Generic Approach for the Generation of Stable Humanized Single-chain Fv Fragments from Rabbit Monoclonal Antibodies

Abstract

Despite their favorable pharmacokinetic properties, singlechain Fv antibody fragments (scFvs) are not commonly used as therapeutics, mainly due to generally low stabilities and poor production yields. In this work, we describe the identification and optimization of a human scFv scaffold, termed FW1.4, which is suitable for humanization and stabilization of a broad variety of rabbit antibody variable domains. A motif consisting of five structurally relevant framework residues that are highly conserved in rabbit variable domains was introduced into FW1.4 to generate a generically applicable scFv scaffold, termed FW1.4gen. Grafting of complementarity determining regions (CDRs) from 15 different rabbit monoclonal antibodies onto FW1.4 and their derivatives resulted in humanized scFvs with binding affinities in the range from 4.7 ؋ 10 ؊9 to 1.5 ؋ 10Interestingly, minimalistic grafting of CDRs onto FW1.4gen, without any substitutions in the framework regions, resulted in affinities ranging from 5.7 ؋ 10 ؊10 to <1.8 ؋ 10 ؊12 M. When compared with progenitor rabbit scFvs, affinities of most humanized scFvs were similar. Moreover, in contrast to progenitor scFvs, which were difficult to produce, biophysical properties of the humanized scFvs were significantly improved, as exemplified by generally good production yields in a generic refolding process and by apparent melting temperatures between 53 and 86°C. Thus, minimalistic grafting of rabbit CDRs on the FW1.4gen scaffold presents a simple and reproducible approach to humanize and stabilize rabbit variable domains.Because of their favorable pharmacokinetic properties, single-chain Fv (scFv) 3 antibody fragments represent an attractive format for therapeutic applications (1, 2). scFvs are often derived from monoclonal antibodies isolated from animal or human lymphocytes. As an alternative to hybridoma screening, in vitro display technologies, e.g. phage and ribosome display, enable the selection of high affinity-binding variable domains from natural or synthetic genetic libraries. Despite the successful use of in vitro randomization and selection systems, generation of antibodies by immunization and subsequent screening of full-size antibodies (e.g. hybridoma supernatants) includes conceptual advantages. For example, in contrast to in vitro display systems, in vivo methods are less prone to preferential selection of well expressed clones, which in many cases results in loss of potentially interesting antibodies. Moreover, in vivo methods are preferred in particular for addressing complex antigens, such as integral membrane proteins that are notoriously difficult to purify. However, reducing a full-length monoclonal antibody to the scFv format frequently is challenging particularly due to solubility and stability problems, which often impair expression and purification. Therefore, technologies to humanize and stabilize the scFv format following isolation of a monoclonal antibody remain critical for the generation of scFv therapeutics. Numerous approaches have been describ...

Novel multispecific heterodimeric antibody format allowing modular assembly of variable domain fragments

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“…We were able to consistently apply λcap technology to V κ 1/V H 3-consensus-derived frameworks to produce functional and stable, humanized Fv modules derived from a discovery platform for rabbit monoclonal antibodies by simple CDR engraftment 37,41,42 . This technology was incorporated in a variety of antibody-based designs, such as scFv, 43 single-chain diabody (scDb) 44 and Fab-scFv 21 formats, with every molecule showing limited aggregation propensity (data not shown).…”

High sensitivity automated multiplexed immunoassays using photonic crystal enhanced fluorescence microfluidic system

“…Four pins (946MP3 Microarray Printing Pins) were used to print the two antibodies at a concentration of 1 mg/mL, which results in 5 × 10 9 molecules/mm 2 . The binding affinity is ~50nM for TNF-alpha antibody while ~ 1nM for IL3 antibody [39]. The positive control was printed at a protein concentration of 10 μg/mL, and the negative control was comprised of printed buffer solution.…”

Production and Characterization of a Humanized Single-chain Antibody against Human Integrin αvβ3 Protein

“…The latter method is of limited use because of the tendency to lose high-affinity binders (26). In this study, we used the former method to engineer a humanized scFv from the mouse monoclonal antibody against human integrin ␣v␤3 by phage antibody display using a predetermined CDR3 gene.…”