Identification of MHC class I phospho-peptide antigens from breast cancer utilizing SHLA technology and complementary enrichment strategies

1. A composition comprising a phosphopeptide having a length of 10 to 30 amino acids and comprising a sequence selected from the group consisting of:

SEQ ID NO: 132;

SEQ ID NO: 132 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 71 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 72 wherein the serine at the eighth position is phosphorylated;

SEQ ID NO: 73 wherein the serine at the sixth position is phosphorylated;

SEQ ID NO: 75 wherein the serine at the sixth position is phosphorylated;

SEQ ID NO: 76 wherein the serine at the eighth position is phosphorylated;

SEQ ID NO: 78 wherein the serine at the sixth position is phosphorylated;

SEQ ID NO: 79 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 80 wherein the threonine at the third position is phosphorylated;

SEQ ID NO: 84 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 87 wherein the serine at the eighth position is phosphorylated;

SEQ ID NO: 93 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 94 wherein the serine at the fifth position is phosphorylated;

SEQ ID NO: 97 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 99 wherein the serine at the sixth position is phosphorylated;

SEQ ID NO: 100 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 101 wherein the serine at the sixth position is phosphorylated;

SEQ ID NO: 102 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 104 wherein the serine at the sixth position is phosphorylated;

SEQ ID NO: 105 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 110 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 112 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 113 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 114 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 115 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 117 wherein the serine at the sixth position is phosphorylated;

SEQ ID NO: 118 wherein the serine at the tenth position is phosphorylated;

SEQ ID NO: 120 wherein the threonine at the fourth position is phosphorylated;

SEQ ID NO: 126 wherein the serine at the sixth position is phosphorylated;

SEQ ID NO: 127 wherein the serine at the sixth position is phosphorylated;

SEQ ID NO: 135 wherein the serine at the sixth position is phosphorylated;

SEQ ID NO: 140 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 143 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 145 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 146 wherein the tyrosine at the fifth position is phosphorylated;

SEQ ID NO: 149 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 151 wherein the serine at the eighth position is phosphorylated;

SEQ ID NO: 153 wherein the serine at the seventh position is phosphorylated;

SEQ ID NO: 154 wherein the serine at the fifth position is phosphorylated;

SEQ ID NO: 160 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 161 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 162 wherein the serine at the fourth position is phosphorylated and the serine at the eighth position is phosphorylated;

SEQ ID NO: 163 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 166 wherein the serine at the seventh position is phosphorylated;

SEQ ID NO: 167 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 168 wherein the serine at the fourth position is phosphorylated;

SEQ ID NO: 169 wherein the serine at the fourth position is phosphorylated; and

SEQ ID NO: 170 wherein the serine at the seventh position is phosphorylated,

wherein:

(a) one or more peptidyl linkages of the phosphopeptide have been replaced by a non-peptidyl linkage comprising a —CH 2 -carbamate linkage (—CH 2 OC(O)NR—), a phosphonate linkage, a —CH 2 -sulfonamide (—CH 2 —S(O) 2 NR—) linkage, a urea (—NHC(O)NH—) linkage, a —CH 2 -secondary amine linkage, or an alkylated peptidyl linkage (—C(O)NR—), in each case wherein R is C 1 -C 4 alkyl; and/or

(b) the N-terminus of the phosphopeptide is derivatized to a NR 1 R 2 group, to a —NR 1 C(O)R 1 group, to a —NR 1 C(O)OR 1 group, to a —NR 1 S(O) 2 R 1 group, or to a —NHC(O)NHR 1 group, in each case wherein R 1 and R 2 are hydrogen or C 1 -C 4 alkyl, and wherein R 1 and R 2 are not both hydrogen; and/or

(c) the C terminus of the phosphopeptide is derivatized to —C(O)R 3 where R 3 is selected from the group consisting of C 1 -C 4 alkoxy, or —NR 4 R 5 , in each case wherein R 4 and R 5 are independently selected from the group consisting of hydrogen and C 1 -C 4 alkyl.

2. The composition of claim 1 , further comprising an adjuvant.

3. The composition of claim 2 , wherein the adjuvant is selected from the group consisting of complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide, lysolecithin, pluronic polyols, dinitrophenol, bacille Calmette-Guerin (BCG), and Corynebacterium parvum , or combinations thereof.

4. The composition of claim 1 , wherein said composition has the ability to stimulate a T cell mediated immune response to at least one of said phosphopeptides.

5. The composition of claim 1 , comprising a phosphopeptide capable of binding to the HLA-B*0702 allele on the MHC class I molecule.

6. A method of inducing an immunogenic response comprising administering to a patient in need thereof a therapeutically effective amount of the composition of claim 1 .

7. A method of treating cancer comprising administering to a patient in need thereof a therapeutically effective amount of the composition of claim 1 .

8. A method of treating breast cancer comprising administering to a patient in need thereof a therapeutically effective amount of the composition of claim 1 .

9. A method for making a cancer vaccine comprising combining the composition of claim 1 with an adjuvant.