Laboratory preparation of a deglycosylated ricin toxin A chain containing immunotoxin directed against a CD7 T lineage differentiation antigen for phase I human clinical studies involving T cell malignancies

Daniel A. Vallera, Linda J. Burns, Arthur E. Frankel, Andrew R. Sicheneder, Roland Gunther, K. Gajl-Peczalska, Christopher A. Pennell, John H. Kersey

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

An immunotoxin consisting of a monoclonal antibody specific for CD7, a cell surface determinant expressed on T acute lymphocytic leukemia (T-ALL) blast cells, was linked to the potent plant toxin deglycosylated ricin toxin A chain (dgRTA) and is currently under evaluation in phase I clinical trials. Scale-up production of this immunotoxin, called DA7, was simplified using a two-step purification protocol that resulted in a highly purified immunotoxin meeting FDA criteria for IND approval. The anti-CD7 antibody, 3A1e, an IgG2b, was coupled to toxin using two different heterobifunctional cross-linkers, (1) N-succinimidyl-3-(2-pyridyl-dithiolproprionate) (SPDP), considered a standard crosslinker and (2) 4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT), designed to hinder the in vivo breakdown of the toxin/antibody disulfide bond. Since experiments revealed that SPDP-DA7 had similar pharmacokinetics and biodistribution in mice and higher yields than DA7 made with a hindered cross-linker, SPDP-DA7 was scaled up for clinical study. Yield of SPDP-DA7 was 25% relative to starting material. Fractions were collected containing a toxin:antibody ratio of 1:1 to 4:1 rather than only a 1:1 ratio since studies showed that this heterogenous fraction was just as toxic to proliferating CD7-expressing leukemia cells as a homogeneous 1:1 fraction. In vitro, the concentration of heterogenous SPDP-DA7 selectively inhibiting 50% activity (IC50) of the CD7+ CEM cell line was 0.01 μg/ml to 0.05 μg/ml for inhibiting activated T cells or T cell lines. In vivo, SPDP-DA7 showed a significant anti-tumor effect against CEM cells administered to scid/scid mice, but even more importantly was effective against primary T cell leukemias taken from patients and injected into scid/scid mice.

Original languageEnglish (US)
Pages (from-to)69-83
Number of pages15
JournalJournal of Immunological Methods
Volume197
Issue number1-2
DOIs
StatePublished - Oct 16 1996

Keywords

  • T cell
  • acute lymphoblastic leukemia
  • cancer theapy
  • human
  • immunotherapy
  • immunotoxin
  • mouse
  • ricin
  • scid/scid

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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