The central theme of Project Ib has been the generation, characterization and application of defined monoclonal antibody (Mab) reagents with anti-glioma reactivity in both in vitro and in vivo settings. Interacting with projects Ia, Ic, the core facilities Ia1, Ib1, and Ic1, IIb, and IVb, Project Ib has as specific aims: 1. Generation of anti-epidermal growth factor receptor (EGFR) murine Mabs vs. both the well-characterized A431 squamous cell carcinoma cell line EGFR and glioma-expressed EGFR for use in vitro (immunohistological, growth control studies) and in vivo (localizing, model immunotherapeutic) applications. With project Ic, Mabs will be prepared using the A431 cell line as source of EGFR; different immunization protocols incorporating trypsinized cells, combination protocols of cells and EGF-affinity purified A431 cell membrane preparations, and gel isolated 170 Kd EGFR-enriched bands will be performed. Optimal method(s) identified will be used to produce Mabs to glioma-associated EGFR. 2. Analysis of the Mab 81C6-defined extracellular matrix antigen tenascin (formerly GMEM) at the genetic, epitopic, molecular, and functional levels, and extended studies of the use of tenascin as a localizable target is the most advanced and extensive aim of Project Ib. Given our established procedures for Mab 81C6 affinity purification of tenascin, we will affinity purify enzymatically cleaved tenascin fragments reactive with anti-tenascin reagents, including polyvalent rabbit and macaque sera, murine Mabs, and serum samples obtained from immunotherapy patients. Reactive polypeptides will undergo epitope restriction analysis, amino acid sequencing, and serve as immunogens for directed immunization vs. epitopes of evolutionarily conserved, organotypic, and potentially fetal brain-glioma restricted expression. Expression clones from a lambdal gt 11 library produced by Dr. Mario Bourdon also will be screened and used to define the genomic profile of tenascin. With Project IIb, the differential capacities of restricted epitope Mabs and their fragments will be assessed in both subcutaneous and intracranial xenograft localization studies. 3. Three new glioma- associated, localizable antigens, C12, D12, and E9 will be developed for potential in vivo application following successful tenascin protocols: affinity purification of antigen, production of second generation Mabs, and Mab fragments for localization studies. 4. Recent data obtained in this laboratory suggest possible phenotypic markers for glial vs. neuronal differentiation pathways in medulloblastoma (Mab); we propose to generate Mabs to the unique bank of four established MED cell lines maintained at Duke, and a fifth available in xenograft form. In conjunction with project IVb, these studies will form the basis for complete phenotypic investigation of medulloblastoma parallel to that ongoing in glioma.
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