The overall goal of the two projects participating in this is to develop and test new monoclonal antibody based strategies for treatment of breast cancer. This project will explore the molecular and cell biology of breast epithelial antigens (BEAs) and their epitope structures that are the best targets for breast tumor localization for radioimmunotherapy (RlT). Project 2 aims to identify human B-lymphocytes recognized by the breast tumor in its human host, produce human monoclonal antibodies secreted by these cells by establishing stable cell lines producing them through genetic engineering, and to develop new strategies for radioimmunotherapy by humanization and fragmentation of existing MoAbs for translation into clinical trials.
The SPECIFIC AIMS of this project are: 1. Isolate and characterize cell-associated forms of the breast mucin and BA46 and distinguish them from secreted forms using the collection of MoAbs that we have already developed. 2. Isolate and characterize the BEA identified by the human MoAbs developed in Project 2. Distinguish cell-associated and secreted forms. 3. Isolate and sequence the cDNAs for the BEA recognized by these human MoAbs for determining the epitope structures that are the best target for RIT. 4. Determine molecular structures of cell-associated BEA that are the best target for RIT, by epitope mapping, construction of chimeric recombinant antigens and oligosaccharide analysis, and by in vitro and in vivo (Project 2) preclinical evaluation. Certain MoAbs against the breast mucin and a 46 kDa breast antigen (BA46) are effective in RIT. Because of the altered processing of the breast mucin some epitopes In the tandem repeat domain are preferentially expressed in breast carcinomas, and thus certain MoAbs recognizing these normally cryptic epitopes are more effective than others in imaging metastases in breast cancer patients. Also, for BA46 antigen, some MoAbs against it are more effective than others in preclinical RIT studies. This may be related to this antigen's possible autocrine/paracrine function and involvement in cell interaction, and the different epitope domains of the molecule. The human MoAbs prepared in Project 2 may identify epitopes on these latter two antigens, If so, the precise epitopes will be determined. If new antigens are identified they will be isolated and characterized, and their cDNAs cloned and sequenced. The epitopes will be analyzed by epitope mapping, oligosaccharide analysis, and production of recombinant antigens and their fragments as chimeric proteins. Molecular characteristics of the epitopes will be correlated with therapeutic effectiveness in in vitro and in vivo (Project 2) preclinical studies, in order to select the best target epitope and MoAb for radioimmunotherapy.
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