Characterization of an anti-CD44 single-chain FV antibody that stimulates natural killer cell activity and induces TNF alpha release.

Abstract

We report the functional characterization of a single-chain Fv (scFv) constructed from an anti-CD44 mAb (S5) that abrogates marrow rejection in a mismatched canine donor transplant model. The variable light chain (VL) and variable heavy chain (VH) domains of the parent anti-CD44 antibody were cloned and exact match PCR primers designed that spliced the mature variable domains together through a 15 amino acid [Gly4Ser]3 linker-encoding sequence. This gene was put under the control of a T7 promoter and expressed in Escherichia coli in insoluble inclusion bodies. The scFv was refolded in a cystine/cysteine redox buffer and purified to homogeneity using anion exchange chromatography. The concentration-dependent binding isotherm of the S5 scFv was determined using both direct binding and competitive inhibition flow cytometry assays. S5 scFv effectively blocked FITC-conjugated MAb S5 binding to canine peripheral blood mononuclear cells (PBMC), possessing a mean EC50 (15 nM) equivalent to Fab' fragments of parental S5 (14.7 nM) and approximately two-fold higher than Mab S5 (6 nM). It also binds directly to canine PBMC and possesses a mean EC50 similar to that of the Fab' fragments (1.01 nM vs 1.03 nM). The recombinant S5 scFv also retains the potent biological activity of the parent Mab, stimulating the activation of natural killer (NK) cell activity and the release of tumor necrosis factor alpha (TNF alpha) in canine PBMC. Like the parent antibody, scFv crossreacted with human CD44 as examined by direct binding to human PBMC in the flow cytometry assay as well as direct binding to human CD44 immunoglobulin fusion protein in an enzyme-linked immunosorbent assay (ELISA). It was also able to induce TNF alpha release in human PBMC. These results support previous work suggesting that monovalent binding is sufficient to generate the in vitro biological activity of S5 (1). The scFv S5 antibody will thus serve as a useful model for elucidating the mechanism of antibody abrogated marrow rejection and may serve as a human therapeutic agent.