Abstract
There are two parts in my dissertation. In the first part, we conducted a phylogenetic study of CεmX segment in primates to elucidate its evolutionary origin. It is hoped that this study will help to understand the function of CεmX in the regulation of IgE production. The study may also help to identify appropriate primate species as animal models to investigate anti-CεmX antibodies as potential therapeutics for IgE-mediated allergic diseases. Membrane-bound IgE (mIgE) on B lymphocytes is essential for IgE production. Earlier studies showed that the ε chain of mIgE (mε) on human B cells has a “long” isoform, with an extra “CεmX” domain of 52 amino acid residues between the CH4 domain and the membrane anchor segment, as compared to the conventional “short” isoform. CεmX is found only in humans and a few primate species that we had examined (unpublished data), not in other species, and its sequence shares no significant homology with sequences in contemporary DNA and protein databases. Comparing membrane exons of each Ig isotype between mouse and human, the mε is the least conservative. Interestingly, 3’ region of gene segments between the CH4 and M1, correspond to the 156bp segment encoding CεmX, have high degrees of sequence conservation in mouse and human, which is with comparable degrees to neighboring coding regions. We analyzed the ε Ig gene; in particular, its membrane exon regions encoding the membrane anchor peptide segment and CεmX domain, of 26 species of the order Primates and 12 species of seven non-Primate orders. Our analyses reveal the unexpected finding that the genes of three extant tarsier species do not contain the membrane exons for mIgE. Another striking finding is that early evolved Strepsirhini primates such as lemurs and lorises do not have gene segments for the long isoform, whereas New World monkeys such as marmosets and squirrel monkeys allow the transcription of only the long isoform. In Old World monkeys and apes, including humans, the ε gene allows the transcription of both isoforms. In the second part of the thesis, I carried out the humanization of an anti-CεmX antibody, 4B12, which has been developed by a research team of which I am a member. After a series of characterizations, 4B12 has been determined as a suitable candidate for clinical development. Academia Sinica has also licensed the technology on 4B12 to a biotech firm based in Taiwan. 6 CεmX provides an antigenic site for targeting IgE expressing B cells. Functional studies have revealed that chimeric anti-CεmX monoclonal antibody (mAb), 4B12, can potentially control IgE production. I humanized it by first grafting its six complementarity-determining regions (CDRs) into human frameworks (FWs). Those residues in the FWs of VH and VL of the parental murine 4B12 that are essential for Fv dimer interaction and CDR conformation were identified by analyzing the binding affinity of a number of designed antibody variants, using competitive ELISA and SPR (surface plasmon resonance) analysis. A humanized 4B12 retaining the affinity of its parent was obtained by adopting all FWs of the selected human Vκ template and all FWs of the selected VH with only one residue change at position 71. The Val of the human VH was replaced by Arg at the corresponding position of the murine VH. The KD of the chimeric and humanized 4B12 are 0.98±0.21 nM and 1.45±0.17 nM, respectively.