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

The high-molecular-weight melanoma-associated antigen (HMW-MAA) is expresse d on a large majority of melanoma tissues but not on most normal or other n eoplastic 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 ca refully, 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 c ells was rapid, and followed by slow loss of surface-bound antibody, probab ly 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 an d degraded. A mathematical model describing these interactions was develope d, and equilibrium and kinetic constants were fitted to the data. To evalua te the utility of 9.2.27 as a toxin-targeting agent, 9.2.27-gelonin immunot oxins were constructed and tested in protein synthesis inhibition assays. T he dependence of the toxicity data for 9.2.27-gelonin on time and concentra tion was quantitatively related to the accumulated intracellular exposure t o 9.2.27-gelonin by a relatively simple equation. This equation had been pr eviously validated for immunotoxins targeted against the transferrin recept or, for which the trafficking kinetics are quite dissimilar from those of t he HMW-MAA. The success of the approach here suggests that this method may be widely applicable for analysis of immunotoxin efficacy.