Generation of single domain antibodies against human melanoma cells.

LACREU, MARÍA LAURA; URRUTIA, MARIELA

Buenos Aires

Congreso; I Congress of LASID-SAI-FAIC; 2015

Eventos Congresos Grupo Uno SRL

GENERATION OF SINGLE DOMAIN ANTIBODIES AGAINST HUMAN MELANOMA CELLS. Background: Cutaneous melanoma is a malignant tumor whose incidence has steadily increased over the past decades in predominantly fair-skinned populations. Although significant progress has been made recently on melanoma immunotherapy, treatment for advanced melanoma is still challenging with limited response rate and poor prognosis. We previously developed a library of antibody fragments constituted by the variable domains of llama heavy-chain antibodies (VHH). After screening, 4 VHHs (44, 50, 54 and 58) were selected based on their reactivity against human melanoma cell lines (hMCL). Methods: We studied the binding of those VHHs against 2 hMCL and 2 human fibroblast cell lines by flow cytometry, ELISA and immunocytochemistry. To detect the VHH-50s antigen (Ag), extracted membrane proteins from hMCL were analyzed by western blot (WB) using the VHH-50. Results: Only VHH-50 recognizes hMCL but not fibroblast cell lines. The epito pe recognized by VHH 50 is peptidic, since binding did not diminish with periodate (carbohydrates oxidation) or methanol (lipids dissolution). A band around 135 kDa was detected by VHH-50 on the hMCL protein extract. When the protein extract was treated with N-glicosidase before SDS-PAGE, the 135 kDa band was not detected but two lower weight bands (60 and 80 kDa) were observed. By immunocytochemistry the Ag was found to be located at the cell membrane and cell extensions. Conclusion: Our results indicate that the single domain antibody VHH-50 generated recognizes a glycoprotein around 135 kDa expressed on the hMCL plasma membrane.