Improving the Developability of an Antigen Binding Fragment by Aspartate Substitutions.

Aggregation can be a major challenge in the development of antibody-based pharmaceuticals as it can compromise the quality of the product during bioprocessing, formulation, and drug administration. To avoid aggregation, developability assessment is often run in parallel with functional optimization in the early screening phases to flag and deselect problematic molecules. As developability assessment can be demanding with regard to time and resources, there is a high focus on the development of molecule design strategies for engineering molecules with a high developability potential. Previously, Dudgeon et al. [(2012) Proc. Natl. Acad. Sci. U. S. A. 109, 10879-10884] demonstrated how Asp substitutions at specific positions in human variable domains and single-chain variable fragments could decrease the aggregation propensity. Here, we have investigated whether these Asp substitutions would improve the developability potential of a murine antigen binding fragment (Fab). A full combinatorial library consisting of 393 Fab variants with single, double, and triple Asp substitutions was first screened in silico with Rosetta; thereafter, 26 variants with the highest predicted thermodynamic stability were selected for production. All variants were subjected to a set of developability studies. Interestingly, most variants had thermodynamic stability on par with or improved relative to that of the wild type. Twenty-five of the variants exhibited improved nonspecificity. Half of the variants exhibited improved aggregation resistance. Strikingly, while we observed remarkable improvement in the developability potential, the Asp substitutions had no substantial effect on the antigenic binding affinity. Altogether, by combining the insertion of negative charges and the in silico screen based on computational models, we were able to improve the developability of the Fab rapidly.

2'-Deoxyadenosine 5'-diphosphoribose is an endogenous TRPM2 superagonist.

Transient receptor potential melastatin 2 (TRPM2) is a ligand-gated Ca2+-permeable nonselective cation channel. Whereas physiological stimuli, such as chemotactic agents, evoke controlled Ca2+ signals via TRPM2, pathophysiological stimuli such as reactive oxygen species and genotoxic stress result in prolonged TRPM2-mediated Ca2+ entry and, consequently, apoptosis. To date, adenosine 5'-diphosphoribose (ADPR) has been assumed to be the main agonist for TRPM2. Here we show that 2'-deoxy-ADPR was a significantly better TRPM2 agonist, inducing 10.4-fold higher whole-cell currents at saturation. Mechanistically, this increased activity was caused by a decreased rate of inactivation and higher average open probability. Using high-performance liquid chromatography (HPLC) and mass spectrometry, we detected endogenous 2'-deoxy-ADPR in Jurkat T lymphocytes. Consistently, cytosolic nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2) and nicotinamide adenine dinucleotide (NAD)-glycohydrolase CD38 sequentially catalyzed the synthesis of 2'-deoxy-ADPR from nicotinamide mononucleotide (NMN) and 2'-deoxy-ATP in vitro. Thus, 2'-deoxy-ADPR is an endogenous TRPM2 superagonist that may act as a cell signaling molecule.

Assessment of Therapeutic Antibody Developability by Combinations of In Vitro and In Silico Methods.

Although antibodies have become the fastest-growing class of therapeutics on the market, it is still challenging to develop them for therapeutic applications, which often require these molecules to withstand stresses that are not present in vivo. We define developability as the likelihood of an antibody candidate with suitable functionality to be developed into a manufacturable, stable, safe, and effective drug that can be formulated to high concentrations while retaining a long shelf life. The implementation of reliable developability assessments from the early stages of antibody discovery enables flagging and deselection of potentially problematic candidates, while focussing available resources on the development of the most promising ones. Currently, however, thorough developability assessment requires multiple in vitro assays, which makes it labor intensive and time consuming to implement at early stages. Furthermore, accurate in vitro analysis at the early stage is compromised by the high number of potential candidates that are often prepared at low quantities and purity. Recent improvements in the performance of computational predictors of developability potential are beginning to change this scenario. Many computational methods only require the knowledge of the amino acid sequences and can be used to identify possible developability issues or to rank available candidates according to a range of biophysical properties. Here, we describe how the implementation of in silico tools into antibody discovery pipelines is increasingly offering time- and cost-effective alternatives to in vitro experimental screening, thus streamlining the drug development process. We discuss in particular the biophysical and biochemical properties that underpin developability potential and their trade-offs, review various in vitro assays to measure such properties or parameters that are predictive of developability, and give an overview of the growing number of in silico tools available to predict properties important for antibody development, including the CamSol method developed in our laboratory.

An accelerated surface-mediated stress assay of antibody instability for developability studies.

High physical stability is required for the development of monoclonal antibodies (mAbs) into successful therapeutic products. Developability assays are used to predict physical stability issues such as high viscosity and poor conformational stability, but protein aggregation remains a challenging property to predict. Among different types of stresses, air-water and solid-liquid interfaces are well known to potentially trigger protein instability and induce aggregation. Yet, in contrast to the increasing number of developability assays to evaluate bulk properties, there is still a lack of experimental methods to evaluate antibody stability against interfaces. Here, we investigate the potential of a hydrophobic nanoparticle surface-mediated stress assay to assess the stability of mAbs during the early stages of development. We evaluate this surface-mediated accelerated stability assay on a rationally designed library of 14 variants of a humanized IgG4, featuring a broad span of solubility values and other developability properties. The assay could identify variants characterized by high instability against agitation in the presence of air-water interfaces. Remarkably, for the set of investigated molecules, we observe strong correlations between the extent of aggregation induced by the surface-mediated stress assay and other developability properties of the molecules, such as aggregation upon storage at 45°C, self-association (evaluated by affinity-capture self-interaction nanoparticle spectroscopy) and nonspecific interactions (estimated by cross-interaction chromatography, stand-up monolayer chromatography (SMAC), SMAC*). This highly controlled surface-mediated stress assay has the potential to complement and increase the ability of the current set of screening techniques to assess protein aggregation and developability potential of mAbs during the early stages of drug development. Abbreviations:AC-SINS: Affinity-Capture Self-Interaction Nanoparticle Spectroscopy; AMS: Ammonium sulfate precipitation; ANS: 1-anilinonaphtalene-8-sulfonate; CIC: Cross-interaction chromatography; DLS: Dynamic light scattering; HIC: Hydrophobic interaction chromatography; HNSSA: Hydrophobic nanoparticles surface-stress assay; mAb: Monoclonal antibody; NP: Nanoparticle; SEC: Size exclusion chromatography; SMAC: Stand-up monolayer chromatography; WT: Wild type.

In vitro and in silico assessment of the developability of a designed monoclonal antibody library.

Despite major advances in antibody discovery technologies, the successful development of monoclonal antibodies (mAbs) into effective therapeutic and diagnostic agents can often be impeded by developability liabilities, such as poor expression, low solubility, high viscosity and aggregation. Therefore, strategies to predict at the early phases of antibody development the risk of late-stage failure of antibody candidates are highly valuable. In this work, we employ the in silico solubility predictor CamSol to design a library of 17 variants of a humanized mAb predicted to span a broad range of solubility values, and we examine their developability potential with a battery of commonly used in vitro and in silico assays. Our results demonstrate the ability of CamSol to rationally enhance mAb developability, and provide a quantitative comparison of in vitro developability measurements with each other and with more resource-intensive solubility measurements, as well as with in silico predictors that offer a potentially faster and cheaper alternative. We observed a strong correlation between predicted and experimentally determined solubility values, as well as with measurements obtained using a panel of in vitro developability assays that probe non-specific interactions. These results indicate that computational methods have the potential to reduce or eliminate the need of carrying out laborious in vitro quality controls for large numbers of lead candidates. Overall, our study provides support to the emerging view that the implementation of in silico tools in antibody discovery campaigns can ensure rapid and early selection of antibodies with optimal developability potential.