Characterization of an Anti-Cd44 Single-Chain F_V Antibody That Stimulates Natural Killer Cell Activity and Induces TNFα 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 (V_L) and variable heavy chain (V_H) 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 concentrationdependent 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 Fab¹ fragments of parental S5 (14.7 nM) and approximately twofold higher than Mab S5 (6 nM). It also binds directly to canine PBMC and possesses a mean EC₅₀ similar to that of the Fab¹ 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 (TNFα) 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 TNFα 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.