Effect of defucosylation on humanized JAA-F11 antibody against TF-antigen and comparison of cytotoxicity with fucosylated counterparts
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JAA-F11 is a mouse monoclonal antibody specific to Thomsen Friedenreich Antigen, which is a carbohydrate antigen expressed on several types of carcinomas and serves as a novel target for development. Humanization of the JAA-F11 was carried out since mouse antibodies have the tendency to produce immune responses in humans. All of the humanized antibodies developed in our laboratory were produced in the CHO-K1 cell line prior to the work described in this thesis. We hypothesized that development of the humanized antibodies in a fucosyltransferase deficient variant of CHO-K1 cell line, namely Lec13, would improve the cytotoxicity levels. Fucosylation is a glycosylation process that involves attachment of fucose to glycolipids and glycoproteins. Absence of fucose improves the binding of tumor antigen bound antibodies to the FcVRIIIA receptor of the natural killer cells, thereby improving the tumor killing ability. In this project, Lec13 cell lines were used to produce antibodies to determine if humanized JAA-F11 constructs produced by these have higher cytotoxicity than the antibodies produced by CHO-K1 cells. Two constructs, H1L3 and H2L3, were developed in CHO-K1 and Lec13 cell lines, and one construct, hJAA-F11-A, was developed in Lec13 cell line as humanized hJAA-F11-A was already available in CHO-K1 cells. Fucose levels were verified by western blot analysis. To further decrease the fucose levels and increase the efficacy of the Lec13 antibodies, one of the samples was passed through an agarose bound Pisum sativum agglutinin lectin column. The flow through was analyzed and the relative % fucose in hJAA-F11-A Lec13 was 2.81%, H1L3 Lec13 (post column) was 83.3% and H2L3 Lec13 was >100% on normalizing with the CHO-K1 antibodies. The internalization potential of the antibodies by EIA and chemical specificity by glycan array analysis were also tested. We observed that the presence of fucose did not affect the ability to internalize or the binding specificity to TF-Ag. Further, % cytotoxicity of all antibodies was determined by antibody dependent cellular cytotoxicity assay, where Lec13 antibodies failed to show the expected increase in cytotoxicity levels using peripheral blood mononuclear cells. Literature has indicated that even 10% presence of fucosylation in the antibodies fails to have a positive effect on cytotoxicity. In addition, Lec13, being a non-robust low producing cell line, and thus alone is not suitable for therapeutic antibody production. Based on this study, other measures of defucosylation must be tested for development of a promising defucosylated antibody for cancer therapy.