Strategic selection and development of immunogenicity binding methods.

The principal components of a meaningful immunogenicity program consist of an initial binding screen followed by confirmation and quantitation of the positive samples. This comprehensive article first describes evolving technologies and assay formats that encourage scientists to start development with appropriate analytical goals that are specific to their clinical program. The selection of the technology and format is based primarily on the product's structure, treatment indication, intended treatment schedule and pharmacokinetic profile. Feasibility studies are described to satisfy specific criteria before proceeding to optimization. Preparation procedures and storage conditions of critical reagents and controls are provided that will render them suitable throughout the length of the project. A multifactor approach to robustness is recommended that assures consistently sensitive, accurate, precise and specific methods that are verified during prevalidation experiments. Finally, a checklist itemizes all the requirements to develop a compliant validation protocol.

An anti-MUC1 antibody-calicheamicin conjugate for treatment of solid tumors. Choice of linker and overcoming drug resistance.

The anti-MUC1 antibody, CTM01, has been chosen to target the potently cytotoxic calicheamicin antitumor antibiotics to solid tumors of epithelial origin that express this antigen. Earlier calicheamicin conjugates relied on the attachment of a hydrazide derivative to the oxidized carbohydrates that occur naturally on antibodies. This produced a "carbohydrate conjugate" capable of releasing active drug by hydrolysis in the lysosomes where the pH is low. Conjugates have now been made that are formed by reacting a calicheamicin derivative containing an activated ester with the lysines of antibodies. This gives an "amide conjugate" that is stable to hydrolysis, leaving the disulfide that is present in all calicheamicin conjugates as the only likely site of drug release from the conjugate. As previously shown for the carbohydrate conjugate, this amide conjugate of CTM01 produces complete regressions of xenograft tumors at doses of 300 microg/kg (calicheamicin equivalents) given three times. This indicates that hydrolytic drug release is not necessary for potent, selective cytotoxicity for calicheamicin conjugates of CTM01. Although the unconjugated calicheamicins are in general less active in cells expressing the multidrug resistance phenotype, both in vitro and in vivo results of studies reported here suggest that the efficacy of the calicheamicins toward such tumors is unexpectedly enhanced by antibody conjugation, especially for the "amide conjugate". These hydrolytically stable conjugates are also active toward cisplatin-resistant ovarian carcinoma cells as well. Such studies indicate that the calicheamicin amide conjugate of CTM01 may have potential for the treatment of MUC1-positive solid tumors, including some types of resistant tumors.

Scleritodermin A, a cytotoxic cyclic peptide from the lithistid sponge Scleritoderma nodosum.

The lithistid sponge Scleritoderma nodosum contains a new cyclic peptide, scleritodermin A (1), the structure of which incorporates l-proline, l-serine, and keto-allo-isoleucine units as well as a novel conjugated thiazole moiety (ACT) and O-methyl-N-sulfoserine. Scleritodermin A (1) inhibited tubulin polymerization and showed significant in vitro cytotoxicity against human tumor cell lines.