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 have studied the contribution of individual germline genes to the pre-immune repertoire and the diversity of the immune response to tetanus toxoid. Our studies on the use of variable region genes have shown that they are not expressed equally. One member of the Vk10 family,Vk10C,has not been detected in functional antibodies. The Vk10C gene can rearrange but is expressed in the spleen at levels 1000 fold lower than the other family members. We have examined the frequency of recombination of this gene. Our studies show that the Vk10C gene is equally accessible to the recombination machinery as the other Vk10 genes as measured by levels of sterile transcripts and, that the frequency of recombination in the preB cell population, the point in development when light chain rearrangement occurs,is also equivalent to the other genes. Examination of the nature of these Vk10C recombinations at different developmental stages shows a selective loss of productive recombinations as the B cell passes from the preB cell stage through the immature B cell in the bone marrow to the mature B cell in the spleen. The reasons for this are currently under study. Antibodies expressed by developing B cells undergo a negative/positive selection process prior to reaching the mature B cell stage. Our studies demonstrating an unequal usage of functional V genes led us to question whether monoclonal antibodies produced by novel technologies such as phage display libraries 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. Such monoclonal antibodies will be more successful therapeutic agents than those which induce a vigorous immune response. The disadvantage of such mAbs is that selection by screening antigen in vitro may not be the same as selection 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 immunized a mouse with tetanus toxoid and made hybridomas from half the spleen and cDNA from the remianing portion. The cDNA is being used to generate a phage display library. The antibody diversity of these mAbs will be compared to that of the hybridoma library. The hybridoma library has been characterized and 11/17 hybridomas are specific for fragment C of tetanus toxin which is responsible for binding to neurons. The fragment C specific mAbs contain 4 different VH-VL family pairs but in some cases, different members of these families are utilized by these mAbs. The combination of VH-VL pair, ability to block other mAbs from binding to fragment C and the ability to block fragment C from binding to gangliside identified 5 epitopes. Three of these epitopes inhibit the activity of tetanus toxin in in vitro spinal cord neuron assays, one does not inhibit and the last one enhances activity. These epitopes are currently being mapped.

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