Measuring Receptor Dimerization To Create Functional Cell-Based Assays for ~85% of the Receptor Family

Scott Gridley, Ph. D.*, Abhishek Saharia, Ph. D., Hyna Dotimas, Sangeetha Gunthuri, Hanako Daino-Laizure, Albert Doan, Phil Achacoso, and Jane Lamerdin Ph. D. DiscoveRx Corporation, Fremont, CA 94538-3142

Abstract Simple, Functional Cell-based Assays for &

Ligand-induced receptor dimerization is an early functional step in receptor activation, representing the most proximal, functional read- A Ustekinumab (Stelara®) B Tocilizumab C Inhibition of IL-6 dependent proliferation of KPMM2 cells by Tocilizumab out for receptor activation. It is well understood that the family of Interleukin receptors will dimerize with the other members of its family IL12RB1/IL23R Assay U-2 OS IL-6R / IL-6ST Assay 140 20000 * * 20000 leading to a complicated signaling cascade that is critically involved in a variety of auto-immune, infl ammatory and oncogenic diseas- IL-6 (ng/mL) es. Surprisingly, existing cellular assays have been unable to faithfully monitor these interactions in a proximal manner to the receptor. 120 15000 0 15000 100 0.01 Here we present a novel application of the Enzyme Fragment Complementation system to monitor receptor-receptor interactions at the * 0.1 80 * 1 surface of intact live cells, applicable to diverse receptor types such as Interleukin receptors, BMP receptors, receptor tyrosine kinases RLU 10000 * 10000 10

RLU * * and receptors, with a specifi c focus on the interleukin family of receptors. The high specifi city, simplicity of the assay protocol, 100 60 * * 5000 * large signal to noise ratio, serum tolerance and reproducibility of these assays has enabled their use in cell-based screening, functional 5000 (%) Cell growth * 40 * * * * * IC ~ 1ug/ml Tocilizumab for characterization, QC lot release assays and neutralizing antibody studies. Examples discussed here include assays for the IL-1, IL-2, IL- * 50 0 0 20 inhibition of proliferation 6, IL-10 receptor families, with assays developed or in development for ~85% of the and their receptors. 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 of KPMM2 cells in presence 0 of 1ng/ml IL-6 Ustekinumab + IL-23 [g/mL] Tocilizumab [g/mL] + IL-6 [g/ml] 0 0.01 0.1 1 10 100 Tocilizumab (μg/mL) EC S/B IC S/B Structural Diversity in Families 50 50 Ustekinumab +100ng/mL IL-23 2.096e-007 12.4 Tocilizumab +3ng/mL IL-6 1.001e-006 4.8 9.591e-007 3.3 Ustekinumab +50mg/mL IL-23 1.750e-007 8.2 Tocilizumab +1ng/mL IL-6 IL-6 1.255e-009 9.9 IL-2 family IL-10 family IL-23 2.570e-008 9.9 One receptor multiple partners Multiple receptors heterodimerize with multiple partners

IL-24 IFN-O3 A. To treat infl ammatory diseases induced by IL-23 or IL-12, antibodies targeting the p40 subunit have been developed as therapeutics, for example Ustekinumab or Stelara® (Stelara is a IL-2 IL-4 IL-7 IL-9 IL-15 IL-21 IL-20 IL-24 IFN-O2 registered trademark of Janssen/Centacor). Response of IL23R assay to the therapeutic antibody Ustekinumab is shown above. The assay has been tested with two different concentrations IL-10 IL-19 IL-20 IL-22 IL-22 IL-26 IFN-O1 of IL-23 agonist, at EC80 and EC50 of the agnoist response. B. Cell-based assay for detecting stimulation and inhibition of IL-6 signaling through the membrane-bound IL-6R. Assay responds IL-2R IL-4R J IL-7R IL-9R J IL-2R IL-21R E D c D Jc D c E Jc Jc to IL-6 with an EC50 of 1.26ng/ml, while tocilizumab (a therapeutic antibody targeting IL-6R) inhibits IL-6-indiuced dimerization. C. Inhibition of IL-6-dependent proliferation of KPMM2 cells Receptors JAK1 JAK3 JAK1JAK3 JAK1JAK3 JAK1 JAK3 JAK1 JAK3 JAK1 JAK3 with Tocilizumab. IC50 of 1ug/ml for Tocilizumab obtained with a stimulation dose of 1ng/ml (red arrow in the fi gure above) is consistent with the IC50 value for tocilizumab in the IL6R di- merization assay (as in fi gure B) run with the same stimulation condition. Figure adopted from Mihara et al., (2005) Int. Immunopharmacology 5: 1731-40.

IL-2RD IL-15RD IL-22BP Robust cell-based assays for IL-34, M-CSF & GM-CSF

A M-CSF receptor GM-CSF receptor B U2-OS CSF1R / CSF1R C U2-OS CSF2RA / CSF2RB IL-10R2 IL-20R2 IL-20R2 IL-10R2 IL-10R2 IL-10R2 1500000 800000 IL-34 M-CSF IL-10R1 IL-20R1 IL-22R1 IL-22R1 IL-20R1 IFN-OR M-CSF GM-CSF 600000 1000000

400000 RLU Structural diversity in interleukin receptors. Receptors for interleukins are arranged in families. Each receptor is composed of at least two subunits. A. All IL-2 family receptors share a sub- RLU 500000 unit, known as the common c subunit, while also encoding a high affi nity receptor subunit that recognizes and binds its specifi c ligand with high affi nity. Both receptor subunits are essen-  200000 tial for transducing signals to downstream pathways. Two receptors (for IL-2 and IL-15) have evolved to require an additional receptor subunit (high specifi city receptor ) for functionality (Figure adapted from Liao et al., 2011. Curr Opin Immun 23(5): 598-604) . B. IL-10 family receptors involve heterodimerization of multiple receptor subunits to generate responses to different 0 0 ligands (Figure adapted from Zdanov A., 2010. Cytokine Growth Factor Rev. 21(5): 325-30). D 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 Ligand [g/mL] GM-CSF [g/mL] ® PathHunter Dimerization Assay Technology EC50 S/B EC50 S/B IL-34 6.355e-008 3.7 6.265e-009 5.3 Ligand E C M-CSF 2.331e-008 3.9 Ligand Ligand-induced Robust cell-based assays for IL-34, M-CSF & GM-CSF. A. Schematic representation of the receptors for macrophage colony-stimulating factor (M-CSF), granulocyte/macrophage CSF Dimerization (GM-CSF). The M-CSF receptor (CSF1R) is a homodimeric ; the GM-CSF receptor (CSF2R) is a heterodimeric receptor. B. The CSF1R homodimerization assay measures a robust response to IL-34 and M-CSF. C. The CSF2R heterodimer assay measures a robust response to GM-CSF.

A Simple Homogenous Protocol With Rapid Results

PK PK Plate Ready-to-Assay Cells Treat with Agonist/Molecule Read Luminescence EA EA Add Detection Add Detection Add compounds Reagent 1 Reagent 2 Substrate Light 0-24 hrs 0.5-16 hrs 15 min 60 min

PathHunter dimerization assay technology has been utilized to construct assays to interrogate an early event in the interleukin pathway signaling cascade. In brief, one receptor in the dimer pair is tagged with the PK fragment of the -gal reporter, while the second subunit is fused to the EA component of -gal. When the ligand engages the high affi nity receptor, it causes it to functionally dimerize with its heterodimer partner, bringing the fragments of the -gal reporter into close proximity which forces them to reconstitute enzymatic function, which can be detected by the addition of enzyme substrate. PathHunter bioassay kits use a simple homogenous protocol with rapid results. Ready-to-assay cells from DiscoveRx are plated on a 96-well plate and incubated for 0-24 hours at 37°C. Highly Specifi c Assays for Interleukin Receptors Across Multiple Families The agonist/test molecule is added to the plate and incubated for 0.5-16 hours. The detection reagents are added sequentially in two addition steps and the chemiluminescent signal can be detected on any plate-reading luminometer. Cells manufactured in bioassay kits are meant for single use in ready-to-assay vials. IL-2 Family IL-10 Family gp130 (IL6) Family IL-2RB / IL-2RG / IL-2RA (U-2 OS) IL-20RA/ IL-20RB (HEK) IL-6R / gp130 (U-2 OS) Phylogram of human interleukins & available assays 8000 50000 150000 IL-20 40000 6000 IL-24 100000 30000 IL18 IL16 IL1F5 IL1F8 IL1A IL1F6 IL17F IL1F10 IL-20 IL-24 IL1F9 IL33 IL1RN IL15 IL1B IL1F7 IL21 TSLP IL25 IL7 IL17B CNTF IL17C IL19 IL24 IL31 4000 IL17D IL12A IL26 IL4 IL34 IL23A IL32 CSF3 RLU IL9 IL2 RLU LIF IL17A IL20 IL28A IL29 RLU IL22 IL27A EC 1.179e-009 20000 EC 1.00e-007 1.263e-008 EC 2.984e-009 IL5 CTF1 IL28B IL10 TXLNA IL11 IL13 50 50 50 IL6 CLCF1 IL3 OSM CSF2

50000 IL8

2000 CSF1 S/B 7.2 10000 S/B 3.6 4.0 S/B 12.8

0 0 0 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 IL-2 [g/mL] Ligand [g/mL] IL-6 [g/mL]

IL-17 Family IL-1 Family CSF2RB Family Available IL-17RA / IL-17RC (U-2 OS) IL-1R / IL-1RAP (U-2 OS) IL-5RA / CSF2RB 60000 400000 120000 IL-17A 6h In Development IL-17F 16h 300000 90000 40000 Not Attempted (Inquire) IL-17A IL-17F 6h 16h 200000 60000 RLU RLU EC 2.221e-008 8.119e-008 EC 3.745e-009 2.603e-009 RLU EC 2.591e-010 20000 50 50 50 S/B 9.7 10.0 100000 S/B 8.9 12.1 30000 S/B 5.7

0 0 0 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 Ligand [g/mL] IL-1D [g/mL] IL-5 [M] Representative examples of assays for interleukin receptors from 6 different families of interleukins / receptors. Each plot shows a dose response for the relevant ligand(s) in a given assay from the indicated family. Data plotted are mean RLU and standard deviation from at least triplicate wells for each dose. These assays are characterized by robust assay windows and low CVs.

Dimerization of infl ammation targets IL-23R and IL-12R is highly specifi c This phylogram of human interleukins and closely related has been adapted from Brocker et al., 2010 which outlines 8 major groups – IL1-like (orange), -chain utilizing ( blue), IL4-like (dark blue), IL6/12-like (purple)), IL10-like (green), IL28-like (pink) and IL17-like (brown), and the non-classifi ed cytokines (black). The blue, green A Binding of heterodimeric IL-12 and IL-23 B IL12RB1 / IL12RB2 (U-2 OS) C IL12RB1/IL23R (U-2 OS) and red dots in front of each name highlights our progress with the development of the assay – green means that we have that assay available, blue means that it is in development and red ligands to the IL-12R and IL-23R dots indicate that we have not yet attempted construction of that assay. 20000 p40 IL-12/IL-23 p40 monomer 25000 [Image adopted from Brocker et al., 2010. Evolutionary divergence and functions of the human interleukin (IL) family. Human Genomics. 5(1): 30-55] p40 IL-23 IL-23 IL12 20000 IL-12 p35 IL23 15000 IL-12 p19 IL-12 / IL-23 p40 Monomer Summary & Conclusions 15000 10000

RLU • Robust cell-based assays for over 85% of human interleukins and related cytokines 10000

5000 RLU • Assays are highly specifi c and based on native biology of receptors 5000 –Proximal readout– monitors early functional activation of interleukin pathway IL12RE-1 IL12R -2 E IL23R IL12RE-1 0 0 –Response specifi c to receptor/ligand (interleukin) pair 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 Ligand [g/mL] Ligand [g/mL] –Applicable to monitoring activation or inhibition of receptor with large molecules • Simple assay protocol STAT 1,3,4,5 STAT 1,3,4,5 –Simple, homogenous, one-step add & read protocol • Scalable for screening or high throughput analysis of samples

Dimerization of IL-23R and IL-12R is highly specifi c. A. IL23R and IL12R share a common subunit (IL12RB1). Each receptor binds a heterodimeric ligand, which share the p40 subunit. Both –Optimized for 96-well and 384-well applications receptors have been linked to Crohn’s disease by GWAS studies. B. Dimerization of IL-12R (IL12RB1/IL12RB2) is stimulated by IL-12, but not by IL-23 or p40 monomer. C. Dimerization of IL-23R (IL23R/ IL12RB1) is stimulated by IL-23 but not by IL-12 or p40 monomer. • Excellent reproducibility and precision

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