Michael Jon Bjorn

Characterization of translational inhibitors from Phytolacca americana Amino-terminal sequence determination and antibody-inhibitor conjugates

Two translational inhibitors (pokeweed antiviral protein and pokeweed antiviral protein II) isolated from the leaves of the pokeweed plant, Phytolacca americana, were characterized as to their behavior during reverse-phase HPLC and their amino-terminal sequences. Alignment of the sequences demonstrated that a substantial degree of homology was present (10 of 29 identical residues). Pokeweed antiviral protein was shown by reverse-phase chromatography to be composed of at least two components, pokeweed antiviral proteina and pokeweed antiviral proteinb, which comigrated on sodium dodecyl sulfate polyacrylamide gel electrophoresis, shared identical N-terminal amino-acid sequences through residue 31, and had similar specific activities in a cell-free translation inhibition assay. Pokeweed antiviral protein II was covalently coupled to a monoclonal antibody that recognizes the transferrin receptor (anti-transferrin receptor). The disulfide-linked conjugate inhibited protein synthesis in the human breast tumor cell line MCF-7, whereas anti-transferrin receptor, pokeweed antiviral protein II, or an immunotoxin composed of an irrelevant antiserum and pokeweed antiviral protein II, were nontoxic. The inhibitory dose 50% of anti-transferrin receptor-pokeweed antiviral protein II for MCF-7 cells was 0.7 nM, whereas the corresponding ricin A chain conjugate (anti-transferrin receptor-ricin A chain) was more potent with a inhibitory dose 50% of 0.1 nM. Pokeweed antiviral protein II can be added to the growing list of translation inhibitors that are effective as components of immunotoxins in vitro. Additional studies will be needed to determine whether pokeweed antiviral protein II immunotoxins provide advantageous properties for in vivo applications.

Breast Cancer Immunotoxins

Mice were immunized with live breast cancer cells or fresh breast cancer membrane extract. The immune splenocytes were fused with SP2/0 myeloma cells and the hybridomas were screened for breast cancer reactive antibody production. Over 100 monoclonal antibodies were purified and characterized. Antibodies were found which bound only a few substructures in normal tissues and which bound the majority of breast cancers. These antibodies were infrequent. A number of these antibodies fall into groups recognizing determinants on several distinct surface or intracellular proteins and cross-react most with endometrial, prostate and ovarian cancer. These antibodies were conjugated to ricin toxin A chain (RTA) and a fraction of these RTA conjugates were able to selectively kill tumor cells in vitro (i.e. low TCID50 on tumor cells but high TCID50 on fibroblasts). Antibodies with higher affinity and antigen copy number produced more active conjugates. Some RTA antibody conjugates showed moderate tumor growth inhibitory activity in vivo at LD10. The poor therapeutic index in vivo may be due to rapid clearance of the conjugate from the plasma to the liver and other organs which results in poor tumor localization of conjugate.

Response of primary human mammary tumor cell cultures to a monoclonal antibody-recombinant ricin A chain immunotoxin.

SummaryMalignant epithelial tumor cells were isolated and cultured from ten human mammary specimens of cancerous origin. The 260F9 monoclonal antibody (MAB) bound to frozen sections of all of the human breast tumors tested and to primary cultured cells from the tumors. Cultured cells from all ten breast tumors were sensitive to the clonal inhibitory effects of immunotoxin 260F9 MAB-recombinant ricin A chain. At an immunotoxin concentration of 200 ng/ml (about 1 nM), inhibition of colony formation was >99% for all ten tumors.