In vivo pharmacology – PK/PD studies in cynomolgus monkey

Results: • non-linear PK after IV administration more than dose-proportional increase in AUC • the terminal half-life is in the range of 5.7 - 7.4 days in the 10 - 100 mg/kg dose groups, but much

shorter in the 1 - 5 mg/kg dose groups • the multi-phasic disposition suggests target-mediated drug disposition • total [sIL-6R] increases as the complex stays longer in circulation via the half-life extension moiety

of ALX-0061 valid PD marker indicating the presence of active drug • Modeling demonstrated almost complete inhibition of the elimination of sIL-6R (Imax= 97%) with

IC50 = 0.068 µg/mL (2.64 nM)

Ablynx’s Nanobodies

Project goal: develop ALX-0061 as an anti-inflammatory treatment for RA

ALX-0061 product description

• affinity matured single domain binding to sIL-6R and mIL-6R

• no induction of ADCC or CDC due to lack of Fc

• half-life extension by binding to Human Serum Albumin (HSA)

Goal of the experiments: comparison between ALX-0061 and

tocilizumab (TCZ) for target affinity and potency

VHH

Ablynx’s Nanobody® • Small (1/10 size of a mAb)

• Flexible formatting

• Highly potent, robust and stable

• Broad target applicability

• Multiple administration routes

• Ease of manufacture

• Speed of discovery

The sensitive GyrolabTM platform was used to determine the KD of ALX-0061 and TCZ for both IL-6R forms

ALX-0061 demonstrated strong binding to the sIL-6R

• the affinity of ALX-0061 for sIL-6R is 2500-fold higher compared to TCZ

ALX-0061 showed preferential binding properties for sIL-6R over mIL-6R

Presence of HSA had no influence on binding of ALX-0061 (data not shown)

Results: • ALX-0061 showed a 10-fold stronger potency compared to TCZ in neutralizing sIL-6R in an IL-6/sIL-6R

complex. • In the highly stable IL-6/sIL-6R/gp130 complex, inhibition IC50 of ALX-0061 remained the same, while

the potency of TCZ decreased 2-fold • Presence of HSA had no influence on the in vitro potencies

Maarten Van Roy, Ariella Van de Sompel, Kristi De Smet, Jasper Jacobs, Tinneke Denayer, Sven Hoefman, Laura Sargentini, Hans Ulrichts

ALX-0061, an Anti-IL-6R Nanobody® for Use in Rheumatoid Arthritis, Demonstrates a Different In Vitro Profile as Compared to Tocilizumab

Nanobody® is a registered trademark of Ablynx nv www.ablynx.com Technologiepark 21, 9052 Zwijnaarde, Belgium

ALX-0061: IL-6R targeting Nanobody Affinity of ALX-0061 and TCZ for sIL-6R and mIL-6R Superior potency of ALX-0061 compared to TCZ on sIL-6R

IL-6R pathway Similar potency of ALX-0061 and TCZ on mIL-6R Ligand replacement

Camelidae family has both forms

VH

VL CL

CH1

CH3

CH2

VHH

CH3

CH2

Conventional antibody • Heavy and light chains • Both chains required for antigen

binding and stability • Large size and relatively low

formatting flexibility

Heavy-chain antibody • Only heavy chains • Full antigen binding

capacity and very stable

Compound IC50 ± SD [nM]

No HSA 1mg/ml HSA

ALX-0061 0.06 ± 0.02 (n=5) 0.06 ± 0.07 (n=3)

TCZ 0.49 ± 0.12 (n=5) 0.45± 0.07 (n=3)

ALX vs TCZ 8.0x 7.5x

Conclusions

ALX-0061 has single domain binding and preferential sIL-6R engagement with a high affinity for sIL-6R (0.2 pM)

Compared to TCZ, ALX-0061 demonstrated superior affinity and potency in multiple in vitro sIL-6R based neutralisation and replacement assays

Presence of HSA had no influence on affinity and potency of ALX-0061

Potency is confirmed in vivo, using sIL-6R as PD biomarker

The difference in target binding profile compared to TCZ may result in:

• a stronger engagement of the disease-driving target form (sIL-6R) in a physiological situation with both forms (sIL-6R and mIL-6R) present

• a different safety profile

• a superior benefit/risk profile

Results of a phase I/II clinical trial with ALX-0061 in RA patients were indicative of a strong potential for disease modifying activity of ALX-0061 (poster presentation #1479)

KD ± SD (n = 3) sIL-6R mIL-6R sIL-6R vs mIL-6R

ALX-0061 0.19 pM ± 0.08 pM 9.1 pM ± 3.6 pM 46x

TCZ 462 pM ± 138 pM 154 pM ± 16 pM 0.31x

ALX-0061 vs TCZ 2,500x 17x

Due to restricted expression of membrane (m) IL-6R, signaling via the classic signaling pathway is confined to only a small population of cell types

IL-6 can activate cells that do not express mIL-6R through a process known as trans-signaling

• IL-6 binds first to free soluble (s)IL-6R the IL-6 / sIL-6R complex binds to the common gp130, which is expressed on the surface of most cells, and this results in a highly stable hexameric complex

Selective inhibition of IL-6 trans-signaling could provide a higher therapeutic efficacy with a better side effect profile than complete IL-6 inhibition (1)

Animal studies with IL-6 inhibitors show that the classic IL-6 signaling pathway via the mIL-6R has important physiological functions, such as metabolic control in the liver,

• whereas blocking the trans-signaling pathway via sIL-6R is sufficient to prevent or treat IL-6-driven diseases

Membrane Soluble Principle

Lt

L

R

Lt

L

R

Measurement of free [R] (= drug) after equilibrium in mixtures with constant [R] and a dilution series of [L] (= target)

Rt

Compound IC50 ± SD [nM]

No HSA 1mg/ml HSA

ALX-0061 0.05 ± 0.01 (n=3) 0.06 (n=1)

TCZ 0.88 ± 0.22 (n=3) 0.77 (n=1)

ALX vs TCZ 18x 13x

Results: • ALX-0061 exhibits comparable potency as TCZ in neutralizing mIL-6R in the presence of HSA • Similar as reported for TCZ (EPAR), ALX-0061 uniformly stained granulocytes and monocytes,

while only a subset of the lymphocytes (CD4+) was stained (indicated with “+” in the graph)

Negative control

Compound

IC50 ± SD [nM] in TF-1 proliferation bio-assay

No HSA 1mg/ml HSA

Tocilizumab 0.72 ± 0.21 (n=3) 0.90 ± 0.07 (n=2)

ALX-0061 0.29 ± 0.03 (n=3) 0.77 ± 0.18 (n=3)

-

+

Binding to human PBMCs

Granulocytes Monocytes

Lymphocytes

ALX-0061

Compound IC50 ± SD [nM], no HSA

IL-6/sIL-6R (3h) IL-6/sIL-6R/gp130 (3h) IL-6/sIL-6R/gp130 (24h)

ALX-0061 0.06 ± 0.003 (n=4) 0.03 ± 0.03 (n=2) 0.04 ± 0.01 (n=2)

TCZ 0.36 ± 0.05 (n=4) 0.61 ± 0.19 (n=2) 3.94 ± 0.76 (n=2)

ALX vs TCZ 6.5x 19x 105x Results: • ALX-0061 and TCZ replaced hIL-6 to a large extent (75%) from a pre-formed ligand/target

complex. The replacement IC50 was comparable to the inhibition IC50 for both compounds. • In the highly stable IL-6/sIL-6R/gp130 complex, maximal inhibition is lower for both

compounds ( 20% after 3h and 40% after 24h incubation). While ALX-0061 maintained potent IC50 in the replacement setting, TCZ showed lower potency.

IL-6 trans-signaling IL-6 classic signaling

Example

experiment Example

experiment

Example

experiment

Example

Experiment (24h)

Example

experiment

Dose-range finding study in cynomolgus monkey with ALX-0061

Poster #1498

(1) Rose-John, S., IL-6 trans-signaling via the soluble IL-6 receptor: importance for the pro-inflammatory activities of IL-6. International journal of biological sciences, 2012. 8(9): p. 1237-47.