Non-hemolytic LLO fusion proteins and methods of utilizing same

• US 20090081248A1
• Filed: 06/23/2008
• Published: 03/26/2009
• Est. Priority Date: 03/26/2001
• Status: Active Grant

• Current Assignee: Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Original Assignee: Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Inventors: Maciag, Paulo Cesar, Paterson, Yvonne
• Granted Patent: US 8,771,702 B2
• Time in Patent Office: Days
• US Class Current: 424/192.100
• CPC Class Codes: A61K 2039/53 DNA (RNA) vaccination
• CPC Class Codes: A61K 2039/55522 Cytokines; Lymphokines; Interferons
• CPC Class Codes: A61K 2239/31 characterized by the route of administration
• CPC Class Codes: A61K 2239/38 characterised by the dose, timing or administration schedule
• CPC Class Codes: A61K 2239/57 Skin; melanoma
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 39/0011 Cancer antigens
• CPC Class Codes: A61K 39/001106 Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ErbB4
• CPC Class Codes: A61K 39/001119 Receptors for interleukins [IL]
• CPC Class Codes: A61K 39/00115 Apoptosis related proteins, e.g. survivin or livin
• CPC Class Codes: A61K 39/001153 Wilms tumor 1 [WT1]
• CPC Class Codes: A61K 39/001156 Tyrosinase and tyrosinase related proteinases [TRP-1 or TRP-2]
• CPC Class Codes: A61K 39/001157 Telomerase or TERT [telomerase reverse transcriptase]
• CPC Class Codes: A61K 39/001182 Carcinoembryonic antigen [CEA]
• CPC Class Codes: A61K 39/001184 Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
• CPC Class Codes: A61K 39/001186 MAGE
• CPC Class Codes: A61K 39/00119 Melanoma antigens
• CPC Class Codes: A61K 39/001192 Glycoprotein 100 [Gp100]
• CPC Class Codes: A61K 39/001193 Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; PAP or PSGR
• CPC Class Codes: A61K 39/001194 Prostate specific antigen [PSA]
• CPC Class Codes: A61K 39/0208 : Specific bacteria not otherwise provided for
• CPC Class Codes: A61K 39/385 : Haptens or antigens, bound to carriers
• CPC Class Codes: A61K 39/461 : characterised by the cell type used
• CPC Class Codes: A61K 39/4644 : Cancer antigens
• CPC Class Codes: A61P 13/08 : of the prostate
• CPC Class Codes: A61P 31/12 : Antivirals
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 35/02 : specific for leukemia
• CPC Class Codes: A61P 37/04 : Immunostimulants
• CPC Class Codes: C07K 14/195 : from bacteria
• CPC Class Codes: C07K 14/705 : Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 16/00 : Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2317/56 : variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2319/40 : containing a tag for immunodetection, or an epitope for immunisation
• CPC Class Codes: C12N 9/52 : derived from bacteria or Archaea

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.

Claims

1. A recombinant protein comprising a listeriolysin O (LLO) protein or N-terminal fragment thereof, wherein said LLO protein or N-terminal fragment comprises a mutation in a cholesterol-binding domain (CBD), wherein said mutation comprises:

a. a substitution of 1-50 μ

amino acid peptide in said CBD as set forth in SEQ ID NO;

18 for a 1-50 amino acid non-LLO peptide;

b. a mutation of residue C484, W491, or W492 of SEQ ID NO;

37 or a combination thereof;

or

c. a deletion of a 1-50 amino acid peptide in said CBD as set for in SEQ ID NO;

18.

wherein said recombinant protein exhibits a greater than 100-fold reduction in hemolytic activity relative to wild-type LLO.

2. The recombinant protein of claim 1, wherein said LLO protein or N-terminal LLO fragment comprises a deletion of the signal peptide sequence thereof.

3. The recombinant protein of claim 1, wherein said LLO protein or N-terminal LLO fragment comprises the signal peptide sequence thereof.

4. The recombinant protein of claim 1, wherein said recombinant protein comprises a heterologous peptide of interest.

5. The recombinant protein of claim 4, wherein said non-LLO peptide comprises said heterologous peptide of interest.

6. The recombinant protein of claim 4, wherein said heterologous peptide of interest is an antigenic peptide.

7. The recombinant protein of claim 6, wherein said antigenic peptide is an NY-ESO-1 peptide.

8. The recombinant protein of claim 6, wherein said antigenic peptide is a Human Papilloma Virus (HPV) E7 peptide.

9. The recombinant protein of claim 6, wherein said antigenic peptide is a B-cell receptor (BCR) peptide.

10. The recombinant protein of claim 6, wherein said antigenic peptide is 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, GP100, or Testisin.

11. A vaccine comprising the recombinant protein of claim 1 and an adjuvant.

12. The vaccine of claim 11, wherein said 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.

13. A composition comprising the recombinant protein of claim 1 and a heterologous peptide of interest, wherein said recombinant protein is not covalently bound to said heterologous peptide of interest.

14. A vaccine comprising the composition of claim 13 and an adjuvant.

15. The vaccine of claim 14, wherein said 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 recombinant vaccine vector encoding the recombinant protein of claim 1.

17. A nucleotide molecule encoding the recombinant protein of claim 1.

18. A vaccine comprising the nucleotide molecule of claim 17 and an adjuvant.

19. A recombinant Listeria strain comprising, the recombinant protein of claim 1.

20. A method for inducing an immune response in a subject, comprising administering to said subject the recombinant protein of claim 6, thereby inducing an immune response against said antigenic peptide.

21. A method for inducing an immune response in a subject, comprising administering to said subject the composition of claim 13, thereby inducing an immune response against said heterologous peptide of interest.

22. A method for inducing an immune response in a subject, comprising administering to said subject the recombinant vaccine vector of claim 16, wherein said non-LLO peptide of said recombinant protein comprises an antigenic peptide of interest, thereby inducing an immune response against said antigenic peptide of interest.

23. A method for inducing an immune response in a subject comprising administering to said subject the recombinant Listeria strain of claim 19, wherein said non-LLO peptide of said recombinant protein comprises an antigenic peptide of interest, thereby inducing an immune response against said antigenic peptide of interest.

24. A method for inducing an immune response in a subject against an NY-ESO-1 expressing cancer cell selected from an ovarian melanoma cell and a lung cancer cell, the method comprising the step of administering to said subject the recombinant protein of claim 7 thereby inducing an immune response against an NY-ESO-1-expressing cancer cell.

25. A method for treating, inhibiting or suppressing an NY-ESO-1-expressing tumor selected from an ovarian melanoma tumor and a lung cancer tumor in a subject, the method comprising the step of administering to said subject the recombinant protein of claim 7, thereby treating, inhibiting, or suppressing an NY-ESO-1-expressing tumor.

26. A method for inducing an immune response in a subject against an HPV E7-expressing cancer cell selected from a cervical cancer cell and a head-and-neck cancer cell, the method comprising the step of administering to said subject the recombinant protein of claim 8, thereby inducing an immune response against an HPV E7-expressing cancer cell selected from a cervical cancer cell and a head-and-neck cancer cell.

27. A method for treating, inhibiting or suppressing an HPV E7 expressing tumor selected from a cervical cancer tumor and a head-and-neck cancer tumor in a subject the method comprising the step of administering to said subject the recombinant protein of claim 8, thereby treating, inhibiting, or suppressing an HPV E7-expressing tumor selected from a cervical cancer tumor and a head-and-neck cancer tumor in a subject.

28. A method for inducing an immune response in a subject against a B-cell receptor (BCR)-expressing lymphoma, the method comprising the step of administering to said subject the recombinant protein of claim 9, thereby inducing an immune response against a BCR-expressing lymphoma.

29. A method for, treating, inhibiting or suppressing a B-cell receptor (BCR)-expressing lymphoma in a subject, the method comprising the step of administering to said subject the recombinant protein of claim 9, thereby treating, inhibiting, or suppressing a BCR-expressing lymphoma in a subject.

60. The recombinant protein of claim 1, wherein the sequence of said LLO protein is as set forth in SEQ ID NO:

37.

30-59. -59. (canceled)

61-81. -81. (canceled)