Kinetics of cellular trafficking and cytotoxicity of 9.2.27-gelonin immunotoxins targeted against the high-molecular-weight melanoma-associated antigen

Abstract

The high-molecular-weight melanoma-associated antigen (HMW-MAA) is expressed on a large majority of melanoma tissues but not on most normal or other neoplastic tissues. Monoclonal antibody 9.2.27 binds with high affinity and specificity to the HMW-MAA, making it an attractive choice as an agent for targeting toxins or chemotherapeutic agents specifically towards melanomas. To characterize the interactions between 9.2.27 and melanoma cells more carefully, data on the kinetics of binding, internalization, and degradation of 9.2.27 by SK-MEL-2 cells were collected. Binding of 9.2.27 to SK-MEL-2 cells was rapid, and followed by slow loss of surface-bound antibody, probably because of loss of surface antigen caused by degradation and/or shedding. A small fraction (approx. 5%) of surface-bound 9.2.27 was internalized and degraded. A mathematical model describing these interactions was developed, and equilibrium and kinetic constants were fitted to the data. To evaluate the utility of 9.2.27 as a toxin-targeting agent, 9.2.27-gelonin immunotoxins were constructed and tested in protein synthesis inhibition assays. The dependence of the toxicity data for 9.2.27-gelonin on time and concentration was quantitatively related to the accumulated intracellular exposure to 9.2.27-gelonin by a relatively simple equation. This equation had been previously validated for immunotoxins targeted against the transferrin receptor, for which the trafficking kinetics are quite dissimilar from those of the HMW-MAA. The success of the approach here suggests that this method may be widely applicable for analysis of immunotoxin efficacy.