“Trends and Innovation in Drug Discovery at Sosei Heptares”

NON – CONFIDENTIAL

“Trends and innovation in drug discovery at Sosei Heptares”

Dr Matt Barnes & Dr Lisa Stott 20th December 2018 |BPS Pharmacology 2018

1 Introduction to Sosei Heptares

2 GPCRs and Immuno-oncology Bridging the Gap Between Recombinant and 3 Primary Cell Assays 4 Overall Summary

3 1

Introduction to Sosei Heptares

4 Introduction to Sosei Heptares

• World-leader in GPCR-focused drug design based on unique IP protected StaR®1 GPCR technology & enabled SBDD2 platform

• A broad and deep pipeline of partnered and in-house drug candidates in multiple therapeutic areas including neurology, immuno-oncology, gastroenterology, inflammation and rare/specialty diseases.

• Our partners and collaborators include: AstraZeneca, Allergan, Pfizer, Novartis, PeptiDream, Kymab, Daiichi-Sankyo and MorphoSys.

• Locations: We are headquartered in Tokyo, Japan, with state- of-the-art R&D facilities in Cambridge, UK and Zürich, Switzerland.

1 Stabilized receptor technology; 2 Structure-based drug design StaR is a Trade Mark of Heptares Therapeutics Limited

5 Why do we target G-Protein-Coupled Receptors (GPCRs)? Huge opportunity to create new drugs, or improve existing drugs

~400 GPCR targets in the body active in disease2 ~400 NEUROLOGICAL DISORDERS GPCR targets active in BEST-IN-CLASS FIRST OR 2 diseases GASTROINTESTINAL DISEASES BEST-IN-CLASS

Drugged METABOLIC DISORDERS In trials ~34% 27% 17% of FDA approvals ONCOLOGY target GPCRs1 As yet undrugged 27% CARDIOVASCULAR 56% of global sales RESPIRATORY are GPCR drugs1 FIRST-IN-CLASS

GPCRs are active in a wide range of disease areas, We are targeting new first-in-class and/or and offer broad therapeutic potential improved best-in-class GPCR medicines

Sources: 1 “Unexplored opportunities in the druggable human genome”, Nature Reviews, 2016 ; 2 “Trends in GPCR in Drug Discovery – new agents, targets and indications”, Nature Reviews, 2017

6 Powerful patent protected StaR® technology Provides unique structural insights into GPCRs that enable better and smarter drug design

7 2

GPCRs and Immuno-oncology

8 GPCRs and Immunology Multiple GPCR families are commonly associated with the immune system

CHEMOKINE

NEUROKININ

ADENOSINE COMPLEMENT

FORMYL PEPTIDE CANNABINOID

LYSOPHOPHOLIPID (S1P)

HISTAMINE PARS

pH-SENSING EICOSINOID

9 Why are we interested in Immunology? Immune mechanisms have potential broad therapeutic utility

Immune mechanisms

Oncology Neuroscience THERAPEUTIC Gastrointestinal AREAS (Immuno-oncology) (Neuroinflammation)

Check-point inhibitors Fingolimod | IFN-b | Tysabri Anti-TNFs | Integrin | JAK inhibitors

VALIDATION

10 Our approach to GPCRs and Immuno-oncology Understanding the relationship between the GPCR target, the immune cell type & the mechanism

GPCR Target Immune Cell Type Mechanism

Antigen Presentation APC Mobilisation effT-cell A2a Recruitment regT-cell CXCR4 Activation

EP2/4 MDSC Suppression

Right cells - Right state - Right place – consideration of checkpoint inhibitors

11 Our approach to GPCRs and Immuno-oncology CXCR4 Example

CXCR4 GPCR Target Expressed in T-cell, T-reg, MDSC Mobilisation, Recruitment, Suppression

12 GPCR Drug Target Approaches Two approaches to identifying new GPCR targets in immuno-oncology

1 GPCR target-based approach 2 Immune cell-based approach

What is the link between the GPCR target Which immune cells are important in TME? and the disease? Which GPCRs are expressed in those cell types?

13 1 GPCR target-based approach Literature/Internal assessment – GPCR target examples as categorised by cell type and mechanism

Target Cell type Mechanism A2a CD8 T-cell ▼ suppression APC ▲ co-stimulation CXCR4 Immune cells ▲ mobilisation CD8 T-cell ▲ recruitment MDSC ▼ recruitment CCR2 MDSC ▼ recruitment

A2b CD8 T-cell ▼ suppression MDSC ▼ activation S1PR1 T-reg ▼ recruitment/accumulation MDSC ▼ recruitment/accumulation EP2/4 MDSC/TAMs ▼ differentiation/activation CD8 T-cell ▼ expression PD-1 Tumor cells ▼ proliferation CXCR2 MDSC ▼ recruitment Neutrophil ▼ mobilisation/recruitment C3a CD8 T-cell ▲/▼ recruitment/suppression Neutrophil/Macrophage ▲/▼ activation Tumor cells ▼ proliferation CCR4 T-reg ▼ recruitment

CCR1 MDSC ▼ recruitment

14 Case Study: A2a Receptor (HTL1071/AZD4635) A2a Biophysical MappingTM with StaR Proteins

1 2

Mutant StaRs Site directed 10-30 Mutations to the Binding screened on mutagenesis site region Biacore chips

4 3 Ligand refined Correlating binding Detection of homology data from binding of model and Biophysical Map multiple ligands different prediction of of with multiple ligands to each protein-ligand binding site StaR proteins mutant StaR binding modes

Source: Zhukov et al., J. Med. Chem. 2011, 54, 4312

15 Case Study: A2a Receptor (HTL1071/AZD4635) A2a StaR - ZM241385 at Sosei Heptares enabled SBDD based drug discovery program

Perfect superposition of Sosei Heptares Excellent definition of ligand, side chains StaR structure (blue) with Stevens et. al. and waters at 1.7 Å resolution chimera structure (4EIY - red) with ZM241385 bound

Now: A2aR is a high-throughput, soakable system

Sources: Doré et al. Structure, 2011, 1283; Rucktooa et al. Sci Rep. 2018, 41

16 Case Study: A2a Receptor (HTL1071/AZD4635) Impact of SBDD on A2A Hit ID → LO → DC

Preladenant Hit 1 Hit 2 HTL1071 / AZD4635

Virtual BPM & SBDD Screen further VS ‘core hop’

A2A pKi 8.8 A2A pKi 8.5 A2A pKi 6.9 A2A pKi 8.8 MW 503, clogP 2.4 MW 310, clogP 3.1 MW 248, clogP 2.7 MW 316, clogP 2.7 LE 0.32, LLE 6.4 LE 0.52, LLE 5.4 LE 0.50, LLE 4.2 LE 0.49, LLE 5.2 CNS MPO 3.3 CNS MPO 4.6 CNS MPO 5.2 CNS MPO 5.1

• Furan containing • Novel non-furan containing • Novel triazene template • Improved LLE • Poor CNS physchem properties • Mod. selectivity (vs A1) • No structural alerts • Improved selectivity • Low selectivity (vs A1) • Improved metabolic stability • Mod. metabolic selectivity

SBDD platform approach significantly impacted • High efficiency leads identified using ‘enhanced homology model’ directed virtual screening identification & design of highly differentiated A2a ligands • SBDD guided approach used to drive LLE & selectivity enhancements

Sources: Langmead et al. J. Med. Chem. 2012, 1904; Congreve et al. J. Med. Chem. 2012, 1898

17 Case Study: A2a Receptor (HTL1071/AZD4635) A2a Receptor (AZD4635 – Phase 2) - Rationale & Evidence in Immuno-oncology

Rationale Supporting Pre-clinical Data

AZD4635 reverses adenosine mediated T-cell suppression AZD4635 enhances gene expression associated with T-cell and APC function

Accumulation of extracellular adenosine within the microenvironment is a strategy exploited by tumors to escape immune surveillance

Adenosine signalling through the high affinity A2a receptor on immune cells elicits a range of immunosuppressive effects

Blockade of the A2a receptor will reverse adenosine mediated immune suppression to enhance anti- tumor immunity AZD4635 exhibits anti-tumor activity & increases intra-tumuroal CD8+ve T-cells

Sources: Borodovsky, et al. AACR 2018; Cancer Research, 2018; Lamb M, GRC – Med Chem, 2018

18 2 Immune cell-based approach Multiple GPCRs and GPCR ligands are expressed in the tumor microenvironment

CCL2 A2b A2a C3a CXCL12 A3 CCL5 A2a CXCR2 CCL17 A2a CXCR4 C3a CXCR5 Adenosine APC FMLP1 A2b CXCL1 FMLP1 A2a A3 CXCL5 C3a CXCL8 EP4 PGE2 A2b EP2 CX3CR1 CXCR4 CCR2 CXCR2 S1PR1 EP4 C5a C3 CCR8 C3a C5a EP2 CCR1 EP4 CCR4 CXCR2 EP2 Neutrophil CXCR4 A2b CXCR1 CCR2

ANTI TUMOR PRO TUMOR

19 2 Immune cell-based approach Focus on T-reg Example

Preclinical tumor model Human meta-analyses

Induction of Tumor Immunity by Removing CD25+CD4+ T Cells: Prognostic value of tumor-infiltrating Fox P3+ regulatory T cells in cancers: A Common Basis Between Tumor Immunity and Autoimmunity A systematic review and meta-analysis J Immunol November 15, 1999, 163 (10) 5211-5218 Sci Rep. 2015 Oct 14;5:15179. doi: 10.1038/srep15179

Results of meta- analyses of OS for each tumor site

High FoxP3+ Tregs densities were associated with significantly Eradication of tumor transplants in nude shorter OS mice by transferring CD25+ cell-depleted splenic cell (CD25 – cell) suspensions

T-reg are a major immunosuppressive cell type in the TME which can suppress effector T-cells . and are associated with a reduction in OS in some cancers

20 2 Immune cell-based approach Focus on T-reg Example

Findings from patient tissue analyses GPCRs/Ligands that are expressed differently

Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness Receptors of Tumor-infiltrating T Regulatory Cells ▲ P2Y14 (UDP-glucose) Immunity. 2016 Nov 15;45(5): 1135-1147. doi: 10.1016/j.immune.2016.10.021 ▲ CCR8 (CCL1/CCL8) • Transcriptomes of Treg cells infiltrating colorectal or non-small-cell lung ▲ cancers were compared to transcriptomes of the same subsets from normal CX3CR1 (CX3CL1/CCL26) tissues and validated at the single-cell level ▲ ETB receptor (Endothelin) ▲ GPR56 (orphan-adhesion)

• Preliminary data from bulk RNA-Seq analysis extracted and differentially ▼ GPR109B (3-hydroxyoctanoic acid)

expressed genes identified - 758 genes were listed as differentially ▼ GPR160 (orphan) expressed between TregC and TregH at the p=0.1 level Ligands

▲ (CCR1, CCR4 and CCR5) CCL3 • GPCRs/Ligands extracted from this list of differentially expressed genes ▲ (CCR8) CCL18 include: ▲ (CXCR5) CXCL13 A number of GPCR and GPCR ligands are differentially expressed tumor associated vs. non-tumor associated T-reg cells

Jens Kleinjung - Bioinformatics 21 GPCR Drug Target Approaches The most interesting GPCRs may be those which overlap the two approaches

1 GPCR target-based approach 2 Immune cell-based approach

GPCR targets which have some degree of validation and differentially expressed in immune cells of the tumor microenvironment

22 3

Bridging the Gap Between Recombinant and Primary Cell Assays

23 Molecular Pharmacology at Sosei Heptares Supporting GPCR Drug Discovery Throughput Recombinant

Target Compound Validation Management

Primary

Target Compound Engagement Screening Relevance In vivo Increasing Lead confidence Characterisation

24 Recombinant and Native Cell Assays Range of assays used to characterise leads in both over- and endogenously expressing cell lines Expression Binding Signalling Assays

Radioligand Ligand 3H / 125I AC Ligand PLC DAG Gαi Gαq PIP ATP cAMP 2 IP3

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Ex: Em: 620nm 2 0 0 0 1 R

T V e h ic le H 335nm 0 A g o n is t Terbium -8 -7 -6 -5 -4 L o g [F o r s k o lin ] (M ) RhoGEF

Fluorophore SNAP Em: 665nm Ligand RhoA

• High throughput • Large S:N 25 • Receptor tags allow multiple assay formats Primary Blood Cell Assays Moving towards therapeutic relevance Blood Cell Isolation Expression Analysis Signalling Assays Functional Responses Flow Cytometry Dynamic Mass Redistribution Chemotaxis

Light Light in Refracted qRT-PCR Western Blot qRT-PCR F-Actin Polymerisation Apoptosis Protein expression Gene expression G ra n u lo c y te S a m p le 1 D a ta 1

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• Low throughput • Limited cell availability • Low endogenous expression = low S:N for traditional signalling assays 26 • Cannot tag receptors so assay formats are limited Chemokine Receptor Data Ligand Characterisation in Recombinant Binding and Signalling Assays Binding – TR-FRET Signalling – cAMP Inhibition

Terbium 1 2 0 1 2 0

Fluorophore e g

n 1 0 0 n

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Ligand s

n e 6 0

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2 0 % % 5 0 5 G proteinGαi 0 6 6

ATP cAMP -1 1 -1 0 -9 -8 -7 -6 -5 -9 -8 -7 -6 -5 -4 L o g [C o m p o u n d ] (M ) L o g [C o m p o u n d ] (M )

• TR-FRET offers safer and higher throughput binding assay to traditional Compound cAMP pIC50 TR-FRET pIC50 iodinated chemokine binding assays 1 - 7.54 ± 0.13 • TR-FRET assay appears more sensitive than cAMP inhibitory assays 2 5.27 ± 0.13 9.10 ± 0.26 • Together these assays allow identification of potential allosteric ligands 3 5.21 ± 0.16 7.81 ± 0.11 • Cpd 5 most effective at inhibiting cAMP response but fails to block 4 5.63 ± 0.23 7.51 ± 0.20 binding of endogenous chemokine 5 6.67 ± 0.23 - • Cpd 1 not fully inhibiting in TR-FRET binding assay 6 - -

Chemotaxis – Primary T Cells

e s

n 1 2 0

Chemokine Receptor Data o

D o n o r 1 p

s 1 0 0

e D o n o r 2 r

Ligand Characterisation in Primary Cell Assays 8 0

e D o n o r 3 Donor 1 -7.775

n i

k 6 0 DMR – Primary T Cells o D o n o r 4 Donor 2 -7.532 m 4 0

e Donor 3 -8.046 h

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s Donor 4 -8.364

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L o g [C h e m o k in e ] (M ) L o g [C o m p o u n d ] (M ) o m

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Donor 1 Donor 2 Donor 3 Donor 4 h C

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-7.982 -7.978 -7.813 -7.94 2 0

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Compound cAMP pIC50 TR-FRET pIC50 DMR pIC50

% %

1 - 7.54 ± 0.13 - -1 0 -9 -8 -7 -1 1 -1 0 -9 -8 -7 2 5.27 ± 0.13 9.10 ± 0.26 6.23 ± 0.28 L o g [C h e m o k in e ] (M ) L o g [C p d 2 ] (M ) 3 5.21 ± 0.16 7.81 ± 0.11 6.13 ± 0.30 4 5.63 ± 0.23 7.51 ± 0.20 6.53 ± 0.22 • Traditional chemotaxis assays more prone to donor 5 6.67 ± 0.23 - - variability than DMR 6 - - - • Schild analysis offers insight into nature of antagonism and • DMR allows characterisation of primary T cell signalling the ability to overcome agonist variability • Agonist responses very consistent across donors

• Antagonist pIC50s correlate with recombinant cAMP assays 28 Iryna Teobald, Susan Brown Summary

• Chemokine receptor responses can be measured in both recombinant and primary cells • TR-FRET allows us to perform high throughput binding assays without the need for iodinated chemokines and allows us to identify allosteric ligands • DMR assays on primary T cells correlate with recombinant functional assays and appear less susceptible to donor variation than traditional chemotaxis assays. • However, chemotaxis assays can be used to bridge between in vitro assays and in vivo tumour models to aid investigation of immune cell infiltration into the tumour microenvironment and increase our confidence in the anti-tumour activity of GPCR modulators.

Future Plans / Aspirations

• Use pathway inhibitors to deconvolute primary T cell signalling • Isolate and test other disease relevant immune cells such as neutrophils, T-regs or MDSCs • Use patient blood to increase translatability

29 4

Overall Summary

30 GPCRs and Immuno-oncology Summary

1 GPCRs are a tractable family of drug targets

2 Multiple GPCR families are associated with various immune functions

Immune mechanisms have potential broad therapeutic utility (Immuno-oncology; Neuroinflammation; 3 Gastrointestinal)

Using both a ‘target-based’ approach and ‘immune cell-based’ approach, a significant number of 4 GPCRs have been found to be associated with immuno-oncology

A combination of both of these approaches may provide GPCR drug targets with the highest 5 probability-of-success

Building an understanding of the molecular pharmacology of GPCRs in primary human immune cells is 6 a key part in understanding how we can influence the make-up of the tumor microenvironment

31 Thank you for your attention

SOSEI HEPTARES

PMO Hanzomon 11F The Steinmetz Building Grabenstrasse 11a, North West House 2-1 Kojimachi, Chiyoda-ku Granta Park, Cambridge CH-8952 Schlieren Zürich, 119 Marylebone Road Tokyo 102-0083 CB21 6DG Switzerland London NW1 5PU Japan United Kingdom United Kingdom