Nicholas Pomato

A carbohydrate-directed heterobifunctional cross-linking reagent for the synthesis of immunoconjugates☆

A novel, highly water-soluble, heterobifunctional cross-linking reagent, S-(2-thiopyridyl)-L-cysteine hydrazide (TPCH), was synthesized which contains a hydrazide moiety for coupling to aldehyde groups generated in the carbohydrate residues of antibodies by mild periodate oxidation, and a pyridyl disulfide moiety for coupling to molecules with a free sulfhydryl group. Since the carbohydrate moieties are distal to the antigen binding region of antibodies, derivatization with this cross-linker minimizes impairment of the antigen binding function. Derivatization of the human monoclonal IgM antibody 16-88 against human colon carcinoma cells with as many as 16 TPCH cross-linker molecules did not impair its antigen binding capability. Using mild oxidation conditions for antibody derivatization, sialic acid residues were identified as attachment sites for the cross-linker molecules, since after desialylation of antibody 16-88 by neuraminidase virtually no cross-linker molecules could be incorporated. Comparison of TPCH with S-(2-thiopyridyl)mercaptopropionic acid hydrazide and S-(2-thiopyridyl)-L-cysteine, two related cross-linking reagents, revealed that TPCH is most efficiently incorporated into periodate-treated antibody. Based on the structural differences of the cross-linkers, the more efficient incorporation of TPCH appears to be a function of the presence of a hydrazide moiety with an adjacent amino group. When three to four molecules of pyridyl disulfide-derivatized barley toxin were coupled to TPCH-derivatized antibody 16-88, the antigen binding capability remained uncompromised. In addition, no significant impairment of toxin activity upon coupling to the antibody was observed. Based on these data, TPCH may be very useful for the synthesis of immuno-conjugates with no or only minimal impairment of the antigen binding function.

Development and Characterization of Human Monoclonal Antibodies and Their Application in the Radioimmunodetection of Colon Carcinoma

Significant progress has been made in the application of monoclonal antibody technology to clinical diagnosis of cancer and to management of the disease. Antibodies labeled with radioactive isotopes have been demonstrated to localize in tumors of the gastrointestinal tract (1–6), ovary (6), breast (6,7), and skin (8) and promise to be very helpful in identifying metastases in patients with these tumors. However, there remain several problems to be resolved before radiolabeled antibody detection of tumor foci will be applied as an accepted and routine diagnostic/prognostic procedure. Problems include antibody cross-reactivity with normal tissues (5,6,9,10), low level penetration of antibody into tumor tissue (5,9,10), and inhibitory effects of specific circulating antigen (11). Current research is focused on selecting antibodies with the most desirable characteristics for tumor detection, improving chemistries to allow use of more appropriate radionuclides, and defining the effective clinical applications with regard to the tumor types, tumor sites and available diagnostic equipment and procedures.