Improved antitumor effects of immunotoxins prepared with deglycosylated ricin A-chain and hindered disulfide linkages.
A monoclonal anti-Thy-1.1 antibody (OX7) was coupled to either native or chemically deglycosylated ricin A-chain (dgA) using one of two different cross-linking agents. One cross-linker, N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)tolu ene (SMPT), generates a sterically hindered disulfide bond which is relatively resistant to reduction, whereas the other, 2-iminothiolane hydrochloride, generates an unhindered disulfide bond with greater lability. A two-compartment pharmacokinetic model was used to analyze the blood levels of each immunotoxin and its breakdown product (free antibody) after i.v. injection into mice. Immunotoxins prepared with SMPT broke down in vivo 6.3-fold more slowly than those prepared with 2-iminothiolane hydrochloride, and immunotoxins containing native A-chain were cleared 2- to 3-fold more rapidly from the bloodstream than those containing dgA. As a result, 24 h after injection, 16% of the OX7-SMPT-dgA remained in the blood as compared with 0.4 to 2.5% of the other immunotoxins. Immunotoxins prepared with dgA were about 3-fold more toxic to mice than those prepared with native A-chain, whereas immunotoxins prepared with SMPT were only slightly more toxic than those prepared with 2-iminothiolane hydrochloride. When equivalent toxic doses of the immunotoxins were administered i.v. to mice which had been given injections of Thy-1.1+ AKR-A/2 lymphoma cells, the OX7-SMPT-dgA gave the best antitumor effect. A dose equivalent to one-seventh of the median lethal dose extended the survival time of the animals by the extent expected if 99.999% of the tumor cells had been eradicated. Furthermore, the tumors that did develop in the mice treated with OX7-SMPT-dgA were mutants which were resistant to all the immunotoxins. Some of the mutants were deficient in Thy-1.1 whereas others were not. In conclusion, both the use of the SMPT cross-linker and deglycosylation of the A-chain significantly improve the therapeutic index of the immunotoxins in AKR-A/2 tumor-bearing mice.
Thorpe
P E
PE
Drug Targeting Laboratory, Imperial Cancer Research Fund, London, England.
Wallace
P M
PM
Knowles
P P
PP
Relf
M G
MG
Brown
A N
AN
Watson
G J
GJ
Blakey
D C
DC
Newell
D R
DR
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D004355:Drug Stability; D005260:Female; D006031:Glycosylation; D000922:Immunotoxins / Q000378:metabolism / Q000494:pharmacology*; D007518:Isoantibodies / Q000276:immunology; D007928:Lethal Dose 50; D008223:Lymphoma / Q000473:pathology / Q000628:therapy; D051379:Mice; D008806:Mice, Inbred AKR; D008807:Mice, Inbred BALB C; D012276:Ricin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D000922:Immunotoxins; D007518:Isoantibodies; C035678:anti-Thy antibody; D012276:Ricin