nanobodies against difficult targets – tackling ion channels · with specificity • difficult...
TRANSCRIPT
Nanobodies® -Inspired by nature
Collaborations in Ion Channel Drug Discovery 19-20th June 2014
Nanobodies against difficult targets – Tackling ion channels
www.ablynx.com
Company highlights
2
Products
Partners
• Pioneer in next generation biologics – Nanobodies®
• >500 granted and pending patents
• ~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
• Drug discovery and development company - Ghent, Belgium• >300 employees
Technology
Corporate
www.ablynx.com
Nanobodies – derived from heavy-chain only antibodies
Camelid heavy chain only antibodies are stable and fully functionalNanobodies represent the next generation of antibody-derived biologics
• utilizes the variable region of naturally occurring heavy-chain only antibodies
3
Conventional antibodies
Heavy chain only antibodies
Ablynx’s Nanobody• 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
VHH
CH2
CH3
CH1
CL
VL
VH
CH2
CH3
VHH
12-15kDa
www.ablynx.com
Nanobodies – pushing the limits of antibody technology
4
lgG Bi-specific, tetra-valentDVD-lg
scFv
Diabody Nanobodies
1st generation• 150 kDa• bi-valent• fixed half-life• mono-specific
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
www.ablynx.com
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
5
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
www.ablynx.com
Delivering Nanobodies against ion channels
Ion channel targets are validated but current approaches often fail to deliverNanobodies 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)
6
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
Kv1.3 ion channel as a first-in-class treatment for a wide range of autoimmune and inflammatory diseases
7
Difficult target• 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.
Kv1.3
NatureRevImmunol2012(12)532
www.ablynx.com
Nanobody development
Conventional antibodies
Selection/screening Nanobodies
Llama Immunisation Blood sampling 6–12 weeks later
VHH
Ablynx’sNanobody®
VHH
CH3
CH2
Nanobody formatting towards desired profile
plus half-lifeextension(HLE)
8
Kv1.3 lead panel
www.ablynx.com
0
50
100
10-12 10-11 10-10 10-9 10-8 10-7 10-6
Conc. (M)
Nor
m.b
indi
ng
0
Kv1.3 lead family – high affinity binding and further improvement through formatting
9
12 1212
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
Sub nM engineerable affinity
www.ablynx.com
Nanobody size and formatting allows combination of different epitopes
10
Despite the limited extracellular exposed region of an ion channel, simultaneousbinding of various Nanobodies is feasible
Flexibility of formatting - Opportunity for heteromultimeric channel complexes
12 1
1
12 12
12
MCF
0
1000
2000
3000
4000
5000
10-12 10-11 10-10 10-9 10-8 10-7
conc. in M
MCF
0
MCF
0
1000
2000
3000
4000
5000
10-12 10-11 10-10 10-9 10-8 10-7
conc. in MM
CF0
MCF
1 1
www.ablynx.com
0
50000
100000
150000
10-12 10-11 10-10 10-9 10-8 10-7 10-6
MC
F
0Conc. (M)
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 fromKv1.3 apart from the first extracellular loop
Leads predominantly bind to the 1st extracellular loop
12 12
11
www.ablynx.com
Kv1.3 Nanobodies show functional activity in an electrophysiological assay
Low frequency, single step depolarization (+40 mV)
Potent gating-dependent channel block
100 ms
1 nA
Dos
e
Activation protocol: 500 ms depolarization, 10 mV increment
1212 12
12 12
12
% re
pson
se
12 12Control
Dose dependent effects• Reduced peak amplitude• Fast current decline during step depolarization
12
www.ablynx.com
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
Monovalent displays wash out rapidly, bivalent does not (within 30 min recording time)
I (nA
)
washout
12 1212
I (nA
)
12 12
Avidity enables long target residence time13
www.ablynx.com
Formatting allows the combination of functional profiles
1212 1212 12
12
12 11 1
1
Engineer desired functional profile14
www.ablynx.com
... and also improvement of the functional potency
1212 1212 12
12
12 11 1
1
Engineer desired functional profile15
www.ablynx.com
Selectivity was evaluated over closest related Kv1 family members and hERG
Kv1.5
16
Exquisite selectivity of Kv1.3 Nanobodies
hERGKv1.6
Greater than 1 000 fold selectivity for Kv1.3
Kv1.3
www.ablynx.com
Potent biological activity of Kv1.3 Nanobodies on primary T cells
17
1212 12
12 12
12Fresh isolated CD45RA-CCR7- T cell subsetStimulated with plate-coated CD3 only72h incubation at 37 °CRead out on IFN release
IFN
(pg/
ml)
IFN
(pg/
ml)
ShK
Functional activity comparable to benchmark toxin
www.ablynx.com
Initial in vivo PoC study - design
18
Day 0 1 2 3 4 5 6
DNFB(in acetone/olive oil)
DNFB(in acetone/olive oil)
Nanobody/ShK s.c. dose(12h and/or 1h pre-challenge)
Sensitization Challenge Read-out
PK sampling(Satelite animals)
Study groups• Vehicle• Dexamethasone topical dose 1hr and 6hr post challenge • ShK s.c. dose (10 µg/kg) 12h and 1h pre-challenge• bivalent, non-HLE: s.c. dose (equimolar) 12h and 1h pre-challenge• trivalent, HLE: s.c. dose (equimolar) 12h and 1h pre-challenge• trivalent, HLE: s.c. dose (equimolar) 1h pre-challenge
Read-outs• Ear thickness• mRNA – cytokines (ear tissue)• PK (satellite animals)
www.ablynx.com
Demonstration of in vivo POC using Kv1.3 Nanobodies
Ear thickness readout
19
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
Incr
ease
in e
ar th
ickn
ess
(mm
)
ShK
VehicleDexamethasone
/
Topical
s.c.
s.c.
s.c.
s.c.
1hr and 6hr post challenge .
12h and 1h pre-challenge
12h and 1h pre-challenge
1h pre-challenge
12h and 1h pre-challenge
www.ablynx.com
Anti-Kv1.3 Nanobody conclusions
Sub nM binding affinity of Kv1.3 Nanobodies demonstrated across speciesFlexibility of formatting allows improvement in activity and represents anopportunity for heteromultimeric channel complexesPotent gating-dependent channel blockAbility to format or combine different Nanobodies to engineer different desiredfunctional profilesGreater than 1 000 fold selectivity for Kv1.3 over other channelsFunctional Kv1.3 blocking activity on cells comparable to reference toxinIn vivo PoC obtained obtained with Kv1.3 Nanobodies
20
www.ablynx.com
Thanks to
Veerle DelanoteDaniel JanssenDiane Van HoorickErik Depla
Ablynx Discovery teamAblynx Pharma team
Helpful discussions and support with electrophysiology
21