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.
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, 3Ale, an IgG2b, was coupled to toxin using two different heterobifunctional cross-linkers, (1) N-succinimidyl-3-(2-pyridyl-dithiolproprionate) (SPDP), considered a standard croslinker and (2) 4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)tolu ene (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 microgram/ml to 0.05 microgram/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.
Department of Medicine, University of Minnesota Cancer Center, Minneapolis, USA.
D A
DA
Vallera
L J
LJ
Burns
A E
AE
Frankel
A R
AR
Sicheneder
R
R
Gunther
K
K
Gajl-Peczalska
C A
CA
Pennell
J H
JH
Kersey
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification; D019011:Antigens, CD7 / Q000276:immunology*; D017321:Clinical Trials, Phase I as Topic; D004306:Dose-Response Relationship, Immunologic; D006801:Humans; D007167:Immunotherapy; D000922:Immunotoxins / Q000302:isolation & purification* / Q000493:pharmacokinetics; D007942:Leukemia, Experimental / Q000628:therapy*; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D016513:Mice, SCID; D008970:Molecular Weight; D009368:Neoplasm Transplantation; D012276:Ricin / Q000737:chemistry*; D013601:T-Lymphocytes / Q000276:immunology*; D014183:Transplantation, Heterologous
D000911:Antibodies, Monoclonal; D019011:Antigens, CD7; D000922:Immunotoxins; D012276:Ricin