Daunorubicin coupled to monoclonal antibodies via a cis-aconitic anhydride linker: biochemical and cytotoxic properties revisited.

Daunorubicin (DNR) was coupled to monoclonal antibodies (Mab) reactive to breast tumor cells using the acid-labile linking agents cis-asonitic anhydride and two other non acid-labile analogs, glutaric anhydride and citraconic anhydride. The acid derivatives of DNR formed by reaction with the anhydrides were converted to their N-hydroxysuccinimide (NHS) active esters for coupling to MAb. The molar input of drug NHS ester to MAb ranged from 1:1 to 100:1. The resulting MAb-DNR conjugates were purified by gel filtration and analyzed by high performance liquid chromatography. Monomeric conjugates contained 0.2 to 11.0 moles of DNR/mole of MAb. No evidence of cell killing was observed up to a concentration of 10 micrograms/ml DNR bound to MAb, while DNR exhibited 50% killing of the breast tumor cell line MCF-7 at a concentration of 1 microgram/ml.

Immunoconjugates of doxorubicin and murine antihuman breast carcinoma monoclonal antibodies prepared via an N-hydroxysuccinimide active ester intermediate of cis-aconityl-doxorubicin: preparation and in vitro cytotoxicity.

Doxorubicin (DXR) conjugated to murine monoclonal antibodies (MoAb) raised against human breast tumor cells demonstrated a MoAb-specific, molar ratio-dependent in vitro cytotoxicity. These conjugates were prepared on a scale sufficient to allow for subsequent clinical trials (1 to 3 g of MoAb per conjugation reaction). The conjugation reaction proceeded via an N-hydroxysuccinimide (NHS) active ester intermediate of cis-aconityl-DXR (CA-DXR), resulting in a cis-aconitate acid-sensitive linker between the DXR and MoAb. Molar ratios of DXR to MoAb ranged from 40 to 45. The immunoreactivity of conjugated MoAb was only slightly decreased from naked MoAb. When immunoconjugates were incubated with MoAb-reactive tumor cells for 3 hours, specific cell-killing was observed. If the exposure time was lengthened to 18 hours, however, nonspecific killing resulted. Incubation of the immunoconjugate with the nonspecific adsorbant Amberlite XAD-2 caused an average 30% decrease in the DXR-to-MoAb molar ratio, suggesting a population of drug that is tightly but noncovalently associated with MoAb.