A recombinant single-chain immunotoxin composed of anti-Tac variable regions and a truncated diphtheria toxin.

Abstract

To kill human or primate cells expressing the p55 subunit of the interleukin 2 receptor, we have constructed a single-chain immunotoxin. DNA sequences encoding the first 388 amino acids of diphtheria toxin (DT) were fused to DNA elements encoding the antigen-binding portion (variable region or Fv) of the anti-Tac monoclonal antibody. The antigenbinding portion consists of 116 amino acids of the heavy-chain variable region connected by a 15-amino acid linker to 106 amino acids of the variable region of the light chain. The single-chain immunotoxin DT388-anti-Tac(Fv) was expressed in Escherichia coli and found in inclusion bodies. The monomeric form was then purified to near homogeneity with a high yield (3-5 mg/liter). Monomeric DT388-anti-Tac(Fv) was highly cytotoxic to cell lines bearing the p55 subunit of the human interleukin 2 receptor but not to cells without this subunit. DT388-anti-Tac(Fv) was also very effective in killing proliferating human T cells produced in a mixed leukocyte reaction.Both bacterial and plant toxins, including Pseudomonas exotoxin (PE) and diphtheria toxin (DT), have been employed in developing targeted toxins as reagents for the treatment of cancer, AIDS, and immunological disorders (see refs. 1-3 for review). Initially, chemical conjugation procedures were used to couple antibodies or growth factors to toxins (4-6). More recently, recombinant DNA techniques have been employed to synthesize chimeric toxins in Escherichia coli; these chimeric toxins are composed ofgrowth factors or lymphokines linked to modified forms of PE or DT (7-12). These fusion proteins selectively kill cells bearing appropriate growth factor receptors or antigens. Both chemically conjugated and recombinant products have been successfully used in animal models (13-15). Several of these are now being evaluated in animal models and in clinical trials (16-18).The immunogenic nature of these reagents currently prohibits their repeated and long-term or prophylactic use due to the production of antibodies to the chimeric toxin. Since the toxin portion is highly immunogenic, it is desirable to prepare reagents that have the same binding portion, but different toxin moieties, so that in the event of development of antibodies to one toxin a similar reagent with a different toxin can be used.Recently In anti-Tac(Fv)-PE40, the toxin is placed at the carboxyl end of the Fv. To determine if a toxin can be placed at the amino end of the single-chain antibody and also to try and circumvent the problem of high titer antibodies to PE40 that may arise upon repeated treatment with anti-Tac(Fv)-PE40, we have now constructed a recombinant immunotoxin [DT388-anti-Tac(Fv)] using a truncated form of DT that is devoid of its own receptor binding site. DT388-anti-Tac(Fv) was expressed in E. coli and was purified to near homogeneity. DT388-anti-Tac(Fv) was very cytotoxic to cell lines bearing the p55 subunit of the human IL-2 receptor. This fusion protein was also very effective in killing proliferating human T cells produced in ...

A circularly permuted recombinant interleukin 4 toxin with increased activity.

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Delivery of drugs, proteins and genes into cells using transferrin as a ligand for receptor-mediated endocytosis

“…Recent developments that present elegant alternatives to chemical conjugation are applications where the conjugate consists exclusively of protein or peptide components. The Pastan group has generated several recombinant immunotoxins containing the truncated toxins PE40 or DT388 linked to single-chain versions of the variable antibody domain directed at the IL-2 receptor [82,83] or the TfR [48]. Comparison of chemical conjugates (antiTfR-LysPE40) with the corresponding fusion protein showed a 750-fold higher activity of the recombinant fusion protein.…”

B3(Fv)-PE38KDEL, a single-chain immunotoxin that causes complete regression of a human carcinoma in mice.

“…Potent single-chain immunotoxins have been made previously by fusing the Fv domains of antibodies directed at the interleukin 2 receptor (6,7) or at the transferrin receptor (8) to truncated forms of PE or diphtheria toxin (10). Receptors often make good immunotoxin targets because they are cell surface proteins that can be rapidly internalized, and toxins must be internalized to kill cells.…”