequences contemplated herein include sequence extensions of relatively short length (e.g., 5-30mer, more typically 8-12mer, or 14-20mer), where such extensions include one or more changes in amino acid sequence. Additional amino acids can be placed upstream or downstream of the altered amino acid, as desired, e.g., to allow additional antigen processing in various compartments of the cell (e.g., for proteasomal processing in the cytosol, or specific protease processing in the endocytic and/or lysosomal compartments).

Thus, it will be appreciated that, depending on the position of the amino acid that is changed, individual amino acid changes may be present in many new epitope sequences that include these changed amino acids. Advantageously, this sequence variability allows for multiple selections of neo-epitopes and thus increases the number of potentially useful targets that can then be selected based on one or more desired characteristics (e.g., highest affinity for the patient's HLA type, highest structural stability, etc.). Most typically, neoepitopes will be calculated to have a length of between 2-50 amino acids, more typically between 5-30 amino acids, and most typically between 8-12 amino acids or between 14-20 amino acids, with the altered amino acids preferably being centered or otherwise positioned in a manner that improves their binding to MHC. For example, in the case of epitopes presented by the MHC-I complex, a typical neoepitope will be about 8-12 amino acids in length, whereas a typical neoepitope presented by the MHC-II complex will be about 14-20 amino acids in length. As will be readily appreciated, since the positions of the altered amino acids in the neoepitope may be non-central, the actual peptide sequence as well as the actual topology of the neoepitope may vary significantly, and sequences of the neoepitope that have the desired binding affinity for MHC-I or MHC-II presentation and/or the desired protease processing will generally determine the particular sequence.

It will, of course, be appreciated that the identification or discovery of new epitopes may begin with a variety of biological materials, including fresh biopsies, frozen or otherwise preserved tissue or cell samples, circulating tumor cells, exosomes, a variety of bodily fluids (and particularly blood), and the like. Thus, suitable omics analysis methods include nucleic acid sequencing, and in particular NGS methods that operate on DNA (e.g., Illumina sequencing, ion torrent sequencing, 454 pyrophosphate sequencing, nanopore sequencing, etc.); RNA sequencing (e.g., RNAseq, reverse transcription based sequencing, etc.); and in some cases protein sequencing or mass spectrometry based sequencing (e.g., SRM, MRM, CRM, etc.).

As such, and particularly for nucleic acid-based sequencing, it should be particularly recognized that high-throughput genomic sequencing of tumor tissue will allow rapid identification of neoepitopes. However, it must be appreciated that normally occurring inter-patient variation (e.g., due to SNPs, short indels (indels), different numbers of repeats, etc.) as well as heterozygosity will result in a relatively large number of potential false positive neo-epitopes when the sequence information so obtained is compared to a standard reference sequence. Notably, this inaccuracy can be eliminated in the case of comparing a patient's tumor sample with a matched normative (i.e., non-tumor) sample of the same patient.

In a particularly preferred aspect of the inventive subject matter, DNA analysis is performed by whole genome sequencing and/or exome sequencing (typically at least 10x, more typically at least 20x by depth of coverage) of tumors and matched normal samples. Alternatively, DNA data can also be provided from established sequence records (e.g., SAM, BAM, FASTA, FASTQ, or VCF files) from previous sequence determinations from the same patient. Thus, data sets suitable for use herein include unprocessed or processed data sets, and exemplary preferred data sets include those having a BAM format, a SAM format, a GAR format, a FASTQ format, or a FASTA format, as well as BAMBAM, SAMBAM, and VCF data sets. However, it is particularly preferred to provide the data sets in different objects in BAM format or bambambambam, as described in US 2012/0059670 a1 and US 2012/0066001 a 1. Furthermore, it should be noted that the data set reflects tumors and matched normative samples of the same patient. Thus, genetic germline changes that do not cause tumors (e.g., silent mutations, SNPs, etc.) can be excluded. Of course, it should be recognized that the tumor sample may be from the original tumor, from the tumor at the beginning of treatment, from a recurrent tumor and/or a metastatic site, and the like. In most cases, the patient's matched normal sample is blood or non-diseased tissue from the same tissue type as the tumor.

Also, computational analysis of the sequence data can be performed in a variety of ways. However, in the most preferred method, for example, as disclosed in US 2012/0059670 and US 2012/0066001, analysis is performed in a computer using BAM files and BAM servers by incremental localization-guided simultaneous alignment of tumor and normal samples. Such analysis advantageously reduces false positive neo-epitopes and significantly reduces the need for storage and computing resources.

It should be noted that any language specific to a computer should be read to include any suitable combination of computing devices, including servers, interfaces, systems, databases, agents, terminals, engines, controllers, or other types of computing devices operating alone or in combination. It should be understood that the computing device includes a processor configured to execute software instructions stored on a tangible, non-transitory computer-readable storage medium (e.g., hard disk drive, solid state drive, RAM, flash memory, ROM, etc.). The software instructions preferably configure the computing device to provide roles, responsibilities, or other functions as discussed below with respect to the disclosed apparatus. Furthermore, the disclosed techniques may be embodied as a computer program product that includes a non-transitory computer-readable medium storing software instructions that cause a processor to perform the disclosed steps associated with a computer-based algorithm, process, method, or other instruction. In a particularly preferred embodiment, the various servers, systems, databases, or interfaces exchange data using standardized protocols or algorithms, possibly based on HTTP, HTTPs, AES, public-private key exchanges, web services APIs, known financial transaction protocols, or other electronic information exchange methods. Data exchange between devices may be through a packet-switched network, the internet, a LAN, a WAN, a VPN, or other type of packet-switched network; a circuit-switched network; a cell switching network; or other type of network.

Viewed from a different perspective, a collection of patient-and cancer-specific in silico sequences can be established that encode new epitopes having a predetermined length of, for example, between 5 and 25 amino acids, and that include at least one altered amino acid. Such a collection will typically include at least two, at least three, at least four, at least five, or at least six members for each altered amino acid, where the positions of the altered amino acids are different. Such a collection advantageously increases the potential candidate molecules for immunotherapy and may then be used for further filtering (e.g., by subcellular localization, transcription/expression levels, MHC-I and/or II affinity, etc.), as described in detail below. Of course, it will be appreciated that these novel epitope sequences can be readily reverse translated into the corresponding nucleic acid sequences to generate nucleic acid sequences encoding the novel epitopes. Most typically, but not necessarily, such reverse translation will take into account the correct codon usage of the organism in which the nucleic acid is expressed.

For example, and using synchronized localization to guide analysis of tumor and matched normative sequence data, the inventors previously identified multiple cancer neoepitopes from multiple cancers and patients, including the following cancer types BLCA, BRCA, CESC, COAD, DLBC, GBM, HNSC, KICH, KIRC, KIRP, LAML, LGG, LIHC, LUAD, lucc, OV, PRAD, READ, SARC, SKCM, STAD, THCA, and UCEC. Exemplary new epitope data for these cancers can be found in international application WO2016/172722, which is incorporated herein by reference.

Depending on the type and stage of the cancer, as well as the immune status of the patient, it will be appreciated that not all of the identified new epitopes necessarily result in a therapeutically equivalent response in the patient. Indeed, it is well known in the art that only a small fraction of neoepitopes will generate an immune response. To increase the likelihood of a therapeutically desirable response, the initially identified new epitopes may be further filtered. Of course, it is to be understood that downstream analysis need not take into account silent mutations for the purposes of the methods presented herein. However, a preferred mutation analysis will provide information on the impact of the mutation (e.g., nonsense, missense, etc.) in addition to the particular type of mutation (e.g., deletion, insertion, transversion, transition, translocation), and may thus serve as a first content filter through which silent mutations are eliminated. For example, a new epitope may be selected for further consideration, where the mutation is a frameshift, nonsense, and/or missense mutation.

In a further filtering approach, the neo-epitopes can also be analyzed in detail for subcellular localization parameters. For example, if a neo-epitope is identified as having a membrane-associated position (e.g., located outside of the cell membrane of a cell) and/or if in silico structural calculations confirm that the neo-epitope may be exposed to a solvent, or present a structurally stable epitope (e.g., J ExpMed [ journal of experimental medicine ]2014), etc., the new epitope sequence may be selected for further consideration.

With respect to filtering neoepitopes, it is generally contemplated that neoepitopes are particularly suitable for use where omic (or other) analysis reveals actual expression of the neoepitope. The expression and expression level of the novel epitope can be identified in all ways known in the art, and preferred methods include quantitative RNA (hnRNA or mRNA) analysis and/or quantitative proteomic analysis. Most typically, the threshold level comprising the neo-epitope will be an expression level that is at least 20%, at least 30%, at least 40%, or at least 50% of the expression level of the corresponding matching normal sequence, thus ensuring that the (neo) epitope is at least potentially 'visible' to the immune system. Therefore, it is generally preferred that omics analysis also include analysis of gene expression (transcriptomics analysis), thereby helping to identify the expression level of genes having mutations.

There are many transcriptome analysis methods known in the art, and all known methods are considered suitable for use herein. For example, preferred materials include mRNA and primary transcripts (hnRNA), and RNA sequence information can be derived from reverse transcribed polyadenylates⁺-RNA(polyA⁺-RNA), the reverse transcription of polyadenylic acid⁺RNA was in turn obtained from tumor samples and matched normal (healthy) samples of the same patient. Also, it should be noted that although polyadenylation is generally preferred⁺RNA as representative of the transcriptome, other forms of RNA (hn-RNA, non-polyadenylated RNA, siRNA, miRNA, etc.) are also considered suitable for use herein. Preferred methods include quantitative RNA (hnRNA or mRNA) analysis and/or quantitative proteomic analysis, especially including RNAseq. In other aspects, RNA quantification and sequencing is performed using RNAseq, qPCR and/or rtPCR based methods, although a variety of alternative methods (e.g., solid phase hybridization based methods) are also considered suitable. From another perspective, transcriptomic analysis (alone or in combination with genomic analysis) may be suitable for identifying and quantifying genes with cancer-specific mutations and patient-specific mutations.

Similarly, proteomic analysis can be performed in a variety of ways to determine the actual translation of the neoepitope's RNA, and all known ways of proteomic analysis are contemplated herein. However, particularly preferred proteomics methods include antibody-based methods and mass spectrometry methods. Furthermore, it should be noted that proteomic analysis can provide not only qualitative or quantitative information about the protein itself, but can also include protein activity data in which the protein has catalytic or other functional activity. One exemplary technique for performing proteomic assays is described in US 7473532, which is incorporated herein by reference. Additional suitable methods of identifying and even quantifying protein expression include a variety of mass spectrometry analyses (e.g., Selective Response Monitoring (SRM), Multiple Response Monitoring (MRM), and Continuous Response Monitoring (CRM)). Thus, it will be appreciated that the above methods will provide patient and tumor specific neoepitopes that can be further filtered by the subcellular location, expression intensity (e.g., over-expressed as compared to a matched normal control for the same patient), etc. of the protein comprising the neoepitope (e.g., membrane location).

In yet another aspect of filtering, the new epitope can be compared to a database containing known human sequences (e.g., sequences of a patient or a set of patients), thereby avoiding the use of sequences identical to human. Moreover, filtering may also include removing new epitope sequences due to SNPs in the patient, where these SNPs are present in both the tumor and the matching normal sequence. For example, dbSNP (single nucleotide polymorphism database) is a free public archive of genetic variation within and between different species developed and hosted by the National Center for Biotechnology Information (NCBI) in collaboration with the national institute of human genome (NHGRI). Although the name of the database implies only a collection of one type of polymorphism (single nucleotide polymorphisms (SNPs)), in practice it contains a relatively broad range of molecular variations: (1) SNP; (2) deficiency and insertion polymorphism (indels/DIPs); (3) microsatellite markers or Short Tandem Repeats (STRs); (4) a polynucleotide polymorphism (MNP); (5) a heterozygous sequence; and (6) the named variants. dbSNP receives a distinct neutral polymorphism, corresponding to a polymorphism of a known phenotype and a region of no variation. Using such a database and other filtering options as described above, patient and tumor specific neo-epitopes can be filtered to remove those known sequences, resulting in a sequence set with multiple new epitope sequences with significantly reduced false positives.

Once the desired level of filtering for neoepitopes is complete (e.g., filtering neoepitopes by tumor versus normal control, and/or expression levels, and/or subcellular location, and/or patient-specific HLA matching, and/or known variants), further filtering steps are contemplated taking into account the gene types affected by the neoepitopes. For example, suitable gene types include cancer driver genes, genes associated with the regulation of cell division, genes associated with apoptosis, and genes associated with signal transduction. However, in particularly preferred aspects, cancer driver genes are particularly preferred (which can persist through the functionalization of a variety of gene types, including receptor genes, signal transduction genes, transcriptional regulatory genes, and the like). In further contemplated aspects, suitable gene types may also be known passenger genes (passenger genes) and genes involved in metabolism.

Various methods and predictive algorithms are known in the art for the identification or other determination (e.g., prediction) of genes that are drivers of cancer, and are considered suitable for use herein. Suitable algorithms include, for example, Mutsig CV (Nature [ Nature ]2014, 505 (7484): 495-501), Activedriver (Mol Syst Biol [ molecular systems biology ]2013, 9: 637), MuSiC (Genome Res [ Genome research ]2012, 22 (8): 1589-. Alternatively or additionally, the identification of cancer driver genes may also employ multiple sources to obtain known cancer driver genes and their association with a particular cancer. For example, the Intogen catalog of driver mutations (2016.5; URL: www.intogen.org) contains the results of a driver analysis by a Cancer genome Interpreter (Cancer genome Interpreter) of 6,792 exons in a pan-Cancer cohort of 28 tumor types.

However, despite the filtering, it will be appreciated that not all neo-epitopes are visible to the immune system, as these neo-epitopes also need to be processed and presented on the patient's MHC complex in a larger context (e.g., within multiple epitopes). In this context, it must be appreciated that only a fraction of all neoepitopes will have sufficient affinity for presentation. Thus, and especially in the context of immunotherapy, it should be apparent that neoepitopes will be more likely to be effective where they are properly processed, bound and presented by MHC complexes. From another perspective, treatment success will increase as the number of neoepitopes that can be presented via MHC complexes increases, where such neoepitopes have minimal affinity for the patient's HLA type. Thus, it is understood that effective binding and presentation is a function of the sequence of the neoepitope in combination with the particular HLA type of the patient. Thus, it is often desirable to determine the HLA type of a patient's tissue. Most typically, the determination of HLA type comprises at least three MHC-I subtypes (e.g., HLA-A, HLA-B, HLA-C) and at least three MHC-II subtypes (e.g., HLA-DP, HLA-DQ, HLA-DR), preferably wherein each subtype is determined to be at least 2-bit or at least 4-bit deep. However, greater depths (e.g., 6 bits, 8 bits) are also contemplated.

Once the HLA type of the patient is determined (using known chemical or in silico assays), structural solutions for the HLA type can be calculated and/or obtained from the database and then used in a in silico docking model to determine the binding affinity of the (typically filtered) neo-epitope to the HLA structural solution. As will be discussed further below, suitable systems for determining binding affinity include NetMHC platforms (see, e.g., Nucleic acids Res. [ Nucleic acids research ] 2008. month 7 1; 36(Web Server Vol.): W509-W512.). Neoepitopes with high affinity (e.g., less than 100nM, less than 75nM, less than 50nM) for the previously identified HLA type are then selected, along with knowledge of the patient's MHC-I/II subtype for treatment.

HLA assays can be performed using a variety of methods in wet chemistry well known in the art, and all of these methods are deemed suitable for use herein. However, in particularly preferred methods, HLA types can also be predicted from computer-simulated omics data using reference sequences that contain most or all known and/or common HLA types. For example, in one preferred method according to the inventive subject matter, a relatively large number of patient sequence reads mapped to zone 1, zone 3, sub-zone (or near/at any other location where an HLA allele is found) of chromosome 6 short arm 2 region are provided by a database or sequencer. Most typically, sequence reads will have a length of about 100-300 bases and contain metadata including read quality, alignment information, orientation, position, etc. For example, suitable formats include SAM, BAM, FASTA, GAR, and the like. Although not limiting to the inventive subject matter, it is generally preferred that the patient sequence reads provide a depth of coverage of at least 5x, more typically at least 10x, even more typically at least 20x, and most typically at least 30 x.

In addition to patient sequence reads, contemplated methods further employ one or more reference sequences comprising sequences of a plurality of known different HLA alleles. For example, a typical reference sequence can be a synthetic (without a corresponding human or other mammalian counterpart) sequence that includes sequence segments of at least one HLA type having multiple HLA alleles of the HLA type. For example, suitable reference sequences include a collection of known genomic sequences of at least 50 different alleles of HLA-a. Alternatively or additionally, the reference sequence may also comprise a collection of known RNA sequences of at least 50 different alleles of HLA-a. Of course, and as discussed in further detail below, the reference sequence is not limited to 50 alleles of HLA-a, but may have alternative compositions with respect to HLA type and number/composition of alleles. Most typically, the reference sequence will be in a computer readable format and will be provided from a database or other data storage device. For example, suitable reference sequence formats include FASTA, FASTQ, EMBL, GCG, or GenBank formats, and may be obtained or constructed directly from data in a common data repository (e.g., IMGT, International ImmunoGeneTiCs (International ImmunoGeneTics) information system, or Allele Frequency network Database (The Allole Frequency Net Database), EUROSTAM, URL: www.allelefrequencies.net). Alternatively, the reference sequence may also be constructed from individual known HLA alleles based on one or more predetermined criteria (e.g., allele frequency, ethnic allele distribution, common or rare allele type, etc.).

Using the reference sequence, the patient sequence can now be read through a dibuglin (de Bruijn) map to identify the allele with the best match. In this context, it should be noted that each individual carries two alleles for each HLA type, and these alleles may be very similar, or in some cases even identical. Such high similarity poses a significant problem for conventional alignment schemes. The inventors have now found that HLA alleles, and even very closely related alleles, can be resolved using a method in which a dibugine map is constructed by breaking down sequence reads into relatively small k-mers (typically of between 10-20 base lengths), and by performing a weighted voting process in which each patient sequence read is based on the k-mer of the sequence read matching the sequence of the allele, providing a vote for each allele ("quantitative read support"). The cumulative highest vote for the allele then indicates the most likely predicted HLA allele. In addition, it is generally preferred that each fragment that matches an allele is also used to calculate the overall coverage and depth of coverage for that allele.

The score can be further refined or refined as needed, especially in cases where many of the top hits (top hits) are similar (e.g., a large portion of their scores are from a highly shared set of k-mers). For example, the score refinement may include a weighting scheme in which alleles that are substantially similar (e.g., > 99%, or other predetermined value) to the current highest hit are removed from consideration. The count of k-mers used for the current highest hit is then re-weighted by a factor (e.g., 0.5) and the score for each HLA allele is recalculated by adding the weighted counts. This selection process is repeated to find a new highest hit. The accuracy of the method can be even further improved using RNA sequence data that allows identification of alleles expressed by the tumor that may sometimes be only 1 out of 2 alleles present in DNA. In a further advantageous aspect of contemplated systems and methods, DNA or RNA, or a combination of both DNA and RNA, can be processed to make highly accurate HLA predictions, and can be derived from tumor or blood DNA or RNA. In further aspects, suitable methods and considerations for high accuracy in computer modeling of HLA typing are described in WO 2017/035392 (incorporated herein by reference).

Once patient and tumor specific neo-epitopes and HLA types are identified, e.g., using NetMHC, optimal binders (e.g., lowest K) can be determined by in silico docking of the neo-epitopes with HLA_DE.g., less than 500nM, or less than 250nM, or less than 150nM, or less than 50nM) for further in silico analysis. It will be appreciated that this approach will not only identify neoepitopes that are truly specific to the patient and tumour, but will also identify those that are most likely to be presented on cells and therefore most likely to elicit an immune response with therapeutic effect. It will also be appreciated, of course, that the so-identified HLA-matched novel epitopes can be biochemically validated in vitro and then the nucleic acid encoding the epitope incorporated into the virus as a payload, as discussed further below.

Of course, it is understood that systems other than NetMHC may be used, and suitable systems include NetMHC II, NetMHCpan, IEDB analysis resources (URL Immunepopic. org), RankPep, PREDEP, SVMHC, Epipredict, HLABinding, etc. (see, e.g., J Immunol Methods [ journal of immunology ] 2011; 374: 1-4), matching the HLA type of the patient to the patient and cancer specific neoepitope. In calculating the highest affinity, it should be noted that a collection of new epitope sequences can be used, in which the position of the changed amino acid is shifted (supra). Alternatively or additionally, modification of the neo-epitopes may be performed by adding N-terminal and/or C-terminal modifications to further increase the binding of the expressed neo-epitopes to the HLA type of the patient. Thus, the new epitope may be native, or may be further modified to better match a particular HLA type. Furthermore, if desired, the binding of the corresponding wild-type sequence (i.e., the new epitope sequence without amino acid changes) can be calculated to ensure high differential affinity. For example, particularly preferred high differential affinities in MHC binding between a novel epitope and its corresponding wild-type sequence are at least 2-fold, at least 5-fold, at least 10-fold, at least 100-fold, at least 500-fold, at least 1000-fold, and the like.

Binding affinities and specific differential binding affinities can also be determined in vitro using a variety of systems and methods. For example, antigen presenting cells of a patient or cells having a matched HLA type can be transfected with nucleic acids (e.g., viruses, plasmids, linear DNA, RNA, etc.) using constructs as described in detail below to express one or more neo-epitopes. After expression and antigen processing, the neoepitope can then be identified in the extracellular MHC complex using a specific binder for the neoepitope or using a cell-based system (e.g., PBMCs of the patient) in which T cell activation or cytotoxic NK cell activity can be observed in vitro. Neoepitopes with differential activity (e.g., eliciting a stronger signal or immune response than the corresponding wild-type epitope) will then be selected for treatment.

Recombinant nucleic acid/polyepitope

Upon appropriate selection of the filtered neoepitope (or tumor-associated antigen or tumor-specific antigen), recombinant nucleic acids can be constructed that form all downstream vaccine compositions. Most typically, the desired nucleic acid sequence (for expression from a virally infected cell) is under the control of suitable regulatory elements well known in the art. It will also be readily appreciated that the choice of regulatory elements will be dictated by the system in which the recombinant nucleic acid is to be expressed. Thus, suitable regulatory elements include constitutively active or inducible bacterial and yeast promoters (and associated inducer sequences and/or repressor sequences as needed), as well as eukaryotic (and preferably mammalian/human) promoter sequences. For example, where recombinant nucleic acids are used in DNA vaccines, suitable promoter elements include constitutively strong promoters (e.g., SV40, CMV, UBC, EF1A, PGK, CAGG promoters). In other aspects, where the recombinant nucleic acid is part of a viral expression vector, contemplated promoters also include inducible promoters (particularly in the case of inducing conditions typical of a tumor microenvironment). For example, inducible promoters include promoters sensitive to hypoxia and promoters sensitive to TGF- β or IL-8 (e.g., via TRAF, JNK, Erk, or other responsive element promoters). In other examples, suitable inducible promoters include tetracycline-inducible promoters, myxovirus resistance 1(Mx1) promoters, and the like.

Similarly, where the recombinant nucleic acid is used to generate a bacterial vaccine and/or a yeast vaccine in which the bacteria or yeast express neo-epitopes or other therapeutic antigens, suitable promoters include strong constitutive or inducible bacterial and yeast promoters. For example, suitable bacterial promoters for expression of the antigen/polyepitope include the T7 promoter, Tac promoter, BAD promoter, Trc promoter, and the like. Likewise, yeast promoters contemplated by yeast include the AOX1 promoter, GAL promoter, GDS promoter, ADH promoter, and the like.

In this context, it should be understood that the inventors have found that the manner in which the new epitopes are arranged and the rationally designed transport of the new epitopes can have a substantial impact on the efficacy of various immunotherapeutic compositions, as will be described in further detail below. For example, a single neo-epitope can be expressed separately from the corresponding recombinant construct delivered as a single plasmid, viral expression construct, or the like. Alternatively, multiple neo-epitopes can be expressed separately from separate promoters to form separate mrnas, which are then separately translated into the respective neo-epitopes; or from a single mRNA containing separate translation initiation points for each new epitope sequence (e.g., using 2A or IRES signals). Notably, while such an arrangement is generally believed to allow controlled delivery of the appropriate neo-epitope peptide, the efficacy of such expression systems has been less than desirable (data not shown).

In contrast, expression, processing and antigen presentation of multiple neoepitopes from a single transcript to form a single transcript, followed by translation of the single transcript into a single polyepitope (i.e., a polypeptide having a series of serially linked neoepitopes, optionally with an inserted linker sequence) is believed to be effective. Notably, expression of polyepitopes requires processing within the cell by suitable proteases (e.g., proteasomes, endocytic proteases, lysosomal proteases) to yield novel epitope sequences, and polyepitopes result in improved antigen processing and presentation of most neoepitopes as compared to expression of single neoepitopes, particularly individual neoepitopes having relatively short lengths (e.g., less than 25 amino acids; results not shown) therein. Moreover, this approach also allows rational design of protease sensitive sequence motifs between the neo-epitope peptide sequences, thereby ensuring or avoiding processing by specific proteases (as proteasome, endocytic and lysosomal proteases have different cleavage preferences). Thus, polyepitopes can be designed that include not only spatially separated linker sequences of new epitopes, but also portions of the sequence that will be preferentially cleaved by specific proteases (e.g., between 3-15 amino acids).

Thus, the inventors contemplate recombinant nucleic acids and expression vectors (e.g., viral expression vectors) comprising a nucleic acid segment encoding a polyepitope, wherein the polyepitope is operably coupled to a desired promoter element, and wherein the individual neoepitopes are optionally separated by a linker and/or a protease cleavage or recognition sequence. For example, FIG. 1 illustrates various contemplated permutations of neoepitopes for expression from an adenoviral expression system (here: AdV5, with deletions of the E1 and E2b genes). Here, construct 1 exemplarily illustrates a neo-epitope arrangement comprising 8 neo-epitopes ('minigenes') with a total length of 15 amino acids in the concatemer sequence without an intervening linker sequence, while construct 2 shows the arrangement of construct 1 but comprising 9 amino acid linkers between each neo-epitope sequence. Of course, and as noted above, it should be recognized that the exact length of the new epitope sequence is not limited to 15 amino acids, and that the exact length may vary widely. However, in most cases, when the new epitope sequence of between 8-12 amino acids is flanked by additional amino acids, the total length will typically not exceed 25 amino acids, or 30 amino acids or 50 amino acids. Also, it should be noted that although fig. 1 indicates a G-S linker, a variety of other linker sequences are also suitable for use herein. Such relatively short neoepitopes are particularly advantageous when the neoepitope is intended to be presented via the MHC-I complex.

In this context, it should be understood that a suitable linker sequence will provide spatial flexibility and separate two adjacent neo-epitopes. However, care must be taken not to select for the linker amino acids that may be immunogenic/form an epitope already present in the patient. Thus, it is often preferable to filter the polyepitope constructs again for the presence of epitopes that can be found in the patient (e.g., as part of the normal sequence or due to SNPs or other sequence variations). Such filtering would apply the same techniques and criteria as already discussed above.

Similarly, construct 3 exemplarily illustrates a neo-epitope arrangement comprising 8 neo-epitopes without an intervening linker sequence in the concatamer sequence, and construct 4 shows the arrangement of construct 3 comprising a 9 amino acid linker between each neo-epitope sequence. As noted above, it should be recognized that the exact length of such new epitope sequences is not limited to 25 amino acids, and that the exact length may vary widely. However, in most cases, when the new epitope sequence of between 14-20 amino acids is flanked by additional amino acids, the total length will typically not exceed 30 amino acids, or 45 amino acids or 60 amino acids. Also, it should be noted that although fig. 1 indicates the G-S linker of these constructs, a variety of other linker sequences are also suitable for use herein. This relatively long neoepitope is particularly advantageous when the neoepitope is intended to be presented via an MHC-II complex.

In this example, it is understood that a 15 amino acid (15-aa) minigene is a MHC class I targeted tumor mutation selected with 7 amino acids of the natural sequence on either side, while a 25 amino acid minigene is a MHC class II targeted tumor mutation selected with 12 amino acids of the natural sequence on either side. An exemplary 9 amino acid linker is believed to be of sufficient length such that "non-native" MHC class I epitopes are not formed between adjacent minigenes. Polyepitopic sequences tend to be processed and presented more efficiently than single neoepitopes (data not shown), and adding more than 12 amino acids for MHC-I presentation, and more than 20 amino acids for MHC-I presentation appears to allow for somewhat improved processing by proteases.

To maximize the likelihood that the tailored protein sequence will remain within the cell for processing and presentation by HLA complexes, the new epitope sequences can be arranged in a manner that minimizes hydrophobic sequences, which can direct trafficking to the cell membrane or extracellular space. Most preferably, detection of the hydrophobic sequence or signal peptide is accomplished by comparing the sequence to a weight matrix (see, e.g., Nucleic Acids Res [ Nucleic Acids research ] 11.6.1986; 14 (11): 4683-plus 4690) or by using a neural network trained on a peptide comprising the signal sequence (see, e.g., Journal of molecular Biology 2004, 338, 5, 1027-plus 1036). FIG. 2 depicts an exemplary scheme for permutation selection in which a plurality of multiple table bit sequences are analyzed. Here, all positional permutations of all neoepitopes are calculated to generate a set of permutations. The collection is then processed by a weight matrix and/or neural network prediction to generate a score representing the likelihood of the presence and/or strength of a hydrophobic sequence or signal peptide. All positional permutations are then ranked by score, and one or more permutations with scores below a predetermined threshold or lowest score (for the likelihood of the presence and/or strength of a hydrophobic sequence or signal peptide) are used to construct a customized new epitope expression cassette.

With respect to the total number of neoepitope sequences in a polyepitope, it is generally preferred that the polyepitope comprises at least two, or at least three, or at least five, or at least eight, or at least ten neoepitope sequences. Indeed, the payload capacity of the host organism of the recombinant DNA is generally expected to be a limiting factor, as well as the availability of filtered and appropriate neo-epitopes. For example, adenoviral expression vectors, and particularly Adv5, are particularly preferred because such vectors can accommodate up to 14kb in recombinant payloads. Also, bacterial and yeast systems can accommodate even larger payloads, typically over 50 kb. In other aspects, when the recombinant DNA is used in a DNA vaccine, the appropriate size will typically range between 5kb and 20 kb.

In aspects of the inventive subject matter that are still further contemplated, it is noted that the neo-epitope/polyepitope can be targeted to a particular subcellular compartment (e.g., cytosol, endocytosis, lysosome) and thus to a particular MHC presentation type. Such targeted expression, processing and presentation is particularly advantageous because it can be prepared to direct an immune response to CD8⁺Type response (in which multiple epitopes are directed to the cytoplasmic space) or to CD4⁺Type response (in which multiple epitopes are directed)To the endocytic/lysosomal compartment). Furthermore, it will be appreciated that multiple epitopes that would normally be presented via the MHC-I pathway may be presented via the MHC-II pathway (and thus mimic cross presentation of neoepitopes). Thus, it will be appreciated that neoepitopes and polyepitope sequences can be designed and directed to one or two MHC presentation pathways using appropriate sequence elements. With respect to delivering such expressed neo-epitopes to the desired MHC system, it should be noted that MHC-I presented peptides will typically be produced from the cytoplasm by proteasomal processing and delivered through the endoplasmic reticulum. Thus, as discussed in more detail below, expression of an epitope intended for MHC-I presentation will typically be directed to the cytoplasm. In other aspects, MHC-II presented peptides will typically be produced from endocytic and lysosomal compartments via degradation and processing by acidic proteases (e.g., legumain, cathepsin L, and cathepsin S) prior to delivery to the cell membrane.

Moreover, it is contemplated that polyepitopic protein degradation may also be enhanced using a variety of methods, and particularly contemplated methods include adding a cleavable or non-cleavable ubiquitin moiety to the N-terminus, and/or placing one or more destabilizing amino acids (e.g., N, K, C, F, E, R, Q) at the N-terminus of the polyepitopic, where presentation is directed to MHC-I. In other aspects, where presentation is directed to MHC-II, the cleavage site for a particular endocytic or lysosomal protease can be engineered to be polyepitopic, thereby helping to facilitate antigen processing.

Thus, in contemplated aspects of the inventive subject matter, the signal and/or leader peptide can be used to transport the neoepitope and/or polyepitope to endocytic and lysosomal compartments, or for retention in the cytoplasmic space. For example, where multiple epitopes are exported to endocytic and lysosomal compartments, a leader peptide (e.g., CD1b leader peptide) can be used to sequester the (nascent) protein from the cytoplasm. Additionally or alternatively, targeting pro-sequences and/or targeting peptides may be employed. The pro sequence of the targeting peptide may be added to the N-terminus and/or C-terminus and typically comprises between 6-136 basic amino acids and hydrophobic amino acids. In the case of peroxisome targeting, the targeting sequence may be at the C-terminus. Other signals (e.g., signal spots) can be used and include sequence elements that are separated in the peptide sequence and function after appropriate peptide folding. In addition, protein modifications such as glycosylation can induce targeting. Among other suitable targeting signals, the inventors contemplate peroxisome targeting signal 1(PTS1) (C-terminal tripeptide), and peroxisome targeting signal 2(PTS2) (nonapeptide located near the N-terminus).

In addition, sorting of proteins into endocytosis and lysosomes can also be mediated by signals within the cytosolic domain of the protein, which typically contain short linear sequences. Some signals are called tyrosine-based sorting signals and conform to NPXY or

Consensus motifs. Other signals, termed dual leucine-based signals, are also suitable [ DE]XXXL[LI]Or dxll consensus motif. All of these signals are recognized by components of the protein coat that are associated with the periphery of the cytosolic surface of the membrane.

And [ DE]XXXL[LI]The signals are recognized by the Adaptor Protein (AP) complexes AP-1, AP-2, AP-3 and AP-4 as having characteristic fine specificity, whereas the DXXLL signal is recognized by another family of adaptor proteins called GGA. The FYVE domain may also be added, which is associated with vacuolar protein sorting and endocytic functions. In a still further aspect, the endocytic compartment can also be targeted using the human CD1 tail sequence (see, e.g., Immunology [ Immunology ]],122, 522-531). For example, as shown in more detail below, lysosomal targeting can be achieved using the LAMP1-TM (transmembrane) sequence; while circulating endocytosis can be targeted via the CD1a tail targeting sequence; and sorting endocytosis can be targeted via the CD1c tail targeting sequence.

Transport to or retention in the cytosolic compartment does not necessarily require one or more specific sequence elements. However, in at least some aspects, an N-or C-terminal cytoplasmic retention signal, including a membrane-anchored protein or a membrane-anchoring domain of a membrane-anchored protein, can be added such that the protein is retained in the cytosol-facing cell. For example, membrane-anchoring proteins include SNAP-25, syntaxin, synaptophysin, vesicle-associated membrane protein (VAMP), synaptophysin (SV2), high affinity choline transporters, neurotoxins, voltage-gated calcium channels, acetylcholinesterase, and NOTCH.

In still further contemplated aspects of the inventive subject matter, the polyepitope may further comprise one or more transmembrane segments that will direct the new epitope to the outside of the cell membrane after processing, thereby rendering it visible to immune competent cells. There are many transmembrane domains known in the art, and all of these are considered suitable for use herein, including those having a single alpha helix, multiple alpha helices, alpha/beta barrel structures, and the like. For example, contemplated transmembrane domains may comprise a T cell receptor, CD epsilon, CD (e.g., CD α, CD β), CD, OX, CD134, CD137, CD154, KIRDS, OX, CD, LFA-1(CD11, CD), ICOS (CD278), 4-1BB (CD137), GITR, CD, BAFFR, HVEM (LIGHTR), SLAMF, NKp (KLRF), CD160, CD, IL2 β, IL2 γ, IL7 α, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITGAL, CD11, ITGAA-1, ITGAM, CD11, ITGAX, CD11, ITGB, LFGB, CD1, ITGAD, ITGAE, ACAR 160, ACAR, CD103, ITGAL, CD2 γ, CD100, CD-100, TAAMB, CD-100, CD-100, ITGAM, CD-6, ITGAD, CD-6, ITGAD, ITGAE, CD-2, CD-100, CD-CD, BLAME (SLAMF8), SELPLG (CD162), LTBR or PAG/one or more transmembrane regions of the alpha, beta or zeta chain of Cbp. Where a fusion protein is desired, it is contemplated that the recombinant chimeric gene has a first portion encoding one or more transmembrane regions, wherein the first portion is cloned in-frame with a second portion encoding a suppressor protein. It should be noted that such presentation will not result in presentation of MHC complexes and thus provide for presentation of neo-epitopes independent of MHC/T cell receptor interactions, which may open further avenues for immune recognition and triggering of antibody production against neo-epitopes.

Alternatively or additionally, the polyepitope may also be designed to include a signal sequence for protein export of one or more neoepitopes, thereby forcing the transfected cells to produce and secrete one or more neoepitopes. For example, the SPARC leader sequence can be added to a neoepitope or polyepitope sequence, resulting in secretion of the neoepitope or polyepitope sequence into the extracellular space in vivo. Advantageously, such secreted neoepitope or polyepitope is then taken up by immune competent cells (and in particular antigen presenting cells and dendritic cells), which in turn typically process and display the neoepitope via the MHC-II pathway.

In still further contemplated aspects, the polyepitope can also be designed as a chimeric polyepitope that includes at least a portion, and more typically the entire tumor associated antigen (e.g., CEA, PSMA, PSA, MUC1, AFP, MAGE, HER2, HCC1, p62, p90, etc.). Most notably, tumor associated antigens are often processed and presented via the MHC-II pathway. Thus, instead of using compartment-specific signal sequences and/or leader sequences, processing mechanisms for tumor-associated antigens can be used for MHC-II targeting.

Thus, it will be appreciated that immunotherapeutic compositions can be prepared that can deliver one or more neo-epitopes to a variety of subcellular locations and generate different immune responses. For example, prior art FIG. 3 schematically illustrates the processing of polyepitopes predominantly in the cytoplasmic proteasome and presentation via the MHC-I complex, which is expressed by CD8⁺T cell receptor recognition by T cells. Thus, targeting polyepitope processing to the cytosolic compartment would direct the immune response to CD8⁺The pattern response is skewed. In other respects, prior art FIG. 4 schematically illustrates the processing of polyepitopes primarily in the endocytic compartment and presentation via the MHC-II complex, which is expressed by CD4⁺T cell receptor recognition by T cells. Thus, targeting multiple epitope processing to either the endocytic or lysosomal compartment would direct the immune response to CD4⁺The pattern response is skewed. In addition, it will be appreciated that such targeting methods allow for multiple epitopes or neogenesisEpitope peptides are specifically delivered to the MHC subtype with the highest affinity for the peptide, even though the peptide will not be presented by the MHC subtype. Thus, as previously mentioned, peptides for MHC-I presentation will typically be designed to have 8-12 amino acids (plus additional amino acids for flexibility of protease processing), while peptides for MHC-II presentation will be designed to have 14-20 amino acids (plus additional amino acids for flexibility of protease processing). In some examples, additional amino acids are added to allow processing flexibility in the cytoplasmic, proteasome, or endocytic compartments.

In still further contemplated aspects of the inventive subject matter, it is noted that transport patterns of neoepitopes or polyepitopes may be combined to suit one or more particular purposes. For example, sequential administration of the same neo-epitope or polyepitope with different targeting is particularly advantageous in a prime-boost regimen, wherein upon first administration, the patient is vaccinated with the recombinant virus to infect the patient's cells, resulting in antigen expression, processing and presentation (e.g., primarily MHC-I presentation), which will result in a first immune response derived intracellularly. A second administration of the same neo-epitopes bound to albumin can then be used as a boost since the protein so delivered is taken up by antigen presenting cells, resulting in a different antigen presentation (e.g., predominantly MHC-II presentation) in most cases. When the same neoepitope or polyepitopes are transported to the cell surface for cell-indicative MHC-independent presentation, ADCC response or NK-mediated cell killing can be promoted. In still further contemplated aspects, and as illustrated by the examples below, the immunogenicity of the neoepitope can be enhanced by cross-presentation or MHC-II directed presentation. Notably, since cancer cell neoepitopes are typically produced and recycled internally and are preferentially presented by the MHC-I system, contemplated systems and methods now allow for the presentation of such neoepitopes via MHC-II, which may be more immunogenic, as shown in more detail below. In addition, due to stimulation of various components of the cellular and humoral immune systems, multiple different trafficking of the same neoepitope or polyepitopes may advantageously augment or complement the immune response.

Of course, it should be understood that a variety of different trafficking of the same neoepitope or polyepitope may be achieved in a variety of ways. For example, different trafficked neo-epitopes or polyepitopes can be administered separately, using the same (e.g., viral expression vector) or different (e.g., viral expression vector and albumin binding) patterns. Similarly, and particularly when the therapeutic agent is an expression system (e.g., viral or bacterial), the recombinant nucleic acid can comprise two different portions that encode the same, although a different trafficked neo-epitope or polyepitope (e.g., a first portion trafficked to a first location (e.g., cytosolic or endocytic or lysosomal), a second portion trafficked to a second, different location (e.g., cytosolic or endocytic or lysosomal, secreted, membrane bound)). Likewise, a first administration may employ cytoplasmic targeted neoepitope or polyepitope viral delivery, while a second administration is typically at least one, two, four, one or two days after the first administration, and may employ endocytosomally or lysosomally targeted or secreted neoepitope or polyepitope viral delivery.

In addition, it will be appreciated that where the recombinant nucleic acid is used in a DNA vaccine, a bacterial vaccine or a yeast vaccine, the pattern of uptake will be at least partially indicative of intracellular trafficking. Most typically, DNA vaccines, bacterial vaccines or yeast vaccines are taken up by endocytosis or related processes and will therefore be preferentially delivered to endocytic or lysosomal compartments. This pathway selection can be further enhanced (at least in the case of DNA vaccines) using appropriate trafficking signals already described above or by using cytoplasmic retention sequences to counteract. However, in other embodiments, it is understood that multiple epitopes delivered via DNA vaccines, bacterial vaccines or yeast vaccines need not have a trafficking signal at all. Such polyepitopes will then be preferentially processed/presented via the MHC-II system.

Additionally, it is contemplated that the expression construct (and in particular a recombinant viral expression vector or DNA plasmid for a DNA vaccine) may further encode at least one, more typically at least two, even more typically at least three, and most typically at least four co-stimulatory molecules to enhance the interaction between infected cells (e.g., antigen presenting cells) and T cells. For example, suitable co-stimulatory molecules include CD80, CD86, CD30, CD40, CD30L, CD40L, ICOS-L, B7-H3, B7-H4, CD70, OX40L, 4-1BBL, while other stimulatory molecules with less defined (or understood) mechanisms of action include GITR-L, TIM-3, TIM-4, CD48, CD58, TL1A, ICAM-1, LFA3, and SLAM family members. However, particularly preferred molecules for the coordinated expression of sequences associated with cancer include CD80(B7-1), CD86(B7-2), CD54(ICAM-1), and CD11 (LFA-1). In addition to costimulatory molecules, the inventors also contemplate that one or more cytokines or cytokine analogs can be expressed from recombinant nucleic acids, and particularly preferred cytokines and cytokine analogs include IL-2, IL-15, and IL-a5 superagonists (ALT-803). Furthermore, it will be appreciated that the expression of the co-stimulatory molecules and/or cytokines will preferably be coordinated such that the neoepitope or polyepitopes are expressed simultaneously with the one or more co-stimulatory molecules and/or cytokines. Thus, it is typically contemplated that co-stimulatory molecules and/or cytokines are produced from a single transcript (which may or may not include portions of the sequence encoding multiple epitopes) or from multiple transcripts, for example, using internal ribosome entry sites or 2A sequences.

Likewise, it is contemplated that the viral vector may further comprise a sequence portion encoding one or more peptide ligands that bind to the checkpoint receptor. Most typically, binding will inhibit or at least reduce signaling through the receptor, and particularly contemplated receptors include CTLA-4 (particularly against CD 8)⁺Cells), PD-1 (especially against CD 4)⁺Cells), TIM1 receptor, 2B4, and CD 160. For example, suitable peptide binding agents may include antibody fragments and particularly scfvs that specifically bind to the receptor, as well as small molecule peptide ligands (e.g., isolated via RNA display or phage panning). Again, it will be appreciated that expression of the peptide molecules will preferably be coordinated such that the neoepitope or polyepitope is expressed simultaneously with one or more peptide ligands. Thus, it is typically contemplated, for example, to use internal ribosome entry sites or 2A sequences, from a single transcript (which may or may not include a sequence portion encoding multiple epitopes) or from multiple transcriptsProducing the peptide ligand.

It is to be understood that all of the co-stimulatory genes and genes encoding inhibitory proteins that interfere with/down-regulate checkpoint inhibition described above are well known in the art, and that sequence information for these genes, isoforms and variants can be retrieved from a variety of public sources, including sequence databases accessible at NCBI, EMBL, GenBank, RefSeq, and the like. Moreover, although the exemplary stimulatory molecules described above are preferably expressed in full-length form for expression in humans, modified and non-human forms are also considered suitable, as long as such forms aid in the stimulation or activation of T cells. Thus, truncated and chimeric forms of the muteins are expressly contemplated herein.

Thus, contemplated expression constructs will preferably comprise a portion of the sequence encoding one or more polyepitopes, wherein at least one, and more typically at least two or all of the polyepitopes will comprise a trafficking signal that will result in preferential trafficking of the polyepitope to at least one, and more typically at least two different subcellular locations. For example, the first polyepitope may be directed to the cytoplasm (and may include additional cleavable or non-cleavable ubiquitin), while the second polyepitope may be directed to the endocytic or lysosomal compartment. Alternatively, the first polyepitope may be directed to an endocytic or lysosomal compartment, while the second polyepitope may be directed to the cell membrane or secreted. As previously described, the encoded polyepitope will comprise at least two neoepitopes (optionally separated by a linker). Moreover, such contemplated expression constructs will also include sequence portions encoding one or more co-stimulatory molecules and/or cytokines, and may also include one or more inhibitory proteins that interfere with/down-regulate checkpoint inhibition. Most typically, the expression construct will also include regulatory sequences operably coupled to the above-described sequence portions to drive simultaneous expression of the polyepitope and co-stimulatory molecules, cytokines and/or profilins. Suitable promoter elements are known in the art, and particularly preferred promoters include the constitutive promoters and inducible promoters discussed above.

Vaccine composition

After the desired neoepitope is identified, the sequence information of the neoepitope can be used to prepare one or more immunotherapeutic agents, which are preferably configured as polyepitopes as described above. Preferably, the immunotherapeutic agent comprises: at least two DNA vaccines comprising recombinant nucleic acids encoding at least one antigen (and more typically at least two, three, four or more antigens) present in a tumor; a bacterial vaccine in which the bacteria express at least one antigen (and more typically at least two, three, four or more antigens) present in the tumor; a yeast vaccine, wherein the bacteria express at least one antigen (and more typically at least two, three, four, or more antigens) present in the tumor; and viral vaccines comprising a viral expression vector comprising a recombinant nucleic acid encoding at least one antigen (and more typically at least two, three, four or more antigens) present in a tumor.

With respect to recombinant nucleic acids for use in expression and DNA vaccination systems, it is contemplated that the recombinant nucleic acid may be RNA or DNA. With respect to the use of RNA, DNA, or other recombinant vectors that result in the expression of tumor antigens and/or neo-epitopes, particularly contemplated nucleic acids include plasmid vectors that can be supercoiled, coiled, relaxed, or even linearized. For example, and in other suitable options, contemplated vectors include vectors for cloning one or more sequence portions for use in preparing viral expression vectors. Thus, particularly contemplated vectors include transfer or shuttle vectors, as well as various general cloning vectors (e.g., having a bacterial origin of replication, a selectable marker (e.g., antibiotic resistance or fluorescent protein), and a multiple cloning site). Suitable vectors are well known in the art and are typically based on plasmids that are replication competent in bacteria for large scale cloning and production. The appropriate carrier selection can be further determined by: by their particular use (e.g., shuttle vectors for adenovirus, lentivirus, or baculovirus, etc.); selection of inducible or constitutive promoters (e.g., CMV, UbC); selection of permanent or transient expression; transfection means (e.g., lipofection, electroporation, etc.), capacity of the recombinant payload, and the like.

It should be further noted that the plasmid may be methylated or unmethylated, which may be controlled by a direct enzymatic in vitro reaction or more simply by replicating the plasmid in vivo in a methylation competent or methylation deficient host. Still further, it is understood that the plasmid may further have one or more nucleic acid portions known to trigger an innate immune response (e.g., CpG islands or other sequence motifs that interact with Toll-like receptors (e.g., TLR3, TLR 7, TLR 8, TLR 9), RIG-I-like receptors, STING, and/or intracellular DNA sensors (e.g., NLRP3/CIAS 1)).

Most typically, and as noted above, contemplated plasmids and other nucleic acids will include one or more sequence elements encoding, preferably patient-and tumor-specific neo-epitopes or polyepitopes, most preferably operably coupled to regulatory elements that permit or drive expression of the neo-epitope or polyepitope in eukaryotic cells, and in particular mammalian (e.g., human) cells. Furthermore, it should be noted that, as also discussed herein, the neo-epitope or multi-epitope can include a trafficking signal for bringing the peptide to a desired subcellular location. Thus, particularly preferred plasmids include plasmids used in the production of viral expression vectors, and thus will already include all regulatory elements required for expression and/or trafficking of polyepitopes in mammalian cells. Therefore, cloning vectors and shuttle vectors are particularly preferred.

As will be appreciated, the plasmids contemplated herein can be administered in a variety of ways known in the art, and suitable modes of delivery include injection (intramuscular, intravenous, intradermal), delivery via gene gun or other ballistic transfer, or transfer mediated by liposomes. Thus, contemplated compositions will include, inter alia, injectable formulations comprising nucleic acid lipid complexes and other DNA-lipid or DNA lipoprotein complexes. Advantageously, the choice of delivery can be used to polarize the immune response against either a Th1 (via injection using saline) or a Th2 (delivered via gene gun) response, as described elsewhere (see, e.g., JImmunol [ journal of immunology ]1997, 3/1, 158(5) 2278-. In particularly preferred aspects, as discussed in more detail below, vaccination with DNA vaccines will preferably be by intravenous injection, however, other routes (including intramuscular, intradermal, subcutaneous, intraarterial) are also considered suitable for use herein, and administration of viral expression vectors (typically using recombinant viruses) will preferably be by subcutaneous injection.

Without wishing to be bound by any particular theory or hypothesis, it is believed that the use of plasmids and other "naked" nucleic acids (e.g., linear DNA RNA) will provide the first opportunity to generate an immune response that is non-specific or specific to the patient's neo-epitope. Non-specific reactions are thought to be caused by components of the innate immune response to foreign nucleic acids (e.g., where the nucleic acids are unmethylated (e.g., via TLR-9)). In addition, expression of neoepitopes or polyepitopes in cells transfected with plasmids will also promote adaptive immune responses.

In addition to using DNA vaccination, contemplated plasmids may also be used to produce viral or yeast expression vectors that can be used to produce recombinant viruses (e.g., lentiviruses, adenoviruses) or yeast for subsequent administration to a patient. When plasmids are used to produce yeast or viral expression systems, all known expression systems are considered suitable for use herein. Suitable materials and protocols can be found, for example, in the non-profit plasmid repository Addgene or in the Adeasy adenovirus vector system, commercially available from Agilent, Inc. (Agilent). Similarly, there are a variety of yeast expression systems known in the art, and all of these are deemed suitable for use herein. This second administration can be considered as a boosting regimen for DNA vaccination, since the mammal is already primed by the plasmid vector. Of course, it will be appreciated that where the primary immunization is a DNA vaccination with a plasmid as described above, the booster immunization may be used in a variety of alternative forms (including vaccination with a neo-epitope peptide or polyepitope using more conventional vaccine formulations). Further suitable DNA vaccines are described in e.g. US 2014/0178438.

With respect to bacterial expression and vaccination systems, it is contemplated that all bacterial strains are considered suitable and include, inter alia, species from the genera Salmonella (Salmonella), Clostridium (Clostridium), Bacillus (Bacillus), Lactobacillus (Lactobacillus), Bifidobacterium (Bifidobacterium) and the like, particularly where such strains are nonpathogenic, genetically engineered to have reduced toxicity, and/or irradiated prior to administration. Historically, most bacterial strains have been considered unsuitable for introduction into the bloodstream or transplantation into organs or tissues, because most bacteria express lipopolysaccharides, which trigger an immune response and elicit an endotoxin response, potentially leading to potentially fatal sepsis (e.g., CD-14 mediated sepsis) in patients. Thus, a particularly preferred bacterial strain is based on genetically modified bacteria that express endotoxin at levels low enough not to elicit an endotoxin response in human cells and/or to induce CD-14 mediated sepsis when introduced into a human.

One preferred bacterial species is the genetically modified e.coli (e.coli) strain because of its rapid growth (e.g., complete one complete cell cycle in 20 minutes), and the availability of many strains optimized for protein overexpression following induction (e.g., induction with the lac promoter of IPTG, etc.). Most typically, the genetic modification will reduce or eliminate the production of most of the lipopolysaccharide components responsible for the endotoxin response. For example, an exemplary bacterial strain with modified lipopolysaccharide synthesis includes

BL21(DE3) electrocompetent cells. The bacterial strain is BL21, and has genotype F-ompT hsdSB (rB-mB-) gal dcm lon lambda (DE3[ lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) msbA148 Δ gutQ Δ kdsD Δ lpxL Δ lpxM Δ pagP Δ lpxP Δ eptA. In this context, it is understood that several specific deletion mutations (Δ gutQ Δ kdsD Δ lpxL Δ lpxM Δ pagP Δ lpxP Δ eptA) encode modifications of lipid IVA by LPS, while one additional compensating mutation (msbA148) enables the cell to maintain viability in the presence of LPS precursor lipid IVA. These mutations result in the deletion of the oligosaccharide chain from LPS. More specifically, two of the six acyl chains are deleted. The six acyl chains of LPS are triggers for myeloid differentiation via Toll-like receptor 4(TLR4)The factor 2(MD-2) complex is identified, thereby causing

Activation of pro-inflammatory cytokines. Lipid IV comprising only four acyl chains_AIs not recognized by TLR4 and therefore does not trigger an endotoxin response. Although electrocompetent BL21 bacteria are provided as examples, the inventors contemplate that the genetically modified bacteria may also be chemically competent bacteria.

Alternatively, the inventors also contemplate that the patient's own endosymbiotic bacteria can be used as a vehicle to express human disease-associated antigens in vivo to elicit an immune response at least locally. As used herein, a patient's endosymbiotic bacteria refers to bacteria that are present in the patient without eliciting any substantial immune response, regardless of the patient's health status. Thus, the endosymbiotic bacteria of the patient are expected to be the normal flora of the patient. For example, the endosymbiotic bacteria of a patient may include the genera escherichia coli or streptococcus, which are commonly found in the intestine or stomach of humans. In these embodiments, the patient's own endosymbiotic bacteria may be obtained from a biopsy or stool sample from a portion of the intestine, stomach, oral mucosa, or conjunctiva. The patient's endosymbiotic bacteria can then be cultured in vitro and transfected with nucleotides encoding one or more human disease-associated antigens.

Thus, it will be appreciated that the bacteria used in the methods presented herein may be derived from a strain that produces LPS, or a strain that is genetically engineered to reduce or eliminate the expression of one or more enzymes that cause the formation of LPS, which is recognized by a TLR (and in particular TLR 4). Most typically, such bacteria will be genetically modified to express at least one human disease-associated antigen for immunotherapy in an inducible manner. In other options, expression may be induced by synthetic compounds not commonly found in mammals (e.g., IPTG, substituted benzene, cyclohexanone related compounds) or by compounds naturally occurring in mammals (e.g., sugars including 1-arabinose, 1-rhamnose, xylose, and sucrose), epsilon-caprolactam, propionates, or peptides), or may be under the control of one or more environmental factors (e.g., temperature or oxygen sensitive promoters).

The desired recombinant nucleic acid encoding the tumor antigen or polyepitope can be inserted into an expression vector having a specific promoter (e.g., inducible promoter, etc.) to drive expression of the antigen or polyepitope in bacteria. The vector is then transfected into bacteria (e.g.,

BL21(DE3) electrocompetent cells) or any other type of competent bacteria expressed at low endotoxin levels insufficient to induce CD-14 mediated sepsis when introduced into humans, or transfected into the patient's own endosymbiotic bacteria optionally cultured in vitro prior to transformation as described above.

With respect to yeast expression and vaccination systems, it is contemplated that all known yeast strains are considered suitable for use herein. Preferably, however, the yeast is a recombinant saccharomyces strain genetically modified with a nucleic acid construct as discussed above, resulting in expression of at least one tumor antigen, thereby eliciting an immune response against the tumor. However, it should be noted that any yeast strain can be used to produce the yeast vehicle of the invention. Yeast are unicellular microorganisms belonging to one of three classes: ascomycetes (Ascomycetes), Basidiomycetes (Basidiomycetes) and imperfect Fungi (Fungi infectins). One consideration in selecting the type of yeast to be used as an immunomodulator is the pathogenicity of the yeast. In a preferred embodiment, the yeast is a non-pathogenic strain (e.g., saccharomyces cerevisiae) because the non-pathogenic yeast strain minimizes any adverse effects on the individual to whom the yeast vehicle is administered. However, if the pathogenicity of the yeast can be eliminated by pharmaceutical intervention, pathogenic yeast can be used.

For example, suitable genera of yeast strains include Saccharomyces (Saccharomyces), Candida (Candida, Cryptococcus), Hansenula (Hansenula), Kluyveromyces (Kluyveromyces), Pichia (Pichia), Rhodotorula (Rhodotorula), Schizosaccharomyces (Schizosaccharomyces), and Yarrowia (Yarrowia). In one aspect, the Saccharomyces is selected from the genera Saccharomyces, Candida, Hansenula, Pichia, or Schizosaccharomyces (Schizosaccharomyces), and in a preferred aspect, Saccharomyces is used. The types of yeast strains that can be used in the present invention include Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida albicans, Candida kefir, Candida tropicalis, Cryptococcus laurentii, Cryptococcus neoformans, Hansenula anomala, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis variants (Kluyveromyces lactis), Pichia pastoris, Rhodotorula rubra, Saccharomyces carlsbergensis, and Yarrowia lipolytica.

It is further understood that many of these species include various subspecies, types, subtypes, etc., which are intended to be included within the aforementioned species. In one aspect, yeast species used in the present invention include saccharomyces cerevisiae (s.cerevisiae), candida albicans (c.albicans), hansenula polymorpha (h.polymorpha), pichia pastoris (p.pastoris), and schizosaccharomyces pombe (s.pombe). Saccharomyces cerevisiae is useful because it is relatively easy to handle and is "generally regarded as safe" or "GRAS" for use as a food additive (GRAS, FDA recommended rules 62FR18938, 4 months and 17 days 1997). Thus, the inventors specifically expect yeast strains (e.g., saccharomyces cerevisiae circular (cir) strains) capable of replicating plasmids to particularly high copy numbers. A saccharomyces cerevisiae strain is a strain that is capable of supporting expression vectors that allow for high levels of expression of one or more target antigens and/or antigen fusion proteins and/or other proteins. In addition, any mutant yeast strain can be used in the present invention, including those that exhibit reduced expression of a target antigen or other post-translational modification of proteins, such as mutations in enzymes that extend N-linked glycosylation.

Expression of the desired antigen/neoepitope in yeast can be accomplished using techniques known to those skilled in the art. Most typically, a nucleic acid molecule encoding at least a neoepitope or other protein is inserted into an expression vector such that the nucleic acid molecule is operably linked to a transcription control sequence that, when converted into a host yeast cell, is capable of effecting constitutive or regulated expression of the nucleic acid molecule. As will be readily appreciated, the nucleic acid molecules encoding one or more antigens and/or other proteins may be on one or more expression vectors operably linked to one or more expression control sequences. Particularly important expression control sequences are those which control the initiation of transcription (e.g., promoters and upstream activating sequences).

Any suitable yeast promoter can be used in the present invention, and a variety of such promoters are known to those of skill in the art and have been generally discussed above. Promoters useful for expression in Saccharomyces cerevisiae include the promoters of the genes encoding the following yeast proteins: alcohol dehydrogenase I (ADH1) or II (ADH2), CUP1, phosphoglycerate kinase (PGK), triosephosphate isomerase (TPI), translational elongation factor EF-1 α (TEF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also known as TDH 3) for triosephosphate dehydrogenase, galactokinase (GAL1), galactose-1-phosphate uracil transferase (GAL7), UDP-galactose epimerase (GAL10), cytochrome c1(CYC1), Sec7 protein (Sec7) and acid phosphatase (PHO5), including hybrid promoters such as ADH2/GAPDH and CYC1/GAL10 promoters, and including ADH2/GAPDH promoter (induced when glucose concentration in the cell is low (e.g., about 0.1% to about 0.2%), and CUP1 promoter and TEF 2. Likewise, a number of Upstream Activating Sequences (UAS) (also known as enhancers) are known. The upstream activating sequences for expression in s.cerevisiae include the UAS of the genes encoding the following proteins: PCK1, TPI, TDH3, CYC1, ADH1, ADH2, SUC2, GAL1, GAL7 and GAL10, and other UAS activated by GAL4 gene product, wherein ADH2UAS is used in one aspect. Since the ADH2UAS is activated by the ADR1 gene product, it may be more preferable to overexpress the ADR1 gene when a heterologous gene is operably linked to the ADH2 UAS. Transcription termination sequences for expression in s.cerevisiae include the termination sequences of the alpha-factor, GAPDH and CYC1 genes. The transcriptional control sequences for expressing genes in methylotrophic yeasts include the transcriptional control regions of the genes encoding alcohol oxidase and formate dehydrogenase.

Likewise, according to the present invention, transfection of nucleic acid molecules into yeast cells can be accomplished by any method by which nucleic acid molecules are administered into cells and includes diffusion, active transport, bath sonication, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. The transfected nucleic acid molecule can be integrated into the yeast chromosome using techniques known to those skilled in the art, or maintained on an extrachromosomal vector. As discussed above, yeast cytoplasm, yeast hulls, and yeast membrane particles or cell wall preparations can also be recombinantly produced by transfecting intact yeast microorganisms or yeast spheroplasts with the desired nucleic acid molecule, producing the antigen therein, and then further processing these microorganisms or spheroplasts using techniques known to those skilled in the art to produce cytoplasm, hull, or subcellular yeast membrane extracts or fractions thereof that contain the desired antigen or other protein. Additional exemplary yeast expression systems, methods and conditions suitable for use herein are described in US 20100196411 a1, US 2017/0246276, or US 2017/0224794 and US 2012/0107347.

With respect to viral expression and vaccination systems, it is contemplated that all therapeutic recombinant viral expression systems are considered suitable for use herein, provided that such viruses are capable of causing expression of the recombinant payload in infected cells. For example, suitable viruses include genetically modified alphaviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, and the like. However, adenoviruses are particularly preferred. For example, genetically modified adenoviruses are preferred, which are suitable not only for multiple vaccinations, but also for vaccination in individuals with pre-existing immunity to adenoviruses (see, e.g., WO 2009/006479 and WO 2014/031178), typically with reduced immunogenicity by deletion of the E2b gene and other complete proteins. Moreover, due to these specific deletions, such genetically modified viruses are replication-defective and allow for use with relatively large recombinant cargos. For example, WO 2014/031178 describes the use of such genetically modified viruses to express CEA (colorectal embryo antigen) to provide an immune response against colon cancer. Moreover, as has been reported, relatively high titers of recombinant virus can be achieved using genetically modified human 293 cells (e.g., J Virol [ J. Virol ]1998 month 2; 72 (2): 926-.

Regardless of the type of recombinant virus, it is contemplated that the virus can be used to infect patient (or non-patient) cells ex vivo or in vivo. For example, the virus may be injected subcutaneously or intravenously, or may be administered intranasally or by inhalation to infect the patient's cells (and in particular antigen presenting cells). Alternatively, immune competent cells (e.g., NK cells, T cells, macrophages, dendritic cells, etc.) of a patient (or from an allogeneic source) can be infected in vivo and then delivered to the patient. Alternatively, immunotherapy need not rely on viruses, but may be effected by transfection with nucleic acids or vaccination using RNA or DNA, or other recombinant vectors that result in expression of the neoepitope (e.g., as a single peptide, in tandem minigenes, etc.) in the desired cells (and particularly in immune competent cells).

As mentioned above, the desired nucleic acid sequence (for expression from a virally infected cell) is under the control of suitable regulatory elements well known in the art. For example, suitable promoter elements include constitutively strong promoters (e.g., SV40, CMV, UBC, EF1A, PGK, CAGG promoters), but inducible promoters are also considered suitable for use herein, particularly under inducing conditions typical for a tumor microenvironment. For example, inducible promoters include promoters sensitive to hypoxia and promoters sensitive to TGF- β or IL-8 (e.g., via TRAF, JNK, Erk, or other responsive element promoters). In other examples, suitable inducible promoters include tetracycline-inducible promoters, myxovirus resistance 1(Mx1) promoters, and the like.

Alternatively or additionally, it will be appreciated that the antigen/neo-epitope or polyepitope may also be administered as a peptide, optionally in conjunction with a carrier protein to thereby act as a peptide vaccine. Among other suitable carrier proteins, human albumin or lactoferrin are particularly preferred. Such carrier proteins may be in a native conformation, or pre-treated to form nanoparticles with exposed hydrophobic domains (see, e.g., Adv Protein Chem Struct Biol. [ advances in Protein chemistry and structure biology ] 2015; 98: 121-43), and a neoepitope or polyepitope may be conjugated to such carrier proteins. Most typically, the conjugation of the neoepitope or polyepitope to the carrier protein will be non-covalent. Similar to the secreted neoepitope or polyepitope, the neoepitope or polyepitope bound to the carrier protein will be taken up by immune competent cells (and especially antigen presenting cells and dendritic cells) which in turn typically process and display the neoepitope via the MHC-II pathway.

Formulations

Where the vaccine is a viral vaccine (e.g., an adenovirus, and particularly AdV with E1 and E2b deletions), it is then contemplated that the recombinant virus may be used as a therapeutic vaccine, alone or in combination, in a pharmaceutical composition, typically formulated as a sterile injectable composition, with a viral titer of 10⁶-10¹³Individual virus particles per dose unit, and more typically at 10⁹-10¹²Individual virus particles per dosage unit. Alternatively, the patient (or other HLA-matched) cells may be infected ex vivo with the virus and then the so-infected cells are delivered to the patient. In a further example, a patient is treated with a virus with T cells that may be accompanied by allogeneic transplantation, or autologous natural killer cells, or T cells in naked form or carrying a chimeric antigen receptor expressing an antibody targeting a neo-epitope, a tumor-associated antigen, or the same payload as the virus. Natural killer cells, including NK-92 cell lines from patients, may also express CD16 and may be conjugated to antibodies.

Similarly, where the vaccine is a bacterial or yeast vaccine, it is preferred to irradiate the bacterial or yeast cells prior to administration to prevent further propagation. Most typically, administration is in the form of a therapeutic vaccine in a pharmaceutical composition, typically formulated as a sterile injectable composition, with a cell titer of 10⁶-10⁹Individual cells per dosage unit, and more typically 10⁸-10¹¹Individual cells per dosage unit. Most preferably, the vaccine formulation is administered intramuscularly, subcutaneously or intratumorally. The DNA vaccine will generally be administered as is well known in the art, preferably by intravenous injection of the DNA as a pharmaceutical composition in a buffered solution. Although not limiting to the inventive subject matter, the total dose/administration of DNA will typically be in the range of 0.1mcg to tens of mg, or in the range of 10mcg to thousands of mcg.

Additional therapeutic modalities based on neoepitopes (e.g., synthetic antibodies directed against neoepitopes as described in WO 2016/172722), alone or in combination with autologous or allogeneic NK cells, and in particular haNK cells or taNK cells (e.g., both commercially available from NantKwest, 9920 jackson great street carrefield City, 90232, california) may be employed, if desired. In the case of using haNK or taNK cells, it is particularly preferred that the haNK cells carry recombinant antibodies on CD16 variants that bind to the neo-epitope of the patient being treated, and in the case of using taNK cells, it is preferred that the chimeric antigen receptor of the taNK cells bind to the neo-epitope of the patient being treated. Additional modes of treatment may also be independent of neoepitopes, and particularly preferred modes include cell-based (e.g., activated NK cells (e.g., aNK cells, commercially available from NantKwest, 9920 jackson great street, cafe, 90232, california)) therapies; and non-cell based therapies (e.g., chemotherapy and/or radiation therapy). In still further contemplated aspects, the immunostimulatory cytokines (and in particular IL-2, IL15, and IL-21) may be administered alone or in combination with one or more checkpoint inhibitors (e.g., ipilimumab, nivolumab, etc.). Similarly, it is still further contemplated that additional pharmaceutical intervention may comprise administration of one or more drugs that suppress immunosuppressive cells (and in particular MDSCs, tregs, and M2 macrophages). Thus, suitable drugs include inhibitors of IL-8 or interferon-gamma or antibodies that bind IL-8 or interferon-gamma; and agents that inactivate MDSCs (e.g., NO inhibitors, arginase inhibitors, ROS inhibitors); drugs that block cell development or differentiate into MDSCs (e.g., IL-12, VEGF inhibitors, bisphosphonates); or an agent that is toxic to MDSCs (e.g., gemcitabine, cisplatin, 5-FU). Also, drugs such as cyclophosphamide, daclizumab, and anti-GITR or anti-OX 40 antibodies can be used to inhibit Treg.

Scheme(s)

Thus, the inventors contemplate a variety of exemplary strategies for treatment with the compositions contemplated herein. Most typically, the treatment will include at least two or at least three different vaccine modes administered in a sequential manner. For example, in some embodiments, the initial administration will be a DNA vaccine administration followed by a bacterial vaccine administration. The bacterial vaccine may optionally follow the yeast vaccine. Following bacterial or yeast vaccine administration, a viral vaccine may then be administered. In another example, the initial administration will be a bacterial vaccine administration, optionally followed by a yeast vaccine administration. Following bacterial or yeast vaccine administration, a viral vaccine may then be administered. In yet another example, the initial administration will be a DNA vaccine administration followed by a viral vaccine administration. As will be readily appreciated, each mode may be administered once or repeatedly as desired. For example, the DNA vaccine may be administered once, while the subsequent bacterial vaccine and/or yeast vaccine may be administered two, three, or more times before the start of administration of the viral vaccine. Similarly, a variety of bacterial or yeast vaccine compositions can be administered prior to a viral vaccine composition. In the same manner, the DNA vaccine, the bacterial vaccine and/or the yeast vaccine may be administered once, and the viral vaccine may be administered multiple times. Thus, it should be noted that in a prime/boost regimen, a different mode of priming vaccination may be used than booster vaccination (e.g., DNA, bacterial or yeast vaccination as priming, viral vaccination as boosting).

It should further be recognized that the administration of a particular pattern is separated by several days to allow the immune response to develop. Most typically, the first administration will be at least two days, more typically at least four days, even more typically at least one week, and most typically two weeks or even longer, apart from the second administration. Furthermore, it should be noted that the patient's immune system may also be pretreated with immunostimulatory cytokines (e.g., IL-2, IL-15, IL-21) or cytokine analogs (e.g., ALT-803); use of checkpoint inhibitors or tregs or M2 macrophage inhibitors to reduce immunosuppression; or the use of cytokines to support the immune response, and the generation of immune memory (e.g., IL-12).

As used herein, the term "administering" a pharmaceutical composition or drug refers to direct and indirect administration of a pharmaceutical composition or drug, wherein direct administration of a pharmaceutical composition or drug is typically by a healthcare professional (e.g., physician, nurse, etc.), and wherein indirect administration includes providing the pharmaceutical composition or drug to the healthcare professional or making the pharmaceutical composition or drug available to the healthcare professional for direct administration (e.g., via injection, infusion, oral delivery, topical delivery, etc.). Most preferably, the recombinant virus is administered via subcutaneous or subdermal injection. However, in other contemplated aspects, administration may also be intravenous injection. Alternatively or additionally, antigen presenting cells may be isolated from or grown in cells of a patient, infected in vitro, and then delivered to the patient. Thus, it should be understood that contemplated systems and methods can be considered complete drug discovery systems (e.g., drug discovery, treatment regimens, verification, etc.) for highly personalized cancer treatment. As will also be appreciated, the intended treatment can be repeated over time, particularly where novel new epitopes have emerged (e.g., as a result of clonal expansion).

Moreover, it should be noted that additional treatment regimens may be implemented to assist in the contemplated methods and compositions. Such additional treatment regimens would preferably be performed to increase the 'visibility' of the tumor to the immune system. For example, to trigger overexpression or transcription of stress signals, it is contemplated that chemotherapy and/or radiation therapy may be employed in a low dose regimen, preferably in a rhythmic manner. For example, it is generally preferred that such treatment will employ a dose effective to affect at least one of protein expression, cell division and cell cycle, preferably to induce apoptosis or at least induce or increase expression of stress-related genes (and in particular NKG2D ligands).Thus, in further contemplated aspects, such treatment will include low dose treatment with one or more chemotherapeutic agents. Most typically, for chemotherapeutic agents, the exposure to low dose therapy will be LD₅₀Or IC₅₀Equal to or less than 70%, equal to or less than 50%, equal to or less than 40%, equal to or less than 30%, equal to or less than 20%, equal to or less than 10%, or equal to or less than 5%. Additionally, in advantageous cases, such low dose regimens may be performed in a rhythmic manner as described, for example, in US 7758891, US 7771751, US 7780984, US 7981445, and US 8034375.

With respect to the particular drugs used in the low dose regimen, it is contemplated that all chemotherapeutic agents are considered suitable. Kinase inhibitors, receptor agonists and antagonists, antimetabolites, cytostatics, and cytotoxic drugs, among other suitable drugs, are contemplated herein. However, particularly preferred agents include those identified as interfering with or inhibiting components of the pathway that drives tumor growth or development. Analysis of the route to the chemical data, for example as described in WO 2011/139345 and WO 2013/062505, can be used to identify suitable drugs. Most notably, expression of the stress-related genes in the tumor cells thus obtained will result in surface presentation of NKG2D, NKP30, NKP44 and/or NKP46 ligands, which in turn activate NK cells to specifically destroy the tumor cells. Thus, it is understood that low dose chemotherapy can be used as a trigger in tumor cells to express and display stress-related proteins, which in turn will trigger NK cell activation and/or NK cell-mediated tumor cell killing. Additionally, NK cell mediated killing will be associated with the release of intracellular tumor specific antigens, which is thought to further enhance the immune response.

Examples of the invention

Exemplary sequence permutations

New epitope sequences were determined by computer simulation by position-guided simultaneous alignment of tumor and normal samples using BAM files and BAM servers as disclosed, for example, in US 2012/0059670 and US 2012/0066001. Specifically, the tumors undergoing DNA analysis were from the B16-F10 mouse melanoma line, and the matched normal control was blood from C57B1/6 parental mouse DNA. These results were filtered for expression by RNA sequencing of the tumor cell line. The newly expressed epitopes were found and further analyzed for binding affinity to murine MHC-I (here Kb) and MHC-II (here I-Ab). After the additional step of dbSNP filtering, the selected binders (with an affinity equal to or less than 200 nM) were further analyzed using positional permutations of all neoepitopes that were then predicted to be processed by weight matrix and neural network to generate scores representing the likelihood of the presence and/or strength of a hydrophobic sequence or signal peptide. The best scoring permutations (lowest probability of hydrophobic sequence or signal peptide) against the polyepitope (not shown) were used for further experiments. The neoepitope is prioritized by detection in an RNAseq or other quantitative system that will generate expression intensities for specific genes with neoepitope mutations.

Table 1 shows exemplary neo-epitopes, identified by RNAseq, expressed along with the gene name and mutated amino acids and positions of mutated amino acids. The new epitope listed by x was discarded after filtering of dbSNP, as it appeared as variant Rs71257443 in 28% of the population.

TABLE 1

Gene	Position of	New epitope-a	New epitope-b
				VIPR2	V73M	GETVTMPCP
LILRB3	T187N	VGPVNPSHR*
				FCRL1	R286C	GLGAQCSEA
FAT4	S1613L	RKLTTELTI	PERRKLTTE
				PIEZO2	T2356M	MDWVWMDTT	VWMDTTLSL
SIGLEC14	A292T	GKTLNPSQT	REGKTLNPS
				SIGLEC1	D1143N	VRNATSYRC	NVTVRNATS
SLC4A11	Q678P	FAMAQIPSL	AQIPSLSLR

Table 2 shows additional examples of novel epitopes in which the positions of the mutated amino acids are altered and additional alternative sequences for MHC-I presentation (9-mer) and MHC-II presentation (15-mer). The novel epitope sequences for MHC-II presentation were reverse translated into the corresponding nucleic acid sequences, also shown in Table 2.

TABLE 2

Sequential transport

Model cancer: tumors were screened in a normal manner relative to that described above using murine B16-F10 melanoma (derived from C57/B16 mice), and expressed mutant epitopes were identified in the B16F10 melanoma cell line. Candidate neoepitopes were further filtered as described above using sequencing data analysis and binding analysis to murine MHC I (H2-Kb, H2-Dd) and MHC II (I-Ab). Nine different polyepitopic constructs were then prepared for testing various transport protocols, and each construct was prepared as a corresponding recombinant nucleic acid under the control of the CMV promoter. Each construct was cloned into an AdV5 expression vector that had deleted the E1 and E2b genes, as discussed further below, and the resulting recombinant viruses were then used to transfect mice.

More specifically, the three polyepitope constructs include MHC I binding neo-epitopes for MHC-I presentation and thus target the cytoplasmic compartment. While one construct has an unmodified N-terminus, another has an N-terminally uncleavable ubiquitin, and yet another has an N-terminally cleavable ubiquitin. Ubiquitination is used to target the proteasome in the cytosol. Three additional polyepitope constructs included MHC I binding neo-epitopes for MHC-II presentation and thus targeted the lysosomal/endocytic compartment. While one construct has a lysosomal targeting sequence, another construct has a circulating endocytosis targeting sequence, and yet another construct has a sorting endocytosis targeting sequence. Three additional polyepitope constructs included MHC II binding neo-epitopes for MHC-II presentation and also targeted the lysosomal/endocytic compartment. Again, one construct has a lysosomal targeting sequence, another construct has a circulating endocytosis targeting sequence, and yet another construct has a sorting endocytosis targeting sequence. These nine constructs have the following sequence arrangement.

In the following exemplary sequences, for MHC-I presentation, ubiquitin (cleavable and non-cleavable) was used for proteasomal targeting, while the CD1b leader peptide was used as the export leader peptide for trafficking the polypeptide out of the cytosol of all MHC-II directed sequences. The LAMP 1-TM/cytoplasmic tail was used as the lysosomal targeting sequence, while the LAMP1-TM/CD1a tail was used as the circulating endocytosis targeting sequence, and the LAMP1-TM/CD1c tail was used as the sorting endocytosis targeting domain.

It should be further noted that various internal controls have also been used in the above polypeptides to illustrate expression and presentation. More specifically, the SIINFEKL peptide was used as an internal control against MHC I-restricted (Kb) peptide epitopes, while the Esat6 peptide was used as an internal control against MHC II-presented secreted proteins. The FLAG tag was used as an internal control epitope for detection of expression, and cMYC was used as an internal control tag for simple protein detection.

Exemplary construction of MHC-I epitopes directed to MHC-I presentation (through proteasome, cytoplasmic targeting) Body:

only multiple epitopes: 12aa-A^m-12aa-AAAA-12aa-B^m-12aa-AAAA-(12aa-X^m-12aa-AAAA)_n-SIINFEKL-AAAA-Esat 6-cMYC. FIG. 5A exemplarily depicts the polypeptide structure of such an arrangement, wherein the SIINFEKL motif is underlined; the Esat6 motif is shown in italics; and wherein the cMY motif is shown in bold font. The nucleotide sequence of fig. 5A is SEQ ID NO: 1, and the polypeptide sequence of fig. 5A is SEQ ID NO: 4.

polyepitope and cleavable ubiquitin GGR N-terminus: ubiquitin-GGR-12 aa-A^m-12aa-AAAA-12aa-B^m-12aa-AAAA-(12aa-X^m-12aa-AAAA)_n-SIIN FEKL-AAAA-Esat 6-cMYC. Fig. 5B exemplarily depicts the polypeptide structure of such an arrangement, wherein cleavable ubiquitin moieties are shown in italics and underlined; the SIINFEKL motif is underlined; the Esat6 motif is shown in italics; and wherein the cMY motif is shown in bold font. The nucleotide sequence of fig. 5B is SEQ id no: 2, and the polypeptide sequence of fig. 5B is SEQ ID NO: 5.

polyepitope and uncleavable ubiquitin G N ends: ubiquitin-G-12 aa-A^m-12aa-AAAA-12aa-B^m-12aa-AAAA-(12aa-X^m-12aa-AAAA)_n-SIINFE KL-AAAA-Esat 6-cMYC. Fig. 5C exemplarily depicts the polypeptide structure of such an arrangement, wherein the cleavable ubiquitin moiety is shown in italics and underlined; the SIINFEKL motif is underlined; the Esat6 motif is shown in italics; and wherein the cMY motif is shown in bold font. The nucleotide sequence of fig. 5C is SEQ id no: 3, and the polypeptide sequence of fig. 5C is SEQ ID NO: 6.

examples for MHC-I epitopes directed to MHC-II presentation (export from the cytoplasm, through the endocytosis/lysosomes) Sex construct:

lysosomal targeting of Kb epitope peptides: (CD1b leader peptide) -20aa-A^m-20aa-GPGPG-20aa B^m-20aa-GPGPG-(20aa-X^m-20aa-GPGPG-)_nEsat6-Flag tag-LAMP 1 TM/cytoplasmic tail. FIG. 6A schematically depicts the polypeptide structure of this arrangement, wherein the CD1b leader peptide portion is shown in bold; the Esat6 motif is underlined; flag tag motif is shown in italics; and wherein LAMP1 TM/cytoplasmic tail is shown in bold/underlined font. The nucleotide sequence of fig. 6A is SEQ ID NO: 7, and the polypeptide sequence of fig. 6A is SEQ ID NO: 10.

recycled lysosomal targeting of Kb epitope peptides: (CD1b leader peptide) -20aa-A^m-20aa-GPGPG-20aa B^m-20aa-GPGPG-(20aa-X^m-20aa-GPGPG-)_nEsat6-Flag tag-LAMP 1TM/CD1a tail. FIG. 6B schematically depicts the polypeptide structure of this arrangement, where CD1B is precededThe leader peptide portion is shown in bold; the Esat6 motif is underlined; flag tag motif is shown in italics; and wherein the LAMP1TM motif is shown in bold/underlined font; and the CD1a targeting motif is underlined/italicized. The nucleotide sequence of fig. 6B is SEQ ID NO: 8, and the polypeptide sequence of fig. 6B is SEQ id no: 11.

sorting endocytosis targeting of Kb epitope peptides: (CD1b leader peptide) -20aa-A^m-20aa-GPGPG-20aa B^m-20aa-GPGPG-(20aa-X^m-20aa-GPGPG-)_nEsat6-Flag tag-LAMP 1TM/CD1c tail. Fig. 6C exemplarily depicts the polypeptide structure of this arrangement, wherein the CD1b leader peptide portion is shown in bold; the Esat6 motif is underlined; flag tag motif is shown in italics; and wherein the LAMP1TM motif is shown in bold/underlined font; and the CD1c targeting motif is underlined/italicized. The nucleotide sequence of fig. 6C is SEQ ID NO: 9, and the polypeptide sequence of fig. 6C is SEQ ID NO: 12.

presentation of MHC-II epitopes directed to MHC-II presentation (export from the cytoplasm, through endocytosis/lysosomes) Exemplary constructs:

lysosomal targeting of IAb epitope peptides: (CD1b leader peptide) -20aa-A^m-20aa-GPGPG-20aa B^m-20aa-GPGPG-(20aa-X^m-20aa-GPGPG-)_nEsat6-Flag tag-LAMP 1 TM/cytoplasmic tail. FIG. 7A schematically depicts the polypeptide structure of this arrangement, wherein the CD1b leader peptide portion is shown in bold; the SIINFEKL and Esat6 motifs are underlined; flag tag motif is shown in italics; and wherein LAMP1 TM/cytoplasmic tail is shown in bold/underlined font. The nucleotide sequence of fig. 7A is SEQ ID NO: 13, and the polypeptide sequence of fig. 7A is SEQ ID NO: 16.

IAb recycled lysosomal targeting of epitope peptides: (CD1b leader peptide) -20aa-A^m-20aa-GPGPG-20aa B^m-20aa-GPGPG-(20aa-X^m-20aa-GPGPG-)_nEsat6-Flag tag-LAMP 1TM/CD1a tail. FIG. 7B schematically depicts the polypeptide structure of this arrangement, wherein the CD1B leader peptide portion is shown in bold; the SIINFEKL and Esat6 motifs are underlined; flag tag motif is shown in italics; wherein the LAMP1TM motif is underlined/boldDisplaying a line font; and wherein the CD1a tails are shown in bold/italics. The nucleotide sequence of fig. 7B is SEQ ID NO: 14, and the polypeptide sequence of fig. 7B is SEQ id no: 17.

sorting endocytosis targeting of IAb epitope peptides: (CD1b leader peptide) -20aa-A^m-20aa-GPGPG-20aa B^m-20aa-GPGPG-(20aa-X^m-20aa-GPGPG-)_nEsat6-Flag tag-LAMP 1TM/CD1c tail. FIG. 7C schematically depicts the polypeptide structure of this arrangement, wherein the CD1b leader peptide portion is shown in bold; the SIINFEKL and Esat6 motifs are underlined; flag tag motif is shown in italics; wherein the LAMP1TM motif is shown in bold/underlined font; and wherein the CD1c tails are shown in bold/italics. The nucleotide sequence of fig. 7C is SEQ ID NO: 15, and the polypeptide sequence of fig. 7C is SEQ id no: 18.

in vivo vaccination

Figure 8 depicts an exemplary in vivo vaccination experiment in which nine groups of C57b1/6 mice were immunized with nine different recombinant Ad5 viruses comprising a sequence arrangement substantially as described above. Immunizations were administered on a biweekly schedule, with different routes (subcutaneous and intravenous) being used for different groups of animals (as schematically shown in figure 8). On day 42, tumor challenge was performed with B16-F10 melanoma cells, followed by administration of M2 macrophage inhibitory drug (RP 182) and IL-15 super agonist (Alt-803). Fig. 9A-9C depict exemplary results of subcutaneous administration, while fig. 10A-10C depict exemplary results of intravenous administration.

Notably, as can be seen from fig. 9A, subcutaneous injection of adenovirus encoding class I polyepitopes that were directed to the cytoplasm and MHC-I presentation failed to provide significant immune protection regardless of the presence of ubiquitination. In other aspects, as is evident from fig. 9B, where class I polyepitopes are directed to endocytic and lysosomal compartments via MHC-II for processing and presentation, some protective immunity directed to circulating endocytic and lysosomal compartments is observed. Even stronger immune protection was observed when class II polyepitopes were directed to endocytic and lysosomal compartments for processing and presentation via MHC-II. Here, as shown in fig. 9C, the strongest protection was observed for the lysosomal and sorted endocytic compartments.

When immunisation was performed with the same viral construct, protection of neoepitope vaccination was observed for class I neoepitopes directed to the cytoplasm (where the polyepitope comprises cleavable ubiquitin), despite intravenous injection, whereas some protection was observed in case the polyepitope comprises non-cleavable ubiquitin, as can be seen from fig. 10A. Notably, as shown in figure 10B, when class I polyepitopes were directed to the endocytic and lysosomal compartments, stronger protection was observed in all vaccinations. Moreover, strong protection is observed when class II polyepitopes are directed to endocytic and lysosomal compartments for processing and presentation via MHC-II. Here, as shown in the graph of fig. 10C, the strongest protection was observed for circulating and sorting endocytic compartments.

Comparison of pathways and vectors

In still further experiments, the inventors further investigated whether the actual route of administration and type of expression vector had an effect on the efficacy of the treatment. To this end, essentially the same mouse model was used as described above, and multiple epitopes were constructed from a novel epitope from B16F10 melanoma cells. FIGS. 11A and 11B show exemplary results of experiments in which the expression vector is an adenovirus. As can be readily seen, as can be seen from fig. 11A, subcutaneous administration of the adenoviral expression system encoding MHC II targeted polyepitopes conferred significant immunoprotection at all subcellular locations, whereas the empty vector failed to provide immunoprotection. Notably, when the same vector constructs were tested using intravenous administration, the immunoprotection was less pronounced as can be seen from fig. 11B.

In contrast, when the expression vector is a plasmid targeting MHC II presentation as described above (here: a shuttle vector for generating an adenovirus expression vector), subcutaneous administration of the plasmid confers significant immunoprotection as compared with the empty vector, as shown in FIG. 11C. Moreover, and unexpectedly, as can be seen from fig. 11D, in the case of administering the same plasmid by intravenous injection, a large degree of immunoprotection was observed even for the empty vector. While not wishing to be bound by any particular theory or hypothesis, the inventors therefore expect that the immunoprotection may be due, at least in part, to innate and adaptive immune responses.

Fig. 11E illustrates a comparison between the use of empty plasmids ('empty') and empty viral ('empty') expression vectors, across different pathways. As can be readily seen from the results, subcutaneous administration did not confer immunoprotection, whereas the strongest immunoprotection was observed by intravenous administration of empty plasmids.

Fig. 12 depicts the data shown in table 3 below, where the type of expression vector is shown as "type" and the route of administration is indicated as "route". Specific MHC targeting and targeting of subcellular locations are shown under "nucleic acids", whereas tumor volume is indicated for the measured date after B16-F10 melanoma cell implantation.

TABLE 3

As can be readily seen from the data provided herein, presentation of MHC-II matched neoepitopes targeting and targeting multiple epitopes to MHC-II is significantly effective via CD1c, LAMP1 and CD1a when administered intravenously in plasmid form and subcutaneously in adenovirus form. Notably, and as reflected in the data, targeting MHC-I is significantly less effective in providing immune protection.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided with respect to certain embodiments herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

It will be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the scope of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. When the claims refer to at least one selected from the group consisting of A, B, c.

The claims (modification according to treaty clause 19)

1. A method of generating a formulation for use in immunotherapy of a mammal, the method comprising:

generating a first recombinant nucleic acid having a sequence encoding a polyepitope, wherein the polyepitope comprises a plurality of filtered neo-epitope sequences;

wherein the first recombinant nucleic acid comprises a first promoter operably linked to a sequence encoding the polyepitope to drive expression of the polyepitope in a mammal;

formulating a first vaccine formulation for booster vaccination using the first recombinant nucleic acid; and

a second vaccine formulation for primary immunization vaccination is formulated using the second nucleic acid.