Cytotoxic ribonuclease chimeras. Targeted tumoricidal activity in vitro and in vivo.
Monoclonal antibodies to the transferrin receptor or to the T cell antigen, CD5, were chemically linked to mammalian RNase A and found to specifically inhibit protein synthesis in antigen-positive cells. Antibody-mediated specificity of these cytotoxic ribonuclease chimeras (CRCs) was demonstrated in three ways. 1) Toxicity was due to the chemical linkage of RNase to antibody, as the individual components added separately or in combination did not inhibit protein synthesis; 2) the anti-transferrin receptor CRCs inhibited protein synthesis in those cells expressing the human transferrin receptor (K562, U251, Jurkat cells) but had no detectable toxicity to cells lacking the human transferrin receptor (Vero or NIH 3T3 cells); 3) free antibody to either the human transferrin receptor (454A12 or 5E-9) or to the T cell antigen, CD5 (T101), blocked the cytotoxicity of the respective CRC. Two CRC species, designated P1 and P2, that differed in size and stoichiometry of RNase A to antibody, were purified by size-exclusion high performance liquid chromatography. The higher molecular weight P1 conjugate had an IC50 of 20-30 nM, whereas the P2 conjugate had a higher IC50 of 300-500 nM. Bioactivity could be reversibly increased more than 10-fold by freezing. The cytotoxicity of the CRCs was examined in vivo in a solid tumor animal model. Intratumoral injections of an anti-transferrin receptor CRC into established U251 human glioblastoma tumors grown in the flanks of nude mice prevented tumor growth, whereas RNase A mixed with antibody was ineffective. CRCs, therefore, express cytotoxicity in vitro and in vivo. Mammalian nucleases coupled to antibodies may be utilized as cell type-selective cytotoxins and have potential as pharmacologic reagents. The systemic toxicity and immunogenicity observed with mammalian derived cytotoxins may be significantly less than that of the currently employed plant- and bacterial-derived immunotoxins.
Newton
D L
DL
National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
Ilercil
O
O
Laske
D W
DW
Oldfield
E
E
Rybak
S M
SM
Youle
R J
RJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000970:Antineoplastic Agents; D002470:Cell Survival / Q000187:drug effects*; D003432:Cross-Linking Reagents; D003603:Cytotoxins; D000922:Immunotoxins / Q000737:chemistry*; D066298:In Vitro Techniques; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D014176:Protein Biosynthesis; D011990:Receptors, Transferrin / Q000276:immunology; D012260:Ribonucleases / Q000737:chemistry / Q000633:toxicity*; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D003432:Cross-Linking Reagents; D003603:Cytotoxins; D000922:Immunotoxins; D011990:Receptors, Transferrin; D012260:Ribonucleases