The collaboration beetween Antitumoral Cellular Technology Laboratory and the Start up "Theraptosis" was based on murin monoclonal antibodies and recombinant antibody fragments (ScFv) (using the Baculovirus-Insect cell vector expression) production. A characterization of these antibodies on human breast tumor lineages, on malignant breast tumors and normal human tissues samples was done. Theraptosis has the mission to develop a chimeric molecule composed of an antibody or ScFV (VH-VL) linked to a mitochondrio-toxic peptide so as to induce tumor cells apoptosis. Several antibodies and recombinant fragments have been produced and analysed. Several clones have been selected upon human tumor lineages specificity and strong membrane labeling. The clone 180D12 of Ig61 class was particularly interesting. The MDA, ZR75 tumor cell line were strongly positive using on indirect immunofluorescence, whereas McF7 was not recognised by this antibody. The clone 180D12 recognized 70 % of breast malignant tumors (including incanalar, invasive and intralobular tumors). The normal breast tissus (from esthetic surgery) remained negative when tested in parallel (immunoenzymatic technic with phosphatase alkalin) (photo 1). Other human normal tissue samples showed only a weak positivity on hepatic tissue, salivary glands, stomach and colon.
Then, internalization tests of this antibody were performed using MDA cells. We could see after 4 hours the antibody was present inside the cells. The negative control was done after the treatment of MDA cell with Desoxy-glucose-Azide (this molecule inhibits the antibody entry). A double labelling was performed on MDA cells with the immunofluorescence antibody and dextran leads. The intracellular colocalisation is visible in photo 2. The next step was to link the antibody to the mitochondrio-toxic peptide derived from Vpr as follows : incorporation of SH on the antibody and linkage of the peptide with a maleimide function. This complex tested on MDA cells did not show an alteration of the membrane specificity, nor the affinity, nor the endocytosis of the parental antibody. Cytotoxic tests realized later showed the death of 50 % of MDA cells tested.
In vivo tests, will be done in the future by injecting the complex antibody-peptide into the peripheral circulation of nude mice MDA tumor transplanted. This model has been developed in the lab.
Keywords: Monoclonal antibodies, ScFv, tumors, peptides, mitochondria