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Advances in Analytical Biophysical Tools to Accelerate the Discovery Of Therapeutics Adv ances in analyt ical biophys ical to ols to ac cel erate the dis covery of t hera peutic a ntibodies The innate ability of antibodies to bind their targets with high affinity and exquisite specificity is leveraged in the discovery of therapeutic antibodies to treat a broad range of diseases including cancer, autoimmune disease and heart disease. In addition, antibodies are commonly used as companion diagnostics and reagents to support the therapeutic pipeline and to probe antigenic surfaces to inform vaccine design. Discovering antibodies with appropriate characteristics for a given purpose relies on their proper selection from the prolific number of clones that are routinely produced by modern antibody generation methods, including hybridoma technology in both normal and transgenic animals, phage display and synthetic libraries. Triaging large panels of antibodies to a few leads is normally followed by substantial engineering to optimise their binding affinity, minimise immunogenicity and improve developability. This article reviews the current and emerging label-free biosensor tools that are used to characterise the binding interactions of antibodies in terms of their kinetics, affinities and epitope specificities from early stage screening to the clinic, with emphasis on throughput. ithin the animal kingdom, antibodies ism and their classical presentation is as a Y-shaped By Dr Yasmina have evolved in jawed vertebrates glycoprotein comprised of heavy and light Noubia Abdiche W (gnathostomes) to protect these organ - polypeptide chains belonging to the immunoglobu - isms against pathogens and parasites 1. Antibodies lin (Ig) superfamily of proteins. However, some bind their targets with high affinity and high speci - species naturally produce other antibody formats, ficity, which makes them appealing as both thera - such as the small heavy chain-only fragments peutic agents and companion diagnostic reagents, known as VHH or nanobodies in camelids 2 and and these products are generating lucrative sales in the Ig new antigen receptor (IgNAR) in sharks 3. the pharmaceutical industry. Antibodies are a class Theoretical possibilities for unique-sequence anti - of specialised molecules produced and secreted by bodies are almost unlimited due to their architec - B lymphocytes in the immune system of an organ - ture, where stretches of conserved framework Drug Discovery World Summer 2018 55 Therapeutics & $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ ' ( Figure 1: SPR kinetics on a 384-array (a) tile view with each tile representing a discrete mAb-coated spot and (b) close-up view of three mAbs with diverse kinetic profiles (App Note, Carterra) References residues descended from a limited set of germlines interactions in terms of their kinetic rate constants 1 Klein, Jan and Nikolaidis, alternate with hypervariable ‘complementary- and affinities 4. Knowledge of an interaction’s affin - Nikolas. The descent of the determining regions’ (CDRs), which are responsi - ity is significant because it dictates the dose or con - antibody-based immune ble for the enormous sequence and structural diver - centration at which an antibody will be effica - system by gradual evolution. 5 PNAS January 4, 2005. 102 (1) sity of an organism’s antibody repertoire. While cious . Oftentimes, selecting an appropriate affini - 169-174. antibody generation in the pharmaceutical indus - ty for a given application is an empiric process and 2 Arbabi-Ghahroudi, M. try is highly commoditised, with modern in vivo can be optimised by engineering. Dissecting a bind - Camelid Single-Domain and in vitro libraries typically producing vast num - ing affinity into its constituent kinetic rate con - Antibodies: Historical bers of clones, the analytical tools used to charac - stants provides useful information that can guide Perspective and Future Outlook Front Immunol. 2017 terise their binding properties in terms of kinetics, the evaluation of an antibody’s performance Nov 20;8:1589. affinity and specificity, which are key parameters throughout screening, optimisation and manufac - 3 Feige, Matthias J, Gräwert, for assessing their quality and functional activity, ture. Label-free methods obviate the need to label Melissa A, Marcinowski, Moritz, lag orders of magnitude behind in throughput. or conjugate either of the interacting species and Hennig, Janosch, Behnke, Julia, With therapeutic antibodies being the largest class the binding event is monitored in real-time. This Ausländer, David, Herold, Eva M, Peschek, Jirka, Castro, of biotherapeutic proteins that are in clinical trials, makes it ideally suited for screening crude antibody Caitlin D, Flajnik, Martin, there is an ever-increasing demand for higher samples that are produced in early-stage research, Hendershot, Linda M, Sattler, throughput analytical methods that can match the where numerous clones are available, but in limit - Michael, Groll, Michael and capacity of antibody production and guide the ed quantities. Buchner, Johannes. The library-to-leads triage. Since taking an antibody Commonly-used commercially-available biosen - structural analysis of shark IgNAR antibodies reveals from bench to the market is estimated to cost sor platforms differ in their throughput, sample evolutionary principles of about $1 billion, there is a need to make antibody consumption and ease of use. They are versatile immunoglobulins. PNAS June screening more efficient and comprehensive to cut tools and are applied to the study of a broad range 3, 2014. 111 (22) 8155-8160. costs and timelines. of biomolecular interactions, from small molecules 4 Hearty, S, Leonard, P, to antibodies. Unlike small molecule analysis, O’Kennedy, R (2012). Measuring Antibody – Antigen Using SPR to characterise the where binding interactions are generally transient Binding Kinetics Using Surface binding kinetics and affinities with weak affinities, as characterised by equilibri - Plasmon Resonance. In: of antibody interactions um dissociation constants (or KD values) in the Chames P. (eds) Antibody Label-free biosensors such as those based on opti - micromolar range, antigen/antibody binding inter - Engineering. Methods in cal detection principles such as surface plasmon actions often exhibit affinities that are a million - Molecular Biology (Methods and Protocols), vol 907. resonance (SPR) or biolayer interferometry (BLI) fold tighter, with KD values in the picomolar range. Humana Press. are routinely employed in the pharmaceutical Accurately measuring the kinetic rate constants of Continued on page 57 industry to characterise antigen/antibody binding high affinity interactions requires long binding 56 Drug Discovery World Summer 2018 Therapeutics cycles to enable sufficient information to be gath - of several wash steps may also compromise the Continued from page 56 ered for both the association and dissociation ability to identify weak affinity binders, which may phases 6. Therefore, biosensor platforms that have desirable epitopes. 5 Rudnick, Stephen I and Adams, Gregory P. Affinity and increase the number of interactions monitored in Another option is the Octet HTX platform from Avidity in Antibody-Based parallel can significantly accelerate the analysis of Pall/ForteBio, which is a 96-channel BLI biosensor Tumor Targeting. Cancer large panels of antibodies. offering versatility in throughput from 8, 16, 32, Biotherapy And A recent study benchmarked the Mass-1 system 48 to 96-channel modes. Since each interaction Radiopharmaceuticals Volume from Sierra Sensors against the Biacore 4000 sys - necessitates the use of single disposable sensors, 24, Number 2, 2009. 6 Katsamba, PS, Navratilova, I, tem from GE Healthcare and found comparable running the system in 96-channel mode is costly on Calderon-Cacia, M, Fan, L, kinetic rate constants for a large panel of sensors and sample consumption scales with the Thornton, K, Zhu, M, Bos, TV, antigen/antibody interactions when chip types size of the assay. Also, the lack of microfluidics Forte, C, Friend, D, Laird- were matched 7. However, with the throughput of affects the accuracy with which binding kinetics Offringa, I, Tavares, G, Whatley, these two state-of-the-art instruments limited to can be measured, because they are often contami - J, Shi, E, Widom, A, Lindquist, KC, Klakamp, S, Drake, A, monitoring only eight interactions at once via par - nated by rebinding artifacts, which confound a Bohmann, D, Roell, M, Rose, L, 8 allel flow channels, these analyses not only con - reliable and detailed analysis . Indeed, a study by Dorocke, J, Roth, B, Luginbühl, sume significant quantities of samples but necessi - Estep et al in 2013 stated that, “When measuring B, Myszka, DG. Kinetic analysis tate long run times when addressing hundreds of affinities based on currently available methods, of a high-affinity interactions, making the analysis of more than 100 one must compromise either on throughput or antibody/antigen interaction performed by multiple Biacore clones rather tedious. As such, SPR is often used as accuracy”, which unfortunately has posed a bottle - users. Anal Biochem. 2006 May 9 a secondary tool, after large antibody panels have neck in analytics for decades . In that paper, they 15;352(2):208-21. Epub 2006 been whittled down to a subset of clones via pre - compared the affinities of antigen/antibody inter - Feb 23. liminary screening by higher throughput, but lower actions measured by orthogonal techniques, name - 7 Kamat, Vishal, Rafique, information content assays,
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