Comparative analysis of humanized antibodies with small changes in either the heavy chain or light chain of the antibody

摘要

Breast cancer was the second leading cause of death in western women in 2015. The overall aim of this research was to find additional therapy to add to the treatment of breast cancer patients by targeting a disaccharide carbohydrate (Galbeta1-3GalNAcalpha) called the Thomsen-Friedenreich Antigen (TF-Ag). This antigen is present on the surface of ~90% of all carcinomas, including breast cancers. The specific aim of this thesis was to produce humanized antibodies and compare the effects of small changes in either the heavy chain or the light chain of the antibody to see if these changes enhance or decrease the efficiency of the antibody in terms of their specificity, affinity, their ability to induce antibody-dependent cellular cytotoxicity (ADCC) and their ability to internalize into tumor cells. The humanized antibodies created were derived from the mouse monoclonal antibody, JAA-F11, which exhibits high specificity for the TF-Ag alpha-linked tumor antigen and has proven to extend median survival time of tumor bearing mice by decreasing tumor metastasis. The three new humanized antibodies were developed: H1L2, H1L3 and H2L3, based on the different heavy and light chains. H1L2 and the H1L3 antibodies were compared for differences in their light chain and the analyses showed that the change affected the ability to internalize and induce ADCC significantly. A slight non-significant increase in affinity was also seen, with the L2 being a better light chain candidate than L3. With respect to chemical specificity, all the humanized constructs showed increased specificity when compared to mouse antibody JAA-F11 and the light chain change slightly affected the specificity to TF-Ag in that H1L2, where it showed an increase in binding to an off target carbohydrate Neu5Acalpha2-6(Galbeta1-3)GalNAcalpha. While comparing the antibodies that differed in their heavy chain, the results of the in vitro analysis showed that the change did not significantly affect ability to internalize, induce ADCC activity or specificity. A slight but not significant increase in affinity of H1L3 antibody compared to H2L3 antibody was observed. The reason for the differences in their biologic properties can be attributed the amino acids that have been changed in the different heavy and light chain variants, especially the framework amino acid closest to the CDR regions of the antibody. The amino acid change from Tyrosine to Phenylalanine at position 31 and from Leucine to Glutamine at position 32 of the light chain are hypothesized to be the ones that generated a potentially more efficacious antibody and all these data together can be used towards optimizing the sequences to create the best version of humanized JAA-F11 antibody.