Comparative cytotoxicity and pharmacokinetics of antimelanoma immunotoxinscontaining either natural or recombinant gelonin

Immunotoxins are a class of targeted therapeutic agents under development b y various research groups. The murine monoclonal antibody designated ZME-01 8 recognizes a high molecular weight glycoprotein present on most human mel anoma cells and biopsy specimens and has been utilized for clinical imaging studies in patients with melanoma. The plant toxin gelonin is a ribosome-i nactivating protein (RIP) with n-glycosidase activity similar to that of ri cin A chain. In previous studies by our group, the gelonin toxin was sequen ced, cloned and expressed in E. coli. The purified recombinant gelonin (RG) was found to have identical protein synthesis inhibitory activity to that of natural gelonin (NG). For comparative purposes, chemical conjugates of a ntibody ZME and either RG or NG were produced using the heterobifunctional crosslinking reagents SPDP and SMPT. The ZME-NG and ZME-RG immunotoxins wer e found to be 10(4)- to 10(5)-fold more cytotoxic to antigen-positive human melanoma cells than free toxin. NG toxin alone was cytotoxic to intact cel ls (IC50 = 100 nM) while RG was nontoxic to cells at doses up to 1 mu M. Bo th ZME-NG and ZME-RG immunoconjugates were nontoxic to antigen-negative (Me -180) cells. ZME-RG immunotoxins constructed with the more stable SMPT reag ent were slightly more effective in culture than conjugates made with SPDP. Tissue distribution studies in tumor-bearing nude mice demonstrated that t umor uptake of the ZME-RG immunotoxin was similar to that of the intact ZME antibody with reduced distribution to normal organs compared to an immunoc onjugate produced with NG. Pharmacokinetic studies showed that the terminal -phase plasma half-life of ZME-RG was similar to that of ZME itself (42 h v s 50 h) and almost threefold higher than that of ZME-NG (11.5 h). The area under the concentration curve (Cxt) for ZME-RG was 50% lower than that for ZME due to an increased apparent volume of distribution (Vd(a)) but was alm ost tenfold higher than the Cxt for ZME-NG. These studies suggest that immu noconjugates comprising RG demonstrate identical in vitro cytotoxic effects to immunoconjugates produced with NG and immunotoxins with RG display impr oved in vivo pharmacodynamics and tissue distribution compared to immunotox ins containing NG.