Small antibody mimetics comprising two complementarity-determining regions and a framework region for tumor targeting

Abstract

Here we show that fusion of two complementarity-determining regions (CDRs), VHCDR1 and VLCDR3, through a cognate framework region (VHFR2) yields mimetics that retain the antigen recognition of their parent molecules, but have a superior capacity to penetrate tumors. The antigen-recognition abilities of these approximately 3 kDa mimetics surpass those of comparable fragments lacking the framework region. In vivo activities of the mimetics suggests that the structural orientation of their CDRs approximates the conformation of the CDRs in the complex of the parent antibody with antigen. We linked the antibody mimetics to the bacterial toxin colicin Ia to create fusion proteins called "pheromonicins," which enable targeted inhibition of tumor growth. In mice bearing human malignant tumors, pheromonicins directed against tumor-specific surface markers show greater capacity to target and penetrate tumors than their parent antibodies. Rational recombination of selected VH/VL binding sites and their framework regions might provide useful targeting moieties for cytotoxic cancer therapies.

Computational Identification of Antigen-Binding Antibody Fragments

“…Some of them had improved affinity, inhibition activity, or solubility. Another study has shown that a toxin-fused drug candidate composed of CDRH1 and CDRL3 fused through VH framework region 2 of an Ab against gp350/220 (EBV envelope protein) has superior capacity to penetrate tumor and inhibit tumor growth (31). Such studies highlight the applicative relevance of CDRderived peptides.…”

“…The use of computational tools has also been explored for the design of Ag-binding peptides and to predict the strength of the peptide-protein interactions (25). In one case, peptide attached to a toxin was shown to inhibit tumor growth (31). These attempts to generate Ag-binding peptides are based on the idea that the interface itself may be modular, with some of its components capable of binding the Ag on their own.…”

“…Attempts to mimic the structure the CDR loop adopts in the context of the full Ab by adding a disulfide bond at the peptide edges has been suggested as a solution to this problem (21,28,29). In another study, stable peptides were produced by creating a peptide dimer and adding a single cysteine residue to one of the peptide termini on each peptide (30), or by joining two CDRs (31). The use of computational tools has also been explored for the design of Ag-binding peptides and to predict the strength of the peptide-protein interactions (25).…”

Cardiac Protection with Targeted Drug Delivery to Ischemic-Reperfused Myocardium

“…Nanobodies seem to be optimal tissue recognition ligands since they have small size and low immunogenicity. Qiu et al (2007) produced low-molecular weight antibody mimetics by virtue of fusion between two complementarily-determining regions of the prototype antibodies. Obtained mimetics with molecular weight of 3 kDa were characterized by the better distribution pattern than corresponding antibodies, suggesting that these mimetics may be used as highly specific targeting ligands.…”

Efficient inhibition of B-cell lymphoma xenografts with a novel recombinant fusion protein: anti-CD20Fab-LDM

“…with FITC-labeled anti-CD20Fab-LDP, anti-CD20Fab or anti-LDP (16 nmol per mouse). 26 The mice were imaged using the previous procedure to monitor the distributions of the fusion protein at different time points. 27 Preparation of energized fusion protein anti-CD20Fab-LDM LDM (10 mg) with high potent activity were suspended in 5 ml methanol and whisked for 5 min at 4 1C, and then the mixture was placed at 20 1C for 1 h. AE was obtained from the supernatant of reaction mixture by centrifugation at 16 000 r.p.m.…”