Beyond affinity: selection of antibody variants with optimal biophysical properties and reduced immunogenicity from mammalian display libraries

Abstract

The early phase of protein drug development has traditionally focused on target binding properties leading to a desired mode of therapeutic action. As more protein therapeutics pass through the development pipeline; however, it is clear that non-optimal biophysical properties can emerge, particularly as proteins are formulated at high concentrations, causing aggregation or polyreactivity. Such late-stage "developability" problems can lead to delay or failure in traversing the development process. Aggregation propensity is also correlated with increased immunogenicity, resulting in expensive, late-stage clinical failures. Using nucleases-directed integration, we have constructed large mammalian display libraries where each cell contains a single antibody gene/ cell inserted at a single locus, thereby achieving transcriptional normalization. We show a strong correlation between poor biophysical properties and display level achieved in mammalian cells, which is not replicated by yeast display. Using two well-documented examples of antibodies with poor biophysical characteristics (MEDI-1912 and bococizumab), a library of variants was created based on surface hydrophobic and positive charge patches. Mammalian display was used to select for antibodies that retained target binding and permitted increased display level. The resultant variants exhibited reduced polyreactivity and reduced aggregation propensity. Furthermore, we show in the case of bococizumab that biophysically improved variants are less immunogenic than the parental molecule. Thus, mammalian display helps to address multiple developability issues during the earliest stages of lead discovery, thereby significantly de-risking the future development of protein drugs.

A single donor is sufficient to produce a highly functional in vitro antibody library

“…If this new BCR has improved surface expression, further receptor editing is prevented by RAG downregulation 95 , 96 . Although it has not been shown for B cells, it is well known that antibody display levels on yeast, another eukaryotic organism, reflect the underlying thermostability and expression levels of the displayed component antibodies 97 , and directed evolution to improve antibody display on mammalian cells results in improved “developability” 98 Similar mechanisms are also likely to be in play during B-cell development, with the immune system ensuring that antibodies have the potential to be expressed at high levels in plasma cells if required.…”

Animal Immunization, in Vitro Display Technologies, and Machine Learning for Antibody Discovery

“…Moreover, a number of new technologies and methodological changes have been introduced recently to mitigate developability risks associated with in vitro display technologies. For example, a novel mammalian cell-based display technology has been shown to facilitate preferential enrichment of antibodies with superior biophysical properties as part of the library selection for function [26]. Similarly, the introduction of predictive developability screens at early stages of antibody discovery significantly de-risks downstream development and manufacturing [27].…”

An end-to-end automated platform process for high-throughput engineering of next-generation multi-specific antibody therapeutics

“…On the other hand, unwanted modification patterns or altered physicochemical properties are avoided ab-initio. Such alterations can result from cell surface display-based selection procedures 30 or at later stages when displayed constructs, typically fragments of the envisioned product, are reformatted into the desired soluble product format. 31 , 32 In addition, mini-bioreactor-like compartments 22 are used for cell cultivation to approximate as best as possible large-scale fermentation processes.…”

“…Different strategies for compartmentalization have been described, including agar plate-, microtiter plate-, water-in oil droplet- or whole cell-based technologies. 30 , 39 , 40 Also, automated microtiter plate-based HT screens of non-deconvoluted variant libraries have proven to be successful in the maturation of antibodies to ultra-high, femtomolar-binding affinities. 25 In our microtiter plate-based approach we explicitly use deconvoluted plasmid libraries instead, as it is a pre-requisite for the systematic pairing of plasmids that code for the individual chains of in-silico designed molecules.…”

“…In conclusion, having this process in place confers considerable advantages, as it allows us not only to screen large sets of MSATs in a very short period of time but at the same time set the foundation for data-driven rational design, more sophisticated virtual screening approaches and various strategies for deep data mining based on machine learning models, amongst others. 30 , 42 , 44 …”