Optimization of an Antibody Light Chain Framework Enhances Expression, Biophysical Properties and Pharmacokinetics

Abstract

Efficacy, safety, and manufacturability of therapeutic antibodies are influenced by their biopharmaceutical and biophysical properties. These properties can be optimized by library approaches or rationale protein design. Here, we employed a protein engineering approach to modify the variable domain of the light chain (VL) framework of an oxidized macrophage migration inhibitory factor (oxMIF)-specific antibody. The amendment of the antibody sequence was based on homology to human germline VL genes. Three regions or positions were identified in the VL domain—L1-4, L66, L79—and mutated independently or in combination to match the closest germline V gene. None of the mutations altered oxMIF specificity or affinity, but some variants improved thermal stability, aggregation propensity, and resulted in up to five-fold higher expression. Importantly, the improved biopharmaceutical properties translated into a superior pharmacokinetic profile of the antibody. Thus, optimization of the V domain framework can ameliorate the biophysical qualities of a therapeutic antibody candidate, and as result its manufacturability, and also has the potential to improve pharmacokinetics.

Antibody Structure and Function: The Basis for Engineering Therapeutics

“…This variant indeed showed greatly improved solubility while maintaining affinity to IL-13 and proved to be the most effective route for enhancing the solubility of CNTO607. Recently, there has been a description of how optimization of the V domain framework can improve the biophysical qualities of a therapeutic antibody candidate [275].…”

Conformational features and interaction mechanisms of VHH antibodies with β‐hairpin CDR3: A case of Nb8‐HigB2 interaction

“…We checked the frequencies of amino acids in heavy chain position 93 using the abYsis software (Swindells et al, 2017 ). This database has previously been employed to examine the amino‐acid frequency at specific positions for the humanization of murine antibodies (Chen et al, 2023 ) and to optimize antibody framework sequences (Douillard et al, 2019 ). The differences in residue features across various organisms were also analyzed in a previous study (Mendoza et al, 2020 ).…”

Beyond Natural Immune Repertoires: Synthetic Antibodies

“…The majority of synthetic antibody libraries are constructed by introducing diversity in the complementarity-determining regions (CDRs), within optimized frameworks, by using degenerate combinations of mono-or trinucleotide units to create highly diverse antigen-binding sites (Shim 2015). Given that only a limited number of residues in the antibody framework can establish interactions with antigens (Kunik et al 2012a,b), such a strategy maximizes the chemical diversity introduced in the CDRs while minimizing the risk of immunogenicity and ensuring high stability and protein production (Tiller et al 2013;Douillard et al 2019). Additionally, the well-defined nature of synthetic antibodies makes them amenable to further library designs, such as in the case of affinity maturation, thereby reducing the time and the optimization required to generate therapeutic antibody candidates (Tiller et al 2017).…”