During B cell development, antibody gene segments undergo multiple recombinations, first at the heavy chain loci followed by recombination at the light chain loci, to produce surface IgM. Throughout B cell development the expression of surface IgM and other markers is modulated and the pattern of expression of these molecules can identify different stages of differentiation or the activation of B cells. We are interested both in antibody and surface marker expression during B cell development. Our studies on the expression of variable region genes have shown that they are not expressed equally. The one member that has not been detected in functional antibodies. We have shown that it is expressed at levels at least 1000 fold lower than the other family members in the spleen. While rearrangements of this gene are detected in both bone marrow and spleen, they occur lower frequencies in both tissues than the other family members. The studies of variable gene expression led us to question whether monoclonal antibodies produced by novel technologies such as phage display libraries or using a """"""""humanized"""""""" mouse, could use unusual VH-VL pairs. The advantage of such technologies is that totally human mAbs are produced and are predicted to be less immunogenic in humans than rodent, non-human primate, chimeric or humanized mAbs. Monoclonal antibodies that are non-immunogenic will be more successful therapeutic agents than immunogenic mAbs. The disadvantage of such mAbs from phage display libraries is that selection by screening antigen in vitro may not be the same as during the course of a normal immune response. In vivo, VH-VL pairs undergo both positive and negative selection. In vitro screening could create VH-VL pairs that are autospecific or could create unusual pairs that might be immunogenic. To study this question we have generated a hybridoma library from a BALB/c mouse immunized with tetanus toxoid. Many of the mAbs are specific for fragment C of tetanus toxin. All but 2 of the mAbs block the binding of fragment C to its receptor, ganglioside GT1b. The fragment C specific mAbs contain 4 different VH-VL family pairs but different members of these families are utilized by these mAbs. The antibody diversity of these mAbs will be compared to that of a phage display library generated from the same mouse. Finally we have been studying the expression of a B cell surface marker defined by a mAb. This activation-induced cell surface molecule of B lymphocytes that is also upregulated on apoptotic cells. (This antigen is not specific to B cells, however.) This surface antigen has homology to the myeloid specific S100 protein MRP 8. Like MRP 8, our antigen is also largely cytoplasmic and is also secreted.
