Synthesis and characterization of an antihuman T-lymphocyte saporin immunotoxin (OKT1-SAP) with in vivo stability into nonhuman primates.
The authors conjugated, by a disulphide bond, the antihuman T-lymphocyte (CD5) monoclonal antibody (MoAb) OKT1 to the saporin-6 (SAP) ribosome-inactivating protein of the plant Saponaria officinalis. The resulting OKT1-SAP immunotoxin bound to CD5-expressing target cells and under standard culture conditions specifically suppressed mitogen-induced-T-lymphocyte DNA and protein synthesis in a dose-related manner. T-lymphocyte killing was achieved by five-minute exposure of the target cells to OKT1-SAP. The concentration inhibiting 50% (IC50) of T-lymphocyte DNA synthesis was 0.32 nmol/L. The potency of OKT1-SAP was moderately enhanced by amantadine (IC50 0.08 nmol/L) but not by ammonium chloride or chloroquine. Whole blood components did not interfere with the efficacy of OKT1-SAP, as in vitro treatment of fresh whole blood resulted in effective elimination of clonable peripheral blood T-lymphocytes assessed by a limiting dilution assay. Because these characteristics of T-lymphocyte killing by OKT1-SAP (ie, rapidity of action, potency also without potentiators) and lack of inhibition by whole blood components may be relevant for the use of an immunotoxin as a therapeutic agent in humans, the authors evaluated the stability in vivo and the circulatory clearance of OKT1-SAP in cynomolgus monkeys. Following a single intravenous (IV) injection of nontoxic dosages (0.16 to 1.3 mg/kg), an initial rapid decline (t1/2 alpha = 1.0 to 4.1 hours) was followed by a long-lasting slower decrease (t1/2 beta = 11.6 to 20.6 hours) of OKT1-SAP plasma concentrations as detected by double-antibody solid phase enzyme-linked immunosorbent assay (ELISA) assay. Not only did OKT1-SAP remain intact immunologically but it also retained its biological activity, as measured by the ability of plasma samples from monkeys given immunotoxin to inhibit DNA synthesis in human T-lymphocytes. Taken together the findings presented in this article indicate the feasibility of using OKT1-SAP as a therapeutic tool and provide information that will facilitate the rational use of immunotoxins as a treatment modality in humans.
D000818:Animals; D000918:Antibody Specificity; D001853:Bone Marrow / Q000276:immunology; D016026:Bone Marrow Transplantation; D004247:DNA / Q000096:biosynthesis; D006801:Humans; D000922:Immunotoxins / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D008213:Lymphocyte Activation; D008252:Macaca fascicularis; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000378:metabolism / Q000494:pharmacology*; D014176:Protein Biosynthesis; D054789:Ribosome Inactivating Proteins, Type 1; D000076985:Saporins; D013601:T-Lymphocytes / Q000276:immunology*
D000922:Immunotoxins; D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D004247:DNA; D009699:N-Glycosyl Hydrolases; D000076985:Saporins