A tunable 100% in vitro selection process
The generation of Nanofitins® consists in identifying the best ligands amongst the vast library of variants. Thanks to in vitro selection with Ribosome Display technology, sublibraries of up to 10E14 ligands are screened with many parameters to tune specificity on the target(s): targeting common epitopes of different antigens (such as human/mouse cross-species binding) or forcing the competition with a ligand, particularly relevant to inhibiting a ligand/receptor interaction. This process is amenable to toxic targets, or non-immunogenic epitopes.
High affinity: nanomolar to single-digit picomolar
The usual selection process takes 2 months and yields a number of different monomeric hits with a high affinity down to single-digit picomolar. However, affinity can be tailored in the 10E-5 – 10E-12 range directly from within the selection process, for affinity chromatography needs, for instance, where release of the target is an essential step.
Affilogic now has experience on 40+ targets, over a wide range:
- Circulating antigens: peptides, proteins…
- Complex entities: Virus-like Particles, bacteria, whole cells…
- Transmembrane receptors for inhibition / modulation (GPCR, ion channels)
A common backbone with intrinsic assets
Very small and extremely stable
Nanofitins® are small single-chain proteins (7 kDa, around 20 times smaller than a monoclonal antibody) exhibiting a higher tissue penetration potential. Nanofitins® derive from a naturally hyperstable scaffold, from which they retain most of the biochemical features such as resistance to temperature (70-80°C) and pH (10-13).
They can be stored several months at room temperature and are not sensitive to repeated freeze-thaw cycles. Nanofitins® spontaneously fold and refold, and can withstand autoclave cycles. They are not stabilized by any disulphide bridge. One major consequence is that they can be synthesised chemically. Nanofitins® have demonstrated a very high protease resistance and can thus survive in gastric fluids, which enables development of orally formulated Nanofitins® for therapeutics applications. They can also be formulated in a large spectrum of buffers and be concentrated >100 mg/mL to reach very high doses in a small volume. The combination of these features make Nanofitins® highly druggable compounds.
A promising safety profile
Neither cellular nor in vivo toxicity has been observed on 20+ cellular and 10+ animal models with Nanofitins®. Nanofitins® immunogenicity has been specifically tested in several in vitro and in vivo prediction models. Cellular immunogenicity potential is comprised between a fully-human antibody and a recombinant protein. The backbone of the Nanofitin® – conserved region among all Nanofitins® – does not bear immunogenic epitopes.
Nanofitins® can be manufactured by simple, scaleable, GMP-compliant bacterial fermentation at very attractive costs (15 to 50 times lower than antibodies). Full chemical synthesis is also possible.
Available N & C termini for simplicity of conjugation
N-/C- termini are not involved in the binding site. Thus, Nanofitins® can be :
- Easily tethered to a support for detection and capture (regioselective conjugation through insertion of one single Cysteine in the sequence),
- Conjugated to other moieties (small molecules, biologics, nanoparticles) by genetic fusion or click chemistry
- Assembled in multimers for enhanced avidity or for multispecificity (up to 5 Nanofitins® as one multivalent compound with retained individual affinity). In every instance, neither the binding property of each single Nanofitin®, nor the function of the conjugate is affected.