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8 • Antibody Structural Dynamics 221
antibodies passed below certain solubility thresholds (calculated with CamSol84) dur‑ ing temperature‑ramp simulations, in which the Fv was heated over the course of the simulation from 300 to 540 K with a temperature increment of 10–20 K and 20 ns of dynamics simulated at each temperature. In general, decreased solubility is observed as temperature increases, indicating partial unfolding that increases the solvent exposure of aggregation‑prone regions.
Compared to experimental data showing the percent change in aggregates detected by size‑exclusion chromatography after 3 months of storage at 40°C (Figure8.3g), we see that the all‑atom simulations are strongly predictive of aggregation. In the case of trastu‑ zumab, a tight transition to less soluble states is seen from ~480–500 K; these simula‑ tions suggest trastuzumab contains a stable Fv that should only begin to lose solubility at high temperatures, consistent with the experimental data showing that trastuzumab indeed has effectively zero aggregation change over a 3‑month period. In contrast, beva‑ cizumab has a broad solubility transition from ~400 to 470 K, and in size‑exclusion chromatography experiments was found to have a ~3% increase in aggregation over 3 months. These results are consistent with analyses showing that the proportion of trastuzumab in a β‑sheet conformation in the all‑atom simulations is effectively con‑ stant at 0.45 until 500 K, when it transitions sharply to an unfolded state by 525 K. Bevacizumab, in contrast, begins to lose its secondary structure at around 400 K and unfolds partially over the course of over 100 K before losing all structure by 525 K.
By correlating their simulation results with experimental assays on aggregation, Berner etal. suggest an intriguing path forward using simple temperature‑ramp simula‑ tions to predict solubility. One of the greatest strengths of their method is its simplic‑ ity– temperature‑ramp simulations are easy to perform, and their analysis relies on standard solubility methods and straightforward calculations of secondary structure content. Future studies considering more of the overall antibody structure (i.e., full Fab or even full‑length antibody sequences) in the simulations could improve the informa‑ tion content of such studies as computational speed continues to improve.

8.5 CONCLUSION

In this chapter, we have described the background and modeling philosophy for simulations of antibodies and described some example case studies in more detail. The applications of MD simulations for antibodies are quickly evolving, undergoing developments in both all‑atom and CG methodologies as well as analysis techniques. The set of studies highlighted here is a small fraction of the complete literature on this topic. For example, in addition to the CG results we described for the predic‑ tion of mAb solution properties, all‑atom simulations have also been employed to predict the inuence of pH and temperature stress on mAbs. poised to enter the development pipeline as a prime tool to predict the behavior of concentrated mAb solutions. All‑atom simulations, on the other hand, will likely be slower to enter industrial use due to their slow speed and high computational cost.
85,86
CG models appear
222 Biopharmaceutical Informatics

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Considerations of Developability During the Early Stages of Antibody Drug Discovery and Design

Maximiliano Vásquez, Bianka Prinz, Eric Krauland, and Tushar Jain
9

9.1 INTRODUCTION

Antibodies have become the main class of biotherapeutics, with over a 100 molecules approved for use in the United States and other major markets. Successful antibody therapies require potent and specic engagement of a disease‑relevant target. Turning an antibody into a drug, however, demands a series of additional criteria to be met, col‑ lectively known as “developability.” This has been increasingly appreciated over the last decade, as numerous studies have been published, illustrating various aspects of the developability problem.
Early examples centered around specic case studies, such as the effort to generate potent, afnity‑enhanced, versions of the anti‑Respiratory Syncytial Virus (RSV) anti‑ body palivizumab. ment in afnity and a 44‑fold enhancement in neutralization of RSV compared to the
228
1,2
A rst attempt1generated an antibody with a 1500‑fold improve‑
9 • Developability in Antibody Discovery 229
parental antibody palivizumab. Subsequent work, however, showed that in vivo potency enhancement was only about 2‑fold,2 which was attributed to unexpectedly poor phar‑ macokinetics in the form of very fast clearance, and traced to increased nonspecic binding across multiple tissues. A re‑examination of the afnity maturation work led to a modied candidate, motavizumab, which had much reduced polyspecicity and showed high potency in vitro and in vivo.2 Motavizumab entered clinical development progressing to phase III and Food and Drug Administration (FDA) submission after having been administered to thousands of patients3; however, it was terminated in late 2010 upon request by the FDA for additional clinical data.
Other work concerned the description of novel assays aimed at evaluating character‑ istics of antibody therapeutics candidates: cross‑interaction chromatography (CIC)4 and afnity‑capture self‑interactionnano‑spectroscopy (AC‑SINS)
5,6
are two such examples. More recently, studies looking at substantial numbers of antibody samples using one or more assays have been published; among them are reports on chemical degradation,
7–9
aggregation propensity,10 viscosity and pharmacokinetics,11multiple biophysical proper‑ ties,12 experimental correlates of viscosity and opalescence,13 and pharmacokinetics.
14,15
It is also worth mentioning papers describing computational metrics that correlate with general developability qualities; representatives include the therapeutic antibody proler,16 the combination of in silico and experimental properties predictive of favor‑ able pharmacokinetics,17 sequence‑based characterization of approved antibody ther‑ apeutics as metric to assess candidate antibody sequences,18 and charge calculations as predictors of viscosity, isoelectric point, pharmacokinetics, and general antibody proling.
19
Assessing developability earlier in the discovery process is ideal because there is more opportunity to avoid choosing a poor molecule when down‑selecting to a lead. However, the challenge with early developability assessment is two‑fold. First, speed, throughput, and minimal use of samples are practical requirements during this stage, where typically large panels of molecules are involved. Secondly, the context within which the antibodies exist is very different than that during clinical development, and therefore, there is a challenge to design assays and then understand and validate their predictive ability. It is crucial to nd a balance between the practical considerations and the ability of an assay, or set of such assays, to correlate with relevant metrics important in development. Metrics of polyspecicity and others relating to pharma‑ cokinetics, approximately “predict” viscosity,
14,15,20,21
or the validation at a larger scale that dynamic light scattering can
13,22
are examples of approaches described that can
potentially meet this challenge.
This chapter is organized into several sections as follows. First, we offer a historical perspective of how ideas and processes emerged over the last decade in our organiza‑ tion and our collaborators, with reference to mostly published material. Second, we review a set of 137 antibody samples constructed using sequences from clinical‑stage molecules12 and revisit previously published results. Next, we examine developability data for 349 antibodies isolated from human B cells,20 followed by an analysis of assay data obtained on over 150 antibody samples constructed using sequences from the lit‑ erature and corresponding to molecules aimed at targets of biomedical interest. These were compiled from antibody controls used across discovery campaigns against over 60
230 Biopharmaceutical Informatics
distinct targets. This is followed by a discussion on how these assays have been used in recent antibody discovery campaigns. We also include a section about the assessment of chemical liabilities via undesirable post‑translational modications. Lastly, we offer conclusions and perspectives for future work.

9.2 HISTORICAL PERSPECTIVE

In early years, we dealt mostly, but not exclusively, with antibodies isolated from our synthetic human‑like antibody diversities harbored by our engineered yeast host as full‑length IgG molecules. Access to yeast‑produced, full‑length, IgGs was subse‑ quently extended to situations where the source of diversity was from human B cells, or from tissues isolated from immunized animals. With IgG material in hand, we were interested in nding assays and workows that could address developability concerns earlier in the antibody discovery process than had been incorporated up to that point.
In 2013, we published our rst two articles on this general subject. article,24 a polyspecicity assay was described for application in both screening and selection. Briey, soluble membrane protein (SMP) and soluble cytosolic protein (SCP) are generated from Chinese Hamster Ovary (CHO) cells and biotinylated; a mix of these two constitutes the polyspecicity reagent (PSR), which is then used as a probe to assess binding by a test antibody. In what would become customary in much of our subsequent work, we included data on 24 control samples generated from variable region sequences from clinical development candidates with international nonproprietary name (INN) designations.
25–28
The results indicated that a small number of the 24 “clinical” anti‑ bodies had high readouts in the PSR binding assay. The PSR metric also showed a good correlation with those obtained from the CIC4 and baculovirus particle (BVP) binding15 assays previously described. This article also demonstrated the ability of the PSR approach to serve as a tool during the selection process utilizing ow cytometry, where millions of antibody variants can be assessed and sorted out of one pot. Lastly, for a subset of the antibody samples studied, a close relation was observed for measure‑ ments using the original soluble membrane preparation (SMP) from CHO cells with preparations of membrane or cytosolic proteins from insect Spodoptera frugiperda (Sf9) cells.24 In the second article,23 a clone self‑interaction assay using bio‑layer inter‑ ferometry (CSI‑BLI) was introduced and, again, tested with a few named (and some reported as mAb1, mAb2, etc.) antibody samples. The CNTO607 antibody, which had been reported to show poor biophysical properties,
29,30
was used as a positive control, and it showed a high response compared to other controls, such as a sample made with the variable regions of adalimumab.
In later work,31 we collaborated with the Tessier group, then at Rensselaer Polytechnic, in an adaptation of this group’s SINS and AC‑SINS assays. named antibodies, including CNTO607, were considered. CNTO607 was conrmed to give high self‑interaction in this assay, but, in addition, we observed that three samples made using INN sequences (corresponding to the clinical‑stage antibodies ganitumab,
23,24
In the rst
6,32
Here, over 30