Immunotoxins are antibody-toxin bifunctional molecules that rely on intracellular toxin action to kill target cells. Target specificity is determined via the binding attributes of the chosen antibody. Mostly, but not exclusively, immunotoxins are purpose-built to kill cancer cells as part of novel treatment approaches. Other applications for immunotoxins include immune regulation and the treatment of viral or parasitic diseases. Here we discuss the utility of protein toxins, of both bacterial and plant origin, joined to antibodies for targeting cancer cells. Finally, while clinical goals are focused on the development of novel cancer treatments, much has been learned about toxin action and intracellular pathways. Thus toxins are considered both medicines for treating human disease and probes of cellular function.

Cancer cells frequently upregulate surface receptors that promote growth and survival. These receptors constitute valid targets for intervention. One strategy involves the delivery of toxic payloads with the goal of killing those cancer cells with high receptor levels. Delivery can be accomplished by attaching a toxic payload to either a receptor-binding antibody or a receptor-binding ligand. Generally, the cell-binding domain of the toxin is replaced with a ligand or antibody that dictates a new binding specificity. The advantage of this “immunotoxin” approach lies in the potency of these chimeric molecules for killing cancer cells. However, receptor expression on normal tissue represents a significant obstacle to therapeutic intervention.

The monoclonal antibody '40H3' binds to EGFRvIII and to full-length EGFR when it is overexpressed on cancer cells. To generate candidate cytotoxic antibody-drug conjugates (ADCs), 40H3 was modified by the addition of small molecular weight payloads that included two tubulin-modifying agents, two topoisomerase inhibitors and a pyrrolobenzodiazepine (PBD) dimer. Conjugates retained antigen binding activity comparable to the unmodified 40H3 antibody. The cytotoxicity of five distinct ADCs was evaluated on a variety of EGFR-expressing cells including three triple negative breast cancer (TNBC) lines. Generally, the 40H3 conjugate with the PBD dimer (40H3-Tesirine) was the most active killing agent. The killing of EGFR-positive cells by 40H3-Tesirine correlated with the number of surface binding sites for 40H3. However, bystander killing was also evident in experiments with antigen-negative cells. In vivo tumor xenograft experiments were conducted on two TNBC tumor lines. Three treatments with the 40H3-Tesirine ADC at 1 mg/kg were sufficient to achieve complete remissions without evidence of mouse toxicity. Data support the development of ADCs derived from the 40H3 antibody for the treatment of cancers that express EGFRvIII or high levels of EGFR.

Background: The dysregulation of epidermal growth factor receptor (EGFR) has been implicated in the oncogenesis of various malignancies including glioblastoma and some epithelial cancers. Oncogenesis occurs from the overexpression of EGFR, often linked to gene amplification or receptor mutagenesis. The 287–302 loop in the extracellular domain is exposed completely on EGFR variant III (EGFRvIII), partially exposed on some cancers but cryptic on normal cells. We report on the generation of antibodies to this loop. Methods: The 286–303 peptide was coupled chemically to keyhole limpet hemocyanin. After immunizations, sera were assayed for reactivity to the peptide. Mice with high titers were used for hybridoma production. Purified antibodies were isolated from hybridoma supernatants, while V regions were cloned and sequenced. Receptor binding was characterized using enzyme-linked immunosorbent assay and flow cytometry. A recombinant immunotoxin was generated from the 40H3 antibody and its cytotoxic activity characterized on relevant cancer cell lines. Results: Seven monoclonal antibodies were generated to the 287–302 loop and characterized further. Each one reacted with EGFRvIII but not wild-type EGFR. Based on reactivity with the immunizing peptide, antibodies were mapped to one of three subgroups. One antibody, 40H3, also exhibited binding to MDA-MB-468 and A431 cells but not to non-cancerous WI-38 cells. Because of its unusual binding characteristics, a recombinant immunotoxin was generated from 40H3, which proved to be cytotoxic to MDA-MB-468, A431 and F98_npEGFRvIII expressing cells. Conclusions: Immunization with a peptide corresponding to a cryptic epitope from EGFR can produce tumor cell-binding antibodies. The 40H3 antibody was engineered as a cytotoxic recombinant immunotoxin and could be further developed as a therapeutic agent.

Background: A feature of many B-cell tumors is a surface-expressed immunoglobulin (sIg). The complementarity-determining regions (CDRs) of the sIg, termed the ‘idiotype’, are unique to each tumor. We report on a phage selection strategy to generate anti-idiotype therapeutics that reacts with sIg CDR3 sequences; the MEC1 B-cell tumor line was used as proof of concept. Methods: To create a mimetic of the MEC1 idiotype, CDR3 sequences from heavy and light chains of the sIg were grafted into a single chain variable fragment (scFv) framework scaffold. Using the Tomlinson I phage library of human scFvs, we enriched for binders to MEC1 CDR3 sequences over unrelated CDR3 sequences. Results: By ELISA we identified 10 binder phages. Of these, five were sequenced, found to be unique and characterized further. By flow cytometry each of the five phages bound to MEC1 cells, albeit with different patterns of reactivity. To establish specificity of binding and utility, the scFv sequences from two of these binders (phages 1 and 7) were converted into antibody-toxin fusion proteins (immunotoxins) and also cloned into a human IgG1 expression vector. Binders 1 and 7 immunotoxins exhibited specific killing of MEC1 cells with little toxicity for non-target B-cell lines. The full-length antibody recreated from the binder-1 scFv also exhibited specific binding. Conclusion: Our results establish the utility of using engrafted CDR3 sequences for selecting phage that recognize the idiotype of B-cell tumors.