The common acute lymphoblastic leukemia antigen (CALLA, CD10) is an important diagnostic marker of acute lymphoblastic leukemia in childhood as well as other lymphoblastic malignancies of the hematopoietic system. Although CD10 was originally thought to be a tumor-specific antigen, CD10 was later identified on normal lymphoid progenitors as well as terminally differentiated granulocytes. This discrete expression pattern has implied that CD10 is important in several stages of hematopoietic development. Despite the recognition for more than a decade of the importance of this surface structure in diagnosis and therapy of human lymphoblastic malignancies, the primary structure of CD10 was unknown until recently. During the last funding interval, the entire primary structure of CD10 was deduced by a combination of protein microsequencing and cDNA cloning in this laboratory. In addition, the genomic organization of CD10 was established: the gene spans more than 8OKb and is composed of 24 exons localized to human chromosome 3q2l-27. Perhaps most importantly, we showed that CD10 is the zinc binding neutral endopeptidase 24.11 which regulates inflammatory responses in both invertebrate hemocytes and mammalian granulocytes. The present proposal is aimed at identifying the role of CD10 in lymphoid differentiation by taking advantage of access to developing organs in the mouse during its gestation and the ease of manipulating gene expression in this organism. First, we will isolate the complete cDNA sequence of murine CD10, determine the intron-exon organization of the murine CD10 gene and its chromosome location. The distribution of the murine molecule will be studied by producing specific MAbs. Second, the effect of CD10 enzymatic organic inhibitors and these MAbs on pre-B cell and B cell growth from murine bone marrow and fetal liver progenitors will be examined in Whitlock-Witte cultures. The effect of these inhibitors on adhesion of pre-B cell progenitors to embryonic stromal cells will also be evaluated. In addition, the influence of known CD10 substrates such as met- and leu-enkephalin on pre-B cell growth, differentiation and migration win be analyzed. Third, the in vivo role of CD10 function will be addressed by studying lymphoid development of a) transgenic mice whose CD10 expression has been purposefully dysregulated, b) CDl0- mice produced by ES cell technology and blastocyst injection, and c) normal mice which have received systemically administered CD10 enzymatic inhibitors. Fourth, we will compare the unrelated ectoenzyme BP1/6C3 aminopeptidase A activity and CD10 activity in pre-B cells with regard to the regulation of expression of these enzymes. Whether one or both enzymes are substrates for IL-7 will be ascertained. Finally, we will produce and purify a secreted, water-soluble recombinant CD10 ectoenzyme in baculovirus vectors for the specific purpose of crystallizing the CD10 molecule and determining its atomic structure.
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