Therapeutics


Table 1: Key features comparison for non-antibody protein scaffolds TEMPLATE PRODUCTION TM SIZE OF


VARIABLE REGION


Affibodies (Affilogic)


DARPins


Z domain of protein A


Natural Ankyrin repeat proteins


Nanobodies (Ablynx)


Single-domain HC- only antibody fragments


Affimer proteins


Human protein Stefin A and plant Cystatin


E. coli or peptide synthesis


E. coli, up to 200mg/mL


E. coli, up to 150mg/mL (post- translational


modifications and disulphide bridges)


E. coli, up to 200 mg/mL


42-100°C 18 12-14 kDa 42-71°C 66-89°C


(AMINO ACIDS) 13


18 (split across three conjugated DARPin units)


60-80°C 9-12 SIZE OF SCAFFOLD 5-6 kDa 18-20 kDa


15 kDa


One of the major disadvantages of anti-ID anti- bodies is the reliance upon animals for their initial generation20. The selection of an appropriate anti- ID antibody relies on the probability of finding a good candidate produced as part of the animal’s natural immune response. Such candidates repre- sent only a small proportion of the antibody reper- toire generated, requiring the screening of a large number of clones, with a variable rate of success. Once a promising candidate has been selected, fur- ther work is often necessary to increase the target affinity of the molecule. Consequently, long devel- opment times and associated costs can become problematic.


Beyond selection and optimisation, these large, complex molecules also present issues when it comes to production, as only costly eukaryotic cell systems can be used. Additionally, the product must be carefully monitored for changes in glyco- sylation and drift in expression throughout pro- duction, to ensure consistency. Furthermore, as monoclonal antibodies are highly sensitive to changes in pH and temperature, their storage and use within complex assay matrices may affect downstream performance.


Recombinant antibodies and antibody fragments There has been a conscious shift across the indus- try toward recombinant antibodies, in order to overcome issues of batch-to-batch reproducibility


associated with standard monoclonal antibodies20, 21. Benefits include the ability to manufacture using in vitro systems, offering increased repro-


12


ducibility between batches of reagents, and the ease of returning to the starting material in the face of genetic drift of the expressing cell line. Recombinant reagents may also be isolated from phage libraries, assuring maintenance of the struc- ture during screening and allowing anti-ID selec- tion to be driven by project aims and requirements, increasing the potential of identifying specific, high-affinity binders to a target antigen. Being able to specifically drive the selection of binders in this manner also ensures that recombinant antibodies can be selected to both free and bound formats of a potential therapeutic antibody, for use in a wide range of bioanalytical PK assays.


Despite the reproducibility advantages of recom- binant antibodies over monoclonal antibodies, the same issues around manufacturability of these complex structures remain. One strategy to pre- vent these problems has been the adoption of anti- body fragments in the place of full antibodies. As these structures are smaller and less complex, it is possible to produce them in prokaryotic culture systems, which are simpler and less expensive. Prominent recombinant antibody suppliers include Bio-Rad and Creative Biolabs. Both com- panies are able to offer rapid initial selection of binders in up to eight weeks, due to the advantages of library screening for recombinant anti-ID binders. However, as Bio-Rad notes within its liter- ature22, further affinity maturation of the selected binder may be required to increase affinity and specificity for the target post-purchase, whether recombinant antibodies or antibody fragments,


Drug Discovery World Winter 2017/18


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