Design and modification of EGF4KDEL 7Mut, a novel bispecific ligand-directed toxin, with decreased immunogenicity and potent anti-mesothelioma activity

Our plain-English summary of Design and modification of EGF4KDEL 7Mut, a novel bispecific ligand-directed toxin, with decreased immunogenicity and potent anti-mesothelioma activity, by B.J. Stish, S. Oh, H. Chen, et al (British Journal of Cancer, 2009).

The study

• Authors: B.J. Stish, S. Oh, H. Chen, A.Z. Dudek, R.A. Kratzke, D.A. Vallera
• Journal: British Journal of Cancer
• Published: 2009;101(7):1114-23
• Cited by: 26 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Potency, immunogenicity, and toxicity are three problems that limit the use of targeted toxins in solid tumour therapy.

Methods: To address potency, we used genetic engineering to develop a novel bispecific ligand-directed toxin (BLT) called EGF4KDEL, a novel recombinant anti-mesothelioma agent created by linking human epidermal growth factor (EGF) and interleukin-4 (IL-4) to truncated pseudomonas exotoxin (PE38) on the same single-chain molecule. Immunogenicity was reduced by mutating seven immunodominant B-cell epitopes on the PE38 molecule to create a new agent, EGF4KDEL 7Mut.

Results: In vitro, bispecific EGF4KDEL showed superior anti-mesothelioma activity compared with its monospecific counterparts. Toxicity in mice was diminished by having both ligands on the same molecule, allowing administration of a 10-fold greater dose of BLT than a mixture of monomeric IL4KDEL and EGFKDEL. EGF4KDEL 7Mut, retained all of its functional activity and induced about 87% fewer anti-toxin antibodies than mice given the parental, non-mutated form. In vivo, intraperitoneal (IP) injection of the BLT showed significant (P<0.01) and impressive effects against two aggressive, malignant IP mesothelioma models when treatment was begun 14-16 days post tumour innoculation.

Conclusion: These data show that EGF4KDEL 7Mut is a promising new anti-mesothelioma agent that was developed to specifically address the obstacles facing clinical utility of targeted toxins.

In plain English

This was a laboratory and mouse study of a recombinant drug, not a study of asbestos exposure. The authors built a bispecific ligand-directed toxin, EGF4KDEL, by linking human epidermal growth factor and circularly permuted interleukin-4 to truncated pseudomonas exotoxin (PE38 with a KDEL ending), then mutated eight amino acids covering seven B-cell epitopes to make EGF4KDEL 7Mut. Against human malignant mesothelioma cell lines, EGF4KDEL inhibited protein synthesis in H2373 cells with an IC50 of 1.3x10 to the minus 5 nM, compared with 4.3x10 to the minus 5 nM for EGFKDEL and 4.0x10 to the minus 3 nM for IL4KDEL; the control BIC3KDEL had no effect. Binding was higher on malignant cells (H2461 MFI 17.03) than on normal mesothelial LP-9 (3.33) and MeT-5A (4.98). In mice, all animals given the bispecific agent survived doses that killed mice given the monomeric mixture. Immunised Balb/c mice averaged serum anti-PE38(KDEL) IgG of 1191 micrograms per millilitre with the parental EGF4KDEL versus 155 with EGF4KDEL 7Mut, 87% fewer (P=0.05). In nude mice, EGF4KDEL reduced H2373 peritoneal colonies (4.8 versus 47.6 untreated, P less than 0.05), and EGF4KDEL 7Mut reduced H513 tumour bioluminescence (P less than 0.001).

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory study: in vitro cytotoxicity, binding and blocking assays in human cell lines, plus mouse toxicity, immunisation and intraperitoneal xenograft experiments
• Who was studied: Human malignant mesothelioma cell lines (H2373, H513, H2461, H2052, H2596), normal mesothelial lines MeT-5A and LP-9, the leukaemic T-cell line HPB-MLT; normal C57BL/6 mice for toxicity, female Balb/c mice (n=6 per group) for immunogenicity, and male nu/nu nude mice bearing intraperitoneal H2373/Luc/GFP (n=6 per group) or H513/Luc/GFP (n=9 per group) tumours; University of Minnesota
• Exposure: Treatment with the bispecific ligand-directed toxin EGF4KDEL and its seven-epitope-mutated form EGF4KDEL 7Mut, compared with monospecific EGFKDEL and IL4KDEL and control agents BIC3KDEL and 2219; intraperitoneal injections in mice (doses given as 4 per injection, the dose unit in the toxicity table being micrograms)
• Main results:
  • Against H2373 mesothelioma cells, EGF4KDEL gave an IC50 of 1.3x10 to the minus 5 nM, lower than the monospecific EGFKDEL (4.3x10 to the minus 5 nM) and IL4KDEL (4.0x10 to the minus 3 nM); BIC3KDEL had no effect
  • Binding of FITC-labelled EGF4KDEL was lower on the normal mesothelial lines LP-9 (MFI 3.33) and MeT-5A (MFI 4.98) than on malignant H2461 (MFI 17.03) and H513 (MFI 12.25) cells
  • In normal mice, half of those given two 0.5 (microgram) injections of the EGFKDEL plus IL4KDEL mixture died, while all mice given 2 (micrograms) of the bispecific EGF4KDEL survived without signs of toxicity
  • Balb/c mice immunised with the parental EGF4KDEL had mean serum anti-PE38(KDEL) IgG of 1191 micrograms per millilitre, versus 155 micrograms per millilitre in mice given EGF4KDEL 7Mut (87% fewer, P=0.05); the micro sign is lost in the OCR, and the figure axis reads 'Serum IgG (g ml-1)'
  • In the H2373 intraperitoneal xenograft model, treated with EGF4KDEL, mean luciferase activity differed significantly from untreated controls (P less than 0.02)
  • Blinded counts of peritoneal tumour colonies in the H2373 model averaged 4.8 per animal in the EGF4KDEL group versus 47.6 in the untreated group (P less than 0.05)
  • In the second model, H513 intraperitoneal tumours, EGF4KDEL 7Mut reduced tumour burden compared with untreated animals (P less than 0.001), with no change in average animal weights over 15 courses of treatment
  • EGF4KDEL 7Mut killed H513 cells with an IC50 of 7.3x10 to the minus 7 nM, which the authors described as nearly identical to the non-mutated molecule
• The authors' conclusion: The authors concluded that EGF4KDEL 7Mut is a promising new anti-mesothelioma agent whose toxicity and immunogenicity were reduced, improving its potential for clinical use
• Limitations the authors state:
  • The authors stated that further pharmacokinetic studies in a full on-target model, such as non-human primates, would be needed to establish the toxicity advantage over the monospecific agents
  • Treatment was given intraperitoneally because of technical difficulty with repeated intravenous injection in mice, and the authors said further experiments are needed to compare local infusion with systemic therapy
  • The authors noted that only about 11% of clinical mesothelioma cases are confined to the peritoneum, the intraperitoneal mouse model being used as a surrogate for pleural mesothelioma

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Mesothelioma.

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