Non-hemolytic LLO fusion proteins and methods of utilizing same

Patent metadata

Field	Value
Publication number	US-10189885-B2
Application number	US-201514789226-A
Country	US
Kind code	B2
Filing date	Jul 1, 2015
Priority date	Jun 23, 2008
Publication date	Jan 29, 2019
Grant date	Jan 29, 2019

Abstract

The present invention provides recombinant proteins or peptides comprising a mutated listeriolysin O (LLO) protein or fragment thereof, comprising a substitution or internal deletion of the cholesterol-binding domain or a portion thereof, fusion proteins or peptides comprising same, nucleotide molecules encoding same, and vaccine vectors comprising or encoding same. The present invention also provides methods of utilizing recombinant proteins, peptides, nucleotide molecules, and vaccine vectors of the present invention to induce an immune response to a peptide of interest.

First claim

What is claimed is:

1. An immunogenic composition comprising a recombinant protein comprising a listeriolysin O (LLO) protein comprising a mutation of amino acid residues on positions 2, 9 and 10 in the cholesterol-binding domain (CBD) of said LLO protein, said CBD is set forth in SEQ ID NO: 18.

2. The immunogenic composition of claim 1 , wherein either said recombinant protein further comprises a heterologous peptide of interest, or wherein said immunogenic composition further comprises a heterologous peptide of interest.

3. The immunogenic composition of claim 1 , wherein said amino acid residues on positions 2, 9 and 10 of SEQ ID NO: 18 correspond to C484, W491 and W492 of an LLO protein, and wherein said LLO protein is set forth in SEQ ID NO: 37 or 46.

4. The immunogenic composition of claim 1 , wherein said mutated CBD is set forth in SEQ ID NO: 53.

5. The immunogenic composition of claim 1 , wherein said LLO protein comprises a deletion of the signal peptide sequence thereof.

6. The immunogenic composition of claim 1 , wherein said LLO protein comprises the signal peptide sequence thereof.

7. The immunogenic composition of claim 2 , wherein said LLO protein and said heterologous peptide of interest are linked together by a peptide bond or are chemically conjugated.

8. The immunogenic composition of claim 2 , wherein said heterologous peptide of interest is an antigenic peptide.

9. The immunogenic composition of claim 8 , wherein said antigenic peptide is a B-cell receptor (BCR) polypeptide, a Human Papilloma Virus (HPV)-16-E6, HPV-16-E7, HPV-18-E6, HPV-18-E7, a Her/2-neu antigen, a Prostate Specific Antigen (PSA), Prostate Stem Cell Antigen (PSCA), a Stratum Corneum Chymotryptic Enzyme (SCCE) antigen, Wilms tumor antigen 1 (WT-1), human telomerase reverse transcriptase (hTERT), Proteinase 3, Tyrosinase Related Protein 2 (TRP2), High Molecular Weight Melanoma Associated Antigen (HMW-MAA), synovial sarcoma, X (SSX)-2, carcinoembryonic antigen (CEA), MAGE-A, interleukin-13 Receptor alpha (IL13-R alpha), Carbonic anhydrase IX (CAIX), survivin, GPIOO, or Testisin, or fragment thereof.

10. The immunogenic composition of claim 9 , wherein said BCR polypeptide or fragment thereof is a single chain fragment of the variable regions (scFV) of said BCR.

11. The immunogenic composition of claim 9 , wherein said polypeptide or fragment thereof comprises an idiotype of said BCR.

12. The immunogenic composition of claim 11 , wherein said idiotype is a 38C13 idiotype of said BCR.

13. The immunogenic composition of claim 12 , wherein the sequence of said 38C13 idiotype comprises SEQ ID NO:62.

14. The immunogenic composition of claim 1 , further comprising an additional adjuvant.

15. The immunogenic composition of claim 14 , wherein said additional adjuvant comprises 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.

16. A method for inducing an immune response in a subject, comprising the step of administering to the subject an immunogenic composition of claim 1 , comprising a recombinant protein, said recombinant protein comprising a listeriolysin O (LLO) protein comprising a mutation of amino acid residues on positions 2, 9 and 10 in the cholesterol-binding domain (CBD) of said LLO protein, said CBD is set forth in SEQ ID NO: 18, wherein either said recombinant protein further comprises an antigenic peptide, or wherein said composition further comprises an antigenic peptide thereby inducing an immune response against said antigenic peptide .

17. The method of claim 16 , wherein said mutated CBD is set forth in SEQ ID NO: 53.

18. The method of claim 16 , wherein said antigenic peptide is a B-cell receptor (BCR) polypeptide, a Human Papilloma Virus (HPV)-16-E6, HPV-16-E7, HPV-18-E6, HPV-18-E7, a Her/2-neu antigen, a Prostate Specific Antigen (PSA), Prostate Stem Cell Antigen (PSCA), a Stratum Corneum Chymotryptic Enzyme (SCCE) antigen, Wilms tumor antigen 1 (WT-1), human telomerase reverse transcriptase (hTERT), Proteinase 3, Tyrosinase Related Protein 2 (TRP2), High Molecular Weight Melanoma Associated Antigen (HMW-MAA), synovial sarcoma, X (SSX)-2, carcinoembryonic antigen (CEA), MAGE-A, interleukin-13 Receptor alpha (IL13-R alpha), Carbonic anhydrase IX (CAIX), survivin, GPIOO, or Testisin, or fragment thereof.

19. The method of claim 16 , wherein said antigenic peptide is expressed in a subject having lymphoma.

20. The Method of claim 19 , wherein said lymphoma is a Non-Hodgkin's Lymphoma.

21. The method of claim 16 , wherein said lymphoma is a B cell lymphoma comprising a BCR idiotype.

22. The method of claim 21 , wherein said BCR idiotype is 38C13 idiotype of said BCR.

23. A recombinant protein comprising a listeriolysin O (LLO) protein comprising a mutation in a cholesterol-binding domain (CBD) set forth in SEQ ID NO: 18, wherein said mutated CBD is selected from the group consisting of SEQ ID NO: 53and SEQ ID NO: 55.

24. The recombinant protein of claim 23 , wherein sequence SEQ ID NO: 53 or SEQ ID NO: 55 replaces the amino acid sequence of SEQ ID NO: 18 in the LLO protein set forth in SEQ ID NO: 37 or SEQ ID NO: 46.

25. The recombinant protein of claim 23 , wherein said LLO protein does not comprise the signal peptide thereof.

26. The recombinant protein of claim 23 , wherein said recombinant protein further comprises a heterologous peptide of interest.

27. The recombinant protein of claim 26 , wherein said heterologous peptide of interest is an antigenic peptide.

28. The recombinant protein of claim 27 , wherein said antigenic peptide is a B-cell receptor (BCR) polypeptide, a Human Papilloma Virus (HPV)-16-E6, HPV-16-E7, HPV-18-E6, HPV- 18-E7, a Her/2-neu antigen, a Prostate Specific Antigen (PSA), Prostate Stem Cell Antigen (PSCA), a Stratum Corneum Chymotryptic Enzyme (SCCE) antigen, Wilms tumor antigen 1 (WT- 1 ), human telomerase reverse transcriptase (hTERT), Proteinase 3, Tyrosinase Related Protein 2 (TRP2), High Molecular Weight Melanoma Associated Antigen (HMW-MAA), synovial sarcoma, X (SSX)-2, carcinoembryonic antigen (CEA), MAGE-A, interleukin-13 Receptor alpha (IL13-R alpha), Carbonic anhydrase IX (CAIX), survivin, GPIOO, or Testisin, or fragment thereof.

29. The recombinant protein of claim 28 , wherein said BCR polypeptide or fragment thereof is a single chain fragment of the variable regions (scFV) of said BCR.

30. The recombinant protein of claim 28 , wherein said polypeptide or fragment thereof comprises an idiotype of said BCR.

31. The recombinant protein of claim 30 , wherein said idiotype is a 38C13idiotype of said BCR.

32. The recombinant protein of claim 31 , wherein the sequence of said 38C13idiotype comprises SEQ ID NO: 62.

33. An immunogenic composition comprising the recombinant protein of claim 30 and an adjuvant.

34. The immunogenic composition of claim 33 , further comprising a heterologous peptide of interest, wherein said recombinant protein is not covalently bound to said LLO protein.

35. The immunogenic composition of claim 33 , wherein said adjuvant comprises a granulocyte/macrophage colony-stimulating factor (GM-CSF) protein, a nucleotide molecule

Assignees

• Univ Pennsylvania
• Advaxis Inc

Inventors

• Paterson Yvonne
• Maciag Paulo

Classifications

• A61P37/04

  Immunostimulants · CPC title

• A61P35/02

  specific for leukemia · CPC title

• A61P31/12

  Antivirals · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• A61P13/08

  of the prostate · CPC title