title: Enhancement of immunotoxin efficacy by acid-cleavable cross-linking agents utilizing diphtheria toxin and toxin mutants.
abstract: We have utilized a new class of acid-cleavable protein cross-linking reagents in the construction of antibody-diphtheria toxin conjugates (Srinivaschar, K., and Neville, D. M., Jr. (1989) Biochemistry 28, 2501-2509). The potency of anti-CD5 conjugates assayed by inhibition of protein synthesis on CD5 bearing cells (Jurkat) is correlated with cross-linker hydrolytic rates. The maximum increase in potency of the cleavable conjugates over non-cleavable conventional conjugates is 50-fold and is specific for the CD5 uptake route as judged by competition with excess anti-CD5. The potency of conjugates made from diphtheria toxin and the anti-high molecular weight melanoma-associated antigen (HMW-MAA) is enhanced 3-10-fold by a cleavable cross-linker. However the potency of transferrin or anti-CD3 diphtheria toxin conjugates is only minimally enhanced (2-3-fold). Mutant diphtheria toxins, CRM103 and CRM9, previously shown to express less than 1/100 of the wild type in binding affinity were substituted into these conjugates as probes for possible intracellular toxin receptor interactions. Both mutants were equally as toxic to Jurkat target cells exhibiting 1/700 the wild-type potency. CRM9 non-cleavable conjugates were equally as potent as wild-type conjugates for transferrin and anti-CD3-mediated uptake but not for anti-CD5-mediated uptake where toxicity was reduced 60-fold over the wild-type analog. The cleavable cross-linker enhanced the toxicity of anti-CD5-CRM103 and anti-CD5-CRM9 conjugates, but potency was only 1/10 that of the analogous wild-type cleavable conjugate. These data are consistent with a model in which potentiation of toxicity of the anti-CD5 and anti-high molecular weight melanoma-associated antigen conjugates by the cleavable cross-linker occurs from an enhanced intracellular toxin-toxin receptor interaction that ultimately results in increased toxin translocation to the cytosol compartment. In contrast, these data indicate that the anti-CD3 and transferrin uptake systems do not require this interaction in agreement with previous work (Johnson, V.G., Wilson, D., Greenfield, L., and Youle, R. J. (1988) J. Biol. Chem. 263, 1295-1300).
authors: Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, Maryland 20892.
authors: D M
authors: DM
authors: Neville
authors: K
authors: K
authors: Srinivasachar
authors: R
authors: R
authors: Stone
authors: J
authors: J
authors: Scharff
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000633:toxicity; D000943:Antigens, Differentiation / Q000276:immunology; D000951:Antigens, Neoplasm; D018956:CD5 Antigens; D002384:Catalysis; D003432:Cross-Linking Reagents; D004167:Diphtheria Toxin / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D000922:Immunotoxins / Q000378:metabolism / Q000633:toxicity*; D008545:Melanoma / Q000276:immunology; D058950:Melanoma-Specific Antigens; D008970:Molecular Weight; D009154:Mutation; D009363:Neoplasm Proteins / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000633:toxicity; D014168:Transferrin / Q000378:metabolism / Q000633:toxicity
chemical_list: D000911:Antibodies, Monoclonal; D000943:Antigens, Differentiation; D000951:Antigens, Neoplasm; D018956:CD5 Antigens; D003432:Cross-Linking Reagents; D004167:Diphtheria Toxin; D000922:Immunotoxins; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins; D011500:Protein Synthesis Inhibitors; D014168:Transferrin