Conjugation of tumor selective antibodies to ribosomal inhibitory proteins (RIP) results in chimeric molecules that are highly toxic to target tumor cells. Since RIPs inactivate sensitive biosynthetic processes within the cell. the conjugates made from them are more cytotoxic than chemotherapeutic drugs. The ability of the conjugates to inhibit tumor cell growth is dependent on : a) binding, b) Internalization and c) translocation of RIP from the endocytotic vesicles to the cytoplasm. This process is enhanced by monocarboxylic ionophores by several orders of magnitude. However, ionophores were not efficacious in vivo due to increased toxicity and faster clearance from the circulation. Therefore, it is important to develop alternative methods to improve the immunotoxin activity. We propose to make the following structural changes in the toxin moiety to achieve this goal: a) addition of the translocation domain from Diphtheria toxin B fragment (CRM-107) which has decreased binding to cells due a point mutation, b) addition of poly-lysine , c) addition of the KDEL translocation signal sequence, and d) incorporation of sequences corresponding to melittin, a bee venom peptide which can penetrate cell membranes and form Ion channels. Other constructs include the generation of fusion proteins containing the bacterial toxin colicin and ribonuclease A. These new chimeric toxin molecules should have increased efficiency in translocation. We have generated two new monoclonal antibodies (OVX1 and OVX2) which recognize a unique determinant on ovarian and breast cancer cells. Immunoconjugates will be prepared between these antibodies and genetically engineered toxins. The efficacy of these conjugates to inhibit ovarian tumor cells will be studied in vitro and in vivo. The overall goal of the project is to generate novel, second generation immunotoxins directed to human ovarian epithelial cancer cells.
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