CONICET | Buscador de Institutos y Recursos Humanos

Immune libraries of single domain antibody-displayed in phages are especially suited for selection of antibodies against surface tumor antigens.One of the most promising strategies for the development of cancer therapies relies on mAbs against surface antigens (Ags). MAb raised against recombinant membrane Ags, often fails to recognize the native Ag on the cell membrane. As tumor Ag presumably not expressed in high copy number, it is important to use immune libraries. Single domain Abs (sdAb) composed of variable domain of llama heavy-chain antibodies posse small size, low immunogenicity and good tissue penetration. Our goal is to obtain sdAbs against surface antigens using sdAb-phage display library enrichment-selection on whole cells. To develop the immune phage display library, we immunized llama with 3 metastatic human melanoma cell lines (MHML) membranes. SdAb sequences from llama periphery blood lymphocytes were cloned into a phage display vector. A sdAb phage display library of reasonable size (107 clones) and good diversity was obtained. For library enrichment in sdAbs that recognize melanoma Ags, we used two cell types: red blood cells (negative steps) to subtract Abs that recognize common Ags to other human cells, and melanoma cells (positive steps) to keep Abs against them. Two rounds of enrichment had negative plus positive step; and the last 3 rounds had just a positive step. Panning evaluation by ELISA shown that the reactivity of the Abs anti-MHML increased meanwhile the rounds advanced (0.19, 0.45, 0.73 and 1.25; for 1st, 3nr, 4th and 5th rounds respectively). In each round of panning, phage-sdAb preincubated with melanoma or control cells were eluted. The 3nr round of panning shown the highest amount of phage-sdAb eluted anti-melanoma respect control cells (3.5 fold). The 3nr round sdAb diversity was evaluated by DNA-digestion, identifying 9 patterns among 20 clones. To select sdAbs anti-melanoma surface Ags, 20 phage-sdAbs from 3nr round were studied in two whole cell assays: dry cell-ELISA and FACS. Selection of 4 phage-sdAbs anti-melanoma and mama tumoral surface Ags are presented.