An adapted consensus protein design strategy for identifying globally optimal biotherapeutics

Abstract

Biotherapeutic optimization, whether to improve general properties or to engineer specific attributes, is a time-consuming process with uncertain outcomes. Conversely, Consensus Protein Design has been shown to be a viable approach to enhance protein stability while retaining function. In adapting this method for a more limited number of protein sequences, we studied 21 consensus single-point variants from eight publicly available CD3 binding sequences with high similarity but diverse biophysical and pharmacological properties. All single-point consensus variants retained CD3 binding and performed similarly in cell-based functional assays. Using Ridge regression analysis, we identified the variants and sequence positions with overall beneficial effects on developability attributes of the CD3 binders. A second round of sequence generation that combined these substitutions into a single molecule yielded a unique CD3 binder with globally optimized developability attributes. In this first application to therapeutic antibodies, adapted Consensus Protein Design was found to be highly beneficial within lead optimization, conserving resources and minimizing iterations. Future implementations of this general strategy may help accelerate drug discovery and improve success rates in bringing novel biotherapeutics to market.

Exploring molecular determinants and pharmacokinetic properties of IgG1-scFv bispecific antibodies

“…Another important factor is the stability of the BsAbs containing scFv in serum and PBS. 36 To determine the serum stability of the three BsAbs, they were incubated in 90% mouse serum or in PBS, and their degradation over time was monitored by their binding to the target antigen(s). The data indicated that neither incubation in mouse serum nor in PBS in a time course over 4 d resulted in a loss of binding, suggesting that all molecules were stable (Table S3).…”

“…The bispecific antibodies IgG1-scFv investigated in this study were produced in-house by mammalian cell culture technology using CHO cells and purified with a standard procedure as described previously. 36 All bispecific antibodies are similar in size with a molecular weight between 195 and 205 kDa. Sample quality was ensured by quantification using analytical size exclusion chromatography passing the acceptance criteria (>97%).…”

Trends in industrialization of biotherapeutics: a survey of product characteristics of 89 antibody-based biotherapeutics

“…Ahmed et al previously showed, through sequence and structural analysis, that non-redundant physicochemical descriptors can be used to flag Fv regions of antibodies for medicine-likeness. 203 There are numerous ways computer-aided drug design can contribute to the marketed antibody-based biotherapeutic landscape, 204 including sequence analysis (for example, see Figures S3 (a, b)), 205 structure analysis, 206 and machine learning. 207 …”

Predicting the Purity of Multispecific Antibodies From Sequence Using Machine Learning: Methods and Applications

“…In order to determine the % monomer of the multispecific antibodies, analytical size exclusion chromatography was carried out using the Agilent HPLC system as previously described 22 . 10&g of sample were injected onto two tandemly connected Acquity BEH200 columns (Waters, 1.7 &m, 4.6 X 150 mm) at a flow rate of 0.25 mL/min.…”

“…All multispecific antibodies were expressed from transfected Chinese hamster ovary (CHO) cells and expressed and purified using Protein A chromatography as previously described 21 .…”