Interaction of clinical-stage antibodies with heme predicts their physiochemical and binding qualities

Abstract

Development of monoclonal antibody therapeutics is a sophisticated process. Early stage detection of negative traits of antibodies, which can impact the production, storage stability or therapeutic efficacy is of critical importance for saving time and resources in drug development programs. Since the developability issues of antibodies are diverse, including expression yield, chemical and physical stability, propensity for aggregation, antigen-binding polyreactivity, etc., a battery of experimental and in silico assays have been developed for thorough examination of drug candidates. Here, by using a set of 113 samples with variable region sequences matching clinical-stage therapeutic antibodies, we demonstrated that unique physicochemical properties of heme allow this cofactor molecule to be applied as a probe for simultaneous detection of different deleterious characteristics of antibodies i.e., antigen-binding polyreactivity, self-association, aggregation, hydrophobicity and expression yield. Moreover, our study demonstrates that similarly as antibody repertoires from healthy individuals, a fraction of therapeutic antibodies acquires use heme as cofactor for recognition of diverse antigens. These results reveal presence of a hidden vulnerability issue for some antibodies, which would not be detectable by standard analytical assays. This liability may be of concern for monoclonal antibodies intended to be use in patients with pathophysiological conditions where extracellular heme is present e.g. hemolysis or tissue damage. We provided evidence about the mechanistic basis of the unique features of heme and offer a powerful analytical tool for rapid and simple detection of important negative traits in candidate therapeutic antibodies.

Interaction with 2,4-dinitrophenol correlates with polyreactivity, self-binding, and stability of clinical-stage therapeutic antibodies

“…In a recent study we investigated the interaction of clinical-stage mAbs to heme -an ironcontaining cofactor molecule (Lecerf et al, 2021). Here we observed a statistically significant correlation in reactivities of mAbs for heme and DNP.…”

“…We used the data from the study of Jain to correlate the binding intensity to DNP-BSA with biophysical properties of importance for developability. We also used the data from a previous study where the reactivity of mAbs towards heme was analysed (Lecerf et al, 2021). The Spearmen's non-parametric correlation analyses revealed strong correlations of capacity of mAbs for binding to immobilized DNP with different developability parameters (Fig.…”

“…4). In a recent study we evaluated the interaction of mAbs with the biological cofactor molecule heme (Lecerf et al, 2021). Here, we analysed the correlation of the reactivities of mAbs to both aromatic compounds.…”

Functional Changes of Therapeutic Antibodies upon Exposure to Pro-Oxidative Agents

“…The experiment was performed as previously described in [23] 96-well polystyrene plates NUNC MaxiSorp™ (Thermo Fisher Scientific) were coated for 1 h at room temperature with human recombinant factor VIII (Advate, Baxter), human plasma-derived C3 (CompTech, Tyler, TX, USA) and recombinant LysM polypeptide of AtlA from Enterococcus faecalis (kindly provided by Dr. Stephan Mesnage, University of Sheffield, Sheffield, UK). The coating was performed with protein diluted to 3 µg/mL in PBS.…”

“…The reactivity of 113 native Abs to the same set of proteins was published in a previous study [23]. As the reactivity of all ferrous ion-exposed 119 native Abs was assessed simultaneously and in same conditions, we used here the previously published data obtained with 113 native Abs combined with 6 additional native Abs as a reference.…”