Humanizing the jaa-f11 mouse monoclonal antibody.

摘要

Thomsen-Friedenreich antigen (TF-Ag) is a carbohydrate antigen that is hidden on normal cells but present on the surface of many types of tumor cells including colon, breast, bladder, prostate, liver, ovary and stomach. Our laboratory has developed JAA-F11, a mouse monoclonal antibody that is highly specific for TF-Ag, which blocks the early stage of metastasis and improves survival in a mouse model. One drawback of mouse monoclonal antibodies is that they trigger the production of human anti-mouse antibody (HAMA) responses in patients which prevent their effective use. For this reason, humanization of the mouse JAA-F11 antibody is required. Our hypothesis was that the humanization of JAA-F11 can generate an antibody with the same or improved biological and chemical specificity as compared to the original mouse JAA-F11 mAb. To address this hypothesis, the first specific aim was to confirm the variable heavy and light regions of JAA-F11 mAb sequences and the second specific aim was to humanize the JAA-F11 antibody. The variable regions of heavy and light chain of JAA-F11 were first cloned and their sequences confirmed. The humanization of JAA-F11 was carried out by the complementary-determining regions (CDRs) grafting approach. Six variants which contained three variable heavy region constructs and three variable light region constructs were synthesized and further grafted respectively onto 6307 pAH human heavy and 6714 pAN light chain constant regions to form the complete antibody gene. The humanized heavy and light chains were co-transfected into CHO-K1 cells through electroporation to express the complete humanized antibody. Culture supernatant from the transfection was positive in ELISA showing that a humanized JAA-F11 antibody was successfully generated.