Immunogenic variant peptides from cancer-associated proteins and methods of use thereof
阅读说明：本技术 来自癌症相关蛋白的免疫原性异变肽及其使用方法 (Immunogenic variant peptides from cancer-associated proteins and methods of use thereof ) 是由 R·珀蒂 麦可·F·宾矽欧塔 B·科德 D·巴里 于 2018-11-08 设计创作，主要内容包括：本文提供了包括异变突变(heteroclitic mutation)的肿瘤相关抗原性肽和包含此类异变肽(heteroclitic peptide)的融合多肽。还提供了编码此类肽和融合多肽的核酸,包括此类肽、融合多肽或核酸的重组的细菌或李斯特菌属(Listeria)菌株,以及包括此类重组的细菌或李斯特菌属菌株的细胞库。本文还提供了产生此类肽、融合多肽、核酸以及重组的细菌或李斯特菌属菌株的方法。还提供了包括此类肽、融合多肽、核酸或重组的细菌或李斯特菌属菌株的免疫原性组合物、药物组合物和疫苗。还提供了在受试者中诱导抗肿瘤相关抗原的免疫应答的方法,在受试者中诱导抗肿瘤或抗癌免疫应答的方法,在受试者中治疗肿瘤或癌症的方法,在受试者中预防肿瘤或癌症的方法,以及使用此类肽、重组融合多肽、核酸、重组的细菌或李斯特菌属菌株、免疫原性组合物、药物组合物或疫苗来保护受试者免于肿瘤或癌症的方法。(Provided herein are tumor-associated antigenic peptides comprising a heteromutation (heterolytic mutation) and fusion polypeptides comprising such heterolytic peptides. Also provided are nucleic acids encoding such peptides and fusion polypeptides, recombinant bacterial or Listeria (Listeria) strains comprising such peptides, fusion polypeptides, or nucleic acids, and cell banks comprising such recombinant bacterial or Listeria strains. Also provided herein are methods of producing such peptides, fusion polypeptides, nucleic acids, and recombinant bacterial or listeria strains. Immunogenic compositions, pharmaceutical compositions and vaccines comprising such peptides, fusion polypeptides, nucleic acids or recombinant bacteria or listeria strains are also provided. Also provided are methods of inducing an immune response against a tumor-associated antigen in a subject, methods of inducing an anti-tumor or anti-cancer immune response in a subject, methods of treating a tumor or cancer in a subject, methods of preventing a tumor or cancer in a subject, and methods of protecting a subject from a tumor or cancer using such peptides, recombinant fusion polypeptides, nucleic acids, recombinant bacteria or listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines.)
1. An isolated peptide comprising an immunogenic fragment of a cancer-associated protein, wherein the fragment comprises a mutated mutation.
2. The isolated peptide according to claim 1, wherein the mutational mutation is a mutation at an anchor position to a preferred amino acid.
3. The isolated peptide of claim 1 or 2, wherein the fragment is from about 7 to about 11 amino acids in length, from about 8 to about 10 amino acids in length, or about 9 amino acids in length.
4. The isolated peptide of any preceding claim, wherein the cancer-associated protein is a cancer testis antigen or a carcinoembryonic antigen.
5. The isolated peptide of any preceding claim, wherein the cancer-associated protein is encoded by one of the following human genes: CEACAM5, GAGE1, TERT, KLHL7, MAGEA3, MAGEA4, MAGEA6, NUF2, NYESO1, PAGE4, PRAME, PSA, PSMA, RNF43, SART3, SSX2, STEAP1, and SURVIVIN.
6. The isolated peptide of claim 5, wherein:
(a) the cancer-associated protein is encoded by CEACAM5, and the fragment comprises SEQ ID NO: 100. 102, 104, 106, and 108;
(b) the cancer-associated protein is encoded by GAGE1, and the fragment comprises SEQ ID NO: 110 and 112;
(c) the cancer-associated protein is encoded by TERT, and the fragment comprises SEQ ID NO: 114, and a carrier;
(d) the cancer-associated protein is encoded by KLHL7, and the fragment comprises SEQ ID NO: 116;
(e) the cancer-associated protein is encoded by MAGEA3, and the fragment comprises SEQ ID NO: 118. 120, 122, and 124;
(f) the cancer-associated protein is encoded by MAGEA4, and the fragment comprises SEQ ID NO: 126;
(g) the cancer-associated protein is encoded by MAGEA6, and the fragment comprises SEQ ID NO: 128;
(h) the cancer-associated protein is encoded by NUF2, and the fragment comprises SEQ ID NO:130 and 132;
(i) the cancer-associated protein is encoded by NYESO1, and the fragment comprises SEQ ID NO: 134 and 136;
(j) the cancer-associated protein is encoded by PAGE4, and the fragment comprises SEQ ID NO: 138;
(k) the cancer-associated protein is encoded by PRAME and the fragment comprises SEQ ID NO: 140 of a solvent;
(l) The cancer-associated protein is encoded by PSA, and the fragment comprises SEQ ID NO: 142;
(m) the cancer-associated protein is encoded by PSMA, and the fragment comprises SEQ ID NO: 144, 144;
(n) the cancer-associated protein is encoded by RNF43, and the fragment comprises SEQ ID NO: 146;
(o) the cancer-associated protein is encoded by SART3, and the fragment comprises SEQ ID NO: 148;
(p) the cancer-associated protein is encoded by SSX2, and the fragment comprises SEQ ID NO: 150;
(q) the cancer associated protein is encoded by STEAP1, and the fragment comprises SEQ ID NO: 152 and 154; or
(r) the cancer-associated protein is encoded by SURVIVIN and the fragment comprises the amino acid sequence of SEQ ID NO: 156 and 158.
7. The isolated peptide of claim 6, wherein:
(a) the cancer-associated protein is encoded by CEACAM5, and the fragment consists of SEQ ID NO: 100. 102, 104, 106, and 108;
(b) the cancer-associated protein is encoded by GAGE1, and the fragment is encoded by SEQ ID NO: 110 and 112;
(c) the cancer-associated protein is encoded by TERT, and the fragment is encoded by SEQ ID NO: 114, and (b);
(d) the cancer-associated protein is encoded by KLHL7, and the fragment is encoded by SEQ ID NO: 116, respectively;
(e) the cancer-associated protein is encoded by MAGEA3, and the fragment is encoded by SEQ ID NO: 118. 120, 122, and 124;
(f) the cancer-associated protein is encoded by MAGEA4, and the fragment is encoded by SEQ ID NO: 126;
(g) the cancer-associated protein is encoded by MAGEA6, and the fragment is encoded by SEQ ID NO: 128 component (b);
(h) the cancer-associated protein is encoded by NUF2, and the fragment is encoded by SEQ ID NO:130 and 132;
(i) the cancer-associated protein is encoded by NYESO1, and the fragment is encoded by SEQ ID NO: 134 and 136;
(j) the cancer-associated protein is encoded by PAGE4, and the fragment is encoded by SEQ ID NO: 138;
(k) the cancer-associated protein is encoded by PRAME and the fragment is encoded by SEQ ID NO: 140 of the composition;
(l) The cancer-associated protein is encoded by PSA, and the fragment is encoded by SEQ ID NO: 142 of a polymer;
(m) the cancer-associated protein is encoded by PSMA, and the fragment is encoded by SEQ ID NO: 144 of the composition;
(n) the cancer-associated protein is encoded by RNF43, and the fragment is encoded by SEQ ID NO: 146;
(o) the cancer-associated protein is encoded by SART3, and the fragment is encoded by SEQ ID NO: 148;
(p) the cancer-associated protein is encoded by SSX2, and the fragment is encoded by SEQ ID NO: 150;
(q) the cancer associated protein is encoded by STEAP1, and the fragment is encoded by SEQ ID NO: 152 and 154; or
(r) the cancer-associated protein is encoded by SURVIVIN and the fragment is encoded by SEQ ID NO: 156 and 158.
8. The isolated peptide of claim 7, wherein:
(a) the cancer-associated protein is encoded by CEACAM5, and the isolated peptide consists of SEQ ID NO: 100. 102, 104, 106, and 108;
(b) the cancer-associated protein is encoded by GAGE1, and the isolated peptide is encoded by SEQ ID NO: 110 and 112;
(c) the cancer-associated protein is encoded by TERT, and the isolated peptide is encoded by SEQ ID NO: 114, and (b);
(d) the cancer related protein is encoded by KLHL7, and the isolated peptide is encoded by SEQ ID NO: 116, respectively;
(e) the cancer-associated protein is encoded by MAGEA3, and the isolated peptide is encoded by SEQ ID NOS: 118. 120, 122, and 124;
(f) the cancer-associated protein is encoded by MAGEA4, and the isolated peptide is encoded by SEQ ID NO: 126;
(g) the cancer-associated protein is encoded by MAGEA6, and the isolated peptide is encoded by SEQ ID NO: 128 component (b);
(h) the cancer-associated protein is encoded by NUF2, and the isolated peptide is encoded by SEQ ID NO:130 and 132;
(i) the cancer-related protein is encoded by NYESO1 and the isolated peptide consists of SEQ ID NO: 134 and 136;
(j) the cancer-associated protein is encoded by PAGE4, and the isolated peptide is encoded by SEQ ID NO: 138;
(k) the cancer-associated protein is encoded by PRAME and the isolated peptide is encoded by SEQ ID NO: 140 of the composition;
(l) The cancer-associated protein is encoded by PSA, and the isolated peptide is encoded by SEQ ID NO: 142 of a polymer;
(m) the cancer-associated protein is encoded by PSMA, and the isolated peptide is encoded by SEQ ID NO: 144 of the composition;
(n) the cancer-associated protein is encoded by RNF43, and the isolated peptide consists of SEQ ID NO: 146;
(o) the cancer-associated protein is encoded by SART3, and the isolated peptide is encoded by SEQ ID NO: 148;
(p) the cancer-associated protein is encoded by SSX2, and the isolated peptide is encoded by SEQ ID NO: 150;
(q) the cancer-associated protein is encoded by STEAP1, and the isolated peptide consists of SEQ ID NO: 152 and 154; or
(r) the cancer-associated protein is encoded by SURVIVIN and the isolated peptide is encoded by SEQ ID NO: 156 and 158.
9. The isolated peptide of any preceding claim, wherein the fragment binds to one or more of the following HLA types: HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02.
10. A nucleic acid encoding the isolated peptide of any preceding claim.
11. The nucleic acid of claim 10, wherein the nucleic acid is codon optimized for expression in humans.
12. The nucleic acid of claim 10, wherein the nucleic acid is codon optimized for expression in Listeria monocytogenes (Listeria monocytogenes).
13. The nucleic acid of any one of claims 10-12, wherein the nucleic acid comprises DNA.
14. The nucleic acid of any one of claims 10-12, wherein the nucleic acid comprises RNA.
15. The nucleic acid of any one of claims 10-14, wherein the nucleic acid comprises a sequence selected from the group consisting of SEQ ID NOs: 223-977 and degenerate variants thereof which encode the same amino acid sequence.
16. The nucleic acid of claim 15, wherein the nucleic acid consists of a sequence selected from the group consisting of SEQ ID NOs: 223-977 and degenerate variants thereof which encode the same amino acid sequence.
17. A pharmaceutical composition comprising:
(a) one or more isolated peptides according to any one of claims 1-9 or one or more nucleic acids according to any one of claims 10-16; and
(b) an adjuvant.
18. The pharmaceutical composition of claim 17, wherein the adjuvant comprises detoxified listeriolysin o (dtllo), a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid a, an unmethylated CpG-containing oligonucleotide, or Montanide ISA 51.
19. The pharmaceutical composition of claim 17 or 18, wherein the pharmaceutical composition comprises a peptide that binds to or a nucleic acid encoding a peptide that binds to each of the following HLA types: HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02.
20. The pharmaceutical composition of any one of claims 17-19, wherein the pharmaceutical composition comprises:
(a) two or more of the peptides shown in table 3 or a nucleic acid encoding two or more of the peptides shown in table 3;
(b) two or more of the peptides shown in table 5 or a nucleic acid encoding two or more of the peptides shown in table 5;
(c) two or more of the peptides shown in table 7 or a nucleic acid encoding two or more of the peptides shown in table 7;
(d) two or more of the peptides set forth in table 9 or a nucleic acid encoding two or more of the peptides set forth in table 9;
(e) two or more of the peptides set forth in table 11 or a nucleic acid encoding two or more of the peptides set forth in table 11;
(f) two or more of the peptides set forth in table 13 or a nucleic acid encoding two or more of the peptides set forth in table 13;
(g) two or more of the peptides set forth in table 15 or a nucleic acid encoding two or more of the peptides set forth in table 15;
(h) two or more of the peptides shown in table 17 or a nucleic acid encoding two or more of the peptides shown in table 17;
(i) two or more of the peptides shown in table 19 or a nucleic acid encoding two or more of the peptides shown in table 19; or
(j) Two or more of the peptides set forth in table 21 or a nucleic acid encoding two or more of the peptides set forth in table 21.
21. The pharmaceutical composition of claim 20, wherein the pharmaceutical composition comprises:
(a) all of the peptides shown in table 3 or nucleic acids encoding all of the peptides shown in table 3;
(b) all of the peptides shown in table 5 or nucleic acids encoding all of the peptides shown in table 5;
(c) all of the peptides shown in table 7 or nucleic acids encoding all of the peptides shown in table 7;
(d) all of the peptides shown in table 9 or nucleic acids encoding all of the peptides shown in table 9;
(e) all of the peptides shown in table 11 or nucleic acids encoding all of the peptides shown in table 11;
(f) all of the peptides shown in table 13 or nucleic acids encoding all of the peptides shown in table 13;
(g) all of the peptides shown in table 15 or nucleic acids encoding all of the peptides shown in table 15;
(h) all of the peptides shown in table 17 or nucleic acids encoding all of the peptides shown in table 17;
(i) all of the peptides shown in table 19 or nucleic acids encoding all of the peptides shown in table 19;
(j) all of the peptides shown in table 21 or nucleic acids encoding all of the peptides shown in table 21.
22. A recombinant bacterial strain comprising a nucleic acid encoding any one of the isolated peptides of claims 1-9.
23. A recombinant bacterial strain comprising one or more nucleic acids encoding two or more of the isolated peptides of claims 1-9.
24. The recombinant bacterial strain of claim 23, wherein the two or more peptides comprise:
(a) two or more of the peptides shown in table 3 or a nucleic acid encoding two or more of the peptides shown in table 3;
(b) two or more of the peptides shown in table 5 or a nucleic acid encoding two or more of the peptides shown in table 5;
(c) two or more of the peptides shown in table 7 or a nucleic acid encoding two or more of the peptides shown in table 7;
(d) two or more of the peptides shown in table 9 or a nucleic acid encoding two or more of the peptides shown in table 9;
(e) two or more of the peptides set forth in table 11 or a nucleic acid encoding two or more of the peptides set forth in table 11;
(f) two or more of the peptides set forth in table 13 or a nucleic acid encoding two or more of the peptides set forth in table 13;
(g) two or more of the peptides set forth in table 15 or a nucleic acid encoding two or more of the peptides set forth in table 15;
(h) two or more of the peptides shown in table 17 or a nucleic acid encoding two or more of the peptides shown in table 17;
(i) two or more of the peptides shown in table 19 or a nucleic acid encoding two or more of the peptides shown in table 19; or
(j) Two or more of the peptides set forth in table 21 or a nucleic acid encoding two or more of the peptides set forth in table 21.
25. The recombinant bacterial strain of claim 24, wherein the two or more peptides comprise:
(a) all of the peptides shown in table 3 or nucleic acids encoding all of the peptides shown in table 3;
(b) all of the peptides shown in table 5 or nucleic acids encoding all of the peptides shown in table 5;
(c) all of the peptides shown in table 7 or nucleic acids encoding all of the peptides shown in table 7;
(d) all of the peptides shown in table 9 or nucleic acids encoding all of the peptides shown in table 9;
(e) all of the peptides shown in table 11 or nucleic acids encoding all of the peptides shown in table 11;
(f) all of the peptides shown in table 13 or nucleic acids encoding all of the peptides shown in table 13;
(g) all of the peptides shown in table 15 or nucleic acids encoding all of the peptides shown in table 15;
(h) all of the peptides shown in table 17 or nucleic acids encoding all of the peptides shown in table 17;
(i) all of the peptides shown in table 19 or nucleic acids encoding all of the peptides shown in table 19; or
(j) All of the peptides shown in table 21 or nucleic acids encoding all of the peptides shown in table 21.
26. The recombinant bacterial strain of any one of claims 23-25, wherein a combination of two or more peptides bind to each of the following HLA types: HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02.
27. The recombinant bacterial strain of any one of claims 22-26, wherein the bacterial strain is a salmonella, listeria, yersinia, shigella, or mycobacterium strain.
28. The recombinant bacterial strain of claim 27, wherein the bacterial strain is a Listeria strain, optionally wherein the Listeria strain is a Listeria monocytogenes (Listeria monocytogenes) strain.
29. A recombinant listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide, wherein the fusion polypeptide comprises a PEST-containing peptide fused to an immunogenic fragment of a cancer-associated protein, wherein the fragment comprises a mutated mutation.
30. The recombinant listeria strain of claim 29, wherein said mutator mutation is a mutation at an anchor position to a preferred amino acid.
31. The recombinant listeria strain of claim 29 or 30, wherein the fragment is from about 7 to about 11 amino acids in length, from about 8 to about 10 amino acids in length, or about 9 amino acids in length.
32. The recombinant listeria strain of any one of claims 29-31, wherein the cancer-associated protein is a cancer testis antigen or a carcinoembryonic antigen.
33. The recombinant listeria strain of any one of claims 29-32, wherein the cancer-associated protein is encoded by one of the following human genes: CEACAM5, GAGE1, TERT, KLHL7, MAGEA3, MAGEA4, MAGEA6, NUF2, NYESO1, PAGE4, PRAME, PSA, PSMA, RNF43, SART3, SSX2, STEAP1 and SURVIVIN.
34. The recombinant listeria strain of claim 33, wherein:
(a) the cancer-associated protein is encoded by CEACAM5, and the fragment comprises SEQ ID NO: 100. 102, 104, 106, and 108;
(b) the cancer-associated protein is encoded by GAGE1, and the fragment comprises SEQ ID NO: 110 and 112;
(c) the cancer-associated protein is encoded by TERT, and the fragment comprises SEQ ID NO: 114, and a carrier;
(d) the cancer-associated protein is encoded by KLHL7, and the fragment comprises SEQ ID NO: 116;
(e) the cancer-associated protein is encoded by MAGEA3, and the fragment comprises SEQ ID NO: 118. 120, 122, and 124;
(f) the cancer-associated protein is encoded by MAGEA4, and the fragment comprises SEQ ID NO: 126;
(g) the cancer-associated protein is encoded by MAGEA6, and the fragment comprises SEQ ID NO: 128;
(h) the cancer-associated protein is encoded by NUF2, and the fragment comprises SEQ ID NO:130 and 132;
(i) the cancer-associated protein is encoded by NYESO1, and the fragment comprises SEQ ID NO: 134 and 136;
(j) the cancer-associated protein is encoded by PAGE4, and the fragment comprises SEQ ID NO: 138;
(k) the cancer-associated protein is encoded by PRAME and the fragment comprises SEQ ID NO: 140 of a solvent;
(l) The cancer-associated protein is encoded by PSA, and the fragment comprises SEQ ID NO: 142;
(m) the cancer-associated protein is encoded by PSMA, and the fragment comprises SEQ ID NO: 144, 144;
(n) the cancer-associated protein is encoded by RNF43, and the fragment comprises SEQ ID NO: 146;
(o) the cancer-associated protein is encoded by SART3, and the fragment comprises SEQ ID NO: 148;
(p) the cancer-associated protein is encoded by SSX2, and the fragment comprises SEQ ID NO: 150;
(q) the cancer associated protein is encoded by STEAP1, and the fragment comprises SEQ ID NO: 152 and 154; or
(r) the cancer-associated protein is encoded by SURVIVIN and the fragment comprises the amino acid sequence of SEQ ID NO: 156 and 158.
35. The recombinant listeria strain of claim 34, wherein:
(a) the cancer-associated protein is encoded by CEACAM5, and the fragment consists of SEQ ID NO: 100. 102, 104, 106, and 108;
(b) the cancer-associated protein is encoded by GAGE1, and the fragment is encoded by SEQ ID NO: 110 and 112;
(c) the cancer-associated protein is encoded by TERT, and the fragment is encoded by SEQ ID NO: 114, and (b);
(d) the cancer-associated protein is encoded by KLHL7, and the fragment is encoded by SEQ ID NO: 116, respectively;
(e) the cancer-associated protein is encoded by MAGEA3, and the fragment is encoded by SEQ ID NO: 118. 120, 122, and 124;
(f) the cancer-associated protein is encoded by MAGEA4, and the fragment is encoded by SEQ ID NO: 126;
(g) the cancer-associated protein is encoded by MAGEA6, and the fragment is encoded by SEQ ID NO: 128 component (b);
(h) the cancer-associated protein is encoded by NUF2, and the fragment is encoded by SEQ ID NO:130 and 132;
(i) the cancer-associated protein is encoded by NYESO1, and the fragment is encoded by SEQ ID NO: 134 and 136;
(j) the cancer-associated protein is encoded by PAGE4, and the fragment is encoded by SEQ ID NO: 138;
(k) the cancer-associated protein is encoded by PRAME and the fragment is encoded by SEQ ID NO: 140 of the composition;
(l) The cancer-associated protein is encoded by PSA, and the fragment is encoded by SEQ ID NO: 142 of a polymer;
(m) the cancer-associated protein is encoded by PSMA, and the fragment is encoded by SEQ ID NO: 144 of the composition;
(n) the cancer-associated protein is encoded by RNF43, and the fragment is encoded by SEQ ID NO: 146;
(o) the cancer-associated protein is encoded by SART3, and the fragment is encoded by SEQ ID NO: 148;
(p) the cancer-associated protein is encoded by SSX2, and the fragment is encoded by SEQ ID NO: 150;
(q) the cancer associated protein is encoded by STEAP1, and the fragment is encoded by SEQ ID NO: 152 and 154; or
(r) the cancer-associated protein is encoded by SURVIVIN and the fragment is encoded by SEQ ID NO: 156 and 158.
36. The recombinant listeria strain of claims 29-35, wherein the fragment binds to one or more of the following HLA types: HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02.
37. The recombinant listeria fungus of any one of claims 29-36, wherein the PEST-containing peptide comprises a bacterial secretion signal sequence, and the fusion polypeptide further comprises a ubiquitin protein fused to the fragment, wherein the PEST-containing peptide, the ubiquitin, and the carboxy-terminal antigenic peptide are arranged in tandem from amino-terminus to carboxy-terminus of the fusion polypeptide.
38. The recombinant listeria strain of any one of claims 29-37, wherein said fusion polypeptide comprises said PEST-containing peptide fused to two or more immunogenic fragments of a cancer-associated protein, wherein each of said two or more fragments comprises a mutated mutation.
39. The recombinant listeria strain of claim 38, wherein the two or more immunogenic fragments are directly fused to each other without an intervening sequence.
40. The recombinant listeria strain of claim 38, wherein the two or more immunogenic fragments are linked to each other by a peptide linker.
41. The recombinant Listeria strain of claim 40, wherein SEQ ID NO: one or more linkers as shown in 209-217 are used to join the two or more immunogenic fragments.
42. The recombinant Listeria strain of claims 38-41, wherein a combination of two or more immunogenic fragments in the fusion polypeptide binds to each of the following HLA types: HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02.
43. The recombinant Listeria strain of any one of claims 38-42, wherein the two or more immunogenic fragments comprise:
(a) two or more of the peptides shown in table 3;
(b) two or more of the peptides shown in table 5;
(c) two or more of the peptides shown in table 7;
(d) two or more of the peptides shown in table 9;
(e) two or more of the peptides shown in table 11;
(f) two or more of the peptides shown in table 13;
(g) two or more of the peptides shown in table 15;
(h) two or more of the peptides shown in table 17;
(i) two or more of the peptides shown in table 19; or
(j) Two or more of the peptides shown in table 21.
44. The recombinant Listeria strain of claim 43, wherein the two or more immunogenic fragments comprise:
(a) all peptides shown in table 3;
(b) all peptides shown in table 5;
(c) all peptides shown in table 7;
(d) all peptides shown in table 9;
(e) all peptides shown in table 11;
(f) all peptides shown in table 13;
(g) all peptides shown in table 15;
(h) all peptides shown in table 17;
(i) all peptides shown in table 19; or
(j) All peptides shown in table 21.
45. The recombinant Listeria strain of any one of claims 29-44, wherein the PEST-containing peptide is on the N-terminus of the fusion polypeptide.
46. The recombinant Listeria strain of claim 45, wherein said PEST-containing peptide is an N-terminal fragment of LLO.
47. The recombinant Listeria strain of claim 46, wherein the N-terminal fragment of LLO has the amino acid sequence of SEQ ID NO:59, or a sequence shown in SEQ ID NO.
48. The recombinant Listeria strain of any one of claims 29-47, wherein the nucleic acid is in an episomal (episomal) plasmid.
49. The recombinant listeria strain of any one of claims 29-48, wherein said nucleic acid does not confer antibiotic resistance to said recombinant listeria strain.
50. The recombinant listeria strain of any one of claims 29-49, wherein the recombinant listeria strain is an attenuated auxotrophic listeria strain.
51. The recombinant Listeria strain of claim 50, wherein the attenuated auxotrophic Listeria strain comprises a mutation in one or more endogenous genes that inactivates the one or more endogenous genes.
52. The recombinant Listeria strain of claim 51, wherein the one or more endogenous genes comprise actA, dal and dat.
53. The recombinant listeria strain of any one of claims 29-52, wherein said nucleic acid comprises a second open reading frame encoding a metabolic enzyme.
54. The recombinant Listeria strain of claim 53, wherein the metabolic enzyme is an alanine racemase enzyme or a D-amino acid aminotransferase.
55. The recombinant listeria strain of any one of claims 29-54, wherein the fusion polypeptide is expressed from an hly promoter.
56. The recombinant Listeria strain of any one of claims 29-55, wherein the recombinant Listeria strain is a Listeria monocytogenes (Listeria monocytogenes) strain.
57. The recombinant Listeria strain of any one of claims 29-56, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes (Listeria monocytogenes) strain comprising deletions or inactivating mutations of actA, dal, and dat, wherein the nucleic acid is in an episomal plasmid and comprises a second open reading frame encoding an alanine racemase or a D-amino acid aminotransferase, and wherein the PEST-containing peptide is an N-terminal fragment of LLO.
58. An immunogenic composition comprising:
(a) the recombinant bacterial strain of any one of claims 22-28 or the recombinant listeria strain of any one of claims 29-57; and
(b) an adjuvant.
59. The immunogenic composition of claim 58, wherein said adjuvant comprises detoxified Listeriolysin O (dtLLO), a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, or an unmethylated CpG-containing oligonucleotide.
60. A method of inducing or enhancing an immune response against a tumor or cancer in a subject, the method comprising administering to the subject the isolated peptide of any one of claims 1-9, the nucleic acid of any one of claims 10-16, the pharmaceutical composition of any one of claims 17-21, the recombinant bacterial strain of any one of claims 22-28, the recombinant Listeria strain of any one of claims 29-57, or the immunogenic composition of any one of claims 58-59.
61. A method of preventing or treating a tumor or cancer in a subject, the method comprising administering to the subject the isolated peptide of any one of claims 1-9, the nucleic acid of any one of claims 10-16, the pharmaceutical composition of any one of claims 17-21, the recombinant bacterial strain of any one of claims 22-28, the recombinant Listeria strain of any one of claims 29-57, or the immunogenic composition of any one of claims 58-59.
62. The method of claim 60 or 61, wherein the cancer is non-small cell lung cancer, prostate cancer, pancreatic cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, lower glioma, colorectal cancer, or head and neck cancer.
Summary of The Invention
Provided herein are methods and compositions for cancer immunotherapy. In one aspect, provided herein is an isolated peptide comprising an immunogenic fragment of a cancer-associated protein, wherein the fragment comprises a heterolytic mutation (heterolytic mutation). In another aspect, provided herein is a recombinant Listeria (Listeria) strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide, wherein the fusion polypeptide comprises a PEST-containing peptide fused to one or more immunogenic fragments of a cancer-associated protein, wherein the fragments comprise a mutated mutation. Also provided herein are such fusion polypeptides and nucleic acids encoding such isolated peptides and fusion polypeptides. Also provided herein are recombinant bacterial strains comprising such nucleic acids.
In another aspect, provided herein is an immunogenic composition, a pharmaceutical composition or a vaccine comprising such an isolated peptide, nucleic acid, fusion polypeptide, recombinant bacterial strain or recombinant listeria strain.
In another aspect, provided herein are methods of inducing or enhancing an immune response against a tumor or cancer in a subject, comprising administering to the subject such isolated peptides, nucleic acids, fusion polypeptides, recombinant bacterial strains, or recombinant listeria strains. Also provided herein are methods of inducing or enhancing an immune response against a tumor or cancer in a subject, the method comprising administering to the subject an immunogenic composition, pharmaceutical composition or vaccine comprising such isolated peptide, nucleic acid, fusion polypeptide, recombinant bacterial strain or recombinant listeria strain.
In another aspect, provided herein are methods of preventing or treating a tumor or cancer in a subject, comprising administering to the subject such isolated peptides, nucleic acids, fusion polypeptides, recombinant bacterial strains, or recombinant listeria strains. Also provided herein are methods of preventing or treating a tumor or cancer in a subject, the method comprising administering to the subject an immunogenic composition, pharmaceutical composition or vaccine comprising such isolated peptide, nucleic acid, fusion polypeptide, recombinant bacterial strain or recombinant listeria strain.
In another aspect, provided herein is a cell bank comprising one or more such recombinant bacteria or recombinant listeria strains.
Brief Description of Drawings
FIGS. 1A and 1B show schematic representations of WT1 minigene (minigene) constructs. Figure 1A shows a WT1 minigene construct designed to express a single WT1 chimeric polypeptide antigen. Figure 1B shows a WT1 minigene construct designed to express three separate WT1 chimeric polypeptide antigens.
FIGS. 2A and 2B show Western blots of Lmdda-WT 1-tLLO-FLAG-Ub-mutator phenylalanine minigene construct (FIG. 2A) and Lmdda-WT 1-tLLO-P1-P2-P3-FLAG-Ub-mutator tyrosine minigene construct (FIG. 2B). In FIG. 2A, lane 1 is a protein molecular weight standard (ladder), lane 2 is an Lmdda-WT 1-tLLO-P1-P2-P3-FLAG-Ub-mutatyrosine minigene construct (68kDa), and lane 3 is a negative control. In FIG. 2B, lane 1 is a protein molecular weight standard, lane 2 is a negative control, and lane 3 is WT 1-tLLO-FLAG-Ub-mutaphe phenylalanine minigene construct (construct No. 1).
Figure 3 shows the colony PCR results of several Lm-minigene constructs expressing a mutator WT1 peptide. The mutated residues are indicated in bold and underlined.
FIG. 4 shows ELISPOT assays performed on splenocytes stimulated ex vivo with WT1 peptides RMFPNAPYL (SEQ ID NO:197) and FMFPNAPYL (SEQ ID NO: 160). Splenocytes were from HLA2 transgenic mice immunized with the WT1-F minigene construct. PBS and LmddA274 were used as negative controls.
FIG. 5 shows ELISPOT assays performed on splenocytes stimulated ex vivo with WT1 peptides RMFPNAPYL (SEQ ID NO:197) and YMFPNAPYL (SEQ ID NO: 169). Splenocytes were from HLA2 transgenic mice immunized with the WT1-AH1-Tyr minigene construct. PBS and LmddA274 were used as negative controls.
FIGS. 6A and 6B show IFN-. gamma.spot-forming cells (SFC) corresponding to each million splenocytes stimulated ex vivo with WT1 peptides RMFPNAPYL (SEQ ID NO: 197; FIG. 6A) and FMFPNAPYL (SEQ ID NO: 160; FIG. 6B). Splenocytes were from HLA2 transgenic mice immunized with the WT1-F minigene construct. PBS and LmddA274 were used as negative controls.
FIGS. 7A and 7B show IFN-. gamma.spot-forming cells (SFC) corresponding to each million splenocytes stimulated ex vivo with WT1 peptides RMFPNAPYL (SEQ ID NO: 197; FIG. 7A) and YMFPNAPYL (SEQ ID NO: 169; FIG. 7B). Splenocytes were from HLA2 transgenic mice immunized with the WT1-AH1-Tyr minigene construct. PBS and LmddA274 were used as negative controls.
Figure 8 shows CT26 tumor volume in mice treated with PBS control or Lm AH1_ HC.
Definition of
The terms "protein," "polypeptide," and "peptide" are used interchangeably herein to refer to polymeric amino acid forms of any length, including coded and non-coded amino acids, as well as chemically or biochemically modified or derivatized amino acids. The term includes polymers that have been modified, such as polypeptides having a modified peptide backbone.
Proteins are said to have an "N-terminus" and a "C-terminus". The term "N-terminus" relates to the initial portion of a protein or polypeptide, terminated by an amino acid having a free amine group (-NH 2). The term "C-terminal" relates to the terminating part of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH).
The term "fusion protein" refers to a protein comprising two or more peptides linked together by peptide or other chemical bonds. The peptides may be linked together directly by peptide bonds or other chemical bonds. For example, the chimeric molecule may be recombinantly expressed as a single chain fusion protein. Alternatively, the peptides may be linked together by a "linker" between two or more peptides, such as one or more amino acids or other suitable linkers.
The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to a polymeric nucleotide form of any length, including ribonucleotides, deoxyribonucleotides or analogs or modified forms thereof. They include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purine bases, pyrimidine bases, or other natural nucleotide bases, chemically modified nucleotide bases, biochemically modified nucleotide bases, non-natural nucleotide bases, or derivatized nucleotide bases.
Nucleic acids are said to have "5 'ends" and "3' ends" because mononucleotides are reacted in such a way that the 5 'phosphate of one mononucleotide pentose ring is linked to the 3' oxygen of its adjacent mononucleotide pentose ring by a phosphodiester bond in one direction to produce an oligonucleotide. If the 5 ' phosphate at the end of the oligonucleotide is not linked to the 3 ' oxygen of the pentose ring of a single nucleotide, said end is called the "5 ' end". If the 3 ' oxygen of the end of the oligonucleotide is not linked to the 5 ' phosphate of another mononucleotide pentose ring, said end is called the "3 ' end". Even within the larger oligonucleotide, the nucleic acid sequence may be said to have a 5 'end and a 3' end. In linear or circular DNA molecules, discrete elements are referred to as being "upstream" or 5 'of "downstream" or 3' elements.
"codon optimization" refers to the process of modifying a nucleic acid sequence to achieve enhanced expression in a particular host cell by replacing at least one codon of the native sequence with a codon in a gene that is more frequently or most frequently used in the host cell, while maintaining the native amino acid sequence. For example, a polynucleotide encoding a fusion polypeptide can be modified to replace codons that have a higher frequency of use in a given listeria cell or any other host cell than the naturally occurring nucleic acid sequence. The Codon Usage table is readily available, for example, at the "Codon Usage Database". The optimal codons for each amino acid utilized by l.monocytogenes (l.monocytogenes) are shown in US2007/0207170, which is incorporated by reference herein in its entirety for all purposes. These tables may be adapted in many ways. See Nakamura et al (2000) Nucleic Acids Research 28:292, which is incorporated by reference herein in its entirety for all purposes. Computer algorithms for codon optimization of specific sequences for expression in a specific host are also available (see e.g. Gene Forge).
The term "plasmid" or "vector" includes any known delivery vector, including bacterial delivery vectors, viral vector delivery vectors, peptide immunotherapy delivery vectors, DNA immunotherapy delivery vectors, episomal plasmids, integrative plasmids, or phage vectors. The term "vector" refers to a construct capable of delivering, and optionally expressing, one or more fusion polypeptides in a host cell.
The term "episomal plasmid" or "extrachromosomal plasmid" refers to a nucleic acid vector that is physically separated from (i.e., episomal or extrachromosomal, and not integrated into the genome of the host cell) chromosomal DNA, and replicates independently of the chromosomal DNA. The plasmid may be linear or circular, and it may be single-stranded or double-stranded. The episomal plasmid may optionally persist in multiple copies in the cytoplasm of the host cell (e.g., listeria cytoplasm), resulting in amplification of any gene of interest within the episomal plasmid.
The term "genomic integration" refers to nucleic acid has been introduced into a cell such that the nucleotide sequence is integrated into the genome of the cell and is capable of being inherited by its progeny. Any protocol can be used to stably incorporate the nucleic acid into the genome of the cell.
The term "stably maintained" refers to the maintenance of a nucleic acid molecule or plasmid for at least 10 generations without detectable loss in the absence of selection (e.g., antibiotic selection). For example, the time period can be at least 15 generations, 20 generations, at least 25 generations, at least 30 generations, at least 40 generations, at least 50 generations, at least 60 generations, at least 80 generations, at least 100 generations, at least 150 generations, at least 200 generations, at least 300 generations, or at least 500 generations. Stable maintenance can refer to a nucleic acid molecule or plasmid that is stably maintained in a cell in vitro (e.g., in culture), in vivo, or both.
An "open reading frame" or "ORF" is a portion of DNA that contains a base sequence that can potentially encode a protein. As an example, the ORF may be located between the start codon sequence (start codon) and the stop codon sequence (stop codon) of the gene.
A "promoter" is a DNA regulatory region that typically comprises a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site of a particular polynucleotide sequence. The promoter may additionally comprise other regions which influence the rate of transcription initiation. The promoter sequences disclosed herein regulate transcription of an operably linked polynucleotide. The promoter can be active in one or more of the cell types disclosed herein (e.g., eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, single cell stage embryos, differentiated cells, or a combination thereof). The promoter can be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally limited promoter (e.g., a developmentally regulated promoter), or a spatially limited promoter (e.g., a cell-specific or tissue-specific promoter). Examples of promoters may be found, for example, in WO 2013/176772, which is incorporated herein by reference in its entirety.
"operably linked" or "operably linked" refers to two or more components (e.g., a promoter and another sequence element) that are adjacent such that both components function normally and such that it is possible that at least one component may mediate a function exerted on at least one other component. For example, a promoter may be operably linked to a coding sequence if it controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. Operably linked can include the sequences being contiguous with each other, or functioning in trans (e.g., a regulatory sequence can function at a distance to control transcription of a coding sequence).
"sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences relates to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage sequence identity is used for proteins, it will be appreciated that residue positions that are not identical often differ by conservative amino acid substitutions, in which an amino acid residue is substituted for another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity), and thus do not alter the functional properties of the molecule. When the sequence difference is a conservative substitution, the percent sequence identity may be adjusted higher to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those skilled in the art. Typically, this involves scoring conservative substitutions as partial rather than complete mismatches, thereby increasing the percentage of sequence identity. Thus, for example, when the same amino acid is given a score of 1 and a non-conservative substitution is given a score of 0, a conservative substitution is given a score between 0 and 1. Conservative substitution scores are calculated, for example, as performed in the program PC/GENE (intelligentics, Mountain View, California).
"percent sequence identity" refers to a value determined by comparing two optimally aligned sequences (with the largest number of perfectly matched residues) over a comparison window, wherein a portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) to achieve optimal alignment of the two sequences. The percentage is calculated by: determining the number of positions at which the same nucleic acid base or amino acid residue is present in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise specified (e.g., the shorter sequence includes a linked heterologous sequence), the comparison window is the full length of the shorter of the two sequences being compared.
Unless stated otherwise, sequence identity/similarity values refer to values obtained using GAP version 10 using the following parameters: percent identity and percent similarity of nucleotide sequences using GAP weight 50 and length weight 3 and nwsgapdna. cmp score matrix; % identity and% similarity of amino acid sequences using GAP weight 8 and length weight 2 and BLOSUM62 scoring matrix; or any equivalent thereof. "equivalent program" includes for any two sequences discussed, when compared to the corresponding alignment generated by GAP version 10, any sequence comparison program that generates an alignment with identical nucleotide or amino acid residue matches and identical percentage of sequence identity.
The term "conservative amino acid substitution" refers to the substitution of an amino acid that is normally present in a sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine or leucine for another non-polar residue. Likewise, examples of conservative substitutions include substitutions of one polar (hydrophilic) residue to another, such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine. Additionally, substitution of a basic residue such as lysine, arginine or histidine for another residue, or substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue, are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine or methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine, and/or the substitution of a polar residue for a non-polar residue. The following summarizes typical amino acid classifications.
"homologous" sequences (e.g., nucleic acid sequences) refer to the following sequences: it is identical or substantially similar to a known reference sequence such that it is, for example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the known reference sequence.
The term "wild-type" refers to an entity having a structure and/or activity as seen in a normal (as opposed to mutated, diseased, altered, etc.) state or situation. Wild-type genes and polypeptides often exist in a variety of different forms (e.g., alleles).
The term "isolated" with respect to proteins and nucleic acids refers to substantially pure preparations of proteins and nucleic acids that are relatively pure with respect to other bacterial, viral, or cellular components that may normally be present in situ. The term "isolated" also includes proteins and nucleic acids that do not have naturally occurring counterparts, have been chemically synthesized and are therefore substantially uncontaminated by other proteins or nucleic acids, or have been separated from or purified from most other cellular components with which they are naturally associated (e.g., other cellular proteins, polynucleotides, or cellular components).
An "exogenous" or "heterologous" molecule or sequence is a molecule or sequence that is not normally expressed in a cell or is not normally present in that form in a cell. Typically present includes reference to a particular developmental stage and environmental conditions of the cell. An exogenous or heterologous molecule or sequence can, for example, include a mutated form of a corresponding endogenous sequence within a cell, or can include a sequence that corresponds to an endogenous sequence within a cell, but is in a different form (i.e., not in a chromosome). An exogenous or heterologous molecule or sequence in a particular cell can also be a molecule or sequence derived from a species different from the reference species of the cell or from a different organism within the same species. For example, in the case of a listeria strain that expresses a heterologous polypeptide, the heterologous polypeptide can be a polypeptide that is not native to, or endogenous to, the listeria strain, is not typically expressed by the listeria strain, is from a source other than the listeria strain, is derived from a different organism within the same species.
In contrast, an "endogenous" molecule or sequence or a "native" molecule or sequence is one that is normally present in that form in a particular cell at a particular developmental stage under particular environmental conditions.
The term "variant" refers to an amino acid or nucleic acid sequence (or organism or tissue) (e.g., a splice variant) that is different from the majority of the population, but still sufficiently similar to the common pattern to be considered one of them.
The term "subtype" refers to a form of a molecule (e.g., a protein) that differs only slightly from another subtype or form (e.g., another subtype or form of the same protein). For example, protein subtypes may arise from different but related genes, they may arise from the same gene by alternative splicing, or they may arise as a result of single nucleotide polymorphisms.
The term "fragment" when referring to a protein means a protein that is shorter or has fewer amino acids than the full-length protein. The term "fragment," when referring to a nucleic acid, means a nucleic acid that is shorter or has fewer nucleotides than the full-length nucleic acid. Fragments may be, for example, N-terminal fragments (i.e., removing a portion of the C-terminus of the protein), C-terminal fragments (i.e., removing a portion of the N-terminus of the protein), or internal fragments. Fragments may also be, for example, functional or immunogenic fragments.
The term "analog" when referring to a protein means a protein that differs from a naturally occurring protein by conservative amino acid differences, by modifications that do not affect the amino acid sequence, or by both.
The term "functional" refers to the innate ability of a protein or nucleic acid (or fragment, subtype or variant thereof) to exhibit biological activity or function. The biological activity or function can include, for example, being capable of eliciting an immune response upon administration to a subject. The biological activity or function may also include, for example, binding to an interaction partner. In the case of functional fragments, subtypes or variants, these biological functions may actually be altered (e.g., in terms of their specificity or selectivity), but retain the basic biological function.
The term "immunogenic" or "immunogenic" refers to the innate ability of a molecule (e.g., a protein, nucleic acid, antigen, or organism) to elicit an immune response in a subject when administered to the subject. Immunogenicity can be measured, for example, by a greater number of antibodies to the molecule, a greater diversity of antibodies to the molecule, a greater number of T cells specific for the molecule, a greater cytotoxic T cell response or helper T cell response to the molecule, and the like.
The term "antigen" is used herein to refer to a substance that, when placed in contact with a subject or organism (e.g., when present in the subject or organism, or when detected by the subject or organism), results in a detectable immune response from the subject or organism. The antigen can be, for example, a lipid, a protein, a carbohydrate, a nucleic acid, or combinations and variations thereof. For example, an "antigenic peptide" refers to a peptide that, when present in or detected by a subject or organism, results in an immune response in the subject or organism. For example, such "antigenic peptides" may encompass the following proteins: which are loaded and presented on MHC class I and/or class II molecules on the surface of the host cell and can be recognized or detected by the host's immune cells, thereby causing the mount of an immune response against the protein. This immune response may also extend to other cells within the host that express the same protein, such as diseased cells (e.g., tumor cells or cancer cells).
The term "epitope" refers to a site on an antigen that is recognized by (e.g., to which an antibody binds) the immune system. Epitopes may be formed of contiguous amino acids or of non-contiguous amino acids that are contiguous by tertiary folding of one or more proteins. Epitopes formed by contiguous amino acids (also known as linear epitopes) are typically retained on exposure to denaturing solvents, while epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost on treatment with denaturing solvents. Epitopes typically comprise at least 3, and more typically at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining the spatial conformation of an epitope include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., epipene Mapping Protocols, in Methods in molecular Biology, vol 66, Glenn e.morris eds (1996), which is incorporated by reference herein in its entirety for all purposes.
The term "mutation" refers to any change in the structure of a gene or protein. For example, the mutation may be caused by a deletion, insertion, substitution, or rearrangement of a chromosome or protein. An "insertion" alters the number of nucleotides in a gene or the number of amino acids in a protein by adding one or more additional nucleotides or amino acids. A "deletion" alters the number of nucleotides in a gene or the number of amino acids in a protein by subtracting one or more additional nucleotides or amino acids.
"frameshift" mutations in DNA occur when the addition or loss of nucleotides changes the reading frame of a gene. The reading frame consists of a group of 3 bases each encoding one amino acid. Frameshift mutations shift the grouping of these bases and alter the coding for amino acids. The resulting protein is typically non-functional. The insertions and deletions may each be frame shift mutations.
A "missense" mutation or substitution refers to a change in one amino acid of a protein, or a point mutation in a single nucleotide resulting in a change in the encoded amino acid. Point mutations in a single nucleotide that result in a change of one amino acid are "non-synonymous" substitutions in the DNA sequence. Non-synonymous substitutions may also result in "nonsense" mutations in which the codon becomes a premature stop codon that results in truncation of the resulting protein. In contrast, "synonymous" mutations in DNA are mutations that do not alter the amino acid sequence of the protein (due to codon degeneracy).
The term "somatic mutation" includes genetic alterations obtained from cells other than germ cells (e.g., sperm or eggs). The mutation may be transmitted to the progeny of the mutant cell during cell division, but may not be inherited. In contrast, germ cell mutations are present in the germ line and can be transmitted to the next generation of offspring.
The term "in vitro" refers to an artificial environment as well as processes or reactions occurring within an artificial environment (e.g., a test tube).
The term "in vivo" refers to the natural environment (e.g., a cell or organism or body) and processes or reactions occurring within the natural environment.
A composition or method that "comprises" or "includes" one or more recited elements may include other elements not expressly recited. For example, a composition that "comprises" or "includes" a protein may contain the protein alone or in combination with other ingredients.
The specification of a range of values includes all integers within or defining the range, as well as all sub-ranges defined by integers within the range.
Unless otherwise apparent from the context, the term "about" encompasses values within the standard measurement error limits (e.g., SEM) of the stated value or ± 0.5%, 1%, 5% or 10% variation from the stated value.
The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "an antigen(s)" or "at least one antigen(s)" may include a plurality of antigens, including mixtures thereof.
Statistical significance means p ≦ 0.05.
Detailed Description
I. Overview
Provided herein are peptides comprising immunogenic fragments of a cancer-associated protein, wherein the fragments comprise a mutated mutation. Some such peptides are recombinant fusion polypeptides comprising one or more immunogenic fragments of a cancer-associated protein, wherein each fragment comprises a mutated mutation (e.g., fused to a PEST-containing peptide). Also provided herein are nucleic acids encoding such peptides; an immunogenic composition, a pharmaceutical composition or a vaccine comprising such a peptide or nucleic acid; recombinant bacteria or listeria strains comprising such peptides or nucleic acids; an immunogenic composition, pharmaceutical composition or vaccine comprising such a recombinant bacterium or listeria strain; as well as methods of producing such peptides, such nucleic acids, and such recombinant bacteria or listeria strains. Also provided herein are methods of inducing an immune response against a tumor-associated antigen in a subject, methods of inducing an anti-tumor or anti-cancer immune response in a subject, methods of treating a tumor or cancer in a subject, methods of preventing a tumor or cancer in a subject, and methods of protecting a subject from a tumor or cancer using such peptides, nucleic acids, recombinant bacteria or listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines.
Designing and using mutated sequences (i.e., sequence optimized peptides) derived from tumor-associated antigen genes (e.g., from Cancer Testis Antigen (CTA) or carcinoembryonic antigen (OFA)) can increase presentation by MHC class I alleles. It has been shown that the mutated sequence is sufficient to elicit a T cell response to overcome central tolerance and elicit a successful cross-reactive immune response against the wild-type peptide. Within cancer indications, OFA and CTA are expressed in up to 100% of patients, but not in healthy tissues of adults (e.g., typically only in embryonic tissues). Many OFA/CTAs play a major role in tumorigenesis. Since the tissue expression of OFA/CTA is highly restricted in cancer, they are a starry target for immunotherapy.
Such variant sequences can be combined such that total patient coverage within one cancer type can approach 100%. The use of multiple sequence-optimized proprietary immunogenic OFA/CTA peptides or tumor-associated antigenic peptides (i.e. sequence-optimized to enhance immunogenicity) can provide additional targets for generating a strong T cell response, eliminating the need to sequence patients prior to treatment, as it can be hypothesized that they will express tumor-associated antigens for which we have designed variant peptides to cover the most prevalent HLA (HLA-a0201, HLA-a0301, HLA-a2402 and HLA-B0702).
In some of the compositions described herein, the mutated peptide is expressed in a Listeria monocytogenes (Lm) vector. Lm technology has mechanisms of action that combine strong innate immune stimulation, delivery of target peptides directly into the cytosol of dendritic cells and antigen presenting cells, generation of targeted T cell responses, and reduction of immunosuppression by regulatory T cells and myeloid-derived suppressor cells in the tumor microenvironment. Multiple treatments can be administered and/or combined without neutralizing antibodies. Lm technology can stimulate the immune system using, for example, live attenuated bioengineered Lm bacteria to treat tumor cells as potentially bacterially infected cells and target them for elimination. The technical process may start with a live attenuated strain of listeria and, for example, multiple copies of a plasmid encoding a fusion protein sequence comprising a fragment, for example, an LLO (listeriolysin o) molecule, linked to an antigen of interest may be added. This fusion protein is secreted by listeria inside antigen presenting cells. This results in stimulation of both the innate and adaptive branches of the immune system, which decreases the tumor defense mechanisms and makes the immune system more prone to attack and destroy cancer cells.
In terms of immunization, Lm-based vectors are a superior platform for generating CD8+ predominant T cell responses compared to peptide vaccines. First, there is no need to add filgrastim (filgrastim) injection adjuvant. This is because live attenuated bacterial vectors inherently trigger numerous innate immune activation triggers including several TLR, PAMP and DAMP receptors and have a powerful ability to agonize STING receptors in the cytosol of antigen presenting cells. This is a more extensive change in the immune microenvironment that primes the patient's immune system to achieve an adaptive immune response. Second, Lm vector was infused intravenously. This allows it to reach significantly more antigen presenting cells than can reside in a limited region of the subcutaneous tissue. This also eliminates the need for subcutaneous injections, the use of filgrastim, and the risk of delayed type hypersensitivity. It is also possible to produce higher T cell titers faster because the optimal CD8+ T cell number typically peaks after 3 treatments, no more than 10 treatments. Third, Lm promotes a primary CD8+ T cell response with CD4+ cross-reactivity to achieve T cell help. CD8+ T cells kill cancer cells most efficiently, and because Lm vectors present their antigens in the cytoplasm of APCs, those peptides are rapidly transferred to the proteasome for processing, complexing with class 1 MHC, and trafficking to the APC surface for presentation to T cells predominantly presenting CD8 +. This should lead to the advantage of producing more CD8+ T cells compared to subcutaneous Montanide presentation of the antigenic peptide. Fourth, Lm vectors increase chemokine and chemokine receptor expression on tumors and peripheral lymph nodes. This helps to attract activated T cells to the vicinity of solid tumors. Fifth, the Lm vector reduces the relative number and containment function of immunosuppressive cells that can protect tumors from T cell attack, better enabling T cell killing of cancer cells. This reduction in the immunosuppressive capacity of regulatory T cells and myeloid-derived suppressor cells would better enable T cells generated against these peptides to have better activity in the case of solid tumors. Sixth, Lm vectors do not produce neutralizing antibodies. Thus, these vectors can be repeatedly administered for extended periods of time without loss of efficacy due to neutralizing antibodies and without developing delayed or acute allergies, which may include allergic reactions.
Lm vectors work through multiple immunotherapeutic mechanisms: potent innate immune stimulation by toll-like receptors (TLRs) and pathogen-associated molecular patterns (PAMPs), including interferon gene Stimulator (STING) receptors, strong CD8+And CD4+T cell responses, epitope spreading, and disabling of immune suppression achieved by tregs and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. In addition, the unique intracellular life cycle of listeria avoids neutralizing antibodies, allowing for repeated dosing. Lm is also advantageous because it has a synergistic effect with checkpoint inhibitors, co-stimulatory agonists and other agents. It also has a large capacity and can be adapted to target many different tumor types. As an example, a live attenuated strain of Lm can be bioengineered to secrete an antigen-adjuvant fusion protein comprising, consisting essentially of, or consisting of: a truncated fragment of listeriolysin O (tLLO) having adjuvant properties and one or more tumor-associated antigens. In thatFollowing infusion into a patient, the bioengineered Lm can be phagocytized by antigen presenting cells where the fusion protein is secreted by Lm, processed, and presented on class I and class II Major Histocompatibility Complex (MHC) molecules. Stimulation of tumor associated antigen specific CD4 by target peptides presented on the surface of antigen presenting cells+T cells and CD8+T cells. Activated CD8+T cells can then search for and kill tumor-associated antigen expressing cancer cells and modulate the tumor microenvironment to overcome immunosuppression.
Lm vectors have several clinical advantages. Any side effects associated with treatment occur within hours immediately following infusion while the patient is still in the clinic, are almost only mild-moderate and respond readily to treatment, and are eliminated on the day of administration without signs of delayed onset, cumulative toxicity or persistent sequelae. Practical advantages include the following facts: there is no need to administer multiple agents and switch to other dosing sites for subsequent administration.
There are several advantages from a manufacturing standpoint. First, there is no need to manufacture individual peptides in high concentrations and purity. Lm bacteria simultaneously transcribe multiple copies of DNA on DNA plasmids inside the bacteria and secrete these peptides directly into the cytoplasm of APC where they are almost immediately transported to the proteasome for processing. Basically, peptides are produced by bacteria just before they are used for antigen processing. Second, Lm carriers are highly scalable. Once the genetic engineering is complete, the bacteria self-replicate in liquid culture. The culture can be scaled up to greatly reduce the cost of the commodity. Third, there is no need to formulate or create an emulsion in a complex carrier such as Montanide. Fourth, the bacteria are extremely stable, for some more than 5 years, without concern for contamination by degradation or decomposition products of the peptide which can result in loss of potency of the peptide preparation.
In some Lm vectors disclosed herein, minigene constructs are used as described in more detail elsewhere herein. The use of the herein disclosed minigene construct approach to expression of specific class I MHC-binding epitopes allows for highly efficient delivery of short peptide sequences into the antigen presentation pathway of professional antigen presenting cells (papcs). A particular advantage of minigene technology is that it does not require proteasome-mediated degradation of larger proteins to release short peptide sequences that can be bound and presented on MHC class I molecules. This results in a much higher efficiency of peptide-MHC class I antigen presentation on the surface of pAPC and, therefore, a much higher level of antigen expression for eliciting an antigen-specific T cell response.
Tumor-associated antigenic peptides comprising a mutator mutation and nucleic acids encoding such peptides
Disclosed herein are peptides comprising immunogenic fragments of a cancer-associated protein, wherein the fragments comprise a mutated mutation.
The term "mutated" refers to a peptide that produces an immune response that recognizes the native peptide from which the mutated peptide is derived (e.g., a peptide that does not contain a mutation in an anchor residue). For example, YLMPVNSEV (SEQ ID NO:130) was generated from YMMPVNSEV (SEQ ID NO:131) by making a residue 2 mutation to methionine. The variant peptide may generate an immune response that recognizes the native peptide from which the variant peptide is derived. For example, the immune response to a native peptide generated by vaccination with a variant peptide may be equal or greater in magnitude than the immune response generated by vaccination with a native peptide. The immune response may be increased, for example, 2-fold, 3-fold, 5-fold, 7-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, 100-fold, 150-fold, 200-fold, 300-fold, 500-fold, 1000-fold, or more.
The variant peptides disclosed herein may bind to one or more Human Leukocyte Antigen (HLA) molecules. HLA molecules, also known as Major Histocompatibility Complex (MHC) molecules, bind peptides and present them to immune cells. The immunogenicity of a peptide may be determined in part by its affinity for HLA molecules. HLA class I molecules interact with CD8 molecules that are normally present on Cytotoxic T Lymphocytes (CTLs). HLA class II molecules interact with CD4 molecules that are normally present on helper T lymphocytes. For example, the variant peptides disclosed herein may bind to HLA molecules with an affinity sufficient to activate T cell precursors, or with an affinity sufficient to mediate recognition by T cells.
The variant peptides disclosed herein may bind to one or more HLA class II molecules. For example, the mutated peptide may bind to an HLA-DRB molecule, an HLA-DRA molecule, an HLA-DQA1 molecule, an HLA-DQB1 molecule, an HLA-DPA1 molecule, an HLA-DPB 1 molecule, an HLA-DMA molecule, an HLA-DMB molecule, an HLA-DOA molecule, or an HLA-DOB molecule.
The native or variant peptides disclosed herein can bind to one or more HLA class I molecules. For example, the mutated peptide may bind to an HLA-A molecule, an HLA-B molecule, an HLA-C molecule, an HLA-A0201 molecule, HLA A1, HLA A2, HLA A2.1, HLAA3, HLA A3.2, HLA A11, HLA A24, HLA B7, HLA B27, or HLA B8. Similarly, the mutated peptides may bind to HLA class I molecules of a superfamily, such as the a2 superfamily, the A3 superfamily, the a24 superfamily, the B7 superfamily, the B27 superfamily, the B44 superfamily, the C1 superfamily, or the C4 superfamily. In a specific example, the variant peptide or fragment binds to one or more of the following HLA types: HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02.
The variant peptides may comprise mutations that enhance binding of the peptide to HLA class II molecules relative to the corresponding native peptide. Alternatively, or in addition, the variant peptides may comprise mutations that enhance binding of the peptide to a class I HLA molecule relative to the corresponding native peptide. For example, the mutant residue can be a HLA class II motif anchor residue. In another embodiment, an "anchor motif or" anchor residue "refers to one or a set of preferred residues at a particular position in an HLA binding sequence (e.g., a class II HLA binding sequence or a class I HLA binding sequence).
Various methods are well known for generating predicted variant epitopes with the potential to elicit cross-reactive immunogenic responses to wild-type epitopes. For example, to design a mutated epitope with the potential to elicit a cross-reactive immunogenic response to a wild-type epitope, the NetMHCpan 3.0 server (www.cbs.dtu.dk/services/NetMHCpan /) can be used to determine the baseline predicted peptide-MHC binding affinity of the wild-type epitope. A peptide-MHC binding affinity percentage scale of less than or equal to 1.0 is considered a powerful binding agent that is likely to elicit an immune response. A potentially mutated epitope is generated by, but not limited to, random substitution of 1 or more amino acids at positions 1,2, 3 or the C-terminal position of the wild-type epitope predicted to be a strong binding agent. The NetMHCpan 3.0 server is then used to estimate the peptide-MHC binding affinity of the potentially variant epitope. The binding affinity ranking percentage is similar to the wild-type epitope, and a variant epitope on the order of less than or equal to 1.0 percent can be considered a potential antigen for future validation.
Other methods for identifying HLA class I and HLA class II residues and for improving HLA binding by mutating residues are well known. See, e.g., US 8,765,687, US 7,488,718, US 9,233,149, and US 7,598,221, each of which is incorporated by reference herein in its entirety for all purposes. For example, methods for predicting MHC class II epitopes are well known. As an example, MHC class II epitopes can be predicted using TEPITOPE (Meister et al (1995) Vaccine 13:581-591, which is incorporated herein by reference in its entirety for all purposes). As another example, MHC class II epitopes can be predicted using EpiMatrix (De Groot et al (1997) AIDS Res. hum. retroviroruses 13:529- > 531, which is incorporated herein by reference in its entirety for all purposes). As another example, class II MHC epitopes can be predicted using the prediction Method (Predict Method) (Yu K et al (2002) mol. Med.8: 137-. As another example, MHC class II epitopes can be predicted using the SYFPEITHI epitope prediction algorithm. SYFPEITHI is a database containing more than 4500 peptide sequences known to bind MHC class I and class II molecules. SYFPEITHI provide scores based on the presence of certain amino acids in certain positions along the MHC binding groove. The ideal amino acid anchor is assigned a score of 10, an unusual anchor value of 6-8, a supplementary anchor value of 4-6, preferably a residue value of 1-4; the effect on bound negative amino acids was scored between-1 and-3. For HLA-a0201, the maximum score is 36. As another example, MHC class II epitopes can be predicted using rankpepep. Rankpep uses a Position Specific Scoring Matrix (PSSM) or profile from each set of aligned peptides known to bind to a given MHC molecule as a predictor of MHC-peptide binding. Rankpep includes information about the score of the peptide and the percentage or percentile score that predicts the optimal value of the peptide relative to the score of the consensus (consensus) sequence that yields the largest score with the selected profile. Rankpep involves selecting 102PSSM and 80PSSM to predict binding of peptides to MHC I molecules and MHC II molecules, respectively. Several PSSMs for predicting different sized peptide binding agents are generally available for each MHC I molecule. As another example, MHC class II epitopes can be identified using SVMHC (Donnes and Elofsson (2002) BMCBbioinformatics 11; 3:25, which is incorporated herein by reference in its entirety for all purposes).
As an example, class I MHC epitopes can be predicted using BIMAS software.BIMAS scoring is based on the assignment of MHC-I/β2-calculation of the theoretical half-life of the microglobulin/peptide complex, which is a measure of the binding affinity of the peptide. The program uses information about HLA-I peptides 8-10 amino acids in length. The higher the binding affinity of a peptide to MHC, the higher the probability that this peptide represents an epitope. The BIMAS algorithm assumes that each amino acid in the peptide contributes independently to binding to class I molecules. Dominant anchor residues critical for binding have a coefficient significantly higher than 1 in the table. The unfavorable amino acids have a positive coefficient of less than 1. If it is not known whether an amino acid contributes favorably or unfavorably to the binding partner, it is assigned the value 1. All values assigned to amino acids were multiplied and the resulting run score was multiplied by a constant to generate an estimate of the dissociation half-life. As another example, MHC class I epitopes can be identified using SYFPEITHI. As another example, MHC class I epitopes can be identified using SVMHC. As another example, class I MHC epitopes can be identified using NetMHC-2.0 (Buus et al (2003) Tissue Antigens 62:378-384, which is incorporated by reference herein in its entirety for all purposes).
Different residues in the HLA binding motif can be mutated to enhance MHC binding. In one example, the mutation that enhances MHC binding is in the residue at position 1 of the HLA class I binding motif (e.g., to tyrosine, glycine, threonine, or phenylalanine). As another example, the mutation may be in position 2 of the class I HLA binding motif (e.g., to leucine, valine, isoleucine, or methionine). As another example, the mutation may be in position 6 of the HLA class I binding motif (e.g., to valine, cysteine, glutamine, or histidine). As another example, the mutation may be in position 9 or in a C-terminal position of the class I HLA binding motif (e.g., to valine, threonine, isoleucine, leucine, alanine, or cysteine). The mutation may be in the primary anchor residue or in the secondary anchor residue. For example, class I HLA primary anchor residues may be positions 2 and 9 and secondary anchor residues may be positions 1 and 8 or positions 1,3, 6,7 and 8. In another example, the point mutation may be in a position selected from positions 4,5 and 8.
Similarly, different residues in the HLA class II binding site can be mutated. For example, HLA class II motif anchor residues may be modified. For example, the P1 position, P2 position, P6 position, or P9 position may be mutated. Alternatively, the P4 position, P5 position, P10 position, P11 position, P12 position, or P13 position may be mutated.
Individual mutator mutations can be selected based on any criteria as discussed in further detail elsewhere herein. For example, individual mutated mutations or peptides may be selected if they are known to produce a CD8+ T lymphocyte response.
After a set of possible mutational mutations has been identified, a mutational immunogenic peptide sequence comprising each mutational mutation can be selected. Peptides of different sizes may be used as disclosed elsewhere herein. For example, a mutator mutant or mutator immunogenic peptide can be concentrated on, for example, a class I MHC epitope consisting of 9 amino acids.
The sequence of the variant antigenic peptide can then be optimized for enhanced binding to MHC class I molecules. To optimize binding to each HLA, peptide MHC binding motifs and amino acid binding profiles can be evaluated from Immune Epitope databases and analytical resources (e.g., iedb. org/MHCalleleid/143). Preferred amino acids at anchor positions may be inserted into the variant antigenic peptide sequences (e.g., NUF 2-wild-type: YMMPVNSEV (SEQ ID NO: 131); and NUF 2-variant: YLMPVNSEV (SEQ ID NO: 130)).
The binding affinity of the sequence-optimized variant antigenic peptide can then be assessed, for example, using one of the following algorithms: a netmhc4.0 server; netmhcpana 4.0 server; and mhcflurry v0.2.0. For example, if the predicted binding affinity for a particular HLA is equal to or stronger than the corresponding native sequence, a heteroantigenic peptide can be considered. Selected sequence optimized heteroantigenic peptides can then be screened for in vitro binding to specific HLA using the REVEAL assay of promimune. For example, a 45% of the heteroantigenic peptides of the positive control peptide of the binding affinity > ═ REVEAL assay are considered binding agents.
The binding affinity (e.g., IC50) of the sequence-optimized variant antigenic peptide can be, for example, less than 1000, 500, 400, 300, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8,7, 6, 5,4, 3,2, or 1 nM. For example, the binding affinity (e.g., IC50) can be about 0.5-500, 0.5-300, 0.5-200, 0.5-100, 0.5-50, 0.5-40, 0.5-30, 0.5-20, 0.5-10, or 0.5-5 nM.
The cancer genomic map (TCGA) RNAseqV2 dataset can also be used to measure the RNA expression levels of the variant antigenic peptides in specific indications. The percentage of TCGA samples with normalized RNA expression reads greater than 0 can be calculated. Heteroantigenic peptides with TCGA expression in most samples can be considered preferentially.
In a specific example, a literature review can be performed to examine the genomic picture of the indication-specific tumor-associated antigen to generate a candidate list of potential TAAs. A second literature review can be performed to determine whether the candidate list TAA contains known immunogenic peptides that generate a CD8+ T lymphocyte response. This approach may for example focus mainly on MHC class I epitopes consisting of 9 amino acids from TAA (9 mer). This step can, for example, identify potential target peptides in 9-mer form that bind to one of the four HLA types (HLA-a 02:01, HLA-a 03:01, HLA-a 24:02, and HLA-B07: 02).
The target peptide may then be sequence optimized for enhanced binding to MHC class I molecules (also known as a variant peptide). To optimize binding to each HLA, peptide MHC binding motifs and amino acid binding profiles can be evaluated from immune epitope databases and analytical resources (e.g., iedb. org/MHCalleleid/143). Preferred amino acids at anchor positions may be inserted into the target peptide sequence (e.g., NUF 2-wild type: YMMPVNSEV (SEQ ID NO: 131); and NUF 2-mutated: YLMPVNSEV (SEQ ID NO: 130)). The binding affinity of the sequence optimized target peptide and the wild type target peptide can then be assessed, for example, using one of the following algorithms: a netmhc4.0 server; netmhcpana 4.0 server; and mhcflurryv0.2.0. For example, if the predicted binding affinity for a particular HLA is equal to or stronger than the wild-type target peptide sequence, then a sequence optimized target peptide may be considered. Selected sequence-optimized target peptides can then be screened for in vitro binding to specific HLA using the REVEAL assay of proammone. For example, 45% of the target peptides of the positive control peptide of the binding affinity > ═ REVEAL assay can be considered as binders. Finally, the TCGA RNAseqV2 data set can be used to measure the RNA expression levels of target peptides in specific indications. For example, the percentage of TCGA samples with normalized RNA expression reads greater than 0 can be calculated. For example, a target peptide with TCGA expression in most samples may be prioritized.
The term "cancer-associated protein" includes proteins having mutations that are present in multiple types of cancer, present in multiple subjects with a particular type of cancer, or associated with the occurrence or progression of one or more types of cancer. For example, a cancer-associated protein can be an oncogenic (i.e., a protein having activity that can promote cancer progression, such as a protein that regulates cell growth), or it can be a tumor suppressor (i.e., a protein that generally functions to moderate the potential for carcinogenesis, such as by negatively regulating the cell cycle or by promoting apoptosis).
The term "cancer-associated protein" in the context of a variant peptide refers to a protein whose expression is associated with the development or progression of one or more types of cancer. Optionally, the protein includes a protein having mutations that are present in multiple types of cancer, present in multiple subjects with a particular type of cancer, or associated with the occurrence or progression of one or more types of cancer. For example, a cancer-associated protein can be an oncogenic protein (i.e., a protein having activity that can promote cancer progression, such as a protein that regulates cell growth), or it can be a tumor suppressor protein (i.e., a protein that generally functions to moderate the potential for carcinogenesis, such as by negatively regulating the cell cycle or by promoting apoptosis). Preferably, the cancer-associated protein from which the variant peptide is derived is a protein that is expressed in a particular type of cancer, but is not normally expressed in healthy adult tissue (i.e., a protein with cancer-specific expression, cancer-restricted expression, tumor-specific expression, or tumor-restricted expression). However, the cancer-associated protein does not necessarily have cancer-specific expression, cancer-restricted expression, tumor-specific expression, or tumor-restricted expression. Examples of proteins considered as cancer specific proteins or cancer limiting proteins are cancer testis antigens or carcinoembryonic antigens. Cancer Testis Antigens (CTA) are a large family of tumor-associated antigens that are expressed in human tumors of different histological origin, but are not expressed in normal tissues except for male germ cells. In cancer, these developmental antigens may be re-expressed and may serve as sites for immune activation. Carcinoembryonic antigen (OFA) is a protein that is normally present only during fetal development, but is found in adults with certain kinds of cancer. The tumor-restricted pattern of expression of CTA and OFA makes them ideal targets for tumor-specific immunotherapy. Most of the OFA/CTA proteins play a key role in tumorigenesis.
For example, the cancer-associated protein can be any of the cancer-associated proteins listed elsewhere herein. For example, a cancer-associated protein may be encoded by one of the following genes: CEACAM5, GAGE1, hTERT, KLHL7, MAGEA3, MAGEA4, MAGEA6, NUF2, NYESO1, PAGE4, PRAME, PSA, PSMA, RNF43, SART3, SSX2, STEAP1 and SURVIVIN.
Each of the variant immunogenic peptides can be a fragment of a cancer-associated protein (i.e., a contiguous amino acid sequence from a cancer-associated protein) that comprises a variant mutation. Each of the variant immunogenic peptides can be of any length sufficient to induce an immune response. For example, the variant immunogenic peptides disclosed herein can be 5-100, 15-50, or 21-27 amino acids in length, or 15-100, 15-95, 15-90, 15-85, 15-80, 15-75, 15-70, 15-65, 15-60, 15-55, 15-50, 15-45, 15-40, 15-35, 15-30, 20-100, 20-95, 20-90, 20-85, 20-80, 20-75, 20-70, 20-65, 20-60, 20-55, 20-50, 20-45, 20-40, 20-35, 20-30, 11-21, 15-21, 21-31, 31-41, 41-51, 51-61, 51-55, 20-50, 20-45, 20-40, 20-35, 20-30, 11-21, 21-31, 31-41, 41-51, 51-61, or 21, 61-71, 71-81, 81-91, 91-101, 101-121, 121-141, 141-161, 161-181, 181-201, 8-27, 10-30, 10-40, 15-30, 15-40, 15-25, 1-10, 10-20, 20-30, 30-40, 1-100, 5-75, 5-50, 5-40, 5-30, 5-20, 5-15, 5-10, 1-75, 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 8-11 or 11-16 amino acids in length. For example, the variant immunogenic peptide can be 15,16,17,18,19,20,21,22,23,24,25,26,27,28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids in length. For example, the variant immunogenic peptide can be 8-100, 8-50, 8-30, 8-25, 8-22, 8-20, 8-15, 8-14, 8-13, 8-12, 8-11, 7-11, or 8-10 amino acids in length. In one example, the length of the variant immunogenic peptide can be 9 amino acids.
In some cases, the variant immunogenic peptides may be hydrophilic, or may be scored up to or below some hydrophilicity threshold that may predict secretion in listeria monocytogenes or other bacteria of interest. For example, the variant immunogenic peptides can be scored through the Kyte and Doolittle hydropathicity index 21 amino acid window, and all scores above the cut-off value (about 1.6) can be excluded because they are unlikely to be secreted by Listeria monocytogenes.
A variant immunogenic peptide may comprise a single variant mutation, or may comprise two or more variant mutations (e.g., two variant mutations). Exemplary mutated mutant peptides consist of, consist essentially of, or comprise the mutated peptide sequences of the following table, which also provides the corresponding wild-type (native) peptides. Residues in the wild-type peptide that have been modified in the corresponding variant peptide are shown in bold underlined text.
Also disclosed herein are nucleic acids encoding such variant peptides. The nucleic acid may be in any form. The nucleic acid may comprise or consist of DNA or RNA and may be single-stranded or double-stranded. The nucleic acid may be in the form of a plasmid, such as an episomal plasmid, a multicopy episomal plasmid, or an integrating plasmid. Alternatively, the nucleic acid may be in the form of a viral vector, a phage vector, or in a bacterial artificial chromosome. Such nucleic acids may have one open reading frame or may have two or more open reading frames. In one example, the nucleic acid can comprise two or more open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between the open reading frames. For example, the nucleic acid can comprise two to four open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. Each open reading frame can encode a different peptide. In some nucleic acids, the codon encoding the carboxy terminus of the fusion polypeptide is followed by two stop codons to ensure termination of protein synthesis.
The nucleic acid may be codon optimized. A nucleic acid is codon optimized if at least one codon in the nucleic acid is replaced with a codon for that amino acid that is more frequently used by a particular organism than the codon in the original sequence (e.g., the codon is optimized for expression in human or listeria monocytogenes). Examples of the nucleic acids encoding the mutated peptides disclosed herein are provided in SEQ ID NO 223-977.
Recombinant fusion polypeptides
Disclosed herein are recombinant fusion polypeptides comprising a PEST-containing peptide fused to one or more tumor-associated antigenic peptides disclosed elsewhere herein that comprise a mutated mutation (i.e., fused to one or more immunogenic fragments of a cancer-associated protein, wherein each fragment comprises a mutated mutation).
Also disclosed herein are recombinant fusion polypeptides comprising one or more tumor-associated antigenic peptides disclosed elsewhere herein (i.e., fused to one or more immunogenic fragments of a cancer-associated protein, wherein each fragment comprises a mutated mutation), and wherein the fusion polypeptides do not comprise a PEST-containing peptide.
Also provided herein are tumor-associated antigenic peptides comprising, from N-terminus to C-terminus, a bacterial secretory sequence, a ubiquitin (Ub) protein, and one or more of the mutations disclosed elsewhere herein (i.e., fused to one or more immunogenic fragments of a cancer-associated protein, wherein each fragment comprises a mutation), i.e., in tandem, such as Ub-peptide 1-peptide 2. Alternatively, a combination of individual fusion polypeptides may be used, wherein each antigenic peptide is fused to its own secretory sequence and to the Ub protein (e.g., Ub 1-peptide 1; Ub 2-peptide 2).
Nucleic acids encoding the recombinant fusion polypeptides (referred to as minigene constructs) are also disclosed. The minigene nucleic acid construct can further comprise two or more open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between the open reading frames. For example, a minigene nucleic acid construct can further comprise two to four open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between the open reading frames. Each open reading frame may encode a different polypeptide. In some nucleic acid constructs, the codon encoding the carboxy terminus of the fusion polypeptide is followed by two stop codons to ensure termination of protein synthesis.
The bacterial signal sequence may be a listeria signal sequence, such as an Hly or ActA signal sequence, or any other known signal sequence. In other cases, the signal sequence may be an LLO signal sequence. An exemplary LLO signal sequence is shown as SEQ ID NO: 97. The signal sequence can be bacterial, can be native to the host bacterium (e.g., listeria monocytogenes, such as the secA1 signal peptide), or can be foreign to the host bacterium. Specific examples of signal peptides include the Usp45 signal peptide from Lactococcus lactis (Lactococcus lactis), the signal peptide from Bacillus anthracis (Bac)illusanthraceis), secA2 signal peptide from listeria monocytogenes such as the p60 signal peptide, and Tat signal peptide such as the bacillus subtilis Tat signal peptide (e.g., PhoD). In particular examples, the secretion signal sequence is from a listerial protein, such as ActA300Secretion signal or ActA100A secretion signal. An exemplary ActA signal sequence is shown as SEQ ID NO 98.
Ubiquitin can be, for example, a full-length protein. An exemplary ubiquitin sequence is shown in SEQ ID NO: 188. Ubiquitin expressed by the nucleic acid constructs provided herein can be cleaved at the carboxy terminus from the remainder of the recombinant fusion polypeptide expressed by the nucleic acid construct by the action of a hydrolase upon entry into the host cell cytosol. This releases the amino terminus of the fusion polypeptide, thereby producing the peptide in the host cell cytosol.
The selection, variations and arrangement of antigenic peptides within a fusion polypeptide are discussed in detail elsewhere herein, and tumor-associated antigenic peptides comprising a mutated mutation are discussed in more detail elsewhere herein.
The recombinant fusion polypeptide may comprise one or more tags. For example, a recombinant fusion polypeptide can comprise one or more peptide tags at the N-terminus and/or C-terminus of one or more antigenic peptides. The tag may be fused directly to the antigenic peptide or linked to the antigenic peptide by a linker, examples of which are disclosed elsewhere herein. Examples of labels include the following: a FLAG label; a 2xFLAG tag; a 3xFLAG tag; his tag, 6xHis tag; and a SIINFEKL tag. An exemplary SIINFEKL tag is shown as SEQ ID NO:16 (encoded by any of the nucleic acids shown as SEQ ID NOS: 1-15). An exemplary 3xFLAG tag is shown as SEQ ID NO:32 (encoded by any of the nucleic acids shown as SEQ ID NO: 17-31). An exemplary variant 3xFLAG tag is shown as SEQ ID NO: 99. Two or more tags may be used together, such as a 2xFLAG tag and a SIINFEKL tag, a 3xFLAG tag and a SIINFEKL tag, or a 6xHis tag and a SIINFEKL tag. If two or more tags are used, they may be positioned anywhere within the recombinant fusion polypeptide and in any order. For example, two tags can be at the C-terminus of the recombinant fusion polypeptide, two tags can be at the N-terminus of the recombinant fusion polypeptide, two tags can be located internally within the recombinant fusion polypeptide, one tag can be at the C-terminus of the recombinant fusion polypeptide and one tag at the N-terminus, one tag can be at the C-terminus and one tag internal within the recombinant fusion polypeptide, or one tag can be at the N-terminus and one tag internal within the recombinant fusion polypeptide. Other tags include Chitin Binding Protein (CBP), Maltose Binding Protein (MBP), glutathione-S-transferase (GST), Thioredoxin (TRX) and poly (NANP). Particular recombinant fusion polypeptides comprise a C-terminal SIINFEKL tag. The tag may allow for easy detection of the recombinant fusion protein, confirmation of secretion of the recombinant fusion protein, or tracking of the immunogenicity of the secreted fusion polypeptide by tracking the immune response to these "tag" sequence peptides. The immune response can be monitored using a number of reagents including, for example, monoclonal antibodies and DNA or RNA probes specific for these tags.
The recombinant fusion polypeptides disclosed herein may be expressed by recombinant listeria strains, or may be expressed and isolated from other vectors and cell systems for protein expression and isolation. Recombinant listeria strains expressing the antigenic peptides can be used, for example, in immunogenic compositions comprising the recombinant listeria and in vaccines comprising the recombinant listeria strains and adjuvants. Expression of one or more antigenic peptides as fusion polypeptides with non-hemolytic truncated forms of LLO, ActA or PEST-like sequences in host cell systems employing listeria strains and host cell systems other than listeria can result in enhanced immunogenicity of the antigenic peptide.
Also disclosed herein are nucleic acids encoding the recombinant fusion polypeptides. The nucleic acid may be in any form. The nucleic acid may comprise or consist of DNA or RNA, and may be single-stranded or double-stranded. The nucleic acid can be in the form of a plasmid, such as an episomal plasmid, a multicopy episomal plasmid, or an integrative plasmid. Alternatively, the nucleic acid may be in the form of a viral vector, a phage vector, or in the form of a bacterial artificial chromosome. The nucleic acid may have one open reading frame or may have two or more open reading frames (e.g., an open reading frame encoding a recombinant fusion polypeptide and a second open reading frame encoding a metabolic enzyme). In one example, the nucleic acid can comprise two or more open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between the open reading frames. For example, the nucleic acid can comprise two to four open reading frames linked by a Shine-Dalgarno ribosome binding site nucleic acid sequence between each open reading frame. Each open reading frame may encode a different polypeptide. In some nucleic acids, the codon encoding the carboxy terminus of the fusion polypeptide is followed by two stop codons to ensure termination of protein synthesis.
A. Antigenic peptides
The recombinant fusion polypeptides disclosed herein comprise one or more tumor-associated antigenic peptides disclosed elsewhere herein that comprise a mutated mutation (i.e., immunogenic fragments of a cancer-associated protein, wherein each fragment comprises a mutated mutation). A fusion polypeptide may comprise a single antigenic peptide, or may comprise two or more antigenic peptides. Each antigenic peptide can be of any length sufficient to induce an immune response, and each antigenic peptide can be of the same length, or the antigenic peptides can be of different lengths. Examples of suitable lengths of the heteroantigenic peptides are disclosed elsewhere herein.
Each antigenic peptide may also be hydrophilic or may be scored up to or below a certain hydrophilicity threshold that may predict secretability in listeria monocytogenes or another target bacterium. For example, antigenic peptides can be scored through the Kyte and Doolittle hydropathic index 21 amino acid window, and all scores above the cutoff value (about 1.6) can be excluded because they are unlikely to be secreted by Listeria monocytogenes. Likewise, a combination of antigenic peptides or fusion polypeptides may be hydrophilic, or may be scored up to or below a certain hydrophilicity threshold that may predict secretability in listeria monocytogenes or another target bacterium.
The antigenic peptides may be linked together in any manner. For example, antigenic peptides can be directly fused to each other without intervening sequences. Alternatively, the antigenic peptides may be indirectly linked to each other through one or more linkers, such as peptide linkers. In some cases, some pairs of adjacent antigenic peptides can be directly fused to each other, and other antigenic peptides can be indirectly linked to each other through one or more linkers. The same linker may be used between each pair of adjacent antigenic peptides, or any number of different linkers may be used between different pairs of adjacent antigenic peptides. In addition, one linker may be used between a pair of adjacent antigenic peptides, or multiple linkers may be used between a pair of adjacent antigenic peptides.
Any suitable sequence may be used for the peptide linker. As an example, the linker sequence may be, for example, 1 to about 50 amino acids in length. Some linkers may be hydrophilic. The joint can meet different purposes. For example, the linker may be used to increase bacterial secretion, facilitate antigen processing, increase the flexibility of the fusion polypeptide, increase the rigidity of the fusion polypeptide, or any other purpose. As a specific example, one or more or all of a flexible linker, a rigid linker, and an immunoproteasome processing linker may be used. Examples of such linkers are provided below. In some cases, different amino acid linker sequences are distributed between antigenic peptides, or different nucleic acids encoding the same amino acid linker sequence are distributed between antigenic peptides (e.g., SEQ ID NOS: 84-94) to minimize repeats. This can also serve to simplify secondary structure, thereby allowing efficient transcription, translation, secretion, maintenance or stabilization of the nucleic acid (e.g., plasmid) encoding the fusion polypeptide within the population of Lm recombinant vector strains. Other suitable peptide linker sequences may be selected, for example, based on one or more of the following factors: (1) they are capable of adopting a flexible extended conformation; (2) they cannot adopt secondary structures that can interact with functional epitopes on antigenic peptides; and (3) lack of hydrophobic or charged residues that may react with a functional epitope. For example, a peptide linker sequence may contain Gly, Asn, and Ser residues. Other near neutral amino acids such as Thr and Ala may also be used in the linker sequence. Amino acid sequences that are suitably used as linkers include Maratea et al (1985) Gene 40: 39-46; murphy et al (1986) Proc Natl Acad Sci USA 83: 8258-8262; US 4,935,233; and those disclosed in US 4,751,180, each of which is incorporated by reference herein in its entirety for all purposes. Specific examples of linkers include those in the tables below (each of which can be used alone as a linker, in linkers comprising repeats of a sequence, in linkers further comprising one or more other sequences in the tables), but other linkers are also contemplated (see, e.g., Reddy Chichili et al (2013) Protein Science 22: 153-. Unless specified, "n" represents an undetermined number of repeats in the listed linker.
Peptide linker
Examples of the invention
SEQ ID NO:
Assumed purpose
(GAS)n
GASGAS
33
Flexibility
(GSA)n
GSAGSA
34
Flexibility
(G)n；n＝4-8
GGGG
35
Flexibility
(GGGGS)n；n＝1-3
GGGGS
36
Flexibility
VGKGGSGG
VGKGGSGG
37
Flexibility
(PAPAP)n
PAPAP
38
Rigidity of the film
(EAAAK)n；n＝1-3
EAAAK
39
Rigidity of the film
(AYL)n
AYLAYL
40
Antigen processing
(LRA)n
LRALRA
41
Antigen processing
(RLRA)n
RLRA
42
Antigen processing
AAY
AAY
N/A
Immunoproteasome processing
ADLVVG
ADLVVG
209
Immunoproteasome processing
ADLIEATAEEVL
ADLIEATAEEVL
210
Immunoproteasome processing
GDGSIVSLAKTA
GDGSIVSLAKTA
211
Immunoproteasome processing
RDGSVADLAKVA
RDGSVADLAKVA
212
Immunoproteasome processing
ADGSVKTLSKVL
ADGSVKTLSKVL
213
Immunoproteasome processing
GDGSIVDGSKEL
GDGSIVDGSKEL
214
Immunoproteasome processing
GDGSIKTAVKSL
GDGSIKTAVKSL
215
Immunoproteasome processing
ADLSVATLAKSL
ADLSVATLAKSL
216
Immunoproteasome processing
ADLAVKTLAKVL
ADLAVKTLAKVL
217
Immunoproteasome processing
The VGKGGSGG linker (SEQ ID NO:37) can be used, for example, to provide flexibility and to charge balance the fusion protein. The EAAAK linker (SEQ ID NO:39) is a rigid/inflexible linker that can be used to facilitate expression and secretion, for example if the fusion protein would otherwise fold upon itself. GGGGS linker (SEQ ID NO:36) is a flexible linker that can be used, for example, to add increased flexibility to a fusion protein to help promote expression and secretion. As the mutant 9-mer designed and disclosed herein, the "i 20" linker (e.g., SEQ ID NO:209-217) is, for example, an immunoproteasome linker designed to help facilitate cleavage of the fusion protein by immunoproteasome and increase the frequency of obtaining the precise minimum binding fragment required. The combination of GGGGS and EAAAK linkers (SEQ ID NOS: 36 and 39, respectively) can be used, for example, to alternate flexibility and rigidity to help balance the construct for expression and secretion improvement, and to help facilitate DNA synthesis by providing more unique codons for selection.
The fusion polypeptide can comprise any number of heteroantigenic peptides. In some cases, the fusion polypeptide comprises any number of heteroantigenic peptides such that the fusion polypeptide is capable of being produced and secreted from a recombinant listeria strain. For example, a fusion polypeptide can comprise at least 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29, or 30 heteroantigenic peptides, or 2-50,2-45,2-40,2-35,2-30,2-25,2-20,2-15,2-10,2-5,5-10,10-15,15-20,20-25,25-30,30-35,35-40,40-45, or 45-50 heteroantigenic polypeptides. In another example, a fusion polypeptide can include a single heteroantigenic peptide. In another example, a fusion polypeptide can be included between about 1-100, 1-5, 5-10,10-15,15-20, 10-20, 20-30, 30-40,40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 5-15, 5-20, 5-25, 15-20, 15-25, 15-30, 15-35, 20-25, 20-35, 20-45, 30-55, 40-65, 50-75, 60-85, 70-95, 80-105, 95-105, 50-100, 1-100, 5-75, 1-100, or a combination thereof, A number of heteroantigenic peptides ranging from 5-50, 5-40, 5-30, 5-20, 5-15, 5-10, 1-100, 1-75, 1-50, 1-40, 1-30, 1-20, 1-15, or 1-10 heteroantigenic peptides. In another example, a fusion polypeptide can include up to about 100, 10, 20, 30, 40, or 50 heteroantigenic peptides. In another example, a fusion polypeptide can comprise about 2, 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 variant antigens.
In addition, a fusion polypeptide can comprise any number of heteroantigenic peptides from the same cancer-related protein (i.e., any number of non-contiguous fragments from the same cancer-related protein). Alternatively, the fusion polypeptide may comprise any number of heteroantigenic peptides from two or more different cancer-associated proteins, such as from 2, 3,4,5,6,7,8,9, or 10 cancer-associated proteins. For example, the fusion polypeptide can comprise a mutation from at least 2, 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19, or 20 cancer-associated proteins, or 2-5,5-10,10-15, or 15-20 cancer-associated proteins. For example, the two or more cancer-associated proteins can be about 2-30, about 2-25, about 2-20, about 2-15, or about 2-10 cancer-associated proteins. For example, a fusion polypeptide can comprise at least 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29, or 30 heteroantigenic peptides from the same cancer-associated protein, or 2-50,2-45,2-40,2-35,2-30,2-25,2-20,2-15,2-10,2-5,5-10,10-15,15-20,20-25,25-30,30-35,35-40,40-45, or 45-50 heteroantigenic polypeptides from the same cancer-associated protein. Likewise, a fusion polypeptide can comprise at least 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29, or 30 heteroantigenic peptides from the same cancer-associated protein, or 2-50,2-45,2-40,2-35,2-30,2-25,2-20,2-15,2-10,2-5,5-10,10-15,15-20,20-25,25-30,30-35,35-40,40-45, or 45-50 antigenic heteropolypeptides from two or more different cancer-associated proteins. In addition, a fusion polypeptide can comprise any number of non-contiguous variant antigenic peptides from the same cancer-related protein (i.e., any number of non-contiguous fragments from the same cancer-related protein). For example, a fusion polypeptide can comprise at least 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29, or 30 non-contiguous, heteroantigenic peptides from the same cancer-associated protein, or 2-50,2-45,2-40,2-35,2-30,2-25,2-20,2-15,2-10,2-5,5-10,10-15,15-20,20-25,25-30,30-35,35-40,40-45, or 45-50 non-contiguous, heteroantigenic peptides from the same cancer-associated protein. In some cases, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or all of the heteroantigenic peptides are non-continuous heteroantigenic peptides from the same cancer-associated protein, or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or all of the heteroantigenic peptides from a single cancer-associated protein are non-continuous heteroantigenic peptides from that cancer-associated protein.
Each heteroantigenic peptide may comprise different (i.e., unique) heteromutation(s). Alternatively, two or more of the variant antigenic peptides in the fusion polypeptide may comprise the same variant mutation. For example, two or more copies of the same variant antigenic polypeptide may be included in a fusion polypeptide (i.e., the fusion polypeptide comprises two or more copies of the same variant antigenic peptide). In some fusion polypeptides, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the heteroantigenic peptides comprise a different (i.e., unique) heteromutation that is not present in any other heteroantigenic peptide.
In some cases, the at least two variant antigenic peptides can comprise overlapping fragments of the same cancer-associated protein. For example, two or more heteroantigenic peptides can comprise different heteromutation at the same amino acid residue of a cancer-associated protein.
Some heteroantigenic peptides can comprise at least two different heteromutations, at least three different heteromutations, or at least four different heteromutations.
Any combination of mutational mutations may be included in the fusion polypeptide. For example, a heteroantigenic peptide that binds to one or more different HLA types can be included. For example, a variant antigenic peptide can be identified that binds to one or more or all of the following HLA types: HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02.
The individual antigenic peptides of the fusion polypeptide can comprise mutational mutations from the same cancer-associated protein, or the combination of antigenic peptides of the fusion polypeptide can comprise mutational mutations from two or more cancer-associated proteins. For example, the fusion polypeptide can comprise a mutation from at least 2, 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19, or 20 cancer-associated proteins or 2-5,5-10,10-15, or 15-20 cancer-associated proteins. For example, the two or more cancer-associated proteins can be about 2-30, about 2-25, about 2-20, about 2-15, or about 2-10 cancer-associated proteins. In one example, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the heteroantigenic peptides comprise a heteromutation from the same cancer-associated protein. In another example, none of the variant antigenic peptides comprise a variant mutation from the same cancer-associated protein.
Exemplary sequences of the variant antigenic peptides are disclosed elsewhere herein. As an example, a heteroantigenic peptide can comprise, consist essentially of, or consist of: a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the antigenic peptide sequences disclosed herein.
As an example, a recombinant fusion polypeptide may comprise a variant peptide encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or all of the following genes: CEACAM5, MAGEA6, MAGEA4, GAGE1, NYESO1, STEAP1, and RNF 43. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, non-small cell lung cancer (NSCLC). The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such antigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all 11 of the heteroantigenic peptides in table 3, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all 11 of the sequences in table 3.
As another example, a recombinant fusion polypeptide can comprise a mutated peptide encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or all of the following genes: CEACAM5, MAGEA4, STEAP1, RNF43, SSX2, SART3, PAGE4, PSMA, and PSA. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, prostate cancer. The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the heteroantigenic peptides in table 5, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the sequences in table 5.
As another example, a recombinant fusion polypeptide may comprise a mutated peptide encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or all of the following genes: CEACAM5, STEAP1, MAGEA3, PRAME, hTERT, and SURVIVIN. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, pancreatic cancer. The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or all 12 of the heteroantigenic peptides in table 7, or peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or all 12 of the sequences in table 7.
As another example, a recombinant fusion polypeptide can comprise a variant peptide encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, GAGE1, NYESO1, RNF43, NUF2, KLHL7, MAGEA3, and PRAME. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, bladder cancer. The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all 14 of the heteroantigenic peptides in table 9, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, or all 13 of the sequences in table 9.
As another example, a recombinant fusion polypeptide may comprise a mutated peptide encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or all of the following genes: CEACAM5, STEAP1, RNF43, MAGEA3, PRAME and hTERT. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, breast cancer (e.g., ER + breast cancer). The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all 11 of the heteroantigenic peptides in table 11, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all 11 of the sequences in table 11.
As another example, a recombinant fusion polypeptide can comprise a variant peptide encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, PRAME, hTERT, STEAP1, RNF43, NUF2, KLHL7 and SART 3. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, uterine cancer. The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all 14 of the heteroantigenic peptides in table 13, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all 14 of the sequences in table 13.
As another example, a recombinant fusion polypeptide can comprise a variant peptide encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, STEAP1, RNF43, SART3, NUF2, KLHL7, PRAME and hTERT. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, ovarian cancer. The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all 14 of the heteroantigenic peptides in table 15, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all 14 of the sequences in table 15.
As another example, a recombinant fusion polypeptide can comprise a variant peptide encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, MAGEA6, STEAP1, RNF43, SART3, NUF2, KLHL7, and hTERT. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, a low-grade glioma. The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the heteroantigenic peptides in table 17, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the sequences in table 17.
As another example, a recombinant fusion polypeptide can comprise a variant peptide encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, MAGEA6, MAGEA4, GAGE1, NYESO1, STEAP1, RNF43, and MAGEA 3. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, colorectal cancer (e.g., MSS colorectal cancer). The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the heteroantigenic peptides in table 19, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the sequences in table 19.
As another example, a recombinant fusion polypeptide may comprise a mutated peptide encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or all of the following genes: CEACAM5, MAGEA4, STEAP1, NYESO1, PRAME and hTERT. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, head and neck cancer. The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the heteroantigenic peptides in table 21, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the sequences in table 21.
B. PEST-containing peptides
The recombinant fusion proteins disclosed herein comprise PEST-containing peptides. The PEST-containing peptide may be at the amino terminus (N-terminus) of the fusion polypeptide (i.e., at the N-terminus of the antigenic peptide), may be at the carboxy terminus (C-terminus) of the fusion polypeptide (i.e., at the C-terminus of the antigenic peptide), or may be embedded within the antigenic peptide. In some recombinant listeria strains and methods, the PEST-containing peptide is not part of the fusion polypeptide and is separate from the fusion polypeptide. Fusion of an antigenic peptide with a PEST-like sequence, such as an LLO peptide, can result in enhanced immunogenicity of the antigenic peptide, and can result in an increased cell-mediated and anti-tumor immune response (i.e., increased cell-mediated and anti-tumor immunity). See, e.g., Singh et al (2005) J Immunol 175(6):3663- & 3673, which is incorporated by reference herein in its entirety for all purposes.
PEST-containing peptides are peptides that comprise a PEST sequence or a PEST-like sequence. PEST sequences in eukaryotic proteins have been identified long ago. For example, proteins containing amino acid sequences rich in proline (P), glutamic acid (E), serine (S) and threonine (T) (PEST), which are typically, but not always, flanked by clusters containing several positively charged amino acids, have rapid intracellular half-lives (Rogers et al (1986) Science 234: 364-. In addition, these sequences have been reported to target proteins to the ubiquitin-proteosome pathway to achieve degradation (Rechsteiner and Rogers (1996) Trends biochem. Sci.21:267-271, which is incorporated by reference herein in its entirety for all purposes). This pathway is also used by eukaryotic cells to produce MHC class I-binding immunogenic peptides, and it has been hypothesized that the PEST sequence is abundant among the eukaryotic proteins that produce the immunogenic peptides (Realini et al (1994) FEBSLett.348:109-113, which is incorporated herein by reference in its entirety for all purposes). Prokaryotic proteins typically do not contain PEST sequences because they do not have this enzymatic pathway. However, PEST-like sequences rich in the amino acids proline (P), glutamic acid (E), serine (S) and threonine (T) have been reported at the amino terminus of LLOs and have been reported to be essential for Listeria monocytogenes pathogenicity (Decatur and Portnoy (2000) Science 290: 992-. The presence of this PEST-like sequence in LLO targets the protein for disruption by the proteolytic machinery of the host cell, such that once LLO has exerted its function and promoted listeria monocytogenes to escape the phagosome or phagolysosomal vesicles, it is disrupted before it can damage the cell.
The identification of PEST and PEST-like sequences is well known in the art and is described, for example, in Rogers et al (1986) Science 234(4774): 364-. PEST or PEST-like sequences can be identified using the PEST-find program. For example, PEST-like sequences may be regions rich in proline (P), glutamic acid (E), serine (S), and threonine (T) residues. Optionally, the PEST-like sequence may be flanked by one or more clusters containing several positively charged amino acids. For example, a PEST-like sequence may be defined as a hydrophilic segment of at least 12 amino acids in length with high local concentrations of proline (P), aspartic acid (D), glutamic acid (E), serine (S), and/or threonine (T) residues. In some cases, PEST-like sequences do not contain positively charged amino acids, i.e., arginine (R), histidine (H), and lysine (K). Some PEST-like sequences may contain one or more internal phosphorylation sites, and phosphorylation at these sites precedes protein degradation.
In one example, the PEST-like sequences conform to the algorithm disclosed by Rogers et al. In another example, PEST-like sequences conform to the algorithms disclosed by Rechsteiner and Rogers. PEST-like sequences can also be identified by initial scanning for positively charged amino acids R, H and K within the designated protein sequence. All amino acids between the positively charged side-linkers were counted and only those motifs containing a number of amino acids equal to or above the window size parameter were further considered. Optionally, a PEST-like sequence must contain at least one P, at least one D or E, and at least one S or T.
The quality of PEST motifs can be improved by means of scoring parameters based on local key amino acid enrichment and motif hydrophobicity. D. E, P, S and T are expressed in mass percent (w/w) and are corrected for one equivalent of D or E, one equivalent of P, and one equivalent of S or T. The calculation of hydrophobicity may also in principle follow the method of Kyte and Doolittle (1982) J.mol.biol.157:105, which are incorporated herein by reference in their entirety for all purposes. For simplified calculations, the Kyte-Doolittle hydrophilicity index, which originally ranged from-4.5 for arginine to +4.5 for isoleucine, was scaled to a positive integer using the following linear transformation that yields a value from 0 for arginine to 90 for isoleucine: hydrophilicity index 10 × Kyte-Doolittle hydrophilicity index + 45.
The hydrophobicity of a potential PEST motif can also be calculated as the sum of the product of the mole percent of each amino acid species and the hydrophobicity index. The desired PEST score is obtained as a combination of a local enrichment term and a hydrophobicity term, as represented by the following equation: PEST score 0.55 DEPST-0.5 hydrophobicity index.
Thus, a PEST-containing peptide may refer to a peptide having a score of at least +5 using the above algorithm. Alternatively, it may refer to a peptide having a score of at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 32, at least 35, at least 38, at least 40, or at least 45.
Any other available method or algorithm known in the art may also be used to identify PEST-like sequences. See, e.g., CaSPredictor (Garay-Malpartida et al (2005) Bioinformatics 21, suppl.1: i169-76, which is incorporated by reference herein in its entirety for all purposes). Another method that can be used is the following: the PEST index of each segment of appropriate length (e.g., a segment of 30-35 amino acids) is calculated by assigning the number 1 to the amino acids Ser, Thr, Pro, Glu, Asp, Asn, or Gln. The Coefficient Value (CV) for each PEST residue is 1, and the CV for each other AA (non-PEST) is 0.
Examples of PEST-like amino acid sequences are those shown in SEQ ID NOS: 43-51. An example of a PEST-like sequence is KENSISSMAPPASPPASPKTPIEKKHADEIDK (SEQ ID NO: 43). Another example of a PEST-like sequence is KENSISSMAPPASPPASPK (SEQ ID NO: 44). However, any PEST or PEST-like amino acid sequence may be used. PEST sequence peptides are known and described, for example, in US 7,635,479; US 7,665,238; and US 2014/0186387, each of which is incorporated by reference herein in its entirety for all purposes.
The PEST-like sequence may be from a listeria species, such as from listeria monocytogenes. For example, the Listeria monocytogenes ActA protein contains at least four of the sequences (SEQ ID NOS: 45-48), any of which is suitable for use in the compositions and methods disclosed herein. Other similar PEST-like sequences include SEQ ID NOS: 52-54. Streptolysin O (streptolysin O) protein from certain species of the genus Streptococcus (Streptococcus sp.) also contains PEST sequences. For example, Streptococcus pyogenes streptolysin O includes PEST-like sequence KQNTASTETTTTNEQPK (SEQ ID NO:49) at amino acids 35-51, and Streptococcus equisimilis streptolysin O includes PEST-like sequence KQNTANTETTTTNEQPK (SEQ ID NO:50) at amino acids 38-54. Another example of a PEST-like sequence is from Listeria seeligeri (Listeria seeligeri) cytolysin (cytolysin) encoded by the lso gene: RSEVTISPAETPESPPATP (e.g., SEQ ID NO: 51).
Alternatively, the PEST-like sequence may be derived from other prokaryotic organisms. Other prokaryotic organisms in which PEST-like amino acid sequences would be expected to be present include, for example, other listeria species.
(1) Listeriolysin O (LLO)
One example of a PEST-containing peptide that may be used in the compositions and methods disclosed herein is a listeriolysin o (llo) peptide. An example of an LLO protein is the protein designated GenBank accession number P13128 (SEQ ID NO: 55; the nucleic acid sequence is shown in GenBank accession number X15127). SEQ ID NO:55 is a proprotein (protein) including a signal sequence. The first 25 amino acids of the original protein are signal sequences and are cleaved from LLO when the LLO is secreted by bacteria, thereby producing a full-length active LLO protein with no signal sequence of 504 amino acids. The LLO peptides disclosed herein may comprise a signal sequence, or may comprise a peptide that does not include a signal sequence. Exemplary LLO proteins that can be used comprise, consist essentially of, or consist of: the sequence shown as SEQ ID NO. 55 or homologues, variants, isoforms, analogues, fragments of homologues, fragments of variants, fragments of analogues, and fragments of isoforms of SEQ ID NO. 55. Any sequence encoding a fragment of an LLO protein or a homolog, variant, isoform, analog, fragment of a homolog, fragment of a variant, or fragment of an analog of an LLO protein can be used. A homologous LLO protein can have, e.g., greater than 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% sequence identity to a reference LLO protein.
Another example of an LLO protein is shown in SEQ ID NO: 56. LLO proteins that can be used can comprise, consist essentially of, or consist of: the sequence shown as SEQ ID NO. 56 or homologues, variants, isoforms, analogs, fragments of homologues, fragments of variants, fragments of analogs, and fragments of isoforms of SEQ ID NO. 56.
Another example of an LLO protein is an LLO protein from listeria monocytogenes strain 10403S, as identified by GenBank accession no: ZP _01942330 or EBA21833, or as indicated by a corresponding reference number such as in GenBank accession No.: NZ _ AARZ01000015 or AARZ 01000015.1. Another example of an LLO protein is an LLO protein from Listeria monocytogenes strain 4b F2365 (see, e.g., GenBank accession No.: YP-012823), EGD-e strain (see, e.g., GenBank accession No.: NP-463733), or any other strain of Listeria monocytogenes. Another example of an LLO protein is an LLO protein from Flavobacterium order (Flavobacterium) bacterium HTCC2170 (see, e.g., GenBank accession No.: ZP-01106747 or EAR01433, or encoded by GenBank accession No.: NZ-AAOC 01000003). LLO proteins that can be used can comprise, consist essentially of, or consist of: any of the above LLO proteins or homologues, variants, isoforms, analogs, fragments of homologues, fragments of variants, fragments of analogs, and fragments of isoforms of the above LLO proteins.
Proteins homologous to LLO or homologues, variants, isoforms, analogs, fragments of homologues, fragments of variants, fragments of analogs, and fragments of isoforms thereof may also be used. One such example is the alveolysin (alveolysin) which can be found, for example, in Paenibacillus alvei (Paenibacillus alvei) (see, for example, GenBank accession No. P23564 or AAA22224, or encoded by GenBank accession No. M62709). Other such homologous proteins are known.
The LLO peptide can be a full-length LLO protein or a truncated LLO protein or an LLO fragment. Likewise, the LLO peptide can be an LLO peptide that retains one or more functionalities of the native LLO protein, or lacks one or more functionalities of the native LLO protein. For example, the retained LLO functionality may be allowing the bacterium (e.g., listeria) to escape from phagosomes or phagolysosomes, or enhancing the immunogenicity of the peptide to which it is fused. The retained functionality may also be a hemolytic function or an antigenic function. Alternatively, the LLO peptide can be a non-hemolytic LLO. Other functions of LLO are known, as are methods and assays for assessing LLO functionality.
The LLO fragment may be a PEST-like sequence or may comprise a PEST-like sequence. The LLO fragment may comprise one or more of an internal deletion, a truncation from the C-terminus, and a truncation from the N-terminus. In some cases, an LLO fragment may contain more than one internal deletion. Other LLO peptides can be full-length LLO proteins with one or more mutations.
Some LLO proteins or fragments have reduced hemolytic activity relative to wild-type LLO, or are non-hemolytic fragments. For example, the LLO protein can be rendered nonhemolytic by deletion or mutation of the activation domain at the carboxy terminus, by deletion or mutation of cysteine 484, or by deletion or mutation at another position.
As detailed in US 8,771,702, which is herein incorporated by reference in its entirety for all purposes, other LLO proteins are rendered nonhemolytic by deletion or mutation of the Cholesterol Binding Domain (CBD). Mutations may include, for example, substitutions or deletions. The entire CBD may be mutated, portions of the CBD may be mutated, or specific residues within the CBD may be mutated. For example, an LLO protein can comprise a mutation of one or more of residues C484, W491 and W492 (e.g., C484, W491, W492, C484 and W491, C484 and W492, W491 and W492, or all three residues) of SEQ ID NO:55 or of the corresponding residue (e.g., the corresponding cysteine or tryptophan residue) when optimally aligned with SEQ ID NO: 55. As an example, a mutant LLO protein can be produced in which residues C484, W491 and W492 of LLO are substituted with alanine residues that will substantially reduce hemolytic activity relative to wild-type LLO. Mutant LLO proteins with C484A, W491A and W492A mutations are referred to as "mutLLO".
As another example, a mutant LLO protein can be produced having an internal deletion comprising a cholesterol binding domain. The sequence of the cholesterol binding domain of SEQ ID NO. 55 is shown as SEQ ID NO. 74. For example, the internal deletion can be a 1-11 amino acid deletion, an 11-50 amino acid deletion, or a longer deletion. Similarly, the mutated region may be 1-11 amino acids, 11-50 amino acids, or longer (e.g., 1-50, 1-11, 2-11, 3-11, 4-11, 5-11, 6-11, 7-11, 8-11, 9-11, 10-11, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 12-50, 11-15, 11-11, 1-11, 2-11, 3-7, 11-20, 11-25, 11-30, 11-35, 11-40, 11-50, 11-60, 11-70, 11-80, 11-90, 11-100, 11-150, 15-20, 15-25, 15-30, 15-35, 15-40, 15-50, 15-60, 15-70, 15-80, 15-90, 15-100, 15-150, 20-25, 20-30, 20-35, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-150, 30-35, 30-40, 30-60, 30-70, 30-80, 30-90, 30-100, or 30-150 amino acids). For example, a mutated region consisting of residues 470-500, 470-510, or 480-500 of SEQ ID NO:55 will result in a deletion sequence comprising a CBD (residue 483-493 of SEQ ID NO: 55). However, the mutated region may also be a fragment of the CBD, or may overlap a portion of the CBD. For example, the mutation region may consist of residues 470-490, 480-488, 485-490, 486-488, 490-500, or 486-510 of SEQ ID NO: 55. For example, a fragment of CBD (residues 484-492) may be replaced by a heterologous sequence that will substantially reduce hemolytic activity relative to wild-type LLO. For example, the CBD (ECTGLAWEWWR; SEQ ID NO:74) may be replaced by a CTL epitope (ESLLMWITQCR; SEQ ID NO:75) from the antigen NY-ESO-1, said CTL epitope comprising the HLA-A2-restricted epitope 157-165 from NY-ESO-1. The resulting LLO is called "ctLLO".
In some mutant LLO proteins, the mutant region can be replaced with a heterologous sequence. For example, a mutant region can be substituted with an equal number of heterologous amino acids, a smaller number of heterologous amino acids, or a larger number of amino acids (e.g., 1-50, 1-11, 2-11, 3-11, 4-11, 5-11, 6-11, 7-11, 8-11, 9-11, 10-11, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-11, 1-4, 12-50, 11-15, 11-20, 11-25, 11-30, 11-35, 11-40, 11-50, 11-60, 11-70, 11-80, 11-90, 11-100, 11-150, 15-20, 15-25, 15-30, 15-35, 15-40, 15-50, 15-60, 15-70, 15-80, 15-90, 15-100, 15-150, 20-25, 20-30, 20-35, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 20-150, 30-35, 30-40, 30-60, 30-70, 30-80, 30-90, 30-100, or 30-150 amino acids). Other mutant LLO proteins have one or more point mutations (e.g., point mutations at 1,2, 3, or more residues). The mutated residues may be contiguous or non-contiguous.
In an example embodiment, the LLO peptide may have a deletion in the signal sequence and a mutation or substitution in the CBD.
Some LLO peptides are N-terminal LLO fragments (i.e., LLO proteins with C-terminal deletions). Some LLO peptides are at least 494, 489, 492, 493, 500, 505, 510, 515, 520, or 525 amino acids in length, or 492 amino acids in length. For example, an LLO fragment can consist of approximately the first 440 or 441 amino acids of an LLO protein (e.g., the first 441 amino acids of SEQ ID NO:55 or 56, or the corresponding fragment of another LLO protein when optimally aligned with SEQ ID NO:55 or 56). Other N-terminal LLO fragments can consist of the first 420 amino acids of an LLO protein (e.g., the first 420 amino acids of SEQ ID NO:55 or 56, or the corresponding fragment of another LLO protein when optimally aligned with SEQ ID NO:55 or 56). Other N-terminal fragments may consist of about amino acids 20-442 of an LLO protein (e.g., amino acids 20-442 of SEQ ID NO:55 or 56, or the corresponding fragment of another LLO protein when optimally aligned with SEQ ID NO:55 or 56). Other N-terminal LLO fragments contain any Δ LLO without an activation domain that contains cysteine 484, and in particular without cysteine 484. For example, the N-terminal LLO fragment can correspond to the first 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 50, or 25 amino acids of the LLO protein (e.g., the first 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 50, or 25 amino acids of SEQ ID NO:55 or 56, or the corresponding fragment of another LLO protein when optimally aligned with SEQ ID NO:55 or 56). Preferably, the fragment comprises one or more PEST-like sequences. The LLO fragments and truncated LLO proteins can contain residues of homologous LLO proteins corresponding to any of the above specified amino acid ranges. The number of residues need not correspond exactly to the number of residues listed above (e.g., if the homologous LLO protein has insertions or deletions relative to the particular LLO protein disclosed herein). Examples of N-terminal LLO fragments include SEQ ID NOS: 57, 58 and 59. LLO proteins that can be used comprise, consist essentially of, or consist of: homologs, variants, subtypes, analogs, fragments of homologs, fragments of variants, fragments of analogs, and fragments of subtypes of SEQ ID NO 57, 58, or 59. In some compositions and methods, the N-terminal LLO fragment shown as SEQ ID NO 59 is used. An example of a nucleic acid encoding the N-terminal LLO fragment shown as SEQ ID NO 59 is SEQ ID NO 60.
(2)ActA
Another example of a PEST-containing peptide that may be used in the compositions and methods disclosed herein is an ActA peptide. ActA is a surface associated protein and serves as a scaffold in infected host cells to promote polymerization, assembly and activation of host actin polymers to push listeria monocytogenes through the cytoplasm. Shortly after entering the cytosol of mammalian cells, listeria monocytogenes induces polymerization of host actin microfilaments and moves using the force generated by actin polymerization, first within the cell and then from cell to cell. ActA is responsible for mediating actin nucleation and actin-based motility. The ActA protein provides multiple binding sites for host cytoskeletal components, thereby acting as a scaffold to assemble the cellular actin polymerization machinery. The N-terminus of ActA binds monomeric actin and acts as a constitutively active nucleation-promoting factor by stimulating intrinsic actin nucleation activity. Both the actA gene and the hly gene are members of a10 kb gene cluster regulated by the transcriptional activator PrfA, and the actA is up-regulated to about 226-fold in the mammalian cytosol. Any sequence encoding an ActA protein or homolog, variant, isoform, analog, fragment of a homolog, fragment of a variant, or fragment of an analog of an ActA protein may be used. The homologous ActA protein may have, for example, greater than 70%, 72%, 75%, 78%, 80%, 82%, 83%, 85%, 87%, 88%, 90%, 92%, 93%, 95%, 96%, 97%, 98% or 99% sequence identity to the reference ActA protein.
An example of an ActA protein comprises, consists essentially of, or consists of: the sequence shown as SEQ ID NO 61. Another example of an ActA protein comprises, consists essentially of, or consists of: the sequence shown as SEQ ID NO: 62. The first 29 amino acids of the proprotein corresponding to either of these sequences are signal sequences and are cleaved from the ActA protein when secreted by bacteria. The ActA peptide may comprise a signal sequence (e.g., amino acids 1-29 of SEQ ID NOS: 61 or 62), or may include a peptide that does not include a signal sequence. Other examples of ActA proteins comprise, consist essentially of, or consist of: 61 or 62, or a fragment of a homologue, variant, isoform, analog, fragment of a homologue, isoform, or analog.
Another example of an ActA protein is an ActA protein from Listeria monocytogenes 10403S strain (GenBank accession No.: DQ054585), NICPBP 54002 strain (GenBank accession No.: EU394959), S3 strain (GenBank accession No.: EU394960), NCTC 5348 strain (GenBank accession No.: EU394961), NICPBP 54006 strain (GenBank accession No.: EU394962), M7 strain (GenBank accession No.: EU394963), S19 strain (GenBank accession No.: EU394964), or any other strain of Listeria monocytogenes. LLO proteins that can be used can comprise, consist essentially of, or consist of: any of the above LLO proteins or homologues, variants, isoforms, analogs, fragments of homologues, fragments of variants, fragments of analogs, and fragments of isoforms of the above LLO proteins.
The ActA peptide may be a full-length ActA protein or a truncated ActA protein or an ActA fragment (e.g., an N-terminal ActA fragment with the C-terminal portion removed). Preferably, the truncated ActA protein comprises at least one PEST sequence (e.g., more than one PEST sequence). In addition, the truncated ActA protein may optionally comprise an ActA signal peptide. Examples of PEST-like sequences contained in truncated ActA proteins include SEQ ID NOS 45-48. Some of the truncated ActA proteins comprise at least two PEST-like sequences shown as SEQ ID NOs 45-48 or homologues thereof, at least three PEST-like sequences shown as SEQ ID NOs 45-48 or homologues thereof, or all four PEST-like sequences shown as SEQ ID NOs 45-48 or homologues thereof. Examples of truncated ActA proteins include those comprising, consisting essentially of, or consisting of: about residues 30-122, about residues 30-229, about residues 30-332, about residues 30-200, or about residues 30-399 of a full-length ActA protein sequence (e.g., SEQ ID NO: 62). Other examples of truncated ActA proteins include those comprising, consisting essentially of, or consisting of: about the first 50, 100, 150, 200, 233, 250, 300, 390, 400, or 418 residues of a full-length ActA protein sequence (e.g., SEQ ID NO: 62). Other examples of truncated ActA proteins include those comprising, consisting essentially of, or consisting of: about residue 200-300 or residue 300-400 of the full-length ActA protein sequence (e.g., SEQ ID NO: 62). For example, a truncated ActA consists of the first 390 amino acids of a wild-type ActA protein as described in US 7,655,238, which is incorporated herein by reference in its entirety for all purposes. As another example, the truncated ActA can be ActA-N100 or a modified form thereof (referred to as ActA-N100), wherein the PEST motif has been deleted and contains a non-conservative QDNKR (SEQ ID NO:73) substitution, as described in US 2014/0186387, which is incorporated by reference herein in its entirety for all purposes. Alternatively, a truncated ActA protein may contain residues corresponding to more than one amino acid range or any of the amino acid ranges of the ActA peptides disclosed herein of the homologous ActA protein. The number of residues need not correspond exactly to the number of residues recited herein (e.g., if the homologous ActA protein has insertions or deletions relative to the ActA protein utilized herein, the number of residues may be adjusted accordingly).
Examples of truncated ActA proteins include, for example, proteins comprising, consisting essentially of, or consisting of: 63, 64, 65 or 66 or a homologue, variant, isoform, analog, fragment of a variant, fragment of an isoform, or fragment of an analog of SEQ ID NO 63, 64, 65 or 66. SEQ ID NO 63 is referred to as ActA/PEST1 and consists of amino acids 30-122 of the full length ActA sequence shown in SEQ ID NO 62. SEQ ID NO:64 is designated ActA/PEST2 or LA229 and consists of amino acids 30-229 of the full-length ActA sequence shown in SEQ ID NO: 62. SEQ ID NO 65 is referred to as ActA/PEST3 and consists of amino acids 30-332 of the full length ActA sequence shown as SEQ ID NO 62. SEQ ID NO 66 is referred to as ActA/PEST4 and consists of amino acids 30-399 of the full-length ActA sequence shown in SEQ ID NO 62. As a specific example, a truncated ActA protein consisting of the sequence shown in SEQ ID NO:64 can be used.
Examples of truncated ActA proteins include, for example, proteins comprising, consisting essentially of, or consisting of: the sequence shown as SEQ ID NO 67, 69, 70 or 72 or a homologue, variant, isoform, analog, fragment of a variant, fragment of an isoform, or fragment of an analog of SEQ ID NO 67, 69, 70 or 72. As a specific example, a truncated ActA protein consisting of the sequence shown as SEQ ID NO:67 (encoded by the nucleic acid shown as SEQ ID NO: 68) can be used. As another specific example, a truncated ActA protein consisting of the sequence shown as SEQ ID NO:70 (encoded by the nucleic acid shown as SEQ ID NO: 71) can be used. 71 is the first 1170 nucleotides encoding ActA in Listeria monocytogenes 10403S strain. In some cases, the ActA fragment may be fused to a heterologous signal peptide. For example, SEQ ID NO 72 shows an ActA fragment fused to an Hly signal peptide.
C. Production of immunotherapy constructs encoding recombinant fusion polypeptides
Also provided herein are methods for producing an immunotherapy construct encoding a recombinant fusion polypeptide disclosed herein or a composition comprising a recombinant fusion polypeptide disclosed herein. For example, the method can include selecting and designing antigenic peptides for inclusion in an immunotherapy construct (and, e.g., testing each antigenic peptide for hydrophilicity and modifying or deselecting it if its score is above a selected hydrophilicity index threshold), designing one or more fusion polypeptides comprising each of the selected antigenic peptides, and generating a nucleic acid construct encoding the fusion polypeptides.
Antigenic peptides can be screened for hydrophobicity or hydrophilicity. For example, an antigenic peptide can be selected if it is hydrophilic or if it scores up to or below a certain hydrophilicity threshold that can predict secretability in a particular target bacterium (e.g., listeria monocytogenes). For example, antigenic peptides can be scored by the Kyte and Doolittle hydropathic indices with a21 amino acid window, and all scores above the cutoff value (about 1.6) are excluded because they are unlikely to be secreted by listeria monocytogenes. See, e.g., Kyte-Doolittle (1982) JMol Biol 157(1): 105-; which is incorporated by reference herein in its entirety for all purposes. Alternatively, the score of the antigenic peptide with respect to the selected cut-off value can be varied (e.g., the length of the antigenic peptide is varied). Other sliding window sizes that may be used include, for example, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or more amino acids. For example, the sliding window size can be 9-11 amino acids, 11-13 amino acids, 13-15 amino acids, 15-17 amino acids, 17-19 amino acids, 19-21 amino acids, 21-23 amino acids, 23-25 amino acids, or 25-27 amino acids. Other cut-off values that may be used include, for example, the following ranges 1.2-1.4, 1.4-1.6, 1.6-1.8, 1.8-2.0, 2.0-2.2, 2.2-2.5, 2.5-3.0, 3.0-3.5, 3.5-4.0, or 4.0-4.5, or the cut-off value may be 1.4, 1.5, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, or 4.5. The cut-off value may vary, for example, depending on the genus or species of the bacterium used to deliver the fusion polypeptide.
Other suitable hydrophilicity profiles or other suitable dimensions include, for example, Rose et al (1993) Annu Rev BiomolStruct22: 381-415; biswas et al (2003) Journal of Chromatography A1000: 637-655; eisenberg (1984) Ann Rev Biochem 53: 595-623; abraham and Leo (1987) Proteins: Structure, Function and Genetics 2: 130-152; sweet and Eisenberg (1983) Mol Biol 171: 479-488; bull and Breese (1974) Arch Biochem Biophys 161: 665-670; guy (1985) Biophys J47: 61-70; miyazawa et al (1985) Macromolecules 18: 534-; roseman (1988) J Mol Biol200: 513-; wolfenden et al (1981) Biochemistry 20: 849-855; wilson (1981) Biochem J199: 31-41; cowan and Whittaker (1990) Peptide Research 3: 75-80; aboderin (1971) Int Jbiochem 2:537- > 544; eisenberg et al (1984) J Mol Biol 179: 125-142; hopp and Woods (1981) Proc Natl Acad Sci USA 78: 3824-; manavalan and Ponnuswamy (1978) Nature 275: 673-674; black and Mould (1991) Anal Biochem 193: 72-82; fauchere and Pliska (1983) Eur JMed Chem 18: 369-375; janin (1979) Nature 277: 491-492; rao and Argos (1986) BiochimBiophys Acta 869: 197-214; tanford (1962) Am Chem Soc84: 4240-4274; welling et al (1985) FEBS Lett 188: 215-218; parker et al (1986) Biochemistry25: 5425-5431; and those reported in Cowan and Whittaker (1990) Peptide Research 3:75-80, each of which is incorporated herein by reference in its entirety for all purposes.
Optionally, the antigenic peptides can be scored for their ability to bind to a subject's Human Leukocyte Antigen (HLA) type (e.g., by using an Immune Epitope Database (IED) available at www.iedb.org, which includes netMHCpan, ANN, SMMPMBEC, SMM, CombLib _ Sidney2008, PickPocket, and netMHCcons), and ranked according to the optimal MHC binding score for each antigenic peptide. Other resources include TEpredict (prediction. source. net/help. html) or other available MHC binding measurement metrics. The cut-off values may be different for different expression vectors, such as Salmonella (Salmonella).
Optionally, the antigenic peptide can be screened for immunosuppressive epitopes (e.g., T-reg epitopes, IL-10 inducible T helper epitopes, etc.) to deselect the antigenic peptide or to avoid immunosuppressive effects.
Optionally, predictive algorithms for the immunogenicity of epitopes can be used to screen antigenic peptides. However, these algorithms are no more than 20% accurate in predicting which peptide will produce a T cell response. Alternatively, no screening/prediction algorithm is used. Alternatively, antigenic peptides can be screened for immunogenicity. For example, this may include contacting one or more T cells with an antigenic peptide, and analyzing an immunogenic T cell response, wherein the immunogenic T cell response identifies the peptide as an immunogenic peptide. This may also include measuring secretion of at least one of CD25, CD44, or CD69 using an immunogenicity assay after contacting the one or more T cells with the peptide, or measuring secretion of a cytokine selected from the group comprising IFN- γ, TNF- α, IL-1, and IL-2, wherein an increase in secretion identifies the peptide as comprising one or more T cell epitopes.
The antigenic peptides of choice may be arranged into one or more candidate sequences for potential fusion polypeptides. If more antigenic peptides are available than can be packaged in a single plasmid, then different antigenic peptides can be prioritized and/or resolved into different fusion polypeptides (e.g., for inclusion in different recombinant listeria strains) as needed/desired. Priority can be determined by factors such as relative size, transcription priority, and/or overall hydrophobicity of the translated polypeptide. The antigenic peptides can be arranged such that they are directly linked together between any number of antigenic peptide pairs without a linker or any combination of linkers as disclosed in more detail elsewhere herein. The number of linear antigenic peptides to be included can be determined based on the following considerations: the number of constructs required relative to the mutation load, the efficiency of translation and secretion of multiple epitopes from a single plasmid, the MOI required for each bacterium or Lm comprising the plasmid.
Combinations of antigenic peptides or the entire fusion polypeptide (i.e., including antigenic peptides and PEST-containing peptides as well as any tags) can also be scored for hydrophobicity. For example, the entire or entire fusion polypeptide of the fusion antigenic peptide can be scored for hydrophilicity by the Kyte and Doolittle hydrophilicity indices with a sliding 21 amino acid window. If any of the regions score above a cut-off value (e.g., about 1.6), the antigenic peptides can be reordered or shuffled within the fusion polypeptide until an acceptable order for the antigenic peptides is obtained (i.e., an order in which no region scores above the cut-off value). Alternatively, any problematic antigenic peptides can be removed or redesigned to have a different size. Alternatively or additionally, one or more linkers between antigenic peptides as disclosed elsewhere herein may be added or modified to alter hydrophobicity. As with the hydrophilicity tests for individual antigenic peptides, other window sizes can be used, or other cut-off values can be used (e.g., depending on the genus or species of bacteria used to deliver the fusion polypeptide). In addition, other suitable hydrophilicity maps or other suitable dimensions may be used.
Optionally, the combination of antigenic peptides or the entire fusion polypeptide may be further screened for immunosuppressive epitopes (e.g., T-reg epitopes, IL-10 inducible T helper epitopes, etc.) to deselect the antigenic peptide or to avoid immunosuppressive effects.
The nucleic acid encoding the candidate antigenic peptide combination or fusion polypeptide can then be designed and optimized. For example, the sequence may be optimized for increased translation levels, expression duration, secretion levels, transcription levels, and any combination thereof. For example, it can be increased to 2-fold to 1000-fold, 2-fold to 500-fold, 2-fold to 100-fold, 2-fold to 50-fold, 2-fold to 20-fold, 2-fold to 10-fold, or 3-fold to 5-fold relative to a control non-optimized sequence.
For example, the fusion polypeptide or nucleic acid encoding the fusion polypeptide may be optimized for a reduced level of secondary structure that may be formed in the oligonucleotide sequence, or alternatively, optimized to prevent attachment of any enzyme that may modify the sequence. Expression in bacterial cells can be hindered, for example, by transcriptional silencing, low mRNA half-life, secondary structure formation, attachment sites for oligonucleotide binding molecules such as repressors and inhibitors, and the availability of rare tRNA pools. The source of many problems in bacterial expression is found within the original sequence. Optimization of the RNA can include modification of the cis-acting element, adapting its GC content, improving codon bias with respect to the non-limiting tRNA pool of the bacterial cell, and avoiding internal regions of homology. Thus, optimizing the sequence may entail, for example, adjusting regions of very high (> 80%) or very low (< 30%) GC content. Optimization of the sequence may also entail, for example, avoiding one or more of the following cis-acting sequence motifs: an internal TATA box, a khosam site and a ribosome entry site; an AT-rich or GC-rich sequence segment; repeat sequences and RNA secondary structures; (cryptic) splice donor and acceptor sites; a branch point; or a combination thereof. Optimizing expression may also entail adding sequence elements to flanking regions of the gene and/or elsewhere in the plasmid.
Optimization of the sequence may also entail, for example, codon bias to adapt codon usage to the host gene (e.g., the listeria monocytogenes gene). For example, the following codons can be used for Listeria monocytogenes.
A＝GCA
G＝GGT
L＝TTA
Q＝CAA
V＝GTT
C＝TGT
H＝CAT
M＝ATG
R＝CGT
W＝TGG
D＝GAT
I＝ATT
N＝AAC
S＝TCT
Y＝TAT
E＝GAA
K＝AAA
P＝CCA
T＝ACA
Termination is TAA
F＝TTC
A nucleic acid encoding the fusion polypeptide can be produced and introduced into a delivery vector, such as a bacterial strain or a listeria strain. Other delivery vectors may be suitable for DNA immunotherapy or peptide immunotherapy, such as vaccinia virus or virus-like particles. Once the plasmid encoding the fusion polypeptide is produced and introduced into the bacterial strain or listeria strain, the bacterial or listeria strain can be cultured and characterized to confirm expression and secretion of the fusion polypeptide comprising the antigenic peptide.
Recombinant bacteria or Listeria strains
Also provided herein are recombinant bacterial strains, such as listeria strains, comprising the mutated peptides or recombinant fusion polypeptides disclosed herein or nucleic acids encoding the mutated peptides or recombinant fusion polypeptides as disclosed elsewhere herein. Preferably, the bacterial strain is a listeria strain, such as a listeria monocytogenes (Lm) strain. However, other bacterial strains, such as Salmonella (Salmonella), Yersinia (Yersinia), Shigella (Shigella) or Mycobacterium (Mycobacterium) strains may also be used. Lm has many inherent advantages as a vaccine carrier. The bacterium grows extremely efficiently in vitro without special requirements, and it lacks LPS, which is the main virulence factor in gram-negative bacteria such as salmonella. Genetically attenuated Lm vectors also provide additional safety because they can be easily eliminated with antibiotics in cases of severe adverse effects, and unlike some viral vectors, integration of genetic material into the host genome does not occur.
The recombinant listeria strain can be any listeria strain. Examples of suitable Listeria strains include Listeria seel, Listeria gregaria (Listeria grayi), Listeria evans (listeriaavanovii), Listeria mullei (Listeria murrayi), Listeria wils (Listeria welshimeri), Listeria monocytogenes (Lm), or any other Listeria species known in the art. Preferably, the recombinant listeria strain is a strain of the species listeria monocytogenes. Examples of listeria monocytogenes strains include the following: listeria monocytogenes 10403S wild-type (see, e.g., Bishop and Hinrichs (1987) J Immunol 139: 2005-412009; Lauer et al (2002) J Bact184: 4177-4186); bacteriophage-depleted Listeria monocytogenes DP-L4056 (see, e.g., Lauer et al (2002) J Bact184: 4177-4186); listeria monocytogenes DP-L4027 with the eliminated phage and with the deletion of the hly gene (see, e.g., Lauer et al (2002) J Bact184: 4177-4186; Jones and Portnoy (1994) infection 65: 5608-5613); listeria monocytogenes DP-L4029 from which the phage has been eliminated and which has a deletion in the actA gene (see, e.g., Lauer et al (2002) J Bact184: 4177-4186; Skoble et al (2000) J Cell Biol 150: 527-538); listeria monocytogenes DP-L4042(Δ PEST) (see, e.g., Brockstedt et al (2004) Proc Natl Acadsi.USA 101:13832-13837 and supporting information); listeria monocytogenes DP-L4097(LLO-S44A) (see, e.g., Brockstedt et al (2004) Proc Natl Acad Sci USA101: 13832-; listeria monocytogenes DP-L4364 (DeltalplA; lipoic acid protein ligase) (see, e.g., Brockstedt et al (2004) Proc Natl Acad Sci USA101: 13832-; listeria monocytogenes DP-L4405(Δ inlA) (see, e.g., Brockstedt et al (2004) Proc Natl Acad SciUSA 101:13832-13837 and supporting information); listeria monocytogenes DP-L4406(Δ inlB) (see, e.g., Brockstedt et al (2004) Proc Natl Acad Sci USA101:13832-13837 and supporting information); listeria monocytogenes CS-LOOOl (. DELTA.acta;. DELTA.inlB) (see, e.g., Brockstedt et al (2004) Proc NatlAcad Sci USA101:13832-13837 and supporting information); listeria monocytogenes CS-L0002(Δ actA; Δ lplA) (see, e.g., Brockstedt et al (2004) Proc Natl Acad Sci USA101: 13832-; listeria monocytogenes CS-L0003(LLO L461T; DeltalplA) (see, e.g., Brockstedt et al (2004) Proc Natl Acad Sci USA101: 13832-; listeria monocytogenes DP-L4038(Δ actA; LLO L461T) (see, e.g., Brockstedt et al (2004) ProcNatl Acad Sci USA101: 13832-; listeria monocytogenes DP-L4384(LLO S44A; LLO L461T) (see, e.g., Brockstedt et al (2004) Proc Natl Acad Sci USA101: 13832-; listeria monocytogenes strains with a deletion of lplA1 (encoding lipoic acid protein ligase LplA1) (see, e.g., O' Riordan et al (2003) Science 302: 462-464); listeria monocytogenes DP-L4017 (10403S with LLO L461T) (see, e.g., US 7,691,393); listeria monocytogenes EGDs (see, e.g., GenBank accession number AL 591824). In another embodiment, the Listeria strain is Listeria monocytogenes EGD-e (see GenBank accession NC-003210; ATCC accession BAA-679); listeria monocytogenes DP-L4029(actA deletion, optionally in combination with a uvrAB deletion (DP-L4029uvrAB)) (see, e.g., US 7,691,393); a listeria monocytogenes actA/inlB double mutant (see, e.g., ATCC accession No. PTA-5562); listeria monocytogenes lplA mutant or hly mutant (see e.g. US 2004/0013690); listeria monocytogenes dal/dat double mutant (see e.g. US 2005/0048081). Other listeria monocytogenes strains include those modified (e.g., by plasmid and/or by genomic integration) to contain a nucleic acid encoding one of the following genes or any combination of the following genes: hly (LLO; Listeriolysin); iap (p 60); inlA; inlB; inlC; dal (alanine racemase); dat (D-amino acid aminotransferase); plcA; plcB; actA; or mediate the growth, distribution, breakdown of single-walled vesicles; decomposition of double-walled vesicles; binding to a host cell; or any nucleic acid that is taken up by the host cell. Each of the above references is incorporated by reference herein in its entirety for all purposes.
The recombinant bacterium or listeria can be wild-type virulent, can have attenuated virulence, or can be avirulent. For example, recombinant listeria can be sufficiently virulent to escape phagosomes or phagolysosomes and enter cytosol. The listeria strain can also be a live attenuated listeria strain comprising at least one attenuating mutation, deletion, or inactivation as disclosed elsewhere herein. Preferably, the recombinant listeria is an attenuated auxotrophic strain. An auxotrophic strain is a strain that is unable to synthesize a particular organic compound required for its growth. Examples of such strains are described in US 8,114,414, which is incorporated herein by reference in its entirety for all purposes.
Preferably, the recombinant listeria strain lacks an antibiotic resistance gene. For example, the recombinant listeria strain can comprise a plasmid that does not encode an antibiotic resistance gene. However, some of the recombinant listeria strains provided herein comprise plasmids that contain nucleic acids encoding antibiotic resistance genes. Antibiotic resistance genes can be used in conventional selection and cloning procedures commonly employed in molecular biology and vaccine preparation. Exemplary antibiotic resistance genes include gene products that confer resistance to ampicillin (ampicilin), penicillin (penicillin), methicillin (methicillin), streptomycin (streptomycin), erythromycin (erythromycin), kanamycin (kanamycin), tetracycline (tetracycline), Chloramphenicol (CAT), neomycin (neomycin), hygromycin (hygromycin), and gentamicin (gentamicin).
A. Bacteria or listeria strains comprising a mutated peptide or a recombinant fusion polypeptide or a nucleic acid encoding a mutated peptide or a recombinant fusion polypeptide
A recombinant bacterial strain disclosed herein (e.g., a listeria strain) comprises a mutated peptide or a recombinant fusion polypeptide disclosed herein or a nucleic acid encoding the mutated peptide or the recombinant fusion polypeptide as disclosed elsewhere herein.
In a bacterium or listeria strain comprising a nucleic acid encoding a mutated peptide or a recombinant fusion protein, the nucleic acid may be codon optimized. The optimal codons for each amino acid utilized by listeria monocytogenes are shown in US2007/0207170, which is incorporated by reference herein in its entirety for all purposes. A nucleic acid is codon optimized if at least one codon in the nucleic acid is replaced with a codon of that amino acid that is more frequently used by listeria monocytogenes than the codon in the original sequence.
The nucleic acid can be present in an episomal plasmid within the bacterium or listeria strain, and/or the nucleic acid can be genomically integrated in the bacterium or listeria strain. Some recombinant bacteria or listeria strains comprise two separate nucleic acids encoding two mutated peptides or recombinant fusion polypeptides as disclosed herein; one nucleic acid is in an episomal plasmid, and one nucleic acid is genomically integrated in a bacterium or listeria strain.
An episomal plasmid can be a plasmid that is stably maintained in vitro (in cell culture), in vivo (in a host), or both in vitro and in vivo. If in an episomal plasmid, the open reading frame encoding the mutated peptide or recombinant fusion polypeptide may be operably linked to promoter/regulatory sequences in the plasmid. If genomically integrated in a bacterial or Listeria strain, the open reading frame encoding the mutated peptide or recombinant fusion polypeptide may be operably linked to exogenous promoter/regulatory sequences or endogenous promoter/regulatory sequences. Examples of promoter/regulatory sequences suitable for driving constitutive expression of a gene are well known and include, for example, the hly, hlyA, actA, prfA, and p60 promoters of listeria, the streptococcal bac promoter, the Streptomyces griseus sgiA promoter, and the bacillus thuringiensis (b.thuringiensis) phaZ promoter. In some cases, the inserted target gene is not interrupted or subject to regulatory constraints, which often occurs as a result of integration into genomic DNA, and in some cases, the presence of the inserted heterologous gene does not result in rearrangement or interruption of an important region of the cell itself.
The recombinant bacteria or listeria strains can be prepared by transforming a bacteria or listeria strain or an attenuated bacteria or listeria strain described elsewhere herein with a plasmid or vector comprising a nucleic acid encoding a mutated peptide or a recombinant fusion polypeptide. The plasmid may be an episomal plasmid that does not integrate into the host chromosome. Alternatively, the plasmid may be an integrative plasmid that integrates into the chromosome of the bacterium or listeria strain. The plasmid used herein may also be a multicopy plasmid. Methods for transforming bacteria are well known and include calcium chloride-based competent cell methods, electroporation methods, phage-mediated transduction, chemical transformation techniques, and physical transformation techniques. See, e.g., de Boer et al (1989) Cell 56: 641-) -649; miller et al (1995) FASEB J.9: 190-; sambrook et al (1989) Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory, New York; ausubel et al (1997) Current Protocols in Molecular Biology, John Wiley & Sons, New York; gerhardt et al, eds, 1994, Methods for General and Molecular Bacteriology, American Society for microbiology, Washington, D.C.; and Miller,1992, a Short Course in bacterial genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, n.y., each of which is incorporated herein by reference in its entirety for all purposes.
Bacterial or listeria strains having genomically integrated heterologous nucleic acids can be prepared, for example, by using site-specific integration vectors, whereby homologous recombination is used to produce bacteria or listeria comprising an integrated gene. The integration vector may be any site-specific integration vector capable of infecting a bacterium or listeria strain. Such an integration vector may comprise, for example, a PSA attPP 'site, a gene encoding PSA integrase, a U153 attPP' site, a gene encoding U153 integrase, an a118 attPP 'site, a gene encoding a118 integrase, or any other known attPP' site or any other phage integrase.
The bacteria or listeria strains comprising an integrated gene can also be produced using any other known method for integrating a heterologous nucleic acid into the bacterial or listeria chromosome. Techniques for homologous recombination are well known and are described, for example, in Baloglu et al (2005) Vet Microbiol 109(1-2): 11-17); jiang et al (2005) Acta Biochim Biophys Sin (Shanghai)37(1):19-24) and US 6,855,320, each of which is incorporated herein by reference in its entirety for all purposes.
Integration into the bacterial or listeria chromosome can also be achieved using transposon insertion. Techniques for transposon insertion are well known and are described, for example, by Sun et al (1990) Infection and Immunity 58:3770-3778, which is incorporated herein by reference in its entirety for all purposes. Transposon mutagenesis achieves stable genome insertion, but the location in the genome where the heterologous nucleic acid has been inserted is unknown.
Integration into the bacterial or Listeria chromosome can also be achieved using a phage integration site (see, e.g., Lauer et al (2002) J Bacteriol 184(15):4177-4186, which is incorporated by reference herein in its entirety for all purposes). For example, the attachment sites for the integrase gene and phage (e.g., U153 or PSA Listeria phage) can be used to insert the heterologous gene into the corresponding attachment site (e.g., the 3' end of the comK or arg tRNA gene), which can be any suitable site in the genome. Endogenous prophages can be eliminated from the attachment sites used prior to integration of the heterologous nucleic acid. The method may, for example, produce a single copy integrant. To avoid the "phage elimination step," a PSA phage integration system based on PSA phage may be used (see, e.g., Lauer et al (2002) J Bacteriol 184:4177-4186, which is incorporated by reference herein in its entirety for all purposes). Maintenance of the integrated gene may require continuous selection, for example by antibiotics. Alternatively, a phage-based chromosomal integration system can be established that does not require selection with antibiotics. Alternatively, the auxotrophic host strain may be supplemented. For example, a phage-based chromosomal integration system for clinical applications can be used, wherein a host strain (e.g., Lm dal (-) dat (-) is used that is auxotrophic for essential enzymes including, for example, D-alanine racemase.
Conjugation can also be used to introduce genetic material and/or plasmids into bacteria. Methods for conjugation are well known and are described, for example, in Nikodinovic et al (2006) Plasmid 56(3): 223-.
In a particular example, a recombinant bacterium or listeria strain can comprise a nucleic acid encoding a mutated peptide or a recombinant fusion polypeptide in the form of an open reading frame with an endogenous actA sequence (encoding an actA protein) or an endogenous hly sequence (encoding an LLO protein) genomically integrated into the bacterium or listeria genome. For example, expression and secretion of the mutated peptide or fusion polypeptide may be under the control of an endogenous actA promoter and actA signal sequence, or may be under the control of an endogenous hly promoter and LLO signal sequence. As another example, a nucleic acid encoding a mutated peptide or a recombinant fusion polypeptide may replace the ActA sequence encoding an ActA protein or the hly sequence encoding an LLO protein.
Selection of the recombinant bacterium or listeria strain can be accomplished by any means. For example, antibiotic selection may be used. Antibiotic resistance genes can be used in conventional selection and cloning procedures commonly employed in molecular biology and vaccine preparation. Exemplary antibiotic resistance genes include gene products that confer resistance to ampicillin, penicillin, methicillin, streptomycin, erythromycin, kanamycin, tetracycline, Chloramphenicol (CAT), neomycin, hygromycin, and gentamicin. Alternatively, auxotrophic strains may be used and exogenous metabolic genes may (also) be used for selection instead of or in addition to antibiotic resistance genes. As an example, to select an auxotrophic bacterium comprising a plasmid encoding a metabolic enzyme or a supplemental gene provided herein, the transformed auxotrophic bacterium can be grown in a medium that will select for expression of the gene encoding a metabolic enzyme (e.g., an amino acid metabolism gene) or the supplemental gene. Alternatively, temperature sensitive plasmids may be used to select recombinants, or any other known means for selecting recombinants.
B. Attenuation of bacteria or Listeria strains
Recombinant bacterial strains disclosed herein (e.g., recombinant listeria strains) can be attenuated. The term "attenuated" encompasses a reduction in the ability of a bacterium to cause disease in a host animal. For example, the pathogenic characteristics of an attenuated listeria strain can be reduced compared to a wild-type listeria, but the attenuated listeria is capable of growing and maintaining in culture. As an example of intravenous inoculation of BALB/c mice with attenuated Listeria, 50% of the inoculated animals survived at a Lethal Dose (LD)50) Preferably to a LD higher than that of wild type Listeria50At least about 10 times, more preferably at least about 100 times, more preferably at least about 1,000 times, even more preferably at least about 10,000 times, and most preferably at least about 100,000 times. Thus, an attenuated listeria strain is one that does not kill the animal to which it is administered, or that kills the animal only when the number of bacteria administered is significantly greater than the number of wild-type, non-attenuated bacteria that would be required to kill the same animal. An attenuated bacterium should also be construed to mean a bacterium that is incapable of replicating in the general environment because the nutrients required for its growth are not present therein. Thus, bacteria are limited to replicating in a controlled environment where the required nutrients are provided. The attenuated strain has environmentSecurity because they cannot achieve uncontrolled copying.
(1) Methods of attenuating bacteria and listeria strains
Attenuation can be achieved by any known means. For example, the attenuated strain may lack one or more endogenous virulence genes or one or more endogenous metabolic genes. Examples of such genes are disclosed herein, and attenuation can be achieved by inactivating any one of the genes disclosed herein or any combination of the genes disclosed herein. Inactivation may be achieved, for example, by deletion or by mutation (e.g., inactivating mutation). The term "mutation" includes any type of mutation or modification to a sequence (nucleic acid or amino acid sequence) and may encompass deletion, truncation, insertion, substitution, disruption, or translocation. For example, mutations can include frameshift mutations, mutations that result in premature termination of a protein, or mutations of regulatory sequences that affect gene expression. Mutagenesis can be accomplished using recombinant DNA techniques, or using traditional mutagenesis techniques using mutagenic chemicals or radiation and subsequent selection of mutants. Deletion mutants may be preferred due to the low likelihood of reversion. The term "metabolic gene" refers to a gene encoding an enzyme involved in or required for the synthesis of a nutrient utilized by or required for the host bacterium. For example, the enzyme may be involved in or required for the synthesis of nutrients required for continued growth of the host bacteria. The term "virulence" gene includes genes whose presence or activity in the genome of an organism promotes pathogenicity of the organism (e.g., enables the organism to achieve colonization of the niche in the host (including attachment to cells), immune evasion (evading the host's immune response), immune suppression (suppressing the host's immune response), entry and exit of cells, or gain nutrition from the host).
A specific example of such an attenuated strain is Listeria monocytogenes (Lm) dal (-) dat (-) (Lmdd). Another example of such an attenuated strain is Lm dal (-) dat (-) Δ acta (LmddA). See, for example, US 2011/0142791, which is incorporated by reference herein in its entirety for all purposes. LmddA is based on a Listeria strain that has been attenuated due to deletion of the endogenous virulence gene, actA. The strain can maintain plasmids for achieving antigen expression in vivo and in vitro by complementing the dal gene. Alternatively, the LmddA may be a Listeria species of dal/dat/actA having mutations in the endogenous dal, dat and actA genes. The mutation may be, for example, a deletion or other inactivating mutation.
Another specific example of an attenuated strain is Lm prfA (-) or a strain with a partial deletion or inactivating mutation in the prfA gene. The PrfA protein controls expression of a regulatory element comprising an essential virulence gene required for Lm to colonize its vertebrate host; thus, prfA mutations strongly impair the ability of prfA to activate expression of prfA-dependent virulence genes.
Another specific example of an attenuated strain is Lm inlB (-) actA (-), in which the two genes essential for the natural virulence of the bacterium, internalizing protein B (internalin B) and act A, are deleted.
Other examples of attenuated bacteria or listeria strains include bacteria or listeria strains that lack one or more endogenous virulence genes. Examples of such genes include actA, prfA, plcB, plcA, inlA, inlB, inlC, inlJ and bsh in listeria. The attenuated listeria strain can also be a double mutant or triple mutant of any of the above mentioned strains. An attenuated listeria strain can comprise a mutation or deletion of each of the genes provided herein, or comprise a mutation or deletion of, for example, up to ten of any of the genes provided herein (e.g., including the actA, prfA, and dal/dat genes). For example, an attenuated listeria strain can comprise a mutation or deletion of an endogenous internalizing protein c (inlc) gene and/or a mutation or deletion of an endogenous actA gene. Alternatively, the attenuated listeria strain may comprise a mutation or deletion of the endogenous internalizing protein b (inlb) gene and/or a mutation or deletion of the endogenous actA gene. Alternatively, the attenuated listeria strain can comprise mutations or deletions of the endogenous inlB, inlC, and actA genes. Translocation of listeria into neighboring cells is inhibited by deletion of the endogenous actA gene and/or the endogenous inlC gene or the endogenous inlB gene involved in the process, thereby producing a high level of attenuation with increased immunogenicity and utility as a strain scaffold. The attenuated listeria strain can also be a double mutant comprising a mutation or deletion of both plcA and plcB. In some cases, strains can be constructed from the EGD listeria backbone.
The bacterium or listeria strain can also be an auxotrophic strain having a mutation in a metabolic gene. As an example, a strain may lack one or more endogenous amino acid metabolism genes. For example, the production of an auxotrophic listeria strain lacking D-alanine, for example, can be accomplished in a number of well-known ways, including deletion mutations, insertion mutations, frameshift mutations, mutations that result in premature termination of the protein, or mutations of regulatory sequences that affect gene expression. Deletion mutants may be preferred due to the low likelihood of reversion of the auxotrophic phenotype. As an example, the ability of D-alanine mutants produced according to the protocol presented herein to grow in the absence of D-alanine can be tested in a simple laboratory culture assay. Those mutants which do not grow in the absence of this compound can be selected.
Examples of endogenous amino acid metabolism genes include a vitamin synthesis gene, a gene encoding pantothenate synthetase, a D-glutamate synthetase gene, a D-alanine aminotransferase (dat) gene, a D-alanine racemase (dal) gene, dga, a gene involved in the synthesis of Diaminopimelic Acid (DAP), a gene involved in the synthesis of cysteine synthetase A (cysK), vitamin B12-independent methionine synthetase, trpA, trpB, trpE, asnB, gltD, gltB, leuA, argG, and thrC. Listeria strains can lack two or more of the genes (e.g., dat and dal). D-glutamate synthesis is controlled in part by the dal genes involved in the conversion of D-glu + pyr to α -ketoglutarate + D-ala and in the reverse reaction.
As another example, an attenuated listeria strain can lack endogenous synthetase genes, such as amino acid synthetic genes. Examples of the genes include folP, a gene encoding a dihydrouridine synthase family protein, ispD, ispF, a gene encoding phosphoenolpyruvate synthase, hisF, hisH, fliI, a gene encoding ribosomal large subunit pseudouridine synthase, ispD, a gene encoding bifunctional GMP synthase/glutaminamidotransferase protein, cobS, cobB, cbiD, a gene encoding uroporphyrin-III C-methyltransferase/uroporphyrinogen-III synthase, cobQ, pS, truB, dxs, mvaS, dapA, ispG, folC, a gene encoding citrate synthase, argJ, a gene encoding 3-deoxy-7-phosphoarabinoheptulosynthase, a gene encoding indole-3-glycero-phosphate synthase, a gene encoding anthranilate synthase/glutaminamidotransferase component, a gene encoding phosphoenolpyruvate synthase, a gene encoding a phosphoenolpyruvate synthase/glutaminamidotransferase component, a gene encoding a phosphoenolpyruvate synthase, a gene encoding a bifunctional enzyme, cobS, a cobB, a cbiD, a gene encoding a ribosyltransferase, a gene encoding a ribosyl, menB, a gene encoding a menadione-specific isochorismate synthase, a gene encoding phosphoribosylcarbonylglycinamidine synthase I or II, a gene encoding phosphoribosylaminoimidazole-butanedioic acid carboxamide synthase, carB, carA, thyA, mgsA, aroB, hepB, rluB, ilvB, ilvN, alsS, fabF, fabH, a gene encoding pseudouridine synthase, pyrG, truA, pabB, and atp synthase genes (e.g., atpC, atpD-2, aptG, atpA-2, etc.).
The attenuated listeria strain may lack endogenous phoP, aroA, aroC, aroD, or plcB. As another example, the attenuated listeria strain can lack an endogenous peptide transporter. Examples include those encoding ABC transporter/ATP-binding/permease protein, oligopeptide ABC transporter/oligopeptide-binding protein, oligopeptide ABC transporter/permease protein, zinc ABC transporter/zinc-binding protein, sugar ABC transporter, phosphate transporter, ZIP zinc transporter, drug-resistant transporter of the EmrB/QacA family, sulfate transporter, proton-dependent oligopeptide transporter, magnesium transporter, formate/nitrite transporter, spermidine/putrescine ABC transporter, Na/Pi cotransporter, sugar phosphate transporter, glutamine ABC transporter, master cotransporter family transporter, glycine betaine/L-proline ABC transporter, molybdenum ABC transporter, teichoic acid ABC transporter, cobalt ABC transporter, ammonium transporter, amino acid ABC transporter, cell division ABC transporter, manganese ABC transporter, Genes for the iron compound ABC transporter, the maltose/maltodextrin ABC transporter, the Bcr/CflA family drug-resistant transporter, and subunits of one or more proteins.
Other attenuated bacteria and listeria strains may lack endogenous metabolic enzymes for the metabolism of amino acids used in bacterial growth processes, replication processes, cell wall synthesis, protein synthesis, fatty acid metabolism, or any other growth or replication process. Likewise, the attenuated strain may lack endogenous metabolic enzymes that can catalyze the formation of, can catalyze the synthesis of, or can be involved in the synthesis of amino acids used in cell wall synthesis. Alternatively, amino acids can be used in cell wall biogenesis. Alternatively, the metabolic enzyme is a D-glutamic acid synthase as a component of the cell wall.
Other attenuated listeria strains may lack the metabolic enzymes encoded by the D-glutamate synthesis gene, dga, alr (alanine racemase) genes, or any other enzyme involved in alanine synthesis. Other examples of metabolic enzymes that listeria strains can lack include enzymes encoded by: serC (phosphoserine aminotransferase), asd (aspartate beta semialdehyde dehydrogenase; involved in the synthesis of the cell wall component diaminopimelic acid), the gene encoding gsaB-glutamate-1-semialdehyde aminotransferase (catalyzing the formation of 5-aminolevulinic acid from (S) -4-amino-5-oxopentanoic acid), hemL (catalyzing the formation of 5-aminolevulinic acid from (S) -4-amino-5-oxopentanoic acid), aspB (aspartate aminotransferase catalyzing the formation of oxaloacetate and L-glutamate from L-aspartate and 2-oxoglutarate), argF-1 (involved in arginine biosynthesis), aroE (involved in amino acid biosynthesis), aroB (involved in 3-dehydroquinic acid biosynthesis), aroD (involved in amino acid biosynthesis), aroC (involved in amino acid biosynthesis), hisB (involved in histidine biosynthesis), hisD (involved in histidine biosynthesis), hisG (involved in histidine biosynthesis), metX (involved in methionine biosynthesis), proB (involved in proline biosynthesis), argR (involved in arginine biosynthesis), argJ (involved in arginine biosynthesis), thil (involved in thiamine biosynthesis), LMOf2365_1652 (involved in tryptophan biosynthesis), aroA (involved in tryptophan biosynthesis), ilvD (involved in valine and isoleucine biosynthesis), ilvC (involved in valine and isoleucine biosynthesis), leuA (involved in leucine biosynthesis), dapF (involved in lysine biosynthesis), and thrB (involved in threonine biosynthesis) (all under GenBank accession No. NC _ 002973).
Attenuated listeria strains can be produced by mutating other metabolic enzymes such as tRNA synthetases. For example, the metabolic enzyme can be encoded by the trpS gene encoding a tryptophanyl tRNA synthetase. For example, the host strain bacteria may be a Δ (trpS aroA) and both markers may be contained in the integration vector.
Other examples of metabolic enzymes that can be mutated to produce an attenuated listeria strain include enzymes encoded by: murE (involved in the synthesis of diaminopimelic acid; GenBank accession No.: NC-003485), LMOf 2365-2494 (involved in teichoic acid biosynthesis), WecE (lipopolysaccharide biosynthesis protein rfFA; GenBank accession No.: AE014075.1), or amiA (N-acetylmuramoyl-L-alanine amidase). Other examples of metabolic enzymes include aspartate aminotransferase, histidinol-phosphate aminotransferase (GenBank accession No. NP _466347), or muramidate glycosylated protein GtcA.
Other examples of metabolic enzymes that can be mutated to produce an attenuated listeria strain include peptidoglycan component or precursor synthetases. The component may be, for example, UDP-N-acetylmuramyl pentapeptide, UDP-N-acetylglucosamine, MurNAc- (pentapeptide) -pyrophosphoryl-undecanol, GlcNAc-p- (1,4) -MurNAc- (pentapeptide) -pyrophosphoryl-undecanol, or any other peptidoglycan component or precursor.
Other examples of metabolic enzymes that can be mutated to produce an attenuated Listeria strain include metabolic enzymes encoded by murG, murD, murA-1 or murA-2 (all shown in GenBank accession NC-002973). Alternatively, the metabolic enzyme may be any other synthetase of a peptidoglycan component or precursor. The metabolic enzyme may also be a transglycosylase, a transpeptidase, a carboxypeptidase, any other class of metabolic enzyme, or any other metabolic enzyme. For example, the metabolic enzyme can be any other listeria metabolic enzyme or any other listeria monocytogenes metabolic enzyme.
Other bacterial strains may be attenuated by mutating the corresponding orthologous genes in other bacterial strains as described above for listeria.
(2) Method of supplementing attenuated bacteria and listeria strains
The attenuated bacteria or listeria strains disclosed herein can further comprise a nucleic acid comprising a complementing gene or encoding a metabolic enzyme that complements the attenuating mutation (e.g., complements an auxotroph of an auxotrophic listeria strain). For example, a nucleic acid having a first open reading frame encoding a fusion polypeptide as disclosed herein can further comprise a second open reading frame comprising a supplemental gene or encoding a supplemental metabolic enzyme. Alternatively, the first nucleic acid may encode a fusion polypeptide and the second nucleic acid alone may comprise a supplemental gene or encode a supplemental metabolic enzyme.
The complementing gene may be extrachromosomal, or may be integrated into the bacterial or listeria genome. For example, an auxotrophic listeria strain can comprise an episomal plasmid that comprises a nucleic acid encoding a metabolic enzyme. The plasmid will be contained episomally or extrachromosomally in the listeria genus. Alternatively, an auxotrophic listeria strain can comprise an integrative plasmid (i.e., an integrative vector) comprising a nucleic acid encoding a metabolic enzyme. The integrative plasmid can be used for integration into the listeria chromosome. Preferably, the episomal plasmid or the integrative plasmid lacks an antibiotic resistance marker.
Instead of or in addition to the antibiotic resistance gene, the metabolic gene(s) can (also) be used for selection. As an example, to select an auxotrophic bacterium comprising a plasmid encoding a metabolic enzyme or a supplemental gene provided herein, the transformed auxotrophic bacterium can be grown in a medium that will select for expression of the gene encoding a metabolic enzyme (e.g., an amino acid metabolism gene) or the supplemental gene. For example, a D-glutamic acid synthesis auxotrophic bacterium may be transformed with a plasmid containing a gene for D-glutamic acid synthesis, and the auxotrophic bacterium will grow in the absence of D-glutamic acid, while an auxotrophic bacterium that has not been transformed with a plasmid or does not express a plasmid encoding a protein for D-glutamic acid synthesis will not grow. Similarly, when transformed and expressing a plasmid comprising a nucleic acid encoding an amino acid metabolizing enzyme for D-alanine synthesis, a D-alanine synthesis auxotrophic bacterium will grow in the absence of D-alanine. Such methods for preparing suitable media containing or lacking essential growth factors, supplements, amino acids, vitamins, antibiotics, and the like are well known and commercially available.
Once an auxotrophic bacterium comprising a plasmid encoding a metabolic enzyme or a complementing gene as provided herein has been selected in an appropriate medium, the bacterium can be propagated in the presence of a selection pressure. The propagating may include growing the bacteria in a medium free of the auxotrophic factor. The presence of a plasmid expressing a metabolic enzyme or a complementing gene in an auxotrophic bacterium ensures that the plasmid will replicate together with the bacterium, thereby continuously selecting bacteria having the plasmid. The production of the bacteria or listeria strains can be readily scaled up by adjusting the volume of medium in which the plasmid-containing auxotrophic bacteria grow.
In a particular example, an attenuated strain is a strain having deletions of or inactivating mutations in dal and dat (e.g., listeria monocytogenes (Lm) dal (-) dat (-) (Lmdd) or Lm dal (-) dat (-) Δ acta (lmdda)), and the complementing gene encodes an alanine racemase (e.g., encoded by the dal gene) or a D-amino acid aminotransferase (e.g., encoded by the dat gene). An exemplary alanine racemase protein may have the sequence shown in SEQ ID NO:76 (encoded by SEQ ID NO: 78; GenBank accession number: AF038438), or may be a homolog, variant, subtype, analog, fragment of a homolog, fragment of a variant, fragment of an analog, or fragment of a subtype of SEQ ID NO: 76. The alanine racemase protein can also be any other listeria alanine racemase protein. Alternatively, the alanine racemase protein may be any other gram-positive alanine racemase protein or any other alanine racemase protein. An exemplary D-amino acid aminotransferase protein may have the sequence shown as SEQ ID NO:77 (encoded by SEQ ID NO: 79; GenBank accession No. AF038439), or may be a homolog, variant, subtype, analog, fragment of a homolog, fragment of a variant, fragment of an analog, or fragment of a subtype of SEQ ID NO: 77. The D-amino acid aminotransferase protein may also be any other Listeria D-amino acid aminotransferase protein. Alternatively, the D-amino acid aminotransferase protein may be any other gram-positive D-amino acid aminotransferase protein or any other D-amino acid aminotransferase protein.
In another particular example, the attenuated strain is a strain having a deletion of prfA or an inactivating mutation in prfA (e.g., Lm prfA (-)), and the complementing gene encodes a prfA protein. For example, the complementing gene may encode a mutant PrfA (D133V) protein that restores partial PrfA function. An example of a wild-type PrfA protein is shown as SEQ ID NO:80 (encoded by the nucleic acid shown as SEQ ID NO: 81), and an example of a D133V mutant PrfA protein is shown as SEQ ID NO:82 (encoded by the nucleic acid shown as SEQ ID NO: 83). The supplemental PrfA protein may be a homolog, variant, isoform, analog, fragment of a homolog, fragment of a variant, fragment of an analog, or fragment of an isoform of SEQ ID NO:80 or 82. The PrfA protein may also be any other listeria PrfA protein. Alternatively, the PrfA protein may be any other gram-positive PrfA protein or any other PrfA protein.
In another example, the bacterial strain or listeria strain can comprise a deletion of the actA gene or an inactivating mutation in the actA gene, and the complementing gene can include the actA gene to complement the mutation and restore function to the listeria strain.
Other auxotrophic strains and supplementation systems may also be employed for use with the methods and compositions provided herein.
C. Preparation and storage of bacteria or Listeria strains
Recombinant bacterial strains (e.g., listeria strains) have optionally been passaged through animal hosts. The generation can maximize the efficacy of the listeria strain as a vaccine carrier, can stabilize the immunogenicity of the listeria strain, can stabilize the virulence of the listeria strain, can increase the immunogenicity of the listeria strain, can increase the virulence of the listeria strain, can remove unstable strains of the listeria strain, or can reduce the prevalence of unstable strains of the listeria strain. Methods for passaging recombinant listeria strains through animal hosts are well known in the art and are described, for example, in US 2006/0233835, which is incorporated by reference herein in its entirety for all purposes.
Recombinant bacterial strains (e.g., listeria strains) can be stored in frozen cell banks or in frozen cell banks. Such a cell bank may be, for example, a master cell bank, a working cell bank, or a Good Manufacturing Practice (GMP) cell bank. Examples of "good manufacturing specifications" include those specified by 21CFR 210-. However, "good manufacturing practice" may also be specified by other standards for the production of clinical grade material or for human consumption, such as standards in countries other than the united states. The cell bank may be intended for the production of clinical grade material or may comply with regulatory regulations regarding human use.
The recombinant bacterial strain (e.g., a listeria strain) can also be from a stock of vaccine agents, from a frozen stock, or from a lyophilized stock.
The cell bank, frozen stock, or batch of vaccine agents may exhibit greater than 90% viability, e.g., after thawing. Thawing may be performed, for example, after cryopreservation or frozen storage for 24 hours. Alternatively, storage may continue, for example, for 2 days, 3 days, 4 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 5 months, 6 months, 9 months, or 1 year.
A cell bank, frozen stock, or batch of vaccine ingredients may be cryopreserved, for example, by a method comprising: growing a culture of a bacterial strain (e.g., a listeria strain) in a nutrient medium, freezing the culture in a solution comprising glycerol, and storing the listeria strain at less than-20 ℃. The temperature may be, for example, about-70 ℃ or between about-70 to about-80 ℃. Alternatively, a cell bank, frozen stock, or batch of vaccine ingredients may be cryopreserved by a method comprising: growing a culture of a listeria strain in a defined medium, freezing the culture in a solution comprising glycerol, and storing the listeria strain at less than-20 ℃. The temperature may be, for example, about-70 ℃ or between about-70 to about-80 ℃. Any defined microbiological culture medium can be used in this method.
Cultures (e.g., cultures of listeria vaccine strains used to produce a batch of listeria vaccine agents) can be inoculated, for example, from a cell bank, from a frozen stock, from a starter culture, or from a colony. The culture can be inoculated, for example, at the middle of log phase growth, at about the middle of log phase growth, or at another phase of growth.
The solution used for freezing may (also) optionally contain another viscous additive or an additive with freeze-resistant properties instead of or in addition to glycerol. Examples of such additives include, for example, mannitol, DMSO, sucrose, or any other viscous additive or additive having freeze-resistant properties.
The nutrient medium used to grow the culture of the bacterial strain (e.g., listeria strain) can be any suitable nutrient medium. Examples of suitable media include, for example, LB; TB; improving the non-animal product top grade broth; or a defined medium.
The growing step may be carried out by any known means of growing bacteria. For example, the growing step can be performed with a shake flask (such as a baffled shake flask), a batch fermentor, a stirred tank or flask, an airlift fermentor, a fed batch reactor, a continuous cell reactor, an immobilized cell reactor, or any other means of growing bacteria.
Optionally, a constant pH is maintained during growth of the culture (e.g., in a batch fermentor). For example, the pH may be maintained at about 6.0, at about 6.5, at about 7.0, at about 7.5, or at about 8.0. Likewise, the pH can be, for example, about 6.5 to about 7.5, about 6.0 to about 8.0, about 6.0 to about 7.0, or about 6.5 to about 7.5.
Optionally, a constant temperature may be maintained during growth of the culture. For example, the temperature may be maintained at about 37 ℃ or at 37 ℃. Alternatively, the temperature may be maintained at 25 deg.C, 27 deg.C, 28 deg.C, 30 deg.C, 32 deg.C, 34 deg.C, 35 deg.C, 36 deg.C, 38 deg.C or 39 deg.C.
Optionally, a constant dissolved oxygen concentration can be maintained during growth of the culture. For example, the dissolved oxygen concentration can be maintained at 20% saturation, 15% saturation, 16% saturation, 18% saturation, 22% saturation, 25% saturation, 30% saturation, 35% saturation, 40% saturation, 45% saturation, 50% saturation, 55% saturation, 60% saturation, 65% saturation, 70% saturation, 75% saturation, 80% saturation, 85% saturation, 90% saturation, 95% saturation, 100% saturation, or near 100% saturation.
Methods for lyophilizing and cryopreserving recombinant bacterial strains, such as listeria strains, are known. For example, a listeria culture can be flash frozen in liquid nitrogen and then stored at a final freezing temperature. Alternatively, the culture may be frozen in a more gradual manner (e.g., by placing the culture in a vial at the final storage temperature). The culture may also be frozen by any other known method for freezing bacterial cultures.
The storage temperature of the culture may for example be between-20 and-80 ℃. For example, the temperature may be significantly lower than-20 ℃ or no warmer than-70 ℃. Alternatively, the temperature may be about-70 ℃, -20 ℃, -30 ℃, -40 ℃, -50 ℃, -60 ℃, -80 ℃, -30 to-70 ℃, -40 to-70 ℃, -50 to-70 ℃, -60 to-70 ℃, -30 to-80 ℃, -40 to-80 ℃, -50 to-80 ℃, -60 to-80 ℃, or-70 to-80 ℃. Alternatively, the temperature may be cooler than 70 ℃ or cooler than-80 ℃.
Immunogenic, pharmaceutical and vaccine compositions
Also provided herein are immunogenic compositions, pharmaceutical compositions or vaccines comprising a mutated peptide as disclosed herein, a recombinant fusion polypeptide as disclosed herein, a nucleic acid encoding a mutated peptide or a recombinant fusion polypeptide as disclosed herein, or a recombinant bacterium or listeria strain as disclosed herein. An immunogenic composition comprising a listeria strain may be inherently immunogenic as it comprises a listeria strain, and/or the composition may also further comprise an adjuvant. Other immunogenic compositions include DNA immunotherapy or peptide immunotherapy compositions.
The term "immunogenic composition" refers to any composition containing an antigen that elicits an immune response against the antigen in a subject following exposure to the composition. The immune response elicited by the immunogenic composition can be directed to a particular antigen or to a particular epitope on an antigen.
The immunogenic composition may comprise a single mutated peptide or recombinant fusion polypeptide as disclosed herein, a nucleic acid encoding a mutated peptide or recombinant fusion polypeptide as disclosed herein, or a recombinant bacterium or listeria strain as disclosed herein, or it may comprise a plurality of different mutated peptides or recombinant fusion polypeptides as disclosed herein, nucleic acids encoding mutated peptides or recombinant fusion polypeptides as disclosed herein, or a recombinant bacterium or listeria strain as disclosed herein. For example, if a first recombinant fusion polypeptide comprises one antigenic peptide that a second recombinant fusion polypeptide does not comprise, then the first recombinant fusion polypeptide is different from the second recombinant fusion polypeptide. The two recombinant fusion polypeptides may include some of the same antigenic peptides and still be considered distinct. The different mutated peptides, recombinant fusion polypeptides, nucleic acids encoding mutated peptides or recombinant fusion polypeptides, or recombinant bacteria or listeria strains may be administered to a subject in parallel or sequentially. Sequential administration can be particularly useful when the drug substance comprising the recombinant listeria strain (or the mutated peptide, the recombinant fusion polypeptide, or the nucleic acid) disclosed herein is in different dosage forms (e.g., one agent is a tablet or capsule and the other agent is a sterile liquid) and/or is administered according to different dosing schedules (e.g., one composition from a mixture is administered at least daily while the other composition is administered less frequently, such as once per week, once every two weeks, or once every three weeks). The plurality of mutated peptides, recombinant fusion polypeptides, nucleic acids encoding mutated peptides or recombinant fusion polypeptides, or recombinant bacteria or listeria strains can each comprise a set of different antigenic peptides. Alternatively, two or more of the mutated peptides, recombinant fusion polypeptides, nucleic acids encoding mutated peptides, recombinant fusion polypeptides, or recombinant bacteria or listeria strains can comprise a set of identical antigenic peptides (e.g., a set of identical antigenic peptides in a different order).
Multiple variant peptides or fragments or recombinant fusion polypeptides can bind multiple different HLA types. For example, they may bind to one or more of the following HLA types: HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02.
As one example, an immunogenic composition may comprise a variant peptide (in the form of, for example, a peptide, nucleic acid, or bacterial vector) encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or all of the following genes: CEACAM5, MAGEA6, MAGEA4, GAGE1, NYESO1, STEAP1, and RNF 43. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, non-small cell lung cancer (NSCLC). The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the heteroantigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such antigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all 11 of the heteroantigenic peptides in table 3, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all 11 of the sequences in table 3.
As another example, an immunogenic composition may comprise a variant peptide (in the form of, for example, a peptide, nucleic acid, or bacterial vector) encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or all of the following genes: CEACAM5, MAGEA4, STEAP1, RNF43, SSX2, SART3, PAGE4, PSMA, and PSA. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, prostate cancer. The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the heteroantigenic peptides in table 5, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the sequences in table 5.
As another example, an immunogenic composition may comprise a variant peptide (in the form of, for example, a peptide, nucleic acid, or bacterial vector) encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or all of the following genes: CEACAM5, STEAP1, MAGEA3, PRAME, hTERT, and SURVIVIN. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, pancreatic cancer. The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or all 12 of the heteroantigenic peptides in table 7, or peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or all 12 of the sequences in table 7.
As another example, an immunogenic composition may comprise a variant peptide (in the form of, for example, a peptide, nucleic acid, or bacterial vector) encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, GAGE1, NYESO1, RNF43, NUF2, KLHL7, MAGEA3, and PRAME. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, bladder cancer. The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all 14 of the heteroantigenic peptides in table 9, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, or all 13 of the sequences in table 9.
As another example, an immunogenic composition may comprise a variant peptide (in the form of, for example, a peptide, nucleic acid, or bacterial vector) encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or all of the following genes: CEACAM5, STEAP1, RNF43, MAGEA3, PRAME and hTERT. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, breast cancer (e.g., ER + breast cancer). The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all 11 of the heteroantigenic peptides in table 11, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all 11 of the sequences in table 11.
As another example, an immunogenic composition may comprise a variant peptide (in the form of, for example, a peptide, nucleic acid, or bacterial vector) encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, PRAME, hTERT, STEAP1, RNF43, NUF2, KLHL7 and SART 3. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, uterine cancer. The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all 14 of the heteroantigenic peptides in table 13, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all 14 of the sequences in table 13.
As another example, an immunogenic composition may comprise a variant peptide (in the form of, for example, a peptide, nucleic acid, or bacterial vector) encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, STEAP1, RNF43, SART3, NUF2, KLHL7, PRAME and hTERT. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, ovarian cancer. The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all 14 of the heteroantigenic peptides in table 15, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all 14 of the sequences in table 15.
As another example, an immunogenic composition may comprise a variant peptide (in the form of, for example, a peptide, nucleic acid, or bacterial vector) encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, MAGEA6, STEAP1, RNF43, SART3, NUF2, KLHL7, and hTERT. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, a low-grade glioma. The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the heteroantigenic peptides in table 17, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the sequences in table 17.
As another example, an immunogenic composition may comprise a variant peptide (in the form of, for example, a peptide, nucleic acid, or bacterial vector) encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, MAGEA6, MAGEA4, GAGE1, NYESO1, STEAP1, RNF43, and MAGEA 3. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, colorectal cancer (e.g., MSS colorectal cancer). The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the heteroantigenic peptides in table 19, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the sequences in table 19.
As another example, an immunogenic composition may comprise a variant peptide (in the form of, for example, a peptide, nucleic acid, or bacterial vector) encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or all of the following genes: CEACAM5, MAGEA4, STEAP1, NYESO1, PRAME and hTERT. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, head and neck cancer. The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the heteroantigenic peptides in table 21, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the sequences in table 21.
The immunogenic composition can further comprise an adjuvant (e.g., two or more adjuvants), a cytokine, a chemokine, or a combination thereof. Optionally, the immunogenic composition may further comprise Antigen Presenting Cells (APCs) that may be autologous or allogenic to the subject.
The term adjuvant includes compounds or mixtures that enhance the immune response to an antigen. For example, an adjuvant can be a non-specific stimulator of an immune response, or a substance that allows for the creation of a depot (depot) in a subject that provides an even more enhanced and/or prolonged immune response when combined with the immunogenic compositions disclosed herein. Adjuvants may, for example, favor an immune response that is primarily Th 1-mediated, a Th 1-type immune response, or a Th 1-mediated immune response. Likewise, adjuvants may favor cell-mediated immune responses over antibody-mediated responses. Alternatively, an adjuvant may facilitate an antibody-mediated response. Some adjuvants may enhance the immune response by slowly releasing the antigen, while others may mediate their effects by any of the following mechanisms: increased cell infiltration, inflammation and migration to the injection site, particularly for Antigen Presenting Cells (APCs); promoting the activation state of APC by upregulating costimulatory signals or Major Histocompatibility Complex (MHC) expression; (ii) enhancing antigen presentation; or inducing cytokine release to achieve an indirect effect.
Examples of adjuvants include saponin (saponin) QS21, CpG-containing oligonucleotides, unmethylated CpG-containing oligonucleotides, MPL, TLR agonists, TLR4 agonists, TLR9 agonists, and the like,Imiquimod, a cytokine or nucleic acid encoding a cytokine, a chemokine or nucleic acid encoding a chemokine, IL-12 or nucleic acid encoding IL-12, IL-6 or nucleic acid encoding IL-6, and lipopolysaccharide. Another example of a suitable adjuvant is Montanide ISA 51. Montanide ISA 51 contains a natural metabolizable oil and a refined emulsifier. Other examples of suitable adjuvants include granulocyte/macrophage colony-stimulating factor (GM-CSF) or a peptide encoding granulocyte-A nucleic acid of macrophage colony stimulating factor and Keyhole Limpet Hemocyanin (KLH) or a nucleic acid encoding keyhole limpet hemocyanin. GM-CSF may be, for example, a human protein produced in a yeast (s.cerevisiae) vector. GM-CSF facilitates clonal expansion and differentiation of hematopoietic progenitor cells, Antigen Presenting Cells (APC), dendritic cells, and T cells.
Another example of a suitable adjuvant is the detoxified Listeria lysin O (dtLLO) protein. Detoxification can be achieved by introducing point mutations into three selected amino acids that are important for LLO binding to cholesterol and for eventual pore formation. The three targeted amino acids are present in the cholesterol binding domain of LLO (ECTGLAWEWWR; SEQ ID NO:74) and can be modified in their sequence by point mutations introduced into the DNA sequence by PCR (EATGLAWEAAR; SEQ ID NO: 96). An example of dtLLO suitable for use as an adjuvant is encoded by SEQ ID NO 95. The detoxified, non-hemolytic form of LLO (dtLLO) is an effective adjuvant in tumor immunotherapy, activating both the innate and cellular immune responses by acting as PAMPs. dtLLO encoded by a sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO 95 is also suitable as an adjuvant.
Other examples of adjuvants include growth factors or nucleic acids encoding growth factors, cell populations, Freund's incomplete adjuvant (Freund's incomplete adjuvant), aluminum phosphate, aluminum hydroxide, BCG (bacille Calmette-Guerin), alum, interleukins or nucleic acids encoding interleukins, quillaja glycosides, monophosphoryl lipid A, liposomes, bacterial mitogens, bacterial toxins, or any other type of known adjuvant (see, e.g., Fundamental Immunology, 5 th edition (8.2003): William E. Paul (eds.; Lippinco Williams & Wilkins Publishers; Chapter 43: vitamins, GJV nosal, which is incorporated herein by reference in its entirety for all purposes).
The immunogenic composition may further comprise one or more immune modulatory molecules. Examples include interferon gamma, cytokines, chemokines and T cell stimulators.
The immunogenic composition may be in the form of a vaccine or pharmaceutical composition. The terms "vaccine" and "pharmaceutical composition" are interchangeable and refer to an immunogenic composition in a pharmaceutically acceptable carrier for in vivo administration to a subject. The vaccine can be, for example, a peptide vaccine (e.g., comprising a mutated peptide or recombinant fusion polypeptide as disclosed herein), a DNA vaccine (e.g., comprising a nucleic acid encoding a mutated peptide or recombinant fusion polypeptide as disclosed herein), or a vaccine contained within and delivered by a cell (e.g., a recombinant listeria as disclosed herein). The vaccine may prevent infection or manifestation of the disease or condition in the subject, and/or the vaccine may be therapeutic for a subject having the disease or condition. Methods for preparing peptide vaccines are well known and are described, for example, in EP 1408048, US 2007/0154953 and Ogasawara et al (1992) Proc. Natl Acadsi USA 89: 8995-. Optionally, peptide evolution techniques can be used to generate antigens with higher immunogenicity. Techniques for peptide evolution are well known and are described, for example, in US 6,773,900, which is incorporated by reference herein in its entirety for all purposes.
By "pharmaceutically acceptable carrier" is meant a vehicle for containing an immunogenic composition that can be introduced into a subject without significant adverse effects as well as without deleterious effects on the immunogenic composition. That is, "pharmaceutically acceptable" means that any formulation is safe and provides for delivery of an effective amount of at least one immunogenic composition for use in the methods disclosed herein, suitable for the desired route of administration. Pharmaceutically acceptable carriers or vehicles or excipients are well known. Descriptions of suitable pharmaceutically acceptable carriers and the factors involved in their selection are found in a variety of readily available sources, such as, for example, Remington's Pharmaceutical Sciences, 18 th edition, 1990, which are incorporated herein by reference in their entirety for all purposes. The carrier may be suitable for any route of administration (e.g. parenteral, enteral (e.g. oral) or topical application). The pharmaceutical composition may be buffered, for example, wherein the pH is maintained at a specific desired value within the range of pH 4.0 to pH 9.0, depending on the stability of the immunogenic composition and the route of administration.
Suitable pharmaceutically acceptable carriers include, for example, sterile water, saline solutions such as saline, dextrose, buffered solutions such as phosphate or bicarbonate buffered solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates (e.g., lactose, amylose, or starch), magnesium stearate, talc, silicic acid, viscous paraffin, white paraffin, glycerol, alginates, hyaluronic acid, collagen, aromatic oils, fatty acid mono-and diglycerides, pentaerythritol fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like. The pharmaceutical composition or vaccine may also include adjuvants that do not deleteriously react with the immunogenic composition, including, for example, diluents, stabilizers (e.g., sugars and amino acids), preservatives, wetting agents, emulsifiers, pH buffering agents, viscosity enhancing additives, lubricants, salts for influencing osmotic pressure, buffers, vitamins, colorants, flavoring agents, aromatic substances, and the like.
For liquid formulations, the pharmaceutically acceptable carrier may be, for example, an aqueous or non-aqueous solution, suspension, emulsion, or oil. Non-aqueous solvents include, for example, propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils include those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil and cod liver oil. Solid carriers/diluents include, for example, gums, starches (e.g., corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, or dextrose), cellulosic materials (e.g., microcrystalline cellulose), acrylates (e.g., polymethyl acrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.
Optionally, sustained or targeted release pharmaceutical compositions or vaccines can be formulated. This can be achieved, for example, by using liposomes or compositions in which the active compound is protected with a differentially degradable coating (e.g., by microencapsulation, multilamellar coating, etc.). The composition may be formulated to achieve immediate or slow release. It is also possible to freeze-dry the composition and use the resulting lyophilisate (e.g. for the preparation of products for injection).
The immunogenic compositions, pharmaceutical compositions, or vaccines disclosed herein may also comprise one or more additional compounds effective in preventing or treating cancer. For example, the additional compound may include compounds suitable for use in chemotherapy, such as amsacrine (amsacrine), bleomycin (bleomycin), busulfan (busulfan), capecitabine (capecitabine), carboplatin (carboplatin), carmustine (carmustine), chlorambucil (chlorambucil), cisplatin (cissplatin), cladribine (cladribine), clofarabine (clofarabine), clinatase (cristatase), cyclophosphamide (cyclophosphamide), cytarabine (cytarabine), dacarbazine (dacarbazine), dactinomycin (dactinomycin), daunorubicin (daunorubicin), docetaxel (docetaxel), doxorubicin (doxorubin), epirubicin (epirubicin), etoposide (ziprasidone), idarubicin (trofloxacin (5), tetrahydropalmatin (leucovorin), fluxapride (5 (tetrahydropalmatin), tetrahydropalmatine (leucovorin), fluxapride (clofaracin), flunomide (flunomide), fluben (fluben-d (a), fluben-d (fluben), fluben-d, fluben (fluben-b), fluben-d-b), fluben-b, Liposomal doxorubicin (lipomaldoxorubicin), liposomal daunorubicin (lipomaldorubicin), lomustine (lomustine), melphalan (melphalan), mercaptopurine (mercaptoprine), mesna (mesna), methotrexate (methotrexate), mitomycin (mitomycin), mitoxantrone (mitoxantrone), oxaliplatin (oxaliplatin), paclitaxel (paclitaxel) (Taxol), pemetrexed (pemetrexed), pentostatin (pentostatin), procarbazine (procarbazine), raltitrexed (triptorexid), satraplatin (saplatatin), streptozotocin (strezocin), tegafur-uracil (tegafur-uracil), temozolomide (temozolomide), teniposide (vincristine), vincristine (vincristine), vinorelbine (vincristine (vinorelbine), vinorelbine (vinorelbine), or a combination thereof. Additional compounds may also include other biological agents, including Against the HER2 antigen(trastuzumab), against VEGF(bevacizumab), or antibodies against EGF receptor such as
Additional compounds may also include immune checkpoint inhibitory antagonists such as PD-1 signaling pathway inhibitors, CD-80/86 and CTLA-4 signaling pathway inhibitors, T cell membrane protein 3(TIM3) signaling pathway inhibitors, adenosine A2a receptor (A2aR) signaling pathway inhibitors, lymphocyte activation gene 3(LAG3) signaling pathway inhibitors, Killer Immunoglobulin Receptor (KIR) signaling pathway inhibitors, CD40 signaling pathway inhibitors, or any other antigen presenting cell/T cell signaling pathway inhibitor. Examples of immune checkpoint inhibitory antagonists include anti-PD-L1/PD-L2 antibody or fragment thereof, anti-PD-1 antibody or fragment thereof, anti-CTLA-4 antibody or fragment thereof, or anti-B7-H4 antibody or fragment thereof. Additional compounds may also include T cell stimulators, such as antibodies or functional fragments thereof that bind to a T cell receptor costimulatory molecule, an antigen presenting cell receptor binding costimulatory molecule, or a member of the TNF receptor superfamily. T cell receptor costimulatory molecules can include, for example, CD28 or ICOS. Antigen presenting cell receptor binding co-stimulatory molecules may include, for example, the CD80 receptor, CD86 receptor, or CD46 receptor. TNF receptor superfamily members can include, for example, glucocorticoid-induced TNF receptor (GITR), OX40(CD134 receptor), 4-1BB (CD137 receptor), or TNFR 25. See, e.g., WO2016100929, WO2016011362, and WO2016011357, each of which is herein incorporated by reference in its entirety for all purposes.
Methods of treatment
The mutated peptides, recombinant fusion polypeptides, nucleic acids encoding the mutated peptides, nucleic acids encoding the recombinant fusion polypeptides, recombinant bacteria or listeria strains, immunogenic compositions, pharmaceutical compositions, and vaccines disclosed herein may be used in a variety of methods. For example, they may be used in a method of inducing or enhancing an immune response against an anti-cancer associated protein or an anti-tumor associated antigen in a subject, a method of inducing or enhancing an anti-tumor or anti-cancer immune response in a subject, a method of treating a tumor or cancer in a subject, a method of preventing a tumor or cancer in a subject, or a method of protecting a subject against a tumor or cancer. They may also be used in methods of increasing the ratio of T effector cells to regulatory T cells (tregs) in the spleen and tumor of a subject, wherein the T effector cells target a tumor-associated antigen. They may also be used in methods of increasing tumor-associated antigen T cells in a subject, increasing survival time of a subject having a tumor or cancer, delaying onset of cancer in a subject, or decreasing size of a tumor or metastasis in a subject.
A method of inducing or enhancing an immune response against a tumor-associated antigen in a subject can comprise, for example, administering to the subject a variegated peptide, a recombinant fusion polypeptide, a nucleic acid encoding a variegated peptide or a recombinant fusion polypeptide, a recombinant bacterium or listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine disclosed herein (e.g., a nucleic acid comprising or encoding a variegated peptide or a recombinant fusion polypeptide comprising the variegated peptide). An anti-tumor associated antigen immune response can thereby be induced or enhanced in a subject. For example, in the case of a recombinant listeria strain, the listeria strain can express the fusion polypeptide, thereby eliciting an immune response in the subject. The immune response may include, for example, a T cell response, such as a CD4+ FoxP3-T cell response, a CD8+ T cell response, or a CD4+ FoxP 3-and CD8+ T cell response. The methods may also increase the ratio of T effector cells to regulatory T cells (tregs) in the spleen and tumor microenvironment of the subject, thereby allowing a deeper anti-tumor response to be achieved in the subject.
A method of inducing or enhancing an anti-tumor or anti-cancer immune response in a subject can comprise, for example, administering to the subject a mutated peptide, a recombinant fusion polypeptide, a nucleic acid encoding a mutated peptide or a recombinant fusion polypeptide, a recombinant bacterium or listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine disclosed herein. An anti-tumor or anti-cancer immune response can thereby be induced in the subject. For example, in the case of a recombinant listeria strain, the listeria strain can express the fusion polypeptide, thereby eliciting an anti-tumor or anti-cancer response in the subject.
A method of treating a tumor or cancer (e.g., wherein the tumor or cancer expresses a particular tumor-associated antigen or cancer-associated protein disclosed elsewhere herein) in a subject can comprise, for example, administering to the subject a mutator peptide, a recombinant fusion polypeptide, a nucleic acid encoding a mutator peptide or a recombinant fusion polypeptide disclosed herein, a recombinant bacterium or listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine. The subject may then mount an immune response against the tumor or cancer expressing the tumor-associated antigen, thereby treating the tumor or cancer in the subject.
A method of preventing a tumor or cancer in a subject or protecting a subject from developing a tumor or cancer (e.g., wherein the tumor or cancer is associated with expression of a particular tumor-associated antigen or cancer-associated protein disclosed elsewhere herein) can comprise, for example, administering to the subject a mutated peptide, a recombinant fusion polypeptide, a nucleic acid encoding a mutated peptide or a recombinant fusion polypeptide disclosed herein, a recombinant bacterium or listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine. The subject may then mount an immune response against the tumor-associated antigen, thereby preventing the tumor or cancer or protecting the subject from developing the tumor or cancer.
In some of the above methods, two or more of the mutated peptides, recombinant fusion polypeptides, nucleic acids encoding the mutated peptides or recombinant fusion polypeptides, recombinant bacteria or listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines are administered. The plurality of mutated peptides, recombinant fusion polypeptides, nucleic acids encoding mutated peptides or recombinant fusion polypeptides, recombinant bacteria or listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines may be administered sequentially in any order or combination, or may be administered simultaneously in any combination. As an example, if four different listeria strains are administered, they can be administered sequentially, they can be administered simultaneously, or they can be administered in any combination (e.g., the first strain and the second strain are administered simultaneously, and then the third strain and the fourth strain are administered simultaneously). Optionally, in case of sequential administration, the compositions may be administered during the same immune response, preferably within 0-10 or 3-7 days of each other. The plurality of mutated peptides, recombinant fusion polypeptides, nucleic acids encoding mutated peptides or recombinant fusion polypeptides, recombinant bacteria or listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines can each comprise a set of different antigenic peptides. Alternatively, two or more may comprise a set of identical antigenic peptides (e.g., a set of identical antigenic peptides in a different order).
Multiple variant peptides or fragments or recombinant fusion polypeptides can bind multiple different HLA types. For example, they may bind to one or more of the following HLA types: HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02.
As an example, a plurality of variant peptides (in the form of, for example, a peptide, a recombinant fusion polypeptide, a nucleic acid, or a bacterial vector) may be encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or all of the following genes: CEACAM5, MAGEA6, MAGEA4, GAGE1, NYESO1, STEAP1, and RNF 43. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, non-small cell lung cancer (NSCLC). The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the heteroantigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such antigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all 11 of the heteroantigenic peptides in table 3, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all 11 of the sequences in table 3.
As another example, a plurality of variant peptides (in the form of, for example, a peptide, a recombinant fusion polypeptide, a nucleic acid, or a bacterial vector) may be encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or all of the following genes: CEACAM5, MAGEA4, STEAP1, RNF43, SSX2, SART3, PAGE4, PSMA, and PSA. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, prostate cancer. The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the heteroantigenic peptides in table 5, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the sequences in table 5.
As another example, a plurality of variant peptides (in the form of, for example, a peptide, a recombinant fusion polypeptide, a nucleic acid, or a bacterial vector) may be encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or all of the following genes: CEACAM5, STEAP1, MAGEA3, PRAME, hTERT, and SURVIVIN. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, pancreatic cancer. The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or all 12 of the heteroantigenic peptides in table 7, or peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or all 12 of the sequences in table 7.
As another example, a plurality of variant peptides (in the form of, for example, a peptide, a recombinant fusion polypeptide, a nucleic acid, or a bacterial vector) may be encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, GAGE1, NYESO1, RNF43, NUF2, KLHL7, MAGEA3, and PRAME. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, bladder cancer. The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all 14 of the heteroantigenic peptides in table 9, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, or all 13 of the sequences in table 9.
As another example, a plurality of variant peptides (in the form of, for example, a peptide, a recombinant fusion polypeptide, a nucleic acid, or a bacterial vector) may be encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or all of the following genes: CEACAM5, STEAP1, RNF43, MAGEA3, PRAME and hTERT. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, breast cancer (e.g., ER + breast cancer). The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all 11 of the heteroantigenic peptides in table 11, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all 11 of the sequences in table 11.
As another example, a plurality of variant peptides (in the form of, for example, a peptide, a recombinant fusion polypeptide, a nucleic acid, or a bacterial vector) may be encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, PRAME, hTERT, STEAP1, RNF43, NUF2, KLHL7 and SART 3. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, uterine cancer. The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all 14 of the heteroantigenic peptides in table 13, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all 14 of the sequences in table 13.
As another example, a plurality of variant peptides (in the form of, for example, a peptide, a recombinant fusion polypeptide, a nucleic acid, or a bacterial vector) may be encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, STEAP1, RNF43, SART3, NUF2, KLHL7, PRAME and hTERT. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, ovarian cancer. The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all 14 of the heteroantigenic peptides in table 15, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, or all 14 of the sequences in table 15.
As another example, a plurality of variant peptides (in the form of, for example, a peptide, a recombinant fusion polypeptide, a nucleic acid, or a bacterial vector) may be encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, MAGEA6, STEAP1, RNF43, SART3, NUF2, KLHL7, and hTERT. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, a low-grade glioma. The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the heteroantigenic peptides in table 17, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the sequences in table 17.
As another example, a plurality of variant peptides (in the form of, for example, a peptide, a recombinant fusion polypeptide, a nucleic acid, or a bacterial vector) may be encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all of the following genes: CEACAM5, MAGEA6, MAGEA4, GAGE1, NYESO1, STEAP1, RNF43, and MAGEA 3. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, colorectal cancer (e.g., MSS colorectal cancer). The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the heteroantigenic peptides in table 19, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the sequences in table 19.
As another example, a plurality of variant peptides (in the form of, for example, a peptide, a recombinant fusion polypeptide, a nucleic acid, or a bacterial vector) may be encoded by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or all of the following genes: CEACAM5, MAGEA4, STEAP1, NYESO1, PRAME and hTERT. The heteroantigenic peptides may bind to, for example, one or more or all of HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02. The cancer-associated protein is associated with, for example, head and neck cancer. The heteroantigenic peptides can be in any order. The heteroantigenic peptides can be fused together directly or linked together through a linker, examples of which are disclosed elsewhere herein. In a particular example, one or more or all of the antigenic peptides can be 9-mers (e.g., 9-mers linked together by a linker). Examples of such heteroantigenic peptides are provided in example 2. The heteroantigenic peptides can include, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the heteroantigenic peptides in table 21, or include peptides comprising, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the sequences in table 21.
Cancer is a physiological condition in mammals that is generally characterized by unregulated cell growth and proliferation. The cancer may be a hematopoietic malignancy or a solid tumor (i.e., a mass of cells resulting from excessive cell growth or proliferation, including precancerous lesions). Metastatic cancer refers to cancer that has spread from the site where it first started to another site in the body. Tumors formed by metastatic cancer cells are referred to as metastatic tumors or metastases, a term also used to refer to the process by which cancer cells spread to other parts of the body. In general, metastatic cancers share the same name and type of cancer cell as the original or primary cancer. Examples of solid tumors include melanoma, carcinoma, blastoma, and sarcoma. Hematologic malignancies include, for example, leukemias or lymphoid malignancies, such as lymphomas. Exemplary classes of cancer include brain cancer, breast cancer, gastrointestinal cancer, urinary tract cancer, gynecological cancer, head and neck cancer, hematological cancer, skin cancer, and chest cancer. Brain malignancies include, for example, glioblastoma, high grade pontine glioma, low grade glioma, medulloblastoma, neuroblastoma and hairy cell astrocytoma. Gastrointestinal cancers include, for example, colorectal cancer, gallbladder cancer, hepatocellular cancer, pancreatic cancer, PNET, gastric cancer, and esophageal cancer. Urinary tract cancers include, for example, adrenocortical carcinoma, bladder carcinoma, renal chromophobe carcinoma, renal (clear cell) carcinoma, renal (papillary) carcinoma, rhabdoid carcinoma, and prostate carcinoma. Gynecological cancers include, for example, uterine carcinosarcoma, uterine endometrial cancer, serous ovarian cancer, and cervical cancer. Head and neck cancers include, for example, thyroid cancer, nasopharyngeal cancer, head and neck cancer, and adenoid cystic cancer. Hematologic cancers include, for example, multiple myeloma, myelodysplasia, mantle cell lymphoma, Acute Lymphoblastic Leukemia (ALL), non-lymphoma, Chronic Lymphocytic Leukemia (CLL), and Acute Myelogenous Leukemia (AML). Skin cancers include, for example, skin melanoma and squamous cell carcinoma. Chest cancers include, for example, squamous lung cancer, small cell lung cancer, and lung adenocarcinoma.
More specific examples of such cancers include squamous cell cancer or carcinoma (e.g., oral squamous cell cancer), myeloma, oral cancer, juvenile nasopharyngeal angiofibroma, neuroendocrine tumor, lung cancer, peritoneal cancer, hepatocellular cancer, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioma, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellular cancer, breast cancer, triple negative breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma or carcinoma, salivary gland carcinoma, kidney cancer (e.g., renal cell carcinoma), prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile cancer, fibrosarcoma, gallbladder cancer, osteosarcoma, mesothelioma, and head and neck cancer. The cancer may also be brain cancer or another type of CNS or intracranial tumor. For example, the subject can have an astrocytoma (e.g., astrocytoma, anaplastic astrocytoma, glioblastoma, hairy astrocytoma, sub-ependymal giant cell astrocytoma, pleomorphic yellow astrocytoma), an oligodendroglioma (e.g., oligodendroglioma, anaplastic oligodendritic glioma), an ependymal tumor (e.g., ependymal, anaplastic ependymal, mucinous ependymal, sub-ependymal), a mixed glioma (e.g., mixed oligoastrocytoma, anaplastic oligoastrocytoma), a neuroepithelial tumor of indeterminate origin (e.g., polar spongiospongioblastoma, astrocytoma, cerebral glioma (gliomastigmatiscedebri)), a tumor (e.g., choroidplexus tumor, chorioplexus tumor), a neuron, or a mixed neuron-glioma tumor (e.g., ganglioneuroblastoma, glioblastoma, gliomas, glioma, Cerebellar dysplasia ganglioneuroma, anaplastic ganglioneuroma, desmoplastic infant ganglioneuroma, central neuroblastoma, dysplastic neuroepithelial tumors, olfactory neuroblastoma), pineal parenchymal tumors (e.g., pineal cytoma, pineal blastoma, mixed pineal cytoma/pineal blastoma), or tumors having mixed neuroblastoma or glioblastic elements (e.g., medullary epithelioma, medulloblastoma, neuroblastoma, retinoblastoma, ependymoblastoma). Other examples of cancers include low-grade glioma, non-small cell lung cancer (NSCLC), estrogen receptor positive (ER +) breast cancer, and DNA mismatch repair-deficient cancers or tumors. If a cancer has a receptor for estrogen, it is said to be estrogen receptor positive. Another example of cancer is microsatellite stabilized (MSS) colorectal cancer.
In particular examples, the cancer is non-small cell lung cancer, prostate cancer, pancreatic cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, lower glioma, colorectal cancer, or head and neck cancer.
The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or ameliorate the targeted tumor or cancer. The treatment may include one or more of the following: directly affecting or curing a symptom associated with a tumor or cancer, suppressing the symptom, inhibiting the symptom, preventing the symptom, lessening the severity of the symptom, delaying the onset of the symptom, slowing the progression of the symptom, stabilizing the progression of the symptom, inducing the remission of the symptom, preventing or delaying metastasis of the symptom, alleviating/ameliorating the symptom, or a combination thereof. For example, treatment may include increasing the expected survival time or decreasing the size of a tumor or metastasis. The effect (e.g., suppression of symptoms, prevention of symptoms, reduction of severity of symptoms, delay in onset of symptoms, slowing of progression of symptoms, stabilizing progression of symptoms, induction of remission of symptoms, prevention or delay of metastasis of symptoms, reduction/amelioration of symptoms, etc.) may be relative to a control subject that is not receiving treatment or that is receiving placebo treatment. The term "treating" can also refer to increasing the probability of percent survival in a subject having a tumor or cancer, or increasing the expected survival time for the subject (e.g., relative to a control subject that did not receive treatment or that received placebo treatment). In one example, "treating" refers to delaying progression, accelerating remission, inducing remission, enhancing remission, accelerating recovery, increasing efficacy of an alternative therapeutic agent, decreasing resistance to an alternative therapeutic agent, or a combination thereof (e.g., relative to a control subject that is not receiving treatment or that is receiving placebo treatment). The term "preventing" or "impeding" can, for example, refer to delaying the onset of symptoms, preventing recurrence of a tumor or cancer, reducing the number or frequency of recurrent episodes, increasing latency between symptomatic episodes, preventing metastasis of a tumor or cancer, or a combination thereof. The term "suppress" or "inhibition" may, for example, refer to a reduction in the severity of a symptom, a reduction in the severity of an acute episode, a reduction in the number of symptoms, a reduction in the occurrence of disease-related symptoms, a reduction in the latency of a symptom, an improvement in a symptom, a reduction in secondary symptoms, a reduction in secondary infection, an extension in the survival of a patient, or a combination thereof.
The term "subject" refers to a mammal (e.g., a human) in need of therapy for or susceptible to developing a tumor or cancer. The term subject also refers to a mammal (e.g., a human) that receives prophylactic or therapeutic treatment. Subjects may include dogs, cats, pigs, cows, sheep, goats, horses, rats, mice, non-human mammals, and humans. The term "subject" does not necessarily exclude individuals that are healthy in all respects and do not have or show signs of cancer or tumor.
If a subject has at least one known risk factor (e.g., genetic, biochemical, family history, and situational exposure), the risk factor places an individual with that risk factor at a statistically significant greater risk of developing a tumor or cancer than an individual without the risk factor, then the individual is at increased risk of developing the tumor or cancer.
"symptoms" or "signs" refer to objective signs of disease as observed by a physician, or subjective signs of disease as perceived by a subject, such as changes in gait. The symptoms or signs may be any manifestation of the disease. The symptoms may be primary or secondary. The term "primary" means that the symptoms are the direct result of a particular disease or disorder (e.g., a tumor or cancer), while the term "secondary" means that the symptoms arise from or occur as a result of a primary cause. The variegated peptides, recombinant fusion polypeptides, nucleic acids encoding the variegated peptides or recombinant fusion polypeptides, immunogenic compositions, pharmaceutical compositions, and vaccines disclosed herein can treat primary or secondary symptoms or secondary complications.
The variant peptide, the recombinant fusion polypeptide, the nucleic acid encoding the variant peptide or the recombinant fusion polypeptide, the recombinant bacterium or listeria strain, the immunogenic composition, the pharmaceutical composition or the vaccine is administered in an effective regime, which means that the dose, the route of administration and the frequency of administration delays the onset of, reduces the severity of, inhibits the further worsening of, and/or ameliorates at least one sign or symptom of a tumor or cancer. Alternatively, the mutated peptide, the recombinant fusion polypeptide, the nucleic acid encoding the mutated peptide or the recombinant fusion polypeptide, the recombinant bacterium or the listeria strain, the immunogenic composition, the pharmaceutical composition or the vaccine is administered in an effective regime, which means that the dose, the route of administration and the frequency of administration induce an immune response to the heterologous antigen in the mutated peptide or the recombinant fusion polypeptide (or encoded by the nucleic acid), the recombinant bacterium or the listeria strain, the immunogenic composition, the pharmaceutical composition or the vaccine, or in the case of the recombinant bacterium or the listeria strain, to the bacterium or the listeria strain itself. If the subject has suffered a tumor or cancer, the regimen may be referred to as a therapeutically effective regimen. A regimen may be referred to as a prophylactically effective regimen if the subject is at elevated risk of developing a tumor or cancer, relative to the general population, but has not experienced symptoms. In some cases, therapeutic or prophylactic efficacy in individual patients may be observed relative to historical controls or past experiences in the same patient. In other instances, therapeutic or prophylactic efficacy may be shown in a population of treated patients relative to a control population of untreated patients in a preclinical or clinical trial. For example, a regimen may be considered therapeutically or prophylactically effective if an individual treated patient achieves a more favorable result than the average result in a control population of similar patients not treated by the methods described herein, or if a more favorable result is shown at a p <0.05 or 0.01 or even 0.001 level in the treated patient relative to the control patient in a control clinical trial (e.g., a phase II, phase II/III, or phase III trial).
Exemplary dosages of the peptides are, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 10-20, 20-40, 30-60, 40-80, 50-100, 50-150, 60-80, 80-100, 100-200, 200-300, 300-400, 400-600, 500-800, 600-800, 800-1000, 1000-1500, or 1500-1200 μ g of peptide per day. Exemplary doses of the peptides are, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 10-20, 20-40, 30-60, 40-80, 50-100, 50-150, 60-80, 80-100, 100-200, 200-300, 300-400, 400-600, 500-800, 600-800, 800-1000, 1000-1500, or 1500-1200mg of peptide per day.
An exemplary dose of recombinant listeria strains is, e.g., 1x106-1x 107CFU、1x 107-1x 108CFU、1x 108-3.31x 1010CFU、1x 109-3.31x 1010CFU、5-500x 108CFU、7-500x 108CFU、10-500x108CFU、20-500x 108CFU、30-500x 108CFU、50-500x 108CFU、70-500x 108CFU、100-500x108CFU、150-500x 108CFU、5-300x 108CFU、5-200x108CFU、5-15x 108CFU、5-100x 108CFU、5-70x 108CFU、5-50x 108CFU、5-30x108CFU、5-20x 108CFU、1-30x 109CFU、1-20x 109CFU、2-30x 109CFU、1-10x 109CFU、2-10x 109CFU、3-10x 109CFU、2-7x 109CFU、2-5x 109CFU, and 3-5x 109And (4) CFU. Other exemplary doses of recombinant listeria strains are, e.g., 1x1071.5X 10 organisms7Individual organism, 2x 108Individual organism, 3x107Individual organism, 4x 107Individual organism, 5x107Individual organism, 6x 107Individual organism, 7x 107Individual organism, 8x 107Individual organism, 10x 1071.5X 10 organisms8Individual organism, 2x 108Organism, 2.5X 108Individual organism, 3x108Individual organism, 3.3x 108Individual organism, 4x 108Individual organism, 5x108Individual organism, 1X1091.5X 10 organisms9Individual organism, 2x 109Individual organism, 3x109Individual organism, 4x 109Individual organism, 5x109Individual organism, 6x 109Individual organism, 7x 109Individual organism, 8x 109Individual organism, 10x 1091.5X 10 organisms10Individual organism, 2x 1010Organism, 2.5X 1010Individual organism, 3x1010Individual organism, 3.3x 1010Individual organism, 4x 1010An organism, and 5x1010An organism. The dosage may depend on the condition of the patient and the response, if any, to prior treatments, whether the treatment is prophylactic or therapeutic, and other factors.
Administration may be by any suitable means. For example, administration may be by: parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intracerebroventricular, intraperitoneal, topical, intranasal, intramuscular, intraocular, intrarectal, conjunctival, transdermal, intradermal, vaginal, rectal, intratumoral, paracancerous, transmucosal, intravascular, intraventricular, inhalation (aerosol), nasal inhalation (spray), sublingual, aerosol, suppository, or a combination thereof. For intranasal administration or administration by inhalation, solutions or suspensions of the mutated peptides, recombinant fusion polypeptides, nucleic acids encoding the mutated peptides or recombinant fusion polypeptides, recombinant bacteria or listeria strains, immunogenic compositions, pharmaceutical compositions or vaccines mixed and aerosolized or nebulized in the presence of a suitable carrier are suitable. Such an aerosol may comprise any of the mutated peptides, recombinant fusion polypeptides, nucleic acids encoding mutated peptides or recombinant fusion polypeptides, recombinant bacteria, or listeria strains described herein. An immunogenic composition, a pharmaceutical composition or a vaccine. Administration may also be in the form of a suppository (e.g., a rectal suppository or a urethral suppository), in the form of a pellet for subcutaneous implantation (e.g., to provide controlled release over a period of time), or in the form of a capsule. Administration can also be by injection into the tumor site or into the tumor. The administration regimen can be readily determined based on factors such as: the exact nature and type of tumor or cancer being treated, the severity of the tumor or cancer, the age and general physical condition of the subject, the weight of the subject, the response of the individual subject, and the like.
The frequency of administration can depend on the half-life of the mutated peptide or recombinant fusion polypeptide, the nucleic acid encoding the mutated peptide or recombinant fusion polypeptide, the recombinant bacterium or listeria strain, the immunogenic composition, the pharmaceutical composition or vaccine in the subject, the condition of the subject and the route of administration, among other factors. In response to a change in the condition of the subject or the progression of the treated tumor or cancer, the frequency can be, for example, daily, weekly, monthly, quarterly, or at irregular intervals. The course of treatment may depend on the condition of the subject and other factors. For example, the course of treatment may be weeks, months, or years (e.g., up to 2 years). For example, repeated administration (dosing) may be performed immediately after the first course of treatment or after an interval of days, weeks, or months to achieve tumor regression or tumor growth arrest. Assessment may be made by any known technique, including diagnostic methods such as imaging techniques, analysis of serum tumor markers, biopsy, or the presence, absence, or amelioration of tumor-related symptoms. As a specific example, a mutated peptide, a recombinant fusion polypeptide, a nucleic acid encoding a mutated peptide or a recombinant fusion polypeptide, a recombinant bacterium or listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine may be administered every 3 weeks for up to 2 years. In one example, the varietal peptides, recombinant fusion polypeptides, nucleic acids encoding the varietal peptides or recombinant fusion polypeptides, recombinant bacteria or listeria strains, immunogenic compositions, pharmaceutical compositions, or vaccines disclosed herein are administered in increasing doses to increase the ratio of T effector cells to regulatory T cells and to generate a more potent anti-tumor immune response. The anti-tumor immune response can be further enhanced by providing the subject with cytokines, including, for example, IFN- γ, TNF- α, and other cytokines known to enhance cellular immune responses. See, for example, US 6,991,785, which is incorporated by reference herein in its entirety for all purposes.
Some methods may further comprise "boosting" the subject with an additional mutated peptide, a recombinant fusion polypeptide, a nucleic acid encoding a mutated peptide or a recombinant fusion polypeptide, a recombinant bacterium or listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine, or multiple administrations of a mutated peptide, a recombinant fusion polypeptide, a nucleic acid encoding a mutated peptide or a recombinant fusion polypeptide, a recombinant bacterium or listeria strain, an immunogenic composition, a pharmaceutical composition, or a vaccine. "boosting" refers to administering an additional dose to a subject. For example, in some methods, 2 boosters are administered (or 3 vaccinations in total), 3 boosters are administered, 4 boosters are administered, 5 boosters are administered, or 6 or more boosters are administered. The number of doses administered may depend, for example, on the response of the tumor or cancer to the treatment.
Optionally, the mutated peptide, the recombinant fusion polypeptide, the nucleic acid encoding the mutated peptide or the recombinant fusion polypeptide, the recombinant bacterium or the listeria strain, the immunogenic composition, the pharmaceutical composition or the vaccine used in the boost vaccination is the same as the mutated peptide, the recombinant fusion polypeptide, the nucleic acid encoding the mutated peptide or the recombinant fusion polypeptide, the recombinant bacterium or the listeria strain, the immunogenic composition, the pharmaceutical composition or the vaccine used in the initial "priming" vaccination. Alternatively, the booster mutated peptide, the recombinant fusion polypeptide, the nucleic acid, the recombinant bacterium or listeria strain, the immunogenic composition, the pharmaceutical composition or the vaccine is different from the prime mutated peptide, the recombinant fusion polypeptide, the nucleic acid, the recombinant bacterium or listeria strain, the immunogenic composition, the pharmaceutical composition or the vaccine. Optionally, the same dose is used in the priming and boosting. Alternatively, a larger dose is used for the reinforcement, or a smaller dose is used for the reinforcement. The period between priming and boosting can be determined experimentally. For example, the period between prime and boost may be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6-8 weeks, or 8-10 weeks.
Heterologous prime boost strategies have been effective in enhancing immune responses and protection against a wide range of pathogens. See, e.g., Schneider et al (1999) Immunol. Rev.170: 29-38; robinson (2002) nat. Rev. Immunol.2: 239-250; gonzalo et al (2002) Vaccine20: 1226-1231; and Tanghe (2001) feed.Immun.69: 3041-3047, each of which is incorporated herein by reference in its entirety for all purposes. Providing the antigen in different forms in the prime and boost injections maximizes the immune response to the antigen. Priming of DNA vaccines followed by boosting with proteins in adjuvants or by delivery of DNA encoding the antigen via viral vectors is an improvement of antigen-specific antibodies and CD4+T cell response or CD8+An efficient way of T cell response. See, e.g., Shiver et al (2002) Nature 415: 331-; gilbert et al (2002) Vaccine20: 1039-1045; Billaut-Mulot et al (2000) Vaccine 19: 95-102; and Sin et al (1999) DNA Cell biol.18:771-779, each of which is incorporated herein by reference in its entirety for all purposes. As an example, when a subject vaccinates with a DNA prime followed by boosting with an adenovirus vector expressing an antigen, the addition of CRL1005 poloxamer (12kDa, 5% POE) to the DNA encoding the antigen can enhance the T cell response. See, e.g., Shiver et al (2002) Nature 415: 331-. As another example, immunization encoding an antigen may be administeredA vector construct for a immunogenic portion and a protein comprising said immunogenic portion of said antigen. See, for example, US 2002/0165172, which is incorporated by reference herein in its entirety for all purposes. Similarly, the immune response of a nucleic acid vaccination may be enhanced by the simultaneous administration (e.g., during the same immune response, preferably within 0-10 or 3-7 days of each other) of the polynucleotide and polypeptide of interest. See, for example, US 6,500,432, which is incorporated by reference herein in its entirety for all purposes.
The methods of treatment disclosed herein may also include administering one or more additional compounds effective in preventing or treating cancer. For example, the additional compound may include a compound suitable for use in chemotherapy, such as amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, critase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil (5-FU), gemcitabine, grignard implants, hydroxyurea, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel (paclitaxel), pemetrexed, pentastatin, procarbazine, and paclitaxel (paclitaxel), Raltitrexed, satraplatin, streptozotocin, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, troosulfan, vinblastine, vincristine, vindesine, vinorelbine or combinations thereof. Alternatively, additional compounds may also include other biologics, including against the HER2 antigen(trastuzumab), against VEGF
The additional compound or treatment can be prior to administration of the mutator peptide, recombinant fusion polypeptide, nucleic acid encoding a mutator peptide or recombinant fusion polypeptide, recombinant bacteria or listeria strain, immunogenic composition, pharmaceutical composition or vaccine disclosed herein, after administration of the mutator peptide, recombinant fusion polypeptide, nucleic acid encoding a mutator peptide or recombinant fusion polypeptide, recombinant bacteria or listeria strain, immunogenic composition, pharmaceutical composition or vaccine disclosed herein, or concurrent with administration of the mutator peptide, recombinant fusion polypeptide, nucleic acid encoding a mutator peptide or recombinant fusion polypeptide, recombinant bacteria or listeria strain, immunogenic composition, pharmaceutical composition or vaccine disclosed herein.
Targeted immunomodulatory therapies have focused primarily on activating costimulatory receptors, for example by using agonist antibodies that target members of the tumor necrosis factor receptor superfamily, including 4-1BB, OX40 and GITR (glucocorticoid-induced TNF receptor-related proteins). Modulation of GITR has shown potential in both anti-tumor and vaccine environments. Another target of agonist antibodies is co-stimulatory signaling molecules for T cell activation. Targeting a costimulatory signaling molecule can lead to enhanced activation of T cells as well as promote a more robust immune response. Co-stimulation may also help prevent inhibitory effects from checkpoint inhibition and increase antigen-specific T cell proliferation.
Listeria-based immunotherapy works by inducing the generation of new (de novo) tumor antigen-specific T cells that infiltrate and destroy tumors, and by reducing the number and activity of immunoregulatory T cells (tregs) and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. Antibodies (or functional fragments thereof) directed against T cell co-inhibitory or co-stimulatory receptors (e.g., checkpoint inhibitors CTLA-4, PD-1, TIM-3, LAG3 and co-stimulators CD137, OX40, GITR and CD40) may have a synergistic effect with listeria-based immunotherapy.
Thus, some methods may comprise further administering a composition comprising an immune checkpoint inhibitory antagonist, such as a PD-1 signaling pathway inhibitor, a CD-80/86 and CTLA-4 signaling pathway inhibitor, a T cell membrane protein 3(TIM3) signaling pathway inhibitor, an adenosine A2a receptor (A2aR) signaling pathway inhibitor, a lymphocyte activation gene 3(LAG3) signaling pathway inhibitor, a Killer Immunoglobulin Receptor (KIR) signaling pathway inhibitor, a CD40 signaling pathway inhibitor, or any other antigen presenting cell/T cell signaling pathway inhibitor. Examples of immune checkpoint inhibitory antagonists include anti-PD-L1/PD-L2 antibody or fragment thereof, anti-PD-1 antibody or fragment thereof, anti-CTLA-4 antibody or fragment thereof, or anti-B7-H4 antibody or fragment thereof. For example, the anti-PD-1 antibody can be administered to the subject at 5-10mg/kg every 2 weeks, 5-10mg/kg every 3 weeks, 1-2mg/kg every 3 weeks, 1-10mg/kg every week, 1-10mg/kg every 2 weeks, 1-10mg/kg every 3 weeks, or 1-10mg/kg every 4 weeks.
Also, some methods may further comprise administering a T cell stimulator, such as an antibody or functional fragment thereof that binds to a T cell receptor costimulatory molecule, an antigen presenting cell receptor binding costimulatory molecule, or a member of the TNF receptor superfamily. T cell receptor costimulatory molecules can include, for example, CD28 or ICOS. Antigen presenting cell receptor binding co-stimulatory molecules may include, for example, the CD80 receptor, CD86 receptor, or CD46 receptor. TNF receptor superfamily members can include, for example, glucocorticoid-induced TNF receptor (GITR), OX40(CD134 receptor), 4-1BB (CD137 receptor), or TNFR 25.
For example, some methods may further comprise administering an effective amount of a composition comprising an antibody or functional fragment thereof that binds to a T cell receptor costimulatory molecule, or an antibody or functional fragment thereof that binds to an antigen presenting cell receptor binding costimulatory molecule. The antibody can be, for example, an anti-TNF receptor antibody or antigen-binding fragment thereof (e.g., a glucocorticoid-induced TNF receptor (GITR), OX40(CD134 receptor), 4-1BB (CD137 receptor), or TNFR25), an anti-OX 40 antibody or antigen-binding fragment thereof, or an anti-GITR antibody or antigen-binding fragment thereof. Alternatively, other agonistic molecules (e.g., GITRL, an active fragment of GITRL, a fusion protein containing an active fragment of GITRL, an Antigen Presenting Cell (APC)/T cell agonist, CD134 or a ligand or fragment thereof, CD137 or a ligand or fragment thereof, or an induced T cell costimulatory (ICOS) protein or a ligand or fragment thereof, or an agonistic small molecule) may be administered.
In a particular example, some methods may further comprise administering an anti-CTLA-4 antibody or functional fragment thereof and/or an anti-CD 137 antibody or functional fragment thereof. For example, the anti-CTLA-4 antibody or functional fragment thereof or the anti-CD 137 antibody or functional fragment thereof can be administered about 72 hours after the first dose of the mutated peptide, recombinant fusion polypeptide, nucleic acid encoding the mutated peptide or recombinant fusion polypeptide, recombinant bacteria or listeria strain, immunogenic composition, pharmaceutical composition, or vaccine, or about 48 hours after the first dose of the mutated peptide, recombinant fusion polypeptide, nucleic acid encoding the mutated peptide or recombinant fusion polypeptide, recombinant bacteria or listeria strain, immunogenic composition, pharmaceutical composition, or vaccine. The anti-CTLA-4 antibody or functional fragment thereof or the anti-CD 137 antibody or functional fragment thereof can be administered, for example, at a dose of about 0.05mg/kg and about 5 mg/kg. Recombinant listeria strains or immunogenic compositions comprising recombinant listeria strains can be, for example, at about 1x109CFU dose. Some of the methods may further comprise administering an effective amount of an anti-PD-1 antibody or functional fragment thereof.
Methods for assessing the efficacy of cancer immunotherapy are well known and are described, for example, in Dzojic et al (2006) protate 66(8): 831-; naruishi et al (2006) Cancer Gene ther.13(7): 658-. As an example, for prostate cancer, a prostate cancer model such as the TRAMP-C2 mouse model, 178-2BMA cell model, PAIII adenocarcinoma cell model, PC-3M model, or any other prostate cancer model can be used to test the methods and compositions disclosed herein.
Alternatively or additionally, immunotherapy can be tested in human subjects, and efficacy can be monitored using known methods. The methods may include, for example, measuring CD4+ and CD8+ T cell responses directly, or measuring disease progression (e.g., by determining the number or size of tumor metastases, or monitoring disease symptoms such as cough, chest pain, weight loss, etc.). Methods for assessing the efficacy of Cancer immunotherapy in human subjects are well known and are described, for example, in Uenaka et al (2007) Cancer immun.7:9 and Thomas-kaskaskel et al (2006) Int J Cancer119(10):2428-2434, each of which is incorporated herein by reference in its entirety for all purposes.
VII. kit
Also provided herein are kits comprising reagents for performing the methods disclosed herein or kits comprising the compositions, tools, or apparatuses disclosed herein.
For example, the kit can include a mutated peptide disclosed herein, a recombinant fusion polypeptide, a nucleic acid encoding a mutated peptide or a recombinant fusion polypeptide disclosed herein, a recombinant bacterium or listeria strain disclosed herein, an immunogenic composition disclosed herein, a pharmaceutical composition disclosed herein, or a vaccine disclosed herein. The kit can additionally include instructional materials describing the use of the peptide or recombinant fusion polypeptide, the nucleic acid encoding the peptide or recombinant fusion polypeptide, the recombinant listeria strain, the immunogenic composition, the pharmaceutical composition, or the vaccine for performing the methods disclosed herein. The kit may optionally further comprise an applicator. Although the model kits are described below, the contents of other suitable kits will be apparent in light of this disclosure.
All patent applications, websites, other publications, accession numbers, and the like, cited above or below are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual item were so specifically and individually indicated to be so incorporated by reference. If at different times there are sequences of different versions associated with an accession number, it is intended that the version associated with the accession number at the time of the filing date of the present application. By valid application date is meant the actual application date or the previous date of application (if applicable) of the priority application to the accession number. Likewise, if different versions of a publication, website, etc. are published at different times, it is intended to refer to the most recently published version at the effective filing date of the application, unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the present invention may be used in combination with any other feature, step, element, embodiment, or aspect, unless expressly stated otherwise. Although the invention has been described in considerable detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.
List of embodiments
The subject matter disclosed herein includes, but is not limited to, the following embodiments.
1. An isolated peptide comprising an immunogenic fragment of a cancer-associated protein, wherein the fragment comprises a mutated mutation.
2. The isolated peptide of embodiment 1, wherein the mutational mutation is a mutation at an anchor position to a preferred amino acid.
3. The isolated peptide of embodiment 1 or 2, wherein the fragment is from about 7 to about 11 amino acids in length, from about 8 to about 10 amino acids in length, or about 9 amino acids in length.
4. The isolated peptide of any preceding embodiment, wherein the cancer-associated protein is a cancer testis antigen or a carcinoembryonic antigen.
5. The isolated peptide of any preceding embodiment, wherein the cancer-associated protein is encoded by one of the following human genes: CEACAM5, GAGE1, TERT, KLHL7, MAGEA3, MAGEA4, MAGEA6, NUF2, NYESO1, PAGE4, PRAME, PSA, PSMA, RNF43, SART3, SSX2, STEAP1, and SURVIVIN.
6. The isolated peptide of embodiment 5, wherein: (a) the cancer-associated protein is encoded by CEACAM5, and the fragment comprises SEQ ID NO: 100. 102, 104, 106, and 108; (b) the cancer-associated protein is encoded by GAGE1, and the fragment comprises SEQ ID NO: 110 and 112; (c) the cancer-associated protein is encoded by TERT, and the fragment comprises SEQ ID NO: 114, and a carrier; (d) the cancer-associated protein is encoded by KLHL7, and the fragment comprises SEQ ID NO: 116; (e) the cancer-associated protein is encoded by MAGEA3, and the fragment comprises seq id NO: 118. 120, 122, and 124; (f) the cancer-associated protein is encoded by MAGEA4, and the fragment comprises SEQ ID NO: 126; (g) the cancer-associated protein is encoded by MAGEA6, and the fragment comprises SEQ ID NO: 128; (h) the cancer-associated protein is encoded by NUF2, and the fragment comprises SEQ ID NO:130 and 132; (i) the cancer-associated protein is encoded by NYESO1, and the fragment comprises SEQ ID NO: 134 and 136; (j) the cancer-associated protein is encoded by PAGE4, and the fragment comprises SEQ ID NO: 138; (k) the cancer-associated protein is encoded by PRAME and the fragment comprises SEQ ID NO: 140 of a solvent; (l) The cancer-associated protein is encoded by PSA, and the fragment comprises SEQ ID NO: 142; (m) the cancer-associated protein is encoded by PSMA, and the fragment comprises SEQ ID NO: 144, 144; (n) the cancer-associated protein is encoded by RNF43, and the fragment comprises SEQ ID NO: 146; (o) the cancer-associated protein is encoded by SART3, and the fragment comprises SEQ ID NO: 148; (p) the cancer-associated protein is encoded by SSX2, and the fragment comprises SEQ ID NO: 150; (q) the cancer associated protein is encoded by STEAP1, and the fragment comprises SEQ ID NO: 152 and 154; or (r) the cancer-associated protein is encoded by SURVIVIN, and the fragment comprises SEQ ID NO: 156 and 158.
7. The isolated peptide of embodiment 6, wherein: (a) the cancer-associated protein is encoded by CEACAM5, and the fragment consists of SEQ ID NO: 100. 102, 104, 106, and 108; (b) the cancer-associated protein is encoded by GAGE1, and the fragment is encoded by SEQ ID NO: 110 and 112; (c) the cancer-associated protein is encoded by TERT, and the fragment is encoded by SEQ ID NO: 114, and (b); (d) the cancer-associated protein is encoded by KLHL7, and the fragment is encoded by SEQ ID NO: 116, respectively; (e) the cancer-associated protein is encoded by MAGEA3, and the fragment is encoded by SEQ ID NO: 118. 120, 122, and 124; (f) the cancer-associated protein is encoded by MAGEA4, and the fragment is encoded by SEQ ID NO: 126; (g) the cancer-associated protein is encoded by MAGEA6, and the fragment is encoded by SEQ ID NO: 128 component (b); (h) the cancer-associated protein is encoded by NUF2, and the fragment is encoded by SEQ ID NO:130 and 132; (i) the cancer-associated protein is encoded by NYESO1, and the fragment is encoded by SEQ ID NO: 134 and 136; (j) the cancer-associated protein is encoded by PAGE4, and the fragment is encoded by SEQ id no: 138; (k) the cancer-associated protein is encoded by PRAME and the fragment is encoded by SEQ ID NO: 140 of the composition; (l) The cancer-associated protein is encoded by PSA, and the fragment is encoded by SEQ ID NO: 142 of a polymer; (m) the cancer-associated protein is encoded by PSMA, and the fragment is encoded by SEQ ID NO: 144 of the composition; (n) the cancer-associated protein is encoded by RNF43, and the fragment is encoded by SEQ ID NO: 146; (o) the cancer-associated protein is encoded by SART3, and the fragment is encoded by SEQ ID NO: 148; (p) the cancer-associated protein is encoded by SSX2, and the fragment is encoded by SEQ ID NO: 150; (q) the cancer associated protein is encoded by STEAP1, and the fragment is encoded by SEQ ID NO: 152 and 154; or (r) the cancer-associated protein is encoded by SURVIVIN and the fragment is encoded by SEQ ID NO: 156 and 158.
8. The isolated peptide of embodiment 7, wherein: (a) the cancer-associated protein is encoded by CEACAM5, and the isolated peptide consists of SEQ ID NO: 100. 102, 104, 106, and 108; (b) the cancer-associated protein is encoded by GAGE1, and the isolated peptide is encoded by SEQ ID NO: 110 and 112; (c) the cancer-associated protein is encoded by TERT, and the isolated peptide is encoded by SEQ ID NO: 114, and (b); (d) the cancer related protein is encoded by KLHL7, and the isolated peptide is encoded by SEQ ID NO: 116, respectively; (e) the cancer-associated protein is encoded by MAGEA3, and the isolated peptide is encoded by SEQ ID NOS: 118. 120, 122, and 124; (f) the cancer-associated protein is encoded by MAGEA4, and the isolated peptide is encoded by SEQ ID NO: 126; (g) the cancer-associated protein is encoded by MAGEA6, and the isolated peptide is encoded by SEQ ID NO: 128 component (b); (h) the cancer-associated protein is encoded by NUF2, and the isolated peptide is encoded by SEQ ID NO:130 and 132; (i) the cancer-related protein is encoded by NYESO1 and the isolated peptide consists of SEQ ID NO: 134 and 136; (j) the cancer-associated protein is encoded by PAGE4, and the isolated peptide is encoded by SEQ ID NO: 138; (k) the cancer-associated protein is encoded by PRAME and the isolated peptide is encoded by SEQ ID NO: 140 of the composition; (l) The cancer-associated protein is encoded by PSA, and the isolated peptide is encoded by SEQ ID NO: 142 of a polymer; (m) the cancer-associated protein is encoded by PSMA, and the isolated peptide is encoded by SEQ ID NO: 144 of the composition; (n) the cancer-associated protein is encoded by RNF43, and the isolated peptide consists of seq id NO: 146; (o) the cancer-associated protein is encoded by SART3, and the isolated peptide is encoded by SEQ ID NO: 148; (p) the cancer-associated protein is encoded by SSX2, and the isolated peptide is encoded by SEQ ID NO: 150; (q) the cancer-associated protein is encoded by STEAP1, and the isolated peptide consists of SEQ ID NO: 152 and 154; or (r) the cancer-associated protein is encoded by SURVIVIN and the isolated peptide is encoded by SEQ ID NO: 156 and 158.
9. The isolated peptide of any preceding embodiment, wherein the fragment binds to one or more of the following HLA types: HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02.
10. A nucleic acid encoding an isolated peptide according to any preceding embodiment.
11. The nucleic acid of embodiment 10, wherein the nucleic acid is codon optimized for expression in a human.
12. The nucleic acid of embodiment 10, wherein the nucleic acid is codon optimized for expression in Listeria monocytogenes (Listeria monocytogenes).
13. The nucleic acid of any one of embodiments 10-12, wherein the nucleic acid comprises DNA.
14. The nucleic acid of any one of embodiments 10-12, wherein the nucleic acid comprises RNA.
15. The nucleic acid according to any one of embodiments 10-14, wherein the nucleic acid comprises a sequence selected from the group consisting of SEQ ID NOs: 223-977 and degenerate variants thereof which encode the same amino acid sequence.
16. The nucleic acid of embodiment 15, wherein the nucleic acid consists of a sequence selected from the group consisting of SEQ ID NOs: 223-977 and degenerate variants thereof which encode the same amino acid sequence.
17. A pharmaceutical composition comprising:
(a) one or more isolated peptides according to any one of embodiments 1-9 or one or more nucleic acids according to any one of embodiments 10-16; and
(b) an adjuvant.
18. The pharmaceutical composition of embodiment 17, wherein said adjuvant comprises detoxified listeriolysin o (dtllo), a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid a, an unmethylated CpG-containing oligonucleotide, or Montanide ISA 51.
19. The pharmaceutical composition of embodiment 17 or 18, wherein the pharmaceutical composition comprises a peptide or a nucleic acid encoding a peptide that binds to each of the following HLA types: HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02.
20. The pharmaceutical composition according to any one of embodiments 17-19, wherein the pharmaceutical composition comprises: (a) two or more of the peptides shown in table 3 or a nucleic acid encoding two or more of the peptides shown in table 3; (b) two or more of the peptides shown in table 5 or a nucleic acid encoding two or more of the peptides shown in table 5; (c) two or more of the peptides shown in table 7 or a nucleic acid encoding two or more of the peptides shown in table 7; (d) two or more of the peptides set forth in table 9 or a nucleic acid encoding two or more of the peptides set forth in table 9; (e) two or more of the peptides set forth in table 11 or a nucleic acid encoding two or more of the peptides set forth in table 11; (f) two or more of the peptides set forth in table 13 or a nucleic acid encoding two or more of the peptides set forth in table 13; (g) two or more of the peptides set forth in table 15 or a nucleic acid encoding two or more of the peptides set forth in table 15; (h) two or more of the peptides shown in table 17 or a nucleic acid encoding two or more of the peptides shown in table 17; (i) two or more of the peptides shown in table 19 or a nucleic acid encoding two or more of the peptides shown in table 19; or (j) two or more of the peptides shown in table 21 or a nucleic acid encoding two or more of the peptides shown in table 21.
21. The pharmaceutical composition of embodiment 20, wherein the pharmaceutical composition comprises: (a) all of the peptides shown in table 3 or nucleic acids encoding all of the peptides shown in table 3; (b) all of the peptides shown in table 5 or nucleic acids encoding all of the peptides shown in table 5; (c) all of the peptides shown in table 7 or nucleic acids encoding all of the peptides shown in table 7; (d) all of the peptides shown in table 9 or nucleic acids encoding all of the peptides shown in table 9; (e) all of the peptides shown in table 11 or nucleic acids encoding all of the peptides shown in table 11; (f) all of the peptides shown in table 13 or nucleic acids encoding all of the peptides shown in table 13; (g) all of the peptides shown in table 15 or nucleic acids encoding all of the peptides shown in table 15; (h) all of the peptides shown in table 17 or nucleic acids encoding all of the peptides shown in table 17; (i) all of the peptides shown in table 19 or nucleic acids encoding all of the peptides shown in table 19; (j) all of the peptides shown in table 21 or nucleic acids encoding all of the peptides shown in table 21.
22. A recombinant bacterial strain comprising a nucleic acid encoding any one of the isolated peptides of embodiments 1-9.
23. A recombinant bacterial strain comprising one or more nucleic acids encoding two or more of the isolated peptides according to embodiments 1-9.
24. The recombinant bacterial strain of embodiment 23, wherein the two or more peptides comprise: (a) two or more of the peptides shown in table 3 or a nucleic acid encoding two or more of the peptides shown in table 3; (b) two or more of the peptides shown in table 5 or a nucleic acid encoding two or more of the peptides shown in table 5; (c) two or more of the peptides shown in table 7 or a nucleic acid encoding two or more of the peptides shown in table 7; (d) two or more of the peptides shown in table 9 or a nucleic acid encoding two or more of the peptides shown in table 9; (e) two or more of the peptides set forth in table 11 or a nucleic acid encoding two or more of the peptides set forth in table 11; (f) two or more of the peptides set forth in table 13 or a nucleic acid encoding two or more of the peptides set forth in table 13; (g) two or more of the peptides set forth in table 15 or a nucleic acid encoding two or more of the peptides set forth in table 15; (h) two or more of the peptides shown in table 17 or a nucleic acid encoding two or more of the peptides shown in table 17; (i) two or more of the peptides shown in table 19 or a nucleic acid encoding two or more of the peptides shown in table 19; or (j) two or more of the peptides shown in Table 21 or a nucleic acid encoding two or more of the peptides shown in Table 21.
25. The recombinant bacterial strain of embodiment 24, wherein the two or more peptides comprise: (a) all of the peptides shown in table 3 or nucleic acids encoding all of the peptides shown in table 3; (b) all of the peptides shown in table 5 or nucleic acids encoding all of the peptides shown in table 5; (c) all of the peptides shown in table 7 or nucleic acids encoding all of the peptides shown in table 7; (d) all of the peptides shown in table 9 or nucleic acids encoding all of the peptides shown in table 9; (e) all of the peptides shown in table 11 or nucleic acids encoding all of the peptides shown in table 11; (f) all of the peptides shown in table 13 or nucleic acids encoding all of the peptides shown in table 13; (g) all of the peptides shown in table 15 or nucleic acids encoding all of the peptides shown in table 15; (h) all of the peptides shown in table 17 or nucleic acids encoding all of the peptides shown in table 17; (i) all of the peptides shown in table 19 or nucleic acids encoding all of the peptides shown in table 19; or (j) all of the peptides shown in Table 21 or nucleic acids encoding all of the peptides shown in Table 21.
26. The recombinant bacterial strain of any one of embodiments 23-25, wherein a combination of two or more peptides bind to each of the following HLA types: HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02.
27. The recombinant bacterial strain of any one of embodiments 22-26, wherein the bacterial strain is a salmonella, listeria, yersinia, shigella, or mycobacterium strain.
28. The recombinant bacterial strain of embodiment 27, wherein said bacterial strain is a Listeria strain, optionally wherein said Listeria strain is a Listeria monocytogenes (Listeria monocytogenes) strain.
29. A recombinant listeria strain comprising a nucleic acid comprising a first open reading frame encoding a fusion polypeptide, wherein said fusion polypeptide comprises a PEST-containing peptide fused to an immunogenic fragment of a cancer-associated protein, wherein said fragment comprises a mutated mutation.
30. The recombinant listeria strain of embodiment 29, wherein said mutator mutation is a mutation at an anchor position to a preferred amino acid.
31. The recombinant listeria strain of embodiment 29 or 30, wherein the fragment is from about 7 to about 11 amino acids in length, from about 8 to about 10 amino acids in length, or about 9 amino acids in length.
32. The recombinant listeria strain of any one of embodiments 29-31, wherein the cancer-associated protein is a cancer testis antigen or a carcinoembryonic antigen.
33. The recombinant listeria strain of any one of embodiments 29-32, wherein the cancer-associated protein is encoded by one of the following human genes: CEACAM5, GAGE1, TERT, KLHL7, MAGEA3, MAGEA4, MAGEA6, NUF2, NYESO1, PAGE4, PRAME, PSA, PSMA, RNF43, SART3, SSX2, STEAP1 and SURVIVIN.
34. The recombinant listeria strain of embodiment 33, wherein:
(a) the cancer-associated protein is encoded by CEACAM5, and the fragment comprises SEQ ID NO: 100. 102, 104, 106, and 108; (b) the cancer-associated protein is encoded by GAGE1, and the fragment comprises SEQ id no: 110 and 112; (c) the cancer-associated protein is encoded by TERT, and the fragment comprises SEQ id no: 114, and a carrier; (d) the cancer-associated protein is encoded by KLHL7, and the fragment comprises SEQ ID NO: 116; (e) the cancer-associated protein is encoded by MAGEA3, and the fragment comprises SEQ ID NO: 118. 120, 122, and 124; (f) the cancer-associated protein is encoded by MAGEA4, and the fragment comprises SEQ ID NO: 126; (g) the cancer-associated protein is encoded by MAGEA6, and the fragment comprises SEQ ID NO: 128; (h) the cancer-associated protein is encoded by NUF2, and the fragment comprises SEQ ID NO:130 and 132; (i) the cancer-associated protein is encoded by NYESO1, and the fragment comprises SEQ ID NO: 134 and 136; (j) the cancer-associated protein is encoded by PAGE4, and the fragment comprises SEQ ID NO: 138; (k) the cancer-associated protein is encoded by PRAME and the fragment comprises SEQ ID NO: 140 of a solvent; (l) The cancer-associated protein is encoded by PSA, and the fragment comprises SEQ ID NO: 142; (m) the cancer-associated protein is encoded by PSMA, and the fragment comprises SEQ ID NO: 144, 144; (n) the cancer-associated protein is encoded by RNF43, and the fragment comprises SEQ ID NO: 146; (o) the cancer-associated protein is encoded by SART3, and the fragment comprises SEQ ID NO: 148; (p) the cancer-associated protein is encoded by SSX2, and the fragment comprises seq id NO: 150; (q) the cancer associated protein is encoded by STEAP1, and the fragment comprises SEQ ID NO: 152 and 154; or (r) the cancer-associated protein is encoded by SURVIVIN and the fragment comprises the amino acid sequence of SEQ ID NO: 156 and 158.
35. The recombinant listeria strain of embodiment 34, wherein:
(a) the cancer-associated protein is encoded by CEACAM5, and the fragment consists of SEQ ID NO: 100. 102, 104, 106, and 108; (b) the cancer-associated protein is encoded by GAGE1, and the fragment is encoded by SEQ ID NO: 110 and 112; (c) the cancer-associated protein is encoded by TERT, and the fragment is encoded by SEQ id no: 114, and (b); (d) the cancer-associated protein is encoded by KLHL7, and the fragment is encoded by SEQ ID NO: 116, respectively; (e) the cancer-associated protein is encoded by MAGEA3, and the fragment is encoded by SEQ ID NO: 118. 120, 122, and 124; (f) the cancer-associated protein is encoded by MAGEA4, and the fragment is encoded by SEQ ID NO: 126; (g) the cancer-associated protein is encoded by MAGEA6, and the fragment is encoded by SEQ ID NO: 128 component (b); (h) the cancer-associated protein is encoded by NUF2, and the fragment is encoded by SEQ ID NO:130 and 132; (i) the cancer-associated protein is encoded by NYESO1, and the fragment is encoded by SEQ ID NO: 134 and 136; (j) the cancer-associated protein is encoded by PAGE4, and the fragment is encoded by SEQ ID NO: 138; (k) the cancer-associated protein is encoded by PRAME and the fragment is encoded by SEQ ID NO: 140 of the composition; (l) The cancer-associated protein is encoded by PSA, and the fragment is encoded by SEQ ID NO: 142 of a polymer; (m) the cancer-associated protein is encoded by PSMA, and the fragment is encoded by SEQ ID NO: 144 of the composition; (n) the cancer-associated protein is encoded by RNF43, and the fragment is encoded by SEQ ID NO: 146; (o) the cancer-associated protein is encoded by SART3, and the fragment is encoded by SEQ ID NO: 148; (p) the cancer-associated protein is encoded by SSX2, and the fragment is encoded by SEQ ID NO: 150; (q) the cancer associated protein is encoded by STEAP1, and the fragment is encoded by SEQ ID NO: 152 and 154; or (r) the cancer-associated protein is encoded by SURVIVIN and the fragment is encoded by SEQ ID NO: 156 and 158.
36. The recombinant listeria strain of embodiments 29-35, wherein said fragment binds to one or more of the following HLA types: HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02.
37. The recombinant listeria fungus of any one of embodiments 29-36, wherein said PEST-containing peptide comprises a bacterial secretion signal sequence, and said fusion polypeptide further comprises a ubiquitin protein fused to said fragment, wherein said PEST-containing peptide, said ubiquitin, and said carboxy-terminal antigenic peptide are arranged in tandem from the amino-terminus to the carboxy-terminus of the fusion polypeptide.
38. The recombinant listeria strain of any one of embodiments 29-37, wherein said fusion polypeptide comprises said PEST-containing peptide fused to two or more immunogenic fragments of a cancer-associated protein, wherein each of said two or more fragments comprises a mutated mutation.
39. The recombinant listeria strain of embodiment 38, wherein the two or more immunogenic fragments are directly fused to each other without an intervening sequence.
40. The recombinant listeria strain of embodiment 38, wherein the two or more immunogenic fragments are linked to each other by a peptide linker.
41. The recombinant listeria strain of embodiment 40, wherein SEQ ID NO: one or more linkers as shown in 209-217 are used to join the two or more immunogenic fragments.
42. The recombinant listeria strain of embodiments 38-41, wherein a combination of two or more immunogenic fragments in the fusion polypeptide bind to each of the following HLA types: HLA-A02: 01, HLA-A03: 01, HLA-A24: 02 and HLA-B07: 02.
43. The recombinant listeria strain of any one of embodiments 38-42, wherein the two or more immunogenic fragments comprise: (a) two or more of the peptides shown in table 3; (b) two or more of the peptides shown in table 5; (c) two or more of the peptides shown in table 7; (d) two or more of the peptides shown in table 9; (e) two or more of the peptides shown in table 11; (f) two or more of the peptides shown in table 13; (g) two or more of the peptides shown in table 15; (h) two or more of the peptides shown in table 17; (i) two or more of the peptides shown in table 19; or (j) two or more of the peptides shown in Table 21.
44. The recombinant listeria strain of embodiment 43, wherein said two or more immunogenic fragments comprise: (a) all peptides shown in table 3; (b) all peptides shown in table 5; (c) all peptides shown in table 7; (d) all peptides shown in table 9; (e) all peptides shown in table 11; (f) all peptides shown in table 13; (g) all peptides shown in table 15; (h) all peptides shown in table 17; (i) all peptides shown in table 19; or (j) all the peptides shown in Table 21.
45. The recombinant listeria strain of any one of embodiments 29-44, wherein the PEST-containing peptide is on the N-terminus of the fusion polypeptide.
46. The recombinant listeria strain of embodiment 45, wherein the PEST-containing peptide is an N-terminal fragment of LLO.
47. The recombinant listeria strain of embodiment 46, wherein the N-terminal fragment of the LLO has the amino acid sequence of SEQ ID NO:59, or a sequence shown in SEQ ID NO.
48. The recombinant listeria strain of any one of embodiments 29-47, wherein the nucleic acid is in an episomal (episomal) plasmid.
49. The recombinant listeria strain of any one of embodiments 29-48, wherein said nucleic acid does not confer antibiotic resistance to said recombinant listeria strain.
50. The recombinant listeria strain of any one of embodiments 29-49, wherein the recombinant listeria strain is an attenuated auxotrophic listeria strain.
51. The recombinant listeria strain of embodiment 50, wherein said attenuated auxotrophic listeria strain comprises a mutation in one or more endogenous genes that inactivates said one or more endogenous genes.
52. The recombinant listeria strain of embodiment 51, wherein the one or more endogenous genes comprise actA, dal, and dat.
53. The recombinant listeria strain of any one of embodiments 29-52, wherein said nucleic acid comprises a second open reading frame encoding a metabolic enzyme.
54. The recombinant listeria strain of embodiment 53, wherein the metabolic enzyme is an alanine racemase enzyme or a D-amino acid aminotransferase.
55. The recombinant listeria strain of any one of embodiments 29-54, wherein the fusion polypeptide is expressed from an hly promoter.
56. The recombinant Listeria strain of any one of embodiments 29-55, wherein the recombinant Listeria strain is a Listeria monocytogenes (Listeria monocytogenes) strain.
57. The recombinant Listeria strain of any one of embodiments 29-56, wherein the recombinant Listeria strain is an attenuated Listeria monocytogenes (Listeria monocytogenes) strain comprising deletions or inactivating mutations of actA, dal, and dat, wherein the nucleic acid is in an episomal plasmid and comprises a second open reading frame encoding an alanine racemase or a D-amino acid aminotransferase, and wherein the PEST-containing peptide is an N-terminal fragment of LLO.
58. An immunogenic composition comprising: (a) the recombinant bacterial strain of any one of embodiments 22-28 or the recombinant listeria strain of any one of embodiments 29-57; and (b) an adjuvant.
59. The immunogenic composition of embodiment 58, wherein said adjuvant comprises detoxified Listeriolysin O (dtLLO), a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein, a nucleotide molecule encoding a GM-CSF protein, saponin QS21, monophosphoryl lipid A, or an unmethylated CpG-containing oligonucleotide.
60. A method of inducing or enhancing an immune response against a tumor or cancer in a subject, the method comprising administering to the subject an isolated peptide according to any one of embodiments 1-9, a nucleic acid according to any one of embodiments 10-16, a pharmaceutical composition according to any one of embodiments 17-21, a recombinant bacterial strain according to any one of embodiments 22-28, a recombinant listeria strain according to any one of embodiments 29-57, or an immunogenic composition according to any one of embodiments 58-59.
61. A method of preventing or treating a tumor or cancer in a subject, the method comprising administering to the subject an isolated peptide according to any one of embodiments 1-9, a nucleic acid according to any one of embodiments 10-16, a pharmaceutical composition according to any one of embodiments 17-21, a recombinant bacterial strain according to any one of embodiments 22-28, a recombinant listeria strain according to any one of embodiments 29-57, or an immunogenic composition according to any one of embodiments 58-59.
62. The method of embodiment 60 or 61, wherein the cancer is non-small cell lung cancer, prostate cancer, pancreatic cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, lower glioma, colorectal cancer, or head and neck cancer.
Brief description of the sequences
The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard nucleotide base letter abbreviations and amino acid three letter codes. The nucleotide sequence follows the standard convention of starting at the 5 'end of the sequence and progressing (i.e., from left to right in each row) to the 3' end. Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the shown strand. When a nucleotide sequence is provided that encodes an amino acid sequence, it will be appreciated that degenerate variants of the codons encoding the same amino acid sequence are also provided. The amino acid sequence follows the standard convention of starting at the amino terminus of the sequence and progressing (i.e., from left to right in each row) to the carboxy terminus.
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