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

Abstract

The genes encoding the heavy-and lightchain Fv regions of the monoclonal murine antibody B3, which recognizes a carbohydrate antigen on the surface of many human carcinomas, were cloned by PCR techniques and used to generate single-chain immunotoxins containing Pseudomonas exotoxin (PE). The light and heavy chains were connected by a flexible linker to form a single-chain antigen-binding protein, B3(Fv), which was in turn fused to truncated forms of PE lacking the cell-binding domain. The single-chain Fv and two different B3(Fv) immunotoxins, B3(Fv)-PE40 and B3(Fv)-PE38KDEL, were expressed in Escherichia coNl and the singlechain immunotoxins were purified to near homogeneity. Both recombinant immunotoxins were shown to be cytotoxic specifically to carcinoma cell lines that express the B3 antigen on their surface; B3(Fv)-PE38KDEL was significantiy more active. Furthermore, intravenous administration of B3(Fv)-PE-38KDEL caused complete regression of human epidermoid carcinomas growing subcutaneously in immunodeflent mice.linked to toxins retain the binding capacity of the scAB as well as the activity of the toxin (6-9).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. Here we describe the cloning of DNA fragments that encode the Fv region of mAb B3, which is directed against a carcinoma-associated cell surface antigen, and the construction of plasmids for expression in Escherichia coli of the B3 single-chain antibody, B3(Fv), fused to two different truncated PE molecules. Further we show that these recombinant B3(Fv) immunotoxins are cytotoxic selectively to cultured human tumor cells bearing the B3 antigen, and they cause complete regression of human tumors growing in immunodeficient mice.

Design, characterization and anti-tumour cytotoxicity of a panel of recombinant, mammalian ribonuclease-based immunotoxins

“…For example, Pseudomonas exotoxin A has been genetically altered to make more effective immunotoxins: the N-terminal cell binding domain has been replaced by alternative ligands (Chaudhary et al, 1989) and the C-terminal REDLK sequence has been mutated to KDEL, which is the consensus human endoplasmic retention sequence (Brinkmann et al, 1991) and which improves cell trafficking. Recombinantly produced fragments of antibodies, selected against tumour-associated antigens are very often used in the construction of immunotoxins to decrease their size and improve their pharmacokinetics, particularly as this technology has advanced so rapidly with the arrival of antibody phage display (reviewed in Winter et al, 1994).…”

Isolation of new anti-CD30 scFvs from DNA-immunized mice by phage display and biologic activity of recombinant immunotoxins produced by fusion with truncatedpseudomonas exotoxin

“…As shown in Figure 5, HRS4 co-incubated with anti-CD30-CL2(Fv)-PE38KDEL blocked its cytotoxicity, indicating that internalization via CD30 is required for cytotoxicity of anti-CD30 IT. Also shown in Figure 5, the T-ALCL cell lines Karpas and SUP-M2 were not significantly sensitive to 10 or 100 ng/ml of the negative control molecules RFB4(dsFv)-PE38KDEL 39 and B3(Fv)-PE38KDEL, 37 which target CD22 and Le Y , respectively. The latter is a positive control molecule for A431-CD30, which expresses Le Y .…”

A novel vascular endothelial growth factor-directed therapy that selectively activates cytotoxic prodrugs

“…Such studies have demonstrated that a wide range of ligands can be regarded as potential antitumour-targeting devices, and a wide range of cellular receptors can be viewed as targets. Furthermore, complete regressions have been recorded (Brinkmann et al, 1991), underlining the success of this approach.…”