LOXO-305, a next generation non-covalent BTK inhibitor, for overcoming acquired resistance to

covalent BTK inhibitors

Presented at: SOHO Annual Meeting 2018 Date: September 12, 2018

LOXO-305, a next generation non-covalent BTK inhibitor, for overcoming acquired resistance to covalent BTK inhibitorsBarbara Brandhuber,1 Eliana Gomez,1 Steven Smith,2 Todd Eary,1 Stacey Spencer,1 S. Michael Rothenberg,1 Steven Andrews1

1Loxo Oncology, Boulder, CO, United States 2Loxo Oncology, South San Francisco, CA, United States Abstract CLL-200

Copyright 2018

n Covalent Bruton’s tyrosinekinase (BTK) inhibitorsincluding ibrutinib andacalabrutinib havetransformed the treatmentlandscape of several BTK-dependentB-cell malignancies,including chroniclymphocytic leukemia,Waldenstrom’smacroglobulinemia, mantle cell lymphoma and marginal zone lymphoma.

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Long-term efficacy of these covalent agents is limited by acquired resistance, most commonly through BTK cysteine-481 (C481) substitution mutations that prevent covalent inhibition of BTK, and toxicity due to inhibition of other non-BTK targets.1-5 Therefore, potent and selective inhibition of BTK without and with BTK C481 mutations may provide clinical benefit to patients with B-cell malignancies who have progressed on covalent BTK inhibitors.LOXO-305 is a next generation, non-covalent BTK inhibitor with equivalent potency against wild-type and C481 mutated BTK.

n To evaluate LOXO-305 potency and selectivity, pharmacologicproperties and multispecies pharmacokinetics, and to assesswhether preclinical characterization supports the advancementof LOXO-305 into human clinical testing.

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Kinase profiling: radiolabeled ATP kinase activity assays wererun at the Km ATP concentration for each enzyme. LOXO-305at 1 µM was tested against 371 wild-type kinases. Biochemicalinhibition in duplicate (or greater replicate number) 10-pointdose response curves were used to determine the biochemicalIC50 values. Z’-Lyte kinase assays (ThermoFisher) were usedto determine EGFR and JAK1 IC50 values.HEK293 cell lines stably expressing BTK and BTK C481Swere generated using standard lentiviral transfection/selectionmethods. Ramos RA1 cell line was obtained from ATCC.TMD8 cell line was licensed from Tokyo Medical and DentalUniversity. In the chemoproteomics study, LOXO-305 was

incubated with human peripheral mononuclear cells (PBMCs) at multiple concentrations for 1h, pelleted, quickly washed to remove unbound LOXO-305, and incubated with biotinylated acyl phosphates of ATP and ADP, which covalently react with protein kinases on lysine residues in the ATP site. Binding of LOXO-305 to 180 kinases in PBMCs was determined by comparing the ATP and ADP probe labelled peptide signals from LOXO-305 treated PBMCs to untreated PBMCs.

n Human liver microsome stability was evaluated in a pool from150 donors. Human hepatocyte stability was evaluated in mixed gender cryopreserved hepatocytes from Bioreclamation IVT.

n In vitro permeability was evaluated at a final chamberconcentration of 10 µM and after a 2 h incubation in bothMDR1-MDCK and Caco-2 cell monolayers.

n All animals were housed as per institutional protocol andexperimental protocols were approved by the Animal Care andUse Committee.

Figure 1: LOXO-305 potently and selectively inhibits BTK and BTK C481S kinase activity

Figure 3: LOXO-305 inhibits BTK autophosphorylation and signaling activity in cells

Figure 4: Effects of LOXO-305 on TMD8 cells

Figure 5: Effect of LOXO-305 on tumor growth in human lymphoma xenograft models

Figure 8: LOXO-305 is predicted to achieve high BTK and BTK C481S target coverageIntroduction

Objectives

Materials and methods

Results

LOXO-305 and ibrutinib effects on BTK autophosphorylation of tyrosine 223 (Y223) and phosphorylation of PLCγ2 tyrosine 1217 (Y1217). HEK293 cells stably expressing BTK (A and B) and BTK C481S (C and D) were treated for 2 h with LOXO-305 or ibrutinib and analyzed by immunoblot (A and C). B and D: BTK Y223 phosphorylation was normalized to total BTK and IC50 values were calculated using a 4-parameter fit in GraphPad Prism 7.04 software. E and F: Ramos RA1 human Burkitt’s lymphoma cells were treated for 2 h with LOXO-305 or ibrutinib followed by 5 min stimulation with anti-IgM. E: Samples were analyzed by immunoblot for phosphorylation of BTK Y223 and PLCγ2 Y1217. F: LOXO-305 dose response curve of BTK Y223 phosphorylation in Ramos RA1 cells. BTK Y223 phosphorylation was normalized to total BTK and IC50 values were calculated using a 4-parameter fit in GraphPad Prism 7.04 software.

Single nucleotide changes (substitution mutations) at C418 render BTK resistant to BTK inhibitors ibrutinib and acalabrutinib

Effect of LOXO-305 on tumor growth in human B-cell lymphoma xenograft models. A–C: LOXO-305 dose-dependent inhibition of tumor growth in OCI-Ly10 DLBCL human xenograft tumor model. OCI-Ly10 cells were implanted subcutaneously into the flanks of male NOD SCID mice and allowed to grow to a volume of approximately 150–200 mm3. Mice were randomized by tumor size and orally dosed with the indicated inhibitors or vehicle. A: Tumor growth with tumor volumes displayed as mean ± SEM. B: Relative tumor volume (mean ± SEM) after dosing cessation. C: Normalized body weight values displayed as mean ± SEM. D–F LOXO-305 dose-dependent inhibition of tumor growth in TMD8 DLBCL human xenograft tumor model. TMD8 cells were implanted subcutaneously into the flanks of female Balb/c SCID mice and allowed to grow for 14 days. Mice were randomized by tumor size and orally dosed with the indicated inhibitors or vehicle. D: Tumor volume displayed as mean ± SEM. E: Tumor weights at the end of the study displayed as mean ± SEM for the 3 groups. F: Mice body weights displayed as mean ± SEM.

Figure 6: Pharmacokinetics of LOXO-305 in animal studies

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Predicted human exposure of LOXO-305 and BTK target coverage. Two planned dose levels of LOXO-305 were modeled using the GastroPlusTM software suite. These illustrate human dose predictions for dose levels planned in the phase 1 dose escalation trial and are not necessarily indicative of the first and second cohorts of the trial. The predicted BTK (wild-type and C481S) target coverage lines were derived from the LOXO-305 inhibition values of autophosphorylation in BTK and BTK C481S HEK293 cells and the plasma protein binding of LOXO-305.

A: LOXO-305 dose-response inhibition on human TMD8 (diffuse large B-cell lymphoma) cell proliferation. Cells were treated with LOXO-305 and proliferation inhibition was analyzed in real-time for 116 h. B: The area under the curve (AUC)(mean ± SD) was plotted vs the inhibitor concentration; IC50 values were calculated using a 4-parameter fit in GraphPad Prism 7.04 software.

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LOXO-305 is a potent and selective inhibitor of BTK and BTK C481S in purified enzyme kinase assays. 371 wild-type kinases were profiled using 1 µM LOXO-305 and an ATP concentration at each enzyme’s KM. Panel A: Kinases with IC50 values <500 nM assayed at KM [ATP] marked with circles. BTK, the most potently inhibited kinase, is denoted with a red circle. Panel B: Percent of control (POC) and IC50 values of kinases inhibited >50 POC by 1 µM LOXO-305 and selected additional kinases of interest.

Figure 2: LOXO-305 potently and selectively binds BTK inhuman peripheral mononuclear cells

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Assay system % remaining at t1/2 CLint CLint60 minutes (min) (µL/min/ (µL/min/

mg protein) 106 cells)Human liver microsomes 85.1% 251 5.51

100% stable stable+ NADPHHuman liver microsomes+ UDPGA Human hepatocytes 89.9% 479 2.89

Kinase Percent of control IC50 Fold@ 1 µM LOXO-305, [ATP] = KM selectivity

[ATP] = KM (%) (nM) over BTKBTK C481S ND 1.42 0.5 XBTK 1.8 3.15 1.0 XERBB4 2.6 13.3 4.2 XBRK 10.3 54.3 17 XMEK2 7.6 82.7 26 XMEK1 12.2 147 47 XYES1 38.6 157 50 XTXK 19.6 209 66 XBMX 70.2 1155 367 XTEC 64.6 1234 392 XBLK 72.8 4100 1302 XEGFR 60.6 >317 X ITK 103SRC 90.5JAK1 96.4JAK2 94.5JAK3 97

>1000>5000>5000>30000 ND ND

>1587 X>1587 X>9524 X ND ND

LOXO-305 is a potent and selective inhibitor of BTK in human peripheral mononuclear cells. Only 2 out of 180 kinases profiled in human peripheral mononuclear cells competitively inhibited the binding of the chemoproteomic nucleotide analogs with IC50 values <5 µM. Panel A: Kinases with IC50 values <5 µM are denoted. Panel B: Kinases denoted in panel A shown with identifying chemoproteomics probe peptide and IC50 values.

A KinaseB Chemoproteomics IC50 Averageidentified peptide (nM) IC50 (nM)

BTK GQYDVAIKMIK 8.6 10.3BTK YVLDDEYTSSVGSKFPVR 12TEC YVLDDQYTSSSGAKFPVK 710 710

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Dosage and Feeding Cmax AUC0-tdose route state (ng/mL) (ng*h/mL)30 mg/kg PO Fasted 18100 16200030 mg/kg PO Fed 10000 105000

Dosage and Feeding Cmax AUC0-tdose route state (ng/mL) (ng*h/mL)35 mg/kg PO Non-fasted 7930 82200

In vitro clearance

Assay system Run Mean Papp A–B Mean Papp B–A Effluxnumber (10-6 cm/s) (10-6 cm/s) ratio

1 17.6 46.3 2.6Caco-2 cells 2 9.18 61.6 6.7

3 7.27 57.9 8.01 9.73 44.4 4.6

MDR1-MDCK 2 7.28 60.2 8.33 9.05 53.1 5.9

In vitro permeability

LOXO-305 is a next generation, non-covalent BTK inhibitorthat can overcome acquired resistance to covalent BTK inhibitors in preclinical models, with minimal off-target kinase and non-kinase inhibitory activity.

• Is a potent, single-digit nanomolar inhibitor of BTK andBTK C481S activity in kinase activity assays

• Is over 300-fold more enzymatically selective for BTKversus 98% of 370 non-BTK wild-type kinases tested

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Potently inhibits BTK-dependent cellular readouts withsingle-digit nanomolar potencySignificantly inhibits tumor growth in two B-cell lymphomatumor xenograft modelsDemonstrates high plasma exposure in rat and dog afteroral dosing and is predicted to achieve high levels of BTKand BTK C481S target coverageDemonstrates low clearance and high permeability inhuman in vitro assaysIs predicted to achieve high BTK and BTK C481S targetcoverage in humans at reasonable doses

n Based on these promising results, the first-in-human clinical trial of LOXO-305 in patients who have progressed on prior covalent BTK inhibitors is planned.

1. Woyach et al. N Engl J Med. 2014; 370:2286–2294.2. Byrd et al. N Engl J Med. 2016; 374:323–332.3. Woyach et al. J Clin Oncol. 2017; 35:1437–1443.4. Xu et al. Blood. 2017; 129:2519–2525.5. Hershkovitz-Rokah et al. Br J Haematol. 2018; 181:306–319.

Conclusions

References

Pharmacokinetics of LOXO-305 in nonclinical animal studies. A: Eight beagle dogs (4 male, 4 female) were administered a single PO dose of 30 mg/kg LOXO-305 on day 1 and day 8 in a fed/fasted cross-over study. Blood samples were collected through 48 h after dosing. LOXO-305 plasma concentrations were determined using LC-MS/MS methodology. B: Four non-fasted Sprague Dawley rats were administered a single PO dose of 35 mg/kg LOXO-305. Blood samples were collected through 24 h after dosing. LOXO-305 plasma concentrations were determined using LC-MS/MS methodology. The predicted BTK (wild-type and C481S) target coverage lines were derived from the LOXO-305 inhibition values of autophosphorylation in BTK and BTK C481S HEK293 cells and the plasma protein binding of LOXO-305. Non-compartmental pharmacokinetic parameters of LOXO-305 in both species were calculated by conventional methods using individual plasma concentration profiles over time and Microsoft Excel.

Figure 7: LOXO-305 is predicted to have low human clearance and high permeability

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n LOXO-305: