Animal-free alternatives and the antibody iceberg
Today, in a market flooded by animal-derived antibodies, the most challenging aspect confronting the ESAC was to identify the major barriers to adoption and development of non-animal-derived antibodies and to understand the erroneous beliefs within the research community that have hindered their widespread use and contributed to their lack of visibility.
None of the negative perceptions has any basis in the presently achievable quality or molecular properties of non-animal-derived antibody reagents; in fact, in vitro recombinant antibody approaches are widely employed in the pharmaceutical industry for creating therapeutics, where products of the highest quality are required. Overall, the ESAC found several factors that contribute to the continued supply and use of animal-derived antibodies in the basic research market, despite the availability of an alternative method of generation: first, there is inertia and a propensity to use existing or familiar methods; second, in most fields of biological research, there is ignorance of the advanced level now attained by non-animal-derived antibody technology; third, there are perceived economic and legal constraints for companies looking to enter the reagent market for in vitro antibody technology; and finally, access to non-animal-derived antibody resources, either from commercial sources or from academic centers, is limited.
The widespread acceptance of mediocre antibody quality by many biologists23 is compounded by the continued commercial supply of animal-derived products and a shortage of companies providing non-animal-derived antibody generation services. The inherent shortcomings of many animal-derived antibodies in supplier catalogs are accepted, while early negative experiences with recombinant prototypes are incorrectly perceived as reasons for basic researchers to avoid non-animal-based alternatives. Despite the abundance of validating publications, a lack of understanding in the use of recombinant methods persists.
A focus by in vitro recombinant antibody technology developers on the more lucrative therapeutics market and intellectual property restrictions surrounding these methods has also inhibited widespread commercial diffusion of the technology. This situation is changing, however. The ESAC report appendix, section III, details the availability of non-animal-derived custom antibody production services and reagents, highlighting a growing number of suppliers (see also http://www.afability.com for updates) and academic consortia that have successfully generated thousands of binding reagents.
Certainly, the establishment of molecular display techniques in laboratories unfamiliar with them can be challenging. Naive recombinant library development (from natural or synthetic sources) requires skilled molecular biology expertise and a substantial initial time investment. However, the diversity of acquired antibody candidates, the longevity of libraries after initial construction and the abolition of ongoing costs of animal care make this effort worthwhile. Once a library is available, the cost of generating and validating non-animal-derived antibodies is in the same range as the cost of animal-derived mAbs, requiring standard molecular biology laboratory equipment and consumables.
As demand increases, high-throughput technologies already solidly established in the therapeutic antibody generation market are expected to be adopted in the life sciences market and further price reductions are expected. Beside cost, there is a major benefit in time as antibodies can be selected from a naive recombinant library in a matter of a few weeks, as impressively demonstrated by the generation of recombinant SARS-CoV-2 binders in as little as four weeks by a host of academic and industrial groups (for example, Antibody Therapy Against Coronavirus, Yumab/Boehringer Ingelheim, CORAT Therapeutics, Molecular Partners, Avant Gen, Chugai Pharmabody Research/Agency for Science, Technology and Research, Centivax/Distributed Bio, Specifica and AstraZeneca; see https://www.antibodysociety.org/covid-19/).
Another commonly expressed misconception is that, to exploit the full potential of the immune repertoire, a whole-animal immune response to an antigen is required. This misconception has been contradicted by the many successfully applied efforts to generate antibodies from universal libraries to a large number of antigens, including SARS-CoV-2. The ESAC report describes how the non-animal-derived antibody development strategy has adopted and adapted the essential principles of the naturally evolving immune system during the generation of antibody candidates against a specific target. Specifically, the underlying mechanistic, functional, structural and diversity-generating similarities are clarified.
Scientific misconceptions surrounding sensitivity and avidity have also influenced a reluctance to adapt to certain formats, such as a monovalent antibody lacking an Fc region. Yet the submerged fraction of the antibody iceberg bestows on us the opportunity to work with a variety of alternative formats, with parameters that render them functionally indistinguishable from animal-derived antibodies in all typical applications. Today, recombinant production of the full immunoglobulin G (IgG) is straightforward, and in certain applications the use of the antibody fragment enjoys its own distinct advantages. Despite decades of development and maturity of a growing list of expression systems, formats, detection methods, picomolar affinities and application to every possible assay, a perception bias against display methods of antibody generation persists in parts of the research community, possibly a holdover from early publications from the 1990s, when the technologies were first introduced and were not as advanced as they are today. In sharp contrast, although not prevailing exclusively, the use of animal-free antibody generation methods by the pharmaceutical industry is now well established, and the clinical success of animal-free mAb products is the ultimate validation of antibody quality24.
Given that any antibody — whether generated by immunization or in vitro display methods — may lack intrinsic specificity for a target; may cross-react as a result of fortuitous interactions with the antibody binding site (lack of specificity); may bind to related epitopes occurring in different antigens, such as homologous proteins (lack of selectivity); or may preferentially bind to folded (conformational) or unfolded (linear) epitopes, the ESAC report stresses the importance of subjecting any affinity reagent to stringent quality control and validation. Sequencing of an antibody, regardless of its source, is important to assure the identity of the reagent and improve reproducibility. The very strict quality control imposed on therapeutic antibodies by the US Food and Drug Administration or European Medicines Agency assures that these goals are met.
The antibody supplier market is, however, a different matter. The inadequate validation of antibodies used in research settings results in a lack of antibody specificity that plagues scientific reproducibility, with a pernicious effect on science in terms of wasted money, opportunity, time, and human or other resources6.
An opportunity exists for antibody manufacturers operating under EU regulation and beyond to develop antibodies by using non-animal-derived antibody technology and for end users to adopt them. The scientific community should also demand a better quality of antibodies from commercial sources worldwide. If improved quality is demanded, manufacturers of existing animal-derived antibodies will have to do the extra time- and cost-consuming sequencing analysis and validation to measure their specificity and performance. As sequencing is routinely part of the process for generating non-animal-derived antibodies, and in conjunction with other advantages such as speed of generation, greater range of targets and opportunities to take advantage of challenging selection conditions, this will give companies using these technologies a clear economic advantage. In return, the scientific community stands to gain improved reproducibility, a readily available and renewable source of antibody reagents, and a reagent with greater versatility. Thus, the adoption of non-animal-derived recombinant antibody technology over immunization-based methods represents a scientific, ethical and societal imperative.