Recombinant Antibodies for Academia: a Practical Approach

Life ScienceS in SwitzerLand CHIMIA 2016, 70, No. 12 893 doi:10.2533/chimia.2016.893 Chimia 70 (2016) 893–897 © Swiss Chemical Society Recombinant Antibodies for Academia: A Practical Approach Pierre Cosson* and Oliver Hartley Abstract: After several decades of optimization, phage display technology enables the routine isolation and production of recombinant monoclonal antibodies in vitro. As such it has the potential to provide the academic community with a vast, inexpensive and renewable supply of well-characterized reagents, reducing bottlenecks in basic science, helping increase reproducibility of experiments, and phasing out the use of animals for production and discovery of antibodies. Yet the overwhelming majority of fundamental research laboratories still use incompletely characterized antibodies developed in animals. In order to promote increased use of recombinant antibodies in academia, we have recently initiated an open source recombinant antibody facility in Geneva (http://www.unige.ch/antibodies). Here we describe our experience at the Geneva Antibody Facility: the various techniques involved in isolation and production of antibodies, the strategic choices that we have made, and what we hope will be a bright future for this project as part of a growing movement in the scientific community to replace all animal-derived antibodies with recombinant antibodies. Keywords: Recombinant antibodies The 3R strategy concerning animal the data generated. Replacement strategies the foreign antigen. It has been known for experiments[1] has three principles: experi- have proven more difficult to implement, centuries that pathogen challenge can be ments involving animals should be refined however, because experimental procedures readily mimicked by immunization, and to decrease animal suffering and increase require constant adaptation as fundamental the natural antibody response in animals the scientific value of the results obtained. research questions evolve. In this respect, has been adopted as a tool to generate an- In doing so, the number of animals should animal immunization for antibody discov- tibodies against a wide range of antigens. be reduced to the minimum necessary to ery and production is a rare exception. The usual strategy is to inject the antigen obtain useful data. Finally, where possible, Antibody generation in animals is no (protein, peptide, or, for non-peptidic anti- animal experiments should be replaced longer either necessary or desirable. Over gens, protein conjugates) into the animal, with alternative non-animal models or the last few decades, new techniques have formulated with adjuvants in order to stim- technologies. been developed to isolate and produce anti- ulate a potent immune response. Notably, In the field of toxicology, replacement bodies entirely in vitro.[3] This provides the the adjuvants generally used for animal im- has recently gathered a great deal of mo- academic community with a unique oppor- munization are not deemed acceptable for mentum: it is likely that integrated in vitro tunity to demonstrate its willingness to ap- use in humans because of the side-effects testing strategies will in the near future re- ply replacement strategies when they are they induce. place much of the mandatory animal tests available. Following immunization, the immune currently performed.[2] For basic science in This article has two main goals. First, system of the animal scans its natural rep- academia, much progress has been made to provide a simple description of the main ertoire of different antibody-producing in refining animal experiments to mini- approaches available for generating anti- cells (107–109, according to the size of the mize suffering and increase the quality of bodies, addressed to non-experts, with a animal) in order to (i) select rare cells that specific focus on antibodies generated in produce antibodies which recognize the vitro. Second, to describe the new Geneva antigen, (ii) induce these cells to multiply, facility dedicated to the in vitro discovery, (iii) stimulate them to evolve higher affin- production and archiving of antibodies for ity binding to the antigen, and (iv) trigger the academic community, stressing the them to produce large quantities of the re- technical choices that guided this project sulting antibodies. and outlining the steps that could be taken From a practical point of view, there to develop its future scalability and sus- are three main methods to generate spe- tainability in the context of other similar cific antibodies. They are referred to here initiatives around the world. as polyclonal, monoclonal or recombinant antibodies (Table 1). First Part: Antibodies Come in Polyclonal Antibodies Three Flavors Polyclonalantibodiesrepresenttheanti- body mixture that can be purified directly *Correspondence: Prof. Dr. P. Cosson Faculty of Medicine When challenged with a pathogen, from an animal’s serum a few months af- University of Geneva innate immunity represents the first line ter immunization. The term ‘polyclonal’ is Centre Médical Universitaire of defence against infections. This is fol- used because at this time the serum will 1 rue Michel Servet CH-1211 Geneva 4 lowed by adaptive immunity, which in- contain a mixture of different antibodies E-mail: [email protected] cludes the generation of antibodies against directed against the antigen, resulting from 894 CHIMIA 2016, 70, No. 12 Life ScienceS in SwitzerLand Table 1. Relative advantages of different types of antibodies sequences, and can be readily produced in cultured cells, in a variety of formats, as POLYCLONAL MONOCLONAL RECOMBINANT described below. Time to isolate and 6 weeks 3–6 months 6 weeks What Constitutes a Useful produce Antibody for Research? Animals required YesYes No A perfect antibody is one that would be suitable for all of the different applications Selection in vivo, by immune in vivo, by immune in vitro, used by the academic research commu- system system independent of nity, (e.g. ELISA, Western blot, immuno- immune system fluorescence microscopy, etc.). According to these criteria there are very few, if any, Defined molecular No YesYes perfect antibodies. All antibodies, whether species polyclonal, monoclonal or recombinant, Reformating No No Yes are initially characterized by testing their possible ability to bind the antigen or antigen fragment that is typically presented in a purified Storage Largest Smaller, requires Smallest, and/or concentrated form. Further charac- volume maintenance no maintenance terization is then necessary to determine, for example, (i) whether an antibody raised Sustainable No Yes, unless Yes against a peptide fragment recognizes the production problems with full-length target protein, (ii) if it is capable storage of detecting the antigen at its endogenous Sequence available No No Yes level (usually low), and (iii) with which de- tection procedures it is compatible. For this reason, catalogs of research antibodies for sale typically specify for which application the combined production of a number of all available monoclonal antibodies are of or applications they are best suited. different antibody producing cell clones rodent origin. By the same criteria, very few adequate- that were amplified during the antibody Monoclonal antibodies are technically ly characterized antibodies are worthless: response. Because more blood can be more difficult to produce than polyclonal for example (i) many antibodies that do not drawn from larger animals, typical sourc- antibodies, but as single molecular spe- recognize their target protein by Western es of polyclonal antibodies are rabbits, cies of defined structure they generally blot recognize it in immunofluorescence sheep and goats rather than rats or mice. represent more valuable research reagents. experiments, (ii) an antibody that recog- Polyclonal antibodies are relatively inex- Furthermore, immortalized hybridoma nizes a peptide epitope within a protein but pensive to produce, but the total amount cells outlive the animals from which they is unable to recognize the full-length pro- that can be produced is limited by the were obtained: they can be stored frozen tein may be useful for certain applications, amount of blood that can be drawn from for many years and then, if needed, ex- such as helping to determine the extent to the immunized animal during its lifetime. panded in culture to produce monoclonal which a protein is folded or degraded, or Once a supply of serum from an animal is antibody in amounts greatly in excess of to report on the post-translational modifi- exhausted, a new immunization must be what the immunized animal could natu- cations it has received. For this reason, all performed, necessitating a new animal ex- rally produce in its lifetime. adequately characterized antibodies with periment and resulting in a polyclonal mix- definable specificity and utility should ture that, because of the stochastic nature Recombinant Antibodies ideally be kept in a searchable archive that of the adaptive immune response, cannot To generate recombinant antibodies, is available for use by the scientific com- possibly be identical to the previous one. the whole process of antibody selection munity. Additionally, calls have been made and production is reconstituted in vitro. for sequence information, which uniquely Monoclonal Antibodies In the most widely used technology, an- defines each monoclonal antibody, to be In 1975, Köhler and Milstein dis- tibody phage display,[3] a large collection

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