PBMCs from 14 patients were incubated with the recombinant toxins for 60 hours, and -leucine incorporation was measured. In DT388-anti- Tac(Fv), anti-Tac(Fv) is fused to the carboxyl terminus of the first 388 amino acids of DT. In anti-Tac(Fv)- PE40KDEL, anti-Tac(Fv) is genetically fused to the amino terminus of PE40KDEL, a recombinant form of PE which contains amino acids 253–608 of PE and the -KDEL mutation at the carboxyl terminus. We now report that two of these single-chain recombinant immunotoxins, anti-Tac(Fv)-PE40KDEL and DT388-anti-Tac(Fv), are cytotoxic toward peripheral blood mononuclear cells (PBMCs) from patients with chronic lymphocytic leukemia (CLL). We have previously shown that the variable domains of the monoclonal antibody anti-Tac can be fused to derivatives of Pseudomonas exotoxin (PE) or diphtheria toxin (DT) to produce recombinant immunotoxins that kill interleukin-2 (IL-2) receptor- bearing cells. Overall, the studies strongly suggest that the alpha-2MR/LRP is responsible for internalizing PE. The concentration of receptor-associated protein that was required to reduce binding and toxicity to 50% was approximately 14 nM, a value virtually identical to the, K(D) measured for the interaction of receptor-associated protein with the purified receptor. The 39-kDa receptor-associated protein, which blocks binding of ligands to alpha-2MR/LRP, also prevents binding and subsequent toxicity of PE for mouse fibroblasts. Furthermore, affinity-purified alpha-2MR/LRP binds specifically to PE but not to a mutant toxin defective in its ability to bind cells. We demonstrate that the alpha-2MR/LRP and the PE-binding glycoprotein have a similar mobility upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and are immunologically indistinguishable. A similar high molecular weight glycoprotein has been identified as a potential receptor for Pseudomonas exotoxin A (PE). The alpha-2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha-2MR/LRP) is a large cell-surface glycoprotein consisting of a 515-kDa and an 85-kDa polypeptide this receptor is thought to be responsible for the binding and endocytosis of activated alpha-2-macroglobulin and apoE-enriched beta-very low density lipoprotein. Some of them show promising results, bringing hope for treatment of chemoresistant cancers. A wide variety of immunotoxins have recently been tested in preclinical and clinical trials. Most common side effects include vascular leak syndrome (VLS) and hepatotoxicity. Unfortunately, the majority of antibodies currently used for immunotoxins preparation recognize antigens that are expressed in both neoplastic and normal cells. The specificity of tumor antigen binding determines the type and severity of side effects, occurring due to immunotoxins binding with non-cancerous cells. Monoclonal antibodies, growth factors or cytokines are used as carrier molecules. The induction of cell death depends on the protein synthesis inhibition due to interactions with various targets such as a ribosomes or EF-2. The most popular among them are: exotoxin A derived from Pseudomonas aeruginosa (PE), diphtheria toxin from Corynebacterium diphtheriae (DT) and ricin from Ricinus communis. Toxins used for immunotoxin preparation have various origin (plant, bacterial, fungal and animal). Immunotoxins consist of two main fragments: protein toxin, which kills target cell after internalization, and carrier molecules which specifically identify and bind cancer cells. The research has been conducted for the past 30-40 years but finding specific surface structures on targeted cells remains a major problem. Immunotoxins are a new group of therapeutics of potential use in targeted tumor therapy.
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