A circularly permuted recombinant interleukin 4 toxin with increased activity.

Abstract

Fusion of ligands such as growth factors to other proteins often dramatically reduces the affinity of the ligand for its receptor. With recombinant DNA techniques, the attachment point between the two proteins has until now been restricted to either the amino or the carboxyl terminus of the d. However, binding may be greatly compromised if both ends are close to the site at which the ligand binds to its receptor. To construct a single-chain growth factor fusion protein with the connection at a new site on the growth factor, we constructed a DNA fria ent encoding circularly permuted interleukin 4 (H4), termed 114(38-37 In many cases, fusion of one protein to another impairs the activity of one or both proteins. This can occur if one or both proteins require one or both termini free for optimal activity. Alternatively, connection of one protein to another can impair the ability ofeither protein to properly fold. It has been reported that transforming growth factor a, IL2, IL3, IL4, IL6, GM-CSF, and insulin-like growth factor I all bind with 20-to 250-fold reduced affinity after fusion to another protein (7,(13)(14)(15)(16). Previously, the options available for improving the function of a fusion protein were limited to switching the order of the two proteins, attaching linkers in between the two proteins, or mutating one of the proteins to decrease junctional effects (13,17).We have previously reported on the properties of chimeric toxins in which IL4 was fused to recombinant forms of Pseudomonas exotoxin (PE). The fusion proteins bind to the IL4 receptor (IL4R) with only "l4% of the affinity of native IL4 (14). Several studies have indicated that the carboxyl terminus of IL4 is important for binding (18,19); therefore, it is likely that the large toxin molecule attached to the carboxyl terminus blocks the binding of 1L4 to the IL4R. We therefore developed a strategy for fusing the two proteins in which the toxin is fused to the new carboxyl terminus of circularly permuted forms of IL4. A circular permuted protein is a mutant protein in which the termini have been fused and new termini created elsewhere in the molecule. Several circularly permuted proteins have been made but none of these have been fused to other proteins (20). We reasoned that circularly permuted IL4 (CP-IL4) could be fused to a toxin so that thejunction would be in an entirely new location and allow the IL4 to bind in a more native fashion. MATERIALS AND METHODSPlasmid Construction. pRKL4EL encodes human IL4 followed by the amino acids ELKA, contains gag-ctc-gaa-gcttga (nucleotide sequences are shown by lowercase letters to avoid confusion with single-letter amino acid symbols) at the end of the coding sequence, and was derived by PCR amplification of pWDMH4 (14) and ligation into the vector pVCDT1-anti-Tac(Fv) (21). pRKL4 encodes the 129 amino acid 14 protein purified from Escherichia coli (22) and was derived from PCR amplification of pRKLAEL and ligation into the vector pVCDT1-anti-Tac(Fv). DNA encoding the toxin used for fusion to CP...

IL-4 receptors on human medulloblastoma tumours serve as a sensitive target for a circular permuted IL-4-Pseudomonas exotoxin fusion protein

“…The absence of g c protein indicated that medulloblastoma cell lines express type II IL-4R as do malignant glioma cells. This configuration is similar to that observed on other tumour cells including renal cell carcinoma, AIDS-Kaposi's tumours, head and neck tumours, colon carcinoma, ovarian carcinoma and breast tumour cells (Husain et al, 1997;Kawakami et al, 2000;Kreitman et al, 1994;Leland et al, 2000;Murata et al, 1997b;Puri et al, 1996b).…”

SV40 Pseudovirion gene delivery of a toxin to treat human adenocarcinomas in mice

“…IL-4 fused with Pseudomonas exotoxin was delivered to brain tumors and destroyed the brain tumor selectively. 31,32 Chen et al 33 attempted to introduce the membrane translocation domain of Pseudomonas exotoxin A into a DNA delivery vehicle to increase the translocation efficiency of DNA into the cytosol. 34 That work, however, was cell-type dependent.…”

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

“…IL-4R is an ideal candidate for targeted toxin therapy because of its rapid internalisation upon ligand binding and limited expression profile in normal tissues (Kawakami et al, 2002a, b). Numerous preclinical studies have shown the potent anti-tumour activity of a monospecific cytotoxin composed of IL-4 linked to PE38 (Kreitman et al, 1994;Shimamura et al, 2007). However, efficacy of this agent in a Phase I clinical trial of patients with renal cell carcinoma and NSCLC yielded no objective tumour responses, likely because of the rapid development of neutralising antibodies (Garland et al, 2005).…”

“…Interleukin-4 is an important cytokine in Th2 differentiation of T cells and IL-4R has proven to be a promising target for treatment of pancreatic cancer and glioblastoma (Rand et al, 2000;Shimamura et al, 2007). To enhance the activity of cytotoxins targeting IL-4R, a circularly permuted IL-4 cytotoxin has been described that improved binding site exposure without altering ligand-receptor specificity (Kreitman et al, 1994) and these alterations were incorportated into EGF4KDEL. Both EGFR and IL-4R are ideal targets for liganddirected toxin therapies as both are cell surface molecules that internalise rapidly upon ligand binding (Haigler et al, 1978;Kawakami et al, 2002a).…”