A tumor-infiltrating lymphocyte from a melanoma metastasis with decreased expression of melanoma differentiation antigens recognizes MAGE-12

Twenty separate tumor infiltrating lymphocyte (TIL) bulk cultures and a tumor cell line were originated simultaneously from a fine needle aspiration biopsy of a metastasis in a patient with melanoma (F001) previously immunized with the HLA·A*0201-associated gp100:209-217(210 M) peptide. None of the TIL recognized gp100. However, 12 recognized autologous (F001-MEL) and allogeneic melanoma cells expressing the HLA haplotype A*0201, B*0702, Cw*0702. Further characterization of F001-MEL demonstrated loss of gp100/PMel17, severely decreased expression of other melanoma differentiation Ags and retained expression of tumor-specific Ags. Transfection of HLA class I alleles into B*0702/Cw*0702-negative melanoma cell lines identified HLA- Cw*0702 as the restriction element for F001-TIL. A cDNA library from F001- MEL was used to transfect IFN-α-stimulated 293 human embryonal kidney (293- HEK) cells expressing HLA-Cw*0702. A 100-gene pool was identified that induced recognition of 293-HEK cells by F001-TIL. Subsequent cloning of the pool identified a cDNA sequence homologous, except for one amino acid (aa 187 D→A), to MAGE-12. Among 25 peptide sequences from MAGE-12 with the HLA- Cw*0702 binding motif, MAGE-12: 170-178 (VRIGHLYIL) induced IFN-γ release by F001-TIL when pulsed on F001-EBV-B cells at concentrations as low as 10 pg/ml. Peptide sequences from MAGE-1, 2, 3, 4a, and 6 aligned to MAGE-12:170- 178 were not recognized by F001-TIL. In summary a TIL recognizing a MAGE protein was developed from an HLA-A*0201 expressing tumor with strongly reduced expression of melanoma differentiation Ags. Persisting tumor-specific Ag expression maintained tumor immune competence suggesting that tumor- specific Ags/melanoma differentiation Ags may complement each other in the context of melanoma Ag-specific vaccination.