PHOSPHOPEPTIDES AS MELANOMA VACCINES

• US 20120177669A1
• Filed: 05/04/2010
• Published: 07/12/2012
• Est. Priority Date: 05/05/2009
• Status: Active Grant

• Current Assignee: Johns Hopkins University, University of Virginia Patent Foundation (University of Virginia)
• Original Assignee: Johns Hopkins University, University of Virginia Patent Foundation (University of Virginia)
• Inventors: Topalian, Suzanne L., Hunt, Donald, Shabanowitz, Jeffrey, Depontieu, Florence, Qian, Jie, Engelhard, Victor, Zaring, Angela
• Granted Patent: US 9,279,011 B2
• Time in Patent Office: Days
• US Class Current: 424/185.1
• CPC Class Codes: A61K 2039/5154 Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
• CPC Class Codes: A61K 2039/5158 Antigen-pulsed cells, e.g. T-cells
• CPC Class Codes: A61K 2239/26 Universal/off- the- shelf cellular immunotherapy; Allogenic cells or means to avoid rejection
• CPC Class Codes: A61K 2239/57 Skin; melanoma
• CPC Class Codes: A61K 39/001191 Melan-A/MART
• CPC Class Codes: A61K 39/39 characterised by the immunostimulating additives, e.g. chemical adjuvants
• CPC Class Codes: A61K 39/4611 T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
• CPC Class Codes: A61K 39/4622 Antigen presenting cells
• CPC Class Codes: A61K 39/464491 Melan-A/MART
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: C07K 14/4748 Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
• CPC Class Codes: G01N 33/5743 of skin, e.g. melanoma

Abstract

We characterized a total of 175 HLA-DR-associated phosphopeptides using sequential affinity isolation, biochemical enrichment, mass spectrometric sequencing and comparative analysis. Many were derived from source proteins which may have roles in cancer development, growth and metastasis. Most were expressed exclusively by either melanomas or transformed B cells, suggesting the potential to define cell type-specific phosphatome “fingerprints”. We generated HLA-DRβ1*0101-restricted CD4⁺ T cells specific for a phospho-MART-1 peptide identified in two melanoma cell lines. These T cells showed specificity for phosphopeptide-pulsed antigen presenting cells as well as for intact melanoma cells. MHC II-restricted phosphopeptides recognizable by human CD4⁺ T cells are potential targets for cancer immunotherapy.

Claims

1. An isolated phosphopeptide comprising between 9 and 30 contiguous amino acids selected from SEQ ID NO:

1 (MART-1) including the serine at position 108 of MART-1, wherein the serine is phosphorylated.

2. A composition comprising the phosphopeptide of claim 1, substantially free of other polypeptides.

3. A composition comprising the phosphopeptide of claim 1, substantially free of human cells.

4. A composition comprising the phosphopeptide of claim 1 in a complex with an HLA-DR1 molecule.

5. The composition of claim 4 wherein the complex is a tetramer.

6. The composition of claim 3 which comprises an admixture with one or more distinct polypeptides.

7. The composition of claim 6 wherein the one or more distinct polypeptides are melanoma-specific peptides.

8. The composition of claim 3 which further comprises an immune adjuvant.

9. The composition of claim 6 which further comprises an immune adjuvant.

10. The composition of claim 4 wherein the HLA-DR1 molecule is HLA-DRB1*0101.

11. The phosphopeptide of claim 1 which comprises an amino acid sequence selected from the group consisting of the following residues of SEQ ID NO:

1;

residues 100-108, residues 101-109, residues 102-110, residues 103-111, residues 104-112, residues 105-113, residues 106-114, residues 107-115, and residues 108-116.

12. The phosphopeptide of claim 1 which consists of an amino acid sequence selected from the group consisting of:

residues 97-116 (SEQ ID NO;

     11), residues 99-110 (SEQ ID NO;

     13), residues 100-111 (SEQ ID NO;

     14), residues 100-114 (SEQ ID NO;

     15), residues 100-115 (SEQ ID NO;

     16), and residues 100-116 (SEQ ID NO;

     17).

13. A method of immunizing a mammal to diminish the risk of, the growth of, or the invasiveness of a melanoma, comprising:

administering to the mammal a composition according to any of claims 2-12, whereby CD4⁺ T cells are activated.

14. The method of claim 13 wherein the mammal is non-human.

15. The method of claim 13 wherein the mammal is a dog.

16. The method of claim 13 wherein the mammal has a melanoma.

17. The method of claim 13 wherein the mammal has metastatic melanoma.

18. The method of claim 13 wherein the mammal has an increased risk of developing a melanoma.

19. A method, comprising:

contacting T cells in vitro with an isolated phosphopeptide according to claim 1, whereby CD4⁺ T cells are stimulated.

20. The method of claim 19 further comprising:

transfusing the stimulated CD4⁺ T cells to a melanoma patient.

21. The method of claim 20 wherein the CD4⁺ T cells are autologous.

22. The method of claim 20 wherein the CD4⁺ T cells are allogeneic.

23. A method comprising:

contacting a sample isolated from a patient with an antibody that specifically binds to the phosphopeptide of claim 1 and does not bind to a polypeptide consisting of the same amino acid sequence but devoid of phosphorylation;

measuring or detecting antibody bound to the sample.

24. The method of claim 23 wherein the sample is blood, serum, plasma, or lymph.

25. The method of claim 23 wherein the sample is a biopsy sample from tumor or normal tissue.

26. The method of claim 23 wherein the sample is from a lymph node.

27. A molecule comprising an antigen-binding region of an antibody, wherein the molecule specifically binds to the phosphopeptide of claim 1 and does not bind to a polypeptide consisting of the same amino acid sequence but devoid of phosphorylation.

28. The molecule of claim 27 which is a monoclonal antibody.

29. The molecule of claim 27 which is a single chain variable region (ScFv).

30. A kit for measuring a phosphopeptide comprising between 9 and 30 contiguous amino acids selected from SEQ ID NO:

1 (MART-1) including the serine at position 108 of MART-1, wherein the serine is phosphorylated, comprising;

a molecule comprising an antigen-binding region of an antibody, wherein the molecule specifically binds to the phosphopeptide and does not bind to a polypeptide consisting of the same amino acid sequence but devoid of phosphorylation.

31. The kit of claim 30 further comprising an antibody which specifically binds to a portion of the molecule which is distinct from the antigen-binding region.

32. The kit of claim 30 further comprising a detectable label.

33. The kit of claim 30 further comprising a solid support on which binding complexes of the molecule and the phosphopeptide can be captured.

34. A method, comprising:

contacting dendritic cells in vitro with an isolated phosphopeptide comprising between 9 and 30 contiguous amino acids selected from SEQ ID NO;

1 (MART-1), including serine at position 108 of MART-1, wherein the serine is phosphorylated, whereby dendritic cells are loaded with the phosphopeptide.

35. The method of claim 34 further comprising:

transfusing or injecting the loaded dendritic cells into a melanoma patient.

36. An in vitro composition comprising dendritic cells made by the method of claim 34.