Affilogic is a French Biotechnology company specialized in the discovery and development of affinity proteins called Nanofitins®. Based on basic research in protein engineering at Pasteur Institute and CNRS, a process has been developed to generate this novel class of alternatives to antibodies. Affilogic has exclusive and worldwide rights to this technology and perform the custom discovery and development of Nanofitins® against nominated targets. A protein scaffold with multiple advantages

Nanofitins® are small proteins (66 Amino Acids) that derive from a naturally hyperstable scaffold, from which they retain most of the biochemical features (Mouratou et al., PNAS (2007).

Nanofitins binding site in red / backbone in grey

Nanofitins®’ average Tm is >80°C (see below). They can withstand autoclave cycles and survive in gastric fluids. They can be stored several months @ RT and are not sensitive to repeated freeze-thaw cycles.

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.

Nanofitins® spontaneously fold and refold. They are not stabilized by any disulphide bridge. One major consequence is that they can be synthetized chemically.

Fusions & conjugates

N-/C- termini are not involved in the binding site. Thus, Nanofitins® can be (i) easily tethered to a support for detection and capture (regioselective conjugation through insertion of one single Cysteine in the sequence), (ii) conjugated to other moieties (small molecules, biologics, nanoparticles) by genetic fusion or click chemistry and (iii) assembled in multimers for enhanced avidity or for multi-specificity (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.

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, sub-libraries 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 cross-species binding human / mouse) or force the competition with a ligand, particularly relevant to inhibit a ligand/receptor interaction. This process is amenable to toxic targets, or non-immunogenic epitopes.

Usual selection process is a 2 months effort and yields to 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.

Nanofitins®, a robust alternative to antibodies

CD Tm measurements

Kon/Koff profiles, different Nanofitin® hits on same target

Binding profile of Nanofitins®

from fermentation from full synthesis

Neutralization or Transport Affilogic has designed Nanofitins® against 40+ targets to date, including a wide range of circulating antigens (peptides, proteins), membrane receptors for inhibition / modulation (GPCR, ion channels) and complex entities (Virus-like Particles, bacteria, whole cells). The process can enable direct screening for inhibitors/competitors and is independent of the toxicity/immunogenicity of the target. Conversely, Nanofitins® can be tailored so as to bind that target without blocking its interaction with one natural ligand. They can thereby be designed as targeted inhibitors, or as vectors for specific transport. In vivo neutralization

Usual systemic routes can be travelled by Nanofitins® aiming at neutralizing relevant interactions. Extremely high concentrations are of particular interest for subcutaneous administration. Their naturally short half-life can be tuned thanks to proprietary albumin-binding technology. Nanofitins® exhibit a very low immunogenic profile both for T-Cell response and ADA.

There is more: intrinsic properties of Nanofitins® make them amenable to other routes, such as topical administration (skin and cornea), up to possible oral route. In vitro permeation assays already demonstrated a high corneal penetration (Ussing chamber) and skin permeation (Frantz cell). We already demonstrated high performances in this approach of non-systemic targeted therapy in inflammatory skin diseases, and a European Consortium is at work for developing oral anti-TNF Nanofitins®.

Bispecifics to pentaspecifics: CMC-friendly

Design of Nanofitin®-based multi-specific compounds also enables to easily address multiple targets with one single molecule (anti-TNF / anti-IL17, anti-TNF / anti-IL23,…) still much smaller than an antibody. Affilogic already demonstrated in vivo efficacy of such bi-specific drugs.

From an engineering standpoint, we demonstrated that up to 5 either identical or different Nanofitins® could be assembled, with various linker possibilities. These oligomeric formats are still under a single protein format that can be manufatured by simple, scaleable, GMP-compliant bacterial fermentation at very attractive costs (15 to 50 times lower than antibodies). Nanofitin-Drug Conjugates

The easy conjugation of a Nanofitin® to another moiety enables to consider it not only as a neutralizing agent but also as a vector to increase target-specificity of the payload or enable BBB-crossing, intra-cellular targeting, …

Previous conjugation experiments include (i) single toxin conjugation via click-chemistry (ii) loaded nanoparticles conjugation for intracellular targeting of 100’s of toxins, (iii) multi-carrier conjugation for signal amplification and (iv) radioisotopes for in-vivo imaging. Along with an R&D tool box: QC, companion diagnostic…

In the course of a drug development, Nanofitins® can be used for detection needs as well. In previous research at Affilogic, tags were added at the N- and/or C-terminus (His-tag, Strep-Tag, Avi-Tag, Flag-Tag), enzymes were chemically and genetically fused to the Nanofitins® (GFP, HRP, AlkPho), and dyes were chemically conjugated either on the various exposed lysines on the Nanofitins®, or at a specific site with the addition of one specific cysteine. It was checked that these modifications did not induce any change in binding efficacy and specificity. Nanofitins® can thus be adapted to a broad range of assay protocols (multiplex lateral flow, immunoassays, FACS, immuno-histochemistry,…), for single target as well as multiplex detection.

Nanofitins, a different approach for targeted therapies

Locally applied Nanofitins® - Psoriasis in vivo efficacy model

Nanofitins® fused with cytotoxic peptide kill cells via intracellular PPI

Collaboration model starts with affordable generation of hits against target of choice Contact: Nadège PREL nadege@affilogic.com / +33 251 125 695

Viability vs. Nanofitins® Concentration