Nanobodies against difficult targets – Tackling ion channels Collaborations in Ion Channel Drug Discovery 19-20th June 2014 Nanobodies® Inspired by nature Company highlights Corporate • • Drug discovery and development company - Ghent, Belgium >300 employees Technology • • Pioneer in next generation biologics – Nanobodies® >500 granted and pending patents Products • • • ~30 programmes – seven in clinical development Two clinical proof-of-concepts >800 healthy volunteers and patients treated with Nanobodies • AbbVie, Boehringer Ingelheim, Eddingpharm, Merck & Co, Merck Serono and Novartis >€320M in non-dilutive cash received to date Partners • www.ablynx.com 2 Nanobodies – derived from heavy-chain only antibodies Camelid heavy chain only antibodies are stable and fully functional Nanobodies represent the next generation of antibody-derived biologics • utilizes the variable region of naturally occurring heavy-chain only antibodies VHH VH CH1 VHH Ablynx’s Nanobody VL CL CH2 CH2 CH3 CH3 Conventional antibodies www.ablynx.com 12-15kDa Heavy chain only antibodies • unique formatting flexibility (up to penta-valent) • speed and ease of generating multi-specifics • nano- to picomolar affinities • favourable biophysical properties (Tm, solubility, viscosity) • tackling intractable targets • multiple administration routes • manufacturing in microbial cells 3 Nanobodies – pushing the limits of antibody technology scFv lgG • • • • 1st generation 150 kDa bi-valent fixed half-life mono-specific www.ablynx.com Diabody Bi-specific, tetra-valent DVD-lg Nanobodies 2nd generation • • • • 30-210 kDa mono- or bi-valent short or long half-life bi-specific 3rd generation • • • • 12-75 kDa valency of choice short or long half-life multi-specific 4 Nanobodies can be made to virtually any target High success rate to many different target classes • ‘easy targets’ - classical antibody targets • historically difficult target classes, including GPCRs and ion channels Typical discovery campaign yields functional leads with affinities ranging from 0.1-10 nM www.ablynx.com Target class Functional Nanobodies In vivo POC / targets tested Ion channels 3 2/2 GPCRs 6 4/4 Growth factors/receptors 22 7/7 TNF superfamily receptors & ligands 7 4/4 Cytokines & receptors 13 4/4 Blood proteins 6 3/3 Brain/neuroloy targets 7 1/1 T-cell coactivators ligands/receptors 6 0/0 Tumour antigens 2 1/1 Immunoglobulins 2 2/2 Viruses 10 7/7 Bacteria 2 2/2 Toxins 9 2/4 Parasites 1 1/1 Miscellaneous 25 0/0 Total 121 39/41 5 Delivering Nanobodies against ion channels Ion channel targets are validated but current approaches often fail to deliver Nanobodies represent a unique solution • • • • selectivity (vs small molecules) developability (vs toxins) target cryptic epitopes/clefts (vs mAbs) target multiple epitopes at once (vs other formats) Ablynx’s five ion channel programmes • P2X7: agonist and antagonist Nanobodies with in vivo POC in glomerulonephritis • Kv1.3: highly potent and selective Nanobodies with in vivo POC • ion-gated: functional Nanobodies discovered • ligand-gated: binders discovered • voltage-gated: lead discovery ongoing www.ablynx.com 6 Kv1.3 ion channel as a first-in-class treatment for a wide range of autoimmune and inflammatory diseases Difficult target Kv1.3 • small molecules have problems with specificity • difficult target for antibodies Kv1.3 channels provide the counterbalancing K+ efflux for Ca2+ entry into TEM cells Wide applicability to autoimmune and inflammatory diseases • MS, psoriasis, type I diabetes, etc. NatureRevImmunol2012(12)532 www.ablynx.com 7 Nanobody development Llama Immunisation Blood sampling 6–12 weeks later Conventional antibodies VHH CH2 CH3 VHH Ablynx’s Nanobody® Selection/screening Nanobodies Kv1.3 lead panel plus half-life extension (HLE) Nanobody formatting towards desired profile www.ablynx.com 8 Kv1.3 lead family – high affinity binding and further improvement through formatting Monovalent Nanobodies show low nanomolar affinity • multiple distinct Nanobodies identified • Nanobody family 1 and 12 represent leads Nanobodies are species cross-reactive • human, cynomolgus monkey, and rat Further ~10-fold improvement in affinity seen with bivalent construct • Also improves rat cross-reactivity 12 12 12 Norm.binding 100 50 0 0 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 Conc. (M) Sub nM engineerable affinity www.ablynx.com 9 Nanobody size and formatting allows combination of different epitopes Despite the limited extracellular exposed region of an ion channel, simultaneous binding of various Nanobodies is feasible 1 MCF MCF 12 12 5000 5000 4000 4000 3000 3000 2000 1000 12 1 12 MCF 1 MCF MCF 1 2000 1000 0 0 0 10 -12 10 -11 10 -10 10 -9 conc. in M 10 -8 10 -7 0 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 conc. in M Flexibility of formatting - Opportunity for heteromultimeric channel complexes www.ablynx.com 10 Lead Kv1.3 Nanobodies bind to a novel epitope Conceivable epitopes for functional effects • Pore domain – E3 region • Top voltage sensing domain Two channel variants were engineered displaying all extracellular residues from Kv1.3 apart from the first extracellular loop Leads predominantly bind to the 1st extracellular loop 150000 12 12 MCF 100000 50000 0 0 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 Conc. (M) www.ablynx.com 11 Kv1.3 Nanobodies show functional activity in an electrophysiological assay Activation protocol: 500 ms depolarization, 10 mV increment Control 12 12 1 nA 100 ms Low frequency, single step depolarization (+40 mV) % repsonse Dose 12 12 12 12 12 12 Dose dependent effects • Reduced peak amplitude • Fast current decline during step depolarization Potent gating-dependent channel block www.ablynx.com 12 Fast onset of block with Kv1.3 Nanobodies and increase in duration with bivalent construct Fast maximal effect during pulse train of 200 ms pulses, every 15 s 12 12 Monovalent displays wash out rapidly, bivalent does not (within 30 min recording time) 12 wash out 12 I (nA) I (nA) 12 Avidity enables long target residence time www.ablynx.com 13 Formatting allows the combination of functional profiles 12 1 1 1 12 1 12 12 12 12 12 Engineer desired functional profile www.ablynx.com 14 ... and also improvement of the functional potency 12 1 1 1 12 1 12 12 12 12 12 Engineer desired functional profile www.ablynx.com 15 Exquisite selectivity of Kv1.3 Nanobodies Selectivity was evaluated over closest related Kv1 family members and hERG Kv1.3 Kv1.5 Kv1.6 hERG Greater than 1 000 fold selectivity for Kv1.3 www.ablynx.com 16 Potent biological activity of Kv1.3 Nanobodies on primary T cells Fresh isolated CD45RA-CCR7- T cell subset 12 Stimulated with plate-coated CD3 only 12 12 12 12 72h incubation at 37 °C ShK IFN (pg/ml) Read out on IFN release IFN (pg/ml) 12 Functional activity comparable to benchmark toxin www.ablynx.com 17 Initial in vivo PoC study - design DNFB DNFB (in acetone/olive oil) (in acetone/olive oil) Sensitization Day 0 1 Challenge 2 3 4 Read-out 5 Nanobody/ShK s.c. dose (12h and/or 1h pre-challenge) 6 PK sampling (Satelite animals) Study groups • • • • • • Vehicle Dexamethasone ShK bivalent, non-HLE: trivalent, HLE: trivalent, HLE: topical dose s.c. dose (10 µg/kg) s.c. dose (equimolar) s.c. dose (equimolar) s.c. dose (equimolar) 1hr and 6hr post challenge 12h and 1h pre-challenge 12h and 1h pre-challenge 12h and 1h pre-challenge 1h pre-challenge Read-outs • Ear thickness • mRNA – cytokines (ear tissue) • PK (satellite animals) www.ablynx.com 18 Demonstration of in vivo POC using Kv1.3 Nanobodies Increase in ear thickness (mm) Ear thickness readout Vehicle Dexamethasone ShK / Topical 1hr and 6hr post challenge . s.c. 12h and 1h pre-challenge s.c. 12h and 1h pre-challenge s.c. 1h pre-challenge s.c. 12h and 1h pre-challenge Comparable effects of all Nb groups and ShK • Effects are moderate but highly significant (p<0.001 vs vehicle) • Both half-life extended (HLE) and non-HLE Kv1.3 Nanobody construct demonstrated efficacy • No differences seen between 1 vs 2 administrations First in vivo PoC obtained www.ablynx.com 19 Anti-Kv1.3 Nanobody conclusions Sub nM binding affinity of Kv1.3 Nanobodies demonstrated across species Flexibility of formatting allows improvement in activity and represents an opportunity for heteromultimeric channel complexes Potent gating-dependent channel block Ability to format or combine different Nanobodies to engineer different desired functional profiles Greater than 1 000 fold selectivity for Kv1.3 over other channels Functional Kv1.3 blocking activity on cells comparable to reference toxin In vivo PoC obtained obtained with Kv1.3 Nanobodies www.ablynx.com 20 Thanks to Veerle Delanote Daniel Janssen Diane Van Hoorick Erik Depla Ablynx Discovery team Ablynx Pharma team Helpful discussions and support with electrophysiology www.ablynx.com 21
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