Display Technologies and Affinity Maturation
At 272BIO, we use Display Technologies that enable us to create the very best VHH biotherapeutics as quickly and cost-effectively as possible.
Display technologies make feasible the simultaneous screening of enormous numbers of clone variants for desirable properties, as opposed to producing and testing investigator-selected clones individually for such properties.
We aim to be able to use large-library screening approaches at every stage of the drug discovery and lead optimization processes.
We use phage (and other) display technology in order to identify VHH binders to desirable targets, as well as to screen for clones with higher binding affinity (which often correlates with increased drug potency).
In addition, at 272BIO we are developing various proprietary display technologies that allow us to use library screening in phases of the drug development pathway where that is traditionally not possible. For example, we aim to be able to perform library screening at the very late stages of biotherapeutic drug development, and to make screening part of the development process of state-of-the-art multiparatopic drugs. Similarly, we are merging new avenues in structural and synthetic biology with display technologies to develop proprietary solutions.
At 272BIO, we believe that new technology development is absolutely essential to meet the unique challenges that veterinary drug development brings. Investment in continual technology development is core to 272BIO’s philosophy, and sets us apart from other companies operating in the veterinary drug development space.
Phage Display: One approach to obtaining high-affinity VHHs that bind a target of interest ( ) is to immunize a llama or alpaca with that target. As part of the animal’s natural immune response, pre-existing antibodies that bind to the target will become enriched in the blood, and higher-affinity variants of these will naturally appear in the blood as well. After several weeks, these antibodies are isolated from the blood, and engineered to be expressed (‘displayed’) on the outside of virus particles of a bacterial virus (a phage). VHHs that bind the target will make the phage particles stick to a plate coated with the target. Analysis of the recovered phage particles reveals the identities of the VHHs that bind the target of interest.
Proprietary phage libraries
Display technologies include phage display, yeast display, ribosome display and mammalian cell display.
We have created various bespoke naïve and semi-synthetic llama VHH libraries for use with the various display technologies, so as to be able to take advantage of the strengths of the various display technologies. We source valuable VHHs both from llama immunization approaches and from in vitro semi-synthetic approaches.
This dual capability enables us to identify VHHs against challenging, immune-tolerant targets. Furthermore, we affinity-mature VHHs where necessary, in order to optimize drug efficacy. Bespoke libraries of VHH clones are routinely created and interrogated for desirable characteristics, targeting both the CDR1/2/3 binding regions as well as framework regions that affect desirable characteristics.
Thus, 272BIO is fully equipped with state-of-the-art display technology and expertise best to engineer VHHs and take advantage of their unique structural characteristics.
Structure and binding characteristics of the VHH domain. The typical organisation of the VHH domain is represented using the structure of a VHH to carbazol (PDB 1U0Q). In framework (FR) 2 (dark grey), the amino acids shown in cyan [in this case Phe42, Glu49, Arg50, and Phe52 (IMGT numbering) represent the hallmark residues that substitute the critical amino acids of VHs that participate in the interaction with the light chain. CDRs 1, 2, abd 3 are coloured in pink, green and red respectively. The loop of FR 3 shown in blue (also known as CDR4) presents significant variability (higher than in VHs of conventional antibodies) and may interact with the antigen (41-43). Frequently, the CDR3 is long and bends over FR 2 shielding its hydrophobic residues and helping to mask Trp 18 (cyan-green), which is key for the interaction of VH with the VL chain. The structure of the long CDR3s, much more frequently in camels, may be stabilised by non-canonical disulfide.