Endocytosis of immunotoxin-791T/36-RTA by tumor cells in relation to its cytotoxic action.

Ricin A chain immunotoxin constructed with monoclonal antibody 791T/36, which recognizes a tumor associated glycoprotein Mr 72,000 antigen present on sarcomas and colon and ovarian cancer cells, is cytotoxic for cell lines from tumors expressing this antigen. Incubation of sarcoma 791T cells with immunotoxin for only 5 min is sufficient to produce greater than 95% inhibition of tumor cell growth. Papain treatment of these cells to remove immunotoxin from the cell surface indicated that the cell surface acts as a reservoir for continued internalization of immunotoxin over several hours, but even so, 50% inhibition of cell survival was produced over a 2- to 3-h period. Analysis of the rate of endocytosis demonstrated that 30-50% of cell bound immunotoxin was internalized over a 180-min period. This was primarily dictated by the antibody moiety, regardless of the degree of conjugation to ricin A chain. This rate is much slower than that of other cell surface ligands such as transferrin. Cell cytosol acidification experiments were performed to determine whether this immunotoxin was internalized by clathrin coated pits, which is relatively rapid, or by smooth pits, which is slower, and the results indicated the latter mechanism is almost exclusively used. Intracellular trafficking of antibody 791T/36, conjugated to human serum albumin-tetramethylrhodamine was investigated by flow cytometry. The movement of the conjugate into the lysosomal compartment was delayed so that degradation products were only detected after a lag phase of 30-60 min. The lack of potentiator dependence of 791T/36 immunotoxin is in keeping with these findings.

Measurement of antibody binding to antigenic peptides conjugated in situ to albumin-coated microtitre plates.

Monoclonal antibodies have been prepared against a synthetic peptide with a sequence corresponding to a repeated hydrophilic region of the protein core of the human MUC-2 gastrointestinal mucin. Peptide conjugates, prepared by glutaraldehyde cross-linking with keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA), were employed as the immunogen and target antigen (for screening by ELISA), respectively. However, for the measurement of antibody binding to peptide by an ELISA procedure, an alternative strategy was developed and is described in this report: peptides were conjugated directly to BSA immobilized by physical adsorption to the surface of microtitre plate wells. This procedure permits peptides to be tested as target antigens by ELISA without prior preparation of peptide-carrier conjugates.

Identification of peptide motifs recognized by a human IgG autoanti-IgE antibody using a phage display library.

BACKGROUND: The potential of murine monoclonal anti-IgE antibodies as long-term therapy for atopic diseases will have to rely, for the time being, on passive antibody administration. There is therefore considerable interest in developing a peptide-based vaccine for active immunization to elicit long-term protective anti-IgE antibodies in the patient. It has been shown that some human IgG autoanti-IgE antibodies have the ability to partially block the binding of IgE to Fc receptors such as Fc epsilonRI. Therefore, the epitopes recognized by such antibodies could have vaccine potential. OBJECTIVE: To determine the epitope specificity of one such human IgG anti-IgE antibody. METHODS: A 15-mer phage-peptide library was used to establish the epitope specificity of an IgG anti-IgE antibody isolated from the serum of an asthma patient. RESULTS: The SRPSP sequence, or part of it (i.e. RPS, RPSP, SPS or PSP), was present in all 18 phage-peptides that have been sequenced. This common motif was found to be within the human epsilon chain sequence Ser341-Thr355 near the N-terminus of the C epsilon3 domain. According to the human Fc epsilon model, the most accessible residues in this sequence are Arg342, Ile350, Arg351, Lys352 and Ser353. CONCLUSIONS: The present data should provide the molecular basis for the rational design of a suitable peptide immunogen (vaccine) for boosting the production of protective autoanti-IgE antibodies.