The effort to design new mAb based therapeutics that will expand the range of therapeutic modalities beyond what is currently possible with traditional mAbs has lead us to design a novel single chain Fc (scFc) molecule. Typically, bivalent mAbs drive antigen cross-linking on the cell surface and this has the potential to elicit unwanted downstream cellular signaling events (anti-cMet) and/or internalization of the mAb-target complex from the cellular surface. The monovalent scFc antibody has the advantage over a typical monoclonal antibody of binding to but not cross linking a cell surface antigen. Additionally, unlike a monovalent F(ab) fragment, the scFc retains both the long circulating half-life and, if desired, FcgR-dependent effector function of mAbs. The scFc provides a convenient and reliable platform for the design of several new Fc-containing binding proteins including: monovalent mAbs, monovalent Fc-fusion proteins and mAbs or fusion proteins with heterodimeric Fc regions that carry functional mutations in either one or the other half of the Fc for developing tunable effector function. Here we describe the reliable production, purification and initial characterization of monovalent scFc antibodies.

Unlike native mAbs where the Fc regions form as a result of the homodimerization of two antibody heavy chains, the single chain Fc is designed to be expressed in a single polypeptide chain with the following domain arrangement: N-terminus-hinge-CH2-CH3-Linker-hinge-CH2-CH3-C-terminus. The linker sequence enables the polypeptide chain to fold back upon itself such that the more C-terminal CH2 and CH3 domains can pair with their corresponding N-terminal CH2 and CH3 domains.

We have evaluated the scFc in a variety of functional assays in vitro and in vivo to assess the stability, pharmacokinetics and Fc functionality of the design. We have found that the monovalent scFc version of a human IgG1 mAb binds antigen with affinities that are comparable to those determined for the corresponding F(ab) preparation. In vitro, the scFc exhibits binding to human FcRn and FcgR I, II and III from human and cyno that is equivalent to WT human IgG1, indicating that the linker sequence in the Fc region does not interfere with the ability of the scFc to engage these receptors. Similarly, no detectable change compared to WT human IgG1 was observed in a C1q binding ELISA and like WT human IgG1, the scFc proteins exhibit a long serum half-life in rats.

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