0 2 4 6 8 10 12 14 Vehicle PWT143, 100 mg/kg BID Mean±SE Neutrophil Count (5 Fields) Therapeutic Neutrophil count in disease paws  PWT143 was tested in cellular assays reflecting activity of PI3K delta, alpha, beta and gamma isoforms –Delta: pAKT (T308) in anti-IgM-stimulated Raji cells –Alpha: pAKT (T308) in IGF1-stimulated MDA-MB-453 cells –Beta: pAKT (S473) in LPA-stimulated PC-3 cells –Gamma: pAKT (S473) in C5a-stimulated RAW264.7 cells PI3K delta is broadly implicated in inflammation signaling in both B and T cells as well as multiple myeloid cell types, which cooperate in the initiation and progression of various inflammatory and autoimmune diseases. We have discovered a series of potent and highly selective PI3K delta inhibitors that potently inhibit signaling in immune cells and have demonstrated compelling efficacy in rodent models of immune disease. One such molecule, PWT143, inhibits AKT phosphorylation with sub-nanomolar potency in cellular assays reflecting PI3K delta function. This activity translates to robust phenotypic activity in multiple cell types, including inhibition of cytokine release from B and T cells. Importantly, PWT143 inhibits basophil activation (as measured by surface CD63 expression in a whole blood assay) with sub- nanomolar potency in response to anti-IgE stimulation but not in response to fMLP stimulation, suggesting a differential effect on autoimmune compared to bacterial antigen- mediated immune cell activation. In vivo, the cellular activities of PWT143 are reflected by dose-dependent pharmacodynamic activity in a mouse passive cutaneous anaphylaxis model. Furthermore, PWT143 completely prevents disease onset and regresses established disease in a mouse collagen-induced arthritis model, suggesting that inhibition of PI3K delta (without inhibition of PI3K gamma) is sufficient for efficacy in this model. Together, these data support the broad utility of PWT143 and related PI3K delta inhibitors for the treatment of autoimmune disease and other pathologies involving dysregulation of the immune response. DISCOVERY OF PI3K DELTA INHIBITORS FOR THE TREATMENT OF INFLAMMATORY AND AUTOIMMUNE DISEASE David J. Matthews, PhD, Marie O’Farrell, PhD, Richard Ventura, S. David Brown, PhD, and Albert Tai Pathway Therapeutics Inc., San Francisco, CA Efficacy Mouse Pharmacokinetics PI3K Isoform Cellular Assays Abstract Compound PI3Kdelta IC 50 (nM) PI3Kalpha IC 50 (nM) PI3Kbeta IC 50 (nM) PI3Kgamma IC 50 (nM) PWT143 5.0 5022 208 2137 CAL‐101 7.1 1122 485 89 In vitro kinase selectivity PWT143 is a potent, selective inhibitor of PI3K delta PWT143 shows no crossreactivity with protein kinases DiscoverX Kinomescan (PWT143 @ 1µM) Effect on B cells Effect on T cells CD4+ human T cells, stimulated with anti-CD3 and anti-CD28 Compound IL‐10 IC 50 (nM) IL‐4 IC 50 (nM) TNF alpha IC 50 (nM) IFN gamma (IC 50 (nM) IL‐2 IC 50 (nM) PWT143 <1.4 13 513 <1.4 120 CAL‐101 ~ 330 ~ 100 1540 ~ 50 ~3000 0 20 40 60 80 100 120 0 1 2 3 TNF alpha (% vehicle) Concentra on (uM) CAL‐101 PW‐A 0 20 40 60 80 100 120 140 0 1 2 3 IL‐10 (% vehicle) Concentra on (uM) CAL‐101 PW‐A PWT143 PWT143 TNFalpha release in Raji B cells (anti-IgM stimulation) Compound B cell pAKT T308 IC 50 (nM) B cell TNF alpha IC 50 (nM) PWT143 0.6 0.6 CAL‐101 5.0 3.9 AKT phosphorylation in Raji B cells (anti-IgM stimulation) Log concentration (nM) pAKT (% control) 0 20 40 60 80 100 120 CAL-101 PWT143 -1 0 1 2 3 -2 Log concentration (nM) TNF alpha (% control) 0 20 40 60 80 100 120 CAL-101 PWT143 -1 0 1 3 -2 2 Compound Anti IgE‐mediated basophil activation IC 50 (nM) fMLP‐mediated basophil activation IC 50 (nM) PWT143 1.6 >3000 CAL‐101 77 >5000 CD63 expression in basophils (whole blood assay, anti-IgE stim.) CD63 expression in basophils (whole blood assay, fMLP stim.) log Concentration (nM) % +CD-63 Cells normalized (control) -3 -2 -1 0 1 2 3 4 5 0 20 40 60 80 100 120 CAL-101 PWT143 log Concentration (nM) % +CD-63 Cells normalized (control) -3 -2 -1 0 1 2 3 4 5 0 20 40 60 80 100 120 CAL-101 PWT143 Effect on Basophils 1 10 100 1000 10000 0 4 8 12 16 20 24 Mean plasma concentration (ng/mL) Time (hr) IV-10 mg/kg PO-30 mg/kg Superior half life and exposure profile in mice compared to clinical POC compound CAL-101 1 10 100 1000 10000 0 4 8 12 16 20 24 Mean plasma concentration (ng/mL) Time (hr) PWT143 in CD1 mice IV-10 mg/kg PO-30 mg/kg CAL-101 in CD1 mice 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 0 1 2 3 4 5 6 7 8 Clinical Arthri s Score (mean +/‐ SE) Dosing Day Vehicle PW‐143 100 mg/kg BID Naïve 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Clinical Arthri s Score (mean +/‐ SE) Arthri s Day Naïve Vehicle Enbrel 10 mg/kg QD Dex 0.2 mg/kg QD PW‐143 50 mg/kg QD PW‐143 100 mg/kg QD PW‐143 100 mg/kg BID 100% prevention of disease in ‘semi-therapeutic’ model Dramatic regression of established disease in therapeutic model Significant impact on histopathological markers of disease Well-tolerated at efficacious doses Semi-therapeutic model Established disease model Day 0 Immunize Day 21 Immunize Day 18 to 33, daily dosing Day 33 End of study Day 0 Immunize Day 21 Immunize Rolling enrollment after paw swelling evident, daily dosing for 11 days Day 33-40 End of study PWT143 PWT143 PWT143 PWT143 Score; 0=Normal, 1=Minimal, 2=Mild, 3=Moderate, 4=Marked, 5=Severe. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Normal Vehicle (Semi-ther) Dex Enbrel PWT143, 50 mg/kg QD PWT143, 100 mg/kg QD PWT143, 100 mg/kg BID Vehicle (ther) 100 mg/kg BID Individual Histopathology Score (Paws, Mean +/- SE) Inflammation Pannus Cartilage Damage Bone Resorption Therapeutic Semi‐therapeutic 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 0 1 2 3 4 5 6 7 8 9 10 11 Clinical Arthritis Score (Mean +/- SE) Arthritis Day Naïve Vehicle PWT143 50 mg/kg QD 10mg/kg dose is fully efficacious in ‘semi-therapeutic’ model Dramatic regression of established disease @ 50mg/kg Semi-therapeutic model Established disease model Day 0 Immunize Day 21 Immunize Day 18 to 33, daily dosing Day 33 End of study Day 0 Immunize Day 21 Immunize Rolling enrollment after significant paw swelling evident, daily dosing for 11 days Day 33‐40 End of study 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Clinical Arthritis Score (Mean +/- SE) Arthritis Day Naïve Vehicle PWT143 10 mg/kg QD PWT143 25 mg/kg QD PWT143 50 mg/kg QD Dex 0.2 mg/kg Conclusions Both Th1-type (IFN) and Th2-type (IL-4, IL-10) cytokine production inhibited We acknowledge and thank Chempartner (ShangPharma) for chemical synthesis, in vitro PI3 kinase assays and mouse pharmacokinetics; DiscoverX for kinome profiling; Caliper Life Sciences for T-cell assays; MD Biosciences for the PCA assay, and Bolder Biopath for CIA model studies. Cellular IC 50 (nM) Delta Alpha Beta Gamma PWT143 0.8 1620 18 428 CAL‐101 5.0 26180 379 1916 Pathway inhibition translates to phenotypic activity (inhibition of TNF alpha release in Raji cells) Pharmacodynamics In cells, PWT143 is highly potent towards PI3K delta and highly selective versus PI3K alpha/gamma, with ~30x selectivity versus PI3K beta Differential response to anti-IgE vs fMLP: potential to inhibit autoimmune function without inhibiting antibacterial activity Salbutamol (β2-adrenergic agonist) PWT143 100 mg/kg PWT143 200 mg/kg vehicle ug of blue dye/g of tissue T=0 hrs sensitize T=24 hrs challenge T=23.5 hrs administer vehicle and PWT143 T=24.5 hrs Termination and sampling T=23.75 hrs administer positive control A 200 mg/kg dose of PWT143 elicits a pharmacodynamic response equivalent to the positive control (salbutamol)  Pharmacodynamic response was assessed at 1 hour post single dose of PWT143 using a murine passive cutaneous anaphylaxis model – Primarily a measurement of mast cell response in vivo – Mice sensitized using anti- DNP IgE, challenged using DNP-BSA – Extravasation of Evans Blue dye measured spectrophotometrically Score; 0=Normal, 1=Minimal, 2=Mild, 3=Moderate, 4=Marked, 5=Severe. Route Dose (mg/kg) Cmax (ng/mL) Tmax (hr) AUCinf (ng·hr/mL) Cl (L/hr/kg) Vss (L/kg) T 1/2 (hr) MRT (hr) F (%) PWT143 IV 10 5860 1.71 6.36 4.29 3.73 PO 30 1977 1.0 13987 3.56 80 CAL-101 IV 10 3088 3.24 0.73 0.433 0.225 PO 30 2903 0.25 3483 0.829 37.6  PWT143 is a potent, selective inhibitor of PI3K delta  >300x vs alpha/gamma, 30x selective vs PI3K beta  Superior activity vs CAL-101 in multiple immune cell types  100% inhibition of disease onset and resolution of established disease in a mouse CIA model