Medullary carcinoma of the breast is the prototype of human tumors which characteristically elicit prominent lymphoplasmacytic cell infiltrates in the tumor stroma.
Our specific aims are to analyze the B cell clonality in these tumors, characterize the major B cell clones, produce corresponding Fab fragments in vitro, and ultimately investigate the distribution and nature of the tumor antigens. Our research design combines two recently developed techniques: 1) the analysis of B cell clonality by PCR amplification of the VDJ region of the immunoglobulin heavy chain gene (IgH); and 2) the synthesis of phagemid combinatorial antibody library, producing immunoreactive Fab fragments in vitro. Our new strategy, if proven successful, will not only address the humoral immune response in medullary carcinoma, but also provide a powerful tool in the future analysis of other human tumors with similar B cell infiltrates. The proposed experiments are: I. Characterization of the lymphoplasmacytic infiltrates in medullary carcinoma: The characteristics of mononuclear cell infiltrates will be evaluated by light microscopy and immunohistochemical assays, and this will be done for two specific reasons: One is to identify tumors with predominantly B cell infiltrates (for Fab production, Aim II-III) and those with T cell infiltrates (as antigenic source for Fab analysis, Aim IV), and the other is to determine the dominant immunoglobulin heavy chain class/subclass in B cells infiltrating tumors. II. Analysis of VDJ heterogeneity of tumor-infiltrating B cells and identification of the major clones: The tumor with predominantly B cell infiltrates will be selected, and the B cell clonality will be analyzed by RT-PCR of the IgH VDJ regions. The dominant B cell clones will be identified, and their CDR3 sequences will be cloned and compared. Oligonucleotide probes specific for these clones will be used for a)in- situ hybridization to evaluate their abundance, and b) DNA blotting to define their heavy chain classes/subclasses. III. Construction of Fab-secreting phagemid clones containing major- clone heavy chains: cDNA encoding IgH VH (Fd) fragments of the major clones will be synthesized and isolated by direct PCR-cloning. These heavy chains with defined sequences will be subcloned into a phagemid library containing random light chains, resulting in combinatorial antibody libraries. Fabs will then be produced from selective clones. IV. Specificity analysis of recombinant Fabs and characterization of tumor antigens: The recombinant Fabs will be analyzed by immunohistochemical methods and by immunoblotting. Immunohistochemical assays will define the tissue distribution and subcellular localization of tumor antigen(s). The biochemical nature of the tumor antigens will be investigated by immunoblotting and by analysis of glycolipid extracts from the tumor.
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