1. 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

2. 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