New coupling agents for the synthesis of immunotoxins containing a hindered disulfide bond with improved stability in vivo

P. E. Thorpe, P. M. Wallace, P. P. Knowles, M. G. Relf, A. N F Brown, G. J. Watson, R. E. Knyba, E. J. Wawrzynczak, D. C. Blakey

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Abstract

Two new coupling agents were synthesized for making immunotoxins containing disulfide bonds with improved stability in vivo: sodium S-4-succinimidyloxycarbonyl-α-methyl benzyl thiosulfate (SMBT) and 4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldithio)toluene (SMPT). Both reagents generate the same hindered disulfide linkage in which a methyl group and a benzene ring are attached to the carbon atom adjacent to the disulfide bond and protect it from attack by thiolate anions. An immunotoxin consisting of monoclonal anti-Thy-1.1 antibody (OX7) linked by means of the SMPT reagent to chemically deglycosylated ricin A-chain had better stability in vivo than an immunotoxin prepared with 2-immunothiolane hydrochloride (2IT) which generates an unhindered disulfide linkage. About 48 h after i.v. injection into mice, one-half of the SMPT-linked immunotoxin present in the blood was in intact form and one-half as released free antibody, whereas equivalent breakdown of the 2IT-linked immunotoxin was seen at about 8 h after injection. Consequently, the blood levels of the SMPT-linked immunotoxin remained higher than those of the 2IT-linked immunotoxin despite loss of immunotoxin from the blood by other mechanisms. Forty-eight h after injection, 10% of the injected dose of the SMPT-linked immunotoxin remained in the bloodstream as compared with only 1.5% of the 2IT-linked immunotoxin. The ability of immunotoxins prepared with the new reagents to inhibit protein synthesis by Thy-1.1-expressing AKR-A/2 lymphoma cells in vitro was identical to that of immunotoxins prepared with 2IT or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Clonogenic assays showed that fewer than 0.01% of AKR-A/2 cells survived exposure to high concentrations of OX7-abrin A-chain immunotoxins prepared with SMBT, 2IT, or SPDP. Twelve clones of cells which had survived treatment with the SMBT-linked immunotoxin were isolated. None of the clones was selectively resistant to the SMBT-linked immunotoxin when retested in cytotoxicity assays. In conclusion, immunotoxins prepared with the new coupling agents should have improved antitumor activity in vivo because they are longer lived and do not break down so readily to release free antibody which could compete for the target antigens.

Original languageEnglish (US)
Pages (from-to)5924-5931
Number of pages8
JournalCancer Research
Volume47
Issue number22
StatePublished - 1987

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Immunotoxins
Disulfides
Toluene
Thiosulfates
Injections
Abrin
Clone Cells
Ricin
Antibodies

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Thorpe, P. E., Wallace, P. M., Knowles, P. P., Relf, M. G., Brown, A. N. F., Watson, G. J., ... Blakey, D. C. (1987). New coupling agents for the synthesis of immunotoxins containing a hindered disulfide bond with improved stability in vivo. Cancer Research, 47(22), 5924-5931.

New coupling agents for the synthesis of immunotoxins containing a hindered disulfide bond with improved stability in vivo. / Thorpe, P. E.; Wallace, P. M.; Knowles, P. P.; Relf, M. G.; Brown, A. N F; Watson, G. J.; Knyba, R. E.; Wawrzynczak, E. J.; Blakey, D. C.

In: Cancer Research, Vol. 47, No. 22, 1987, p. 5924-5931.

Research output: Contribution to journalArticle

Thorpe, PE, Wallace, PM, Knowles, PP, Relf, MG, Brown, ANF, Watson, GJ, Knyba, RE, Wawrzynczak, EJ & Blakey, DC 1987, 'New coupling agents for the synthesis of immunotoxins containing a hindered disulfide bond with improved stability in vivo', Cancer Research, vol. 47, no. 22, pp. 5924-5931.
Thorpe PE, Wallace PM, Knowles PP, Relf MG, Brown ANF, Watson GJ et al. New coupling agents for the synthesis of immunotoxins containing a hindered disulfide bond with improved stability in vivo. Cancer Research. 1987;47(22):5924-5931.
Thorpe, P. E. ; Wallace, P. M. ; Knowles, P. P. ; Relf, M. G. ; Brown, A. N F ; Watson, G. J. ; Knyba, R. E. ; Wawrzynczak, E. J. ; Blakey, D. C. / New coupling agents for the synthesis of immunotoxins containing a hindered disulfide bond with improved stability in vivo. In: Cancer Research. 1987 ; Vol. 47, No. 22. pp. 5924-5931.
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AU - Thorpe, P. E.

AU - Wallace, P. M.

AU - Knowles, P. P.

AU - Relf, M. G.

AU - Brown, A. N F

AU - Watson, G. J.

AU - Knyba, R. E.

AU - Wawrzynczak, E. J.

AU - Blakey, D. C.

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N2 - Two new coupling agents were synthesized for making immunotoxins containing disulfide bonds with improved stability in vivo: sodium S-4-succinimidyloxycarbonyl-α-methyl benzyl thiosulfate (SMBT) and 4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldithio)toluene (SMPT). Both reagents generate the same hindered disulfide linkage in which a methyl group and a benzene ring are attached to the carbon atom adjacent to the disulfide bond and protect it from attack by thiolate anions. An immunotoxin consisting of monoclonal anti-Thy-1.1 antibody (OX7) linked by means of the SMPT reagent to chemically deglycosylated ricin A-chain had better stability in vivo than an immunotoxin prepared with 2-immunothiolane hydrochloride (2IT) which generates an unhindered disulfide linkage. About 48 h after i.v. injection into mice, one-half of the SMPT-linked immunotoxin present in the blood was in intact form and one-half as released free antibody, whereas equivalent breakdown of the 2IT-linked immunotoxin was seen at about 8 h after injection. Consequently, the blood levels of the SMPT-linked immunotoxin remained higher than those of the 2IT-linked immunotoxin despite loss of immunotoxin from the blood by other mechanisms. Forty-eight h after injection, 10% of the injected dose of the SMPT-linked immunotoxin remained in the bloodstream as compared with only 1.5% of the 2IT-linked immunotoxin. The ability of immunotoxins prepared with the new reagents to inhibit protein synthesis by Thy-1.1-expressing AKR-A/2 lymphoma cells in vitro was identical to that of immunotoxins prepared with 2IT or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Clonogenic assays showed that fewer than 0.01% of AKR-A/2 cells survived exposure to high concentrations of OX7-abrin A-chain immunotoxins prepared with SMBT, 2IT, or SPDP. Twelve clones of cells which had survived treatment with the SMBT-linked immunotoxin were isolated. None of the clones was selectively resistant to the SMBT-linked immunotoxin when retested in cytotoxicity assays. In conclusion, immunotoxins prepared with the new coupling agents should have improved antitumor activity in vivo because they are longer lived and do not break down so readily to release free antibody which could compete for the target antigens.

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