Recombinant anti-erbB2 immunotoxins containing Pseudomonas exotoxin.

Abstract

Immunotoxins were made using five different murine monoclonal antibodies to the human erbB2 gene product and LysPE40, a 40-kDa recombinant form ofPseudomonas exotoxin (PE) lacking its cell-binding domain. All five conjugates were specifically cytotoxic to cancer cell lines overexpressing erbB2 protein. The most active conjugate was e23-LysPE40, generated by chemical crosslinking of anti-erbB2 monoclonal antibody e23 to LysPE40. In addition, a recombinant immunotoxin, e23(Fv)PE40, was constructed that consists of the light-chain variable domain of e23 connected through a peptide linker to its heavy-chain variable domain, which in turn is fused to PE40. The recombinant protein was made in Escherichia coli, purified to near homogeneity, and shown to selectively kill cells expressing the erbB2 protooncogene. To improve the cytotoxic activity of e23(Fv)PE40, PE40 was replaced with a variant, PE38KDEL, in which the carboxyl end of PE is changed from Arg-Glu-Asp-Leu-Lys to Lys-Asp-GluLeu and amino acids 365-380 of PE are deleted. The e23(Fv)PE38KDEL protein inhibits the growth of tumors formed by the human gastric cancer cell line N87 in immunodeficient mice.

Antineoplastic effect of anti-erbB-2 intrabody is not correlated with scFv affinity for its target

“…Chief among these advantages is the facile means whereby anti-oncoprotein scFvs may be derived. Such methods have included genetic techniques of deriving cDNA directly from hybridomas [21][22][23][24][25]34 as well as biopanning of phage scFv 33,35 libraries and others. 38 These methods have allowed direct isolation of scFvs against virtually any subcellular target protein.…”

“…In this regard, various techniques have been used to improve the affinity of scFvs for cognate targets. [33][34][35] Such efforts have largely been endeavored in the context of the comparison of recombinant immunotoxins, whereby the affinity of the antibody clearly correlated with tumor targeting and with the overall therapeutic efficacy. In our studies, increasing the binding affinity of the scFv did not translate into an improvement in intrabody-mediated tumor cell cytotoxicity induction.…”

“…In this regard, screening of a phage display library and sitedirected mutagenesis has been used to obtain an antierbB-2 scFv such that this new derivative anti-erbB-2 scFv, C6.5, possesses an affinity for its target that is 1000 times greater than that for e23. [33][34][35] We thus used this scFv to determine the degree to which affinity differential predicated utility for intrabody knockout approaches. Our results demonstrated that affinity enhancement of the parent scFv does not necessarily predict an improvement in intrabody-mediated antineoplastic effects.…”

Targeted inhibition of tumour cell growth by a bispecific single-chain toxin containing an antibody domain and TGF α

“…Several recombinant Pseudomonas exotoxin A fusion proteins binding to EGFR or ErbB-2 have been described (Chaudhary et al, 1987;Wels et al, 1992Wels et al, , 1995Batra et al, 1992). Since many tumour cells express both ErbB-2 and EGFR, therapeutic reagents binding to both receptor proteins might offer advantages over monospecific molecules by inducing the formation of receptor heterodimers which in turn might lead to more rapid uptake of toxin-receptor complexes.…”

“…Therefore, recombinant fragments of ErbB-2-specific MAbs have been employed to direct target cell specificity. ScFv-ETA fusion proteins containing ErbB-2-specific binding domains have demonstrated highly selective anti-tumour activity in vitro and in vivo (Wels et al, , 1995Batra et al, 1992).…”