Monoclonal antibody 45-2D9 conjugated to the A chain of ricin is specifically toxic to c-Ha-ras-transfected NIH 3T3 cells expressing gp74.

Monoclonal antibody 45-2D9 recognizes a 74K Mr glycoprotein determinant on a c-Ha-ras oncogene-transfected cell line (45-342). An immunotoxin was made by conjugating this antibody to the A chain of ricin toxin (RTA). The immunotoxin could mediate essentially complete inhibition of leucine and thymidine incorporation by 45-342 cells prepared as single cell suspensions from tumors grown in vivo. Addition of ammonium chloride to the culture medium potentiated this inhibition, but the magnitude of this effect was dependent on incubation time and cell concentration. The immunotoxin effects were noted at concentrations 100-fold lower than similar effects caused by unconjugated RTA, and the 45-2D9 antibody had no effect in the assay system. Immunotoxins directed against antigens not expressed by 45-342 were not effective, and the 45-2D9 immunotoxin was not specifically toxic to other transfected cells not expressing the gp74 antigen. After a 72-hr incubation, lysis of 80% of the 45-342 cells was demonstrated by trypan blue exclusion. Complete inhibition of 45-342 colony formation was achieved at 10 days with a 10(-9) M concentration of the specific immunotoxin. These results indicate that an immunotoxin with specific reactivity towards an oncogene-transformed cell can be made, and that such cells derived from fresh tumors are susceptible to immunoconjugate-mediated toxicity.