Preclinical characterization of LOXO-435 (LOX-24350), a potent and highly isoform-

selective FGFR3 inhibitor

Presented at: AACR-NCI-EORTC VIRTUAL INTERNATIONAL CONFERENCE ON MOLECULAR TARGETS AND CANCER

THERAPEUTICS Date: October 7, 2021

Preclinical characterization of LOXO-435 (LOX-24350), a potent and highly isoform-selective FGFR3 inhibitor

Joshua A. Ballard1, Tim Kercher1, David Abraham1, Ryan Brecht1, Nathan Brooks3, Thomas Buckles1, Desta Bume1, Dave Busha1, Peder Cedervall1, Kevin Condroski1, Andrew Dilger1, Kevin Ebata1, Severine Isabelle Gharbi2, Robert Hazlitt1, Tony Morales1, Nisha Patel1, Jessica Podoll1, Kaveri Urkalan1, Sandra Gomez Villalain2, Shane Walls1, Faith Watson1, Peiyi Yang1, Barbara J. Brandhuber1, Steven W. Andrews1

1Loxo Oncology at Lilly, Boulder, CO, USA; 2Eli Lilly and Company, Alcobendas, Spain; 3Eli Lilly and Company, Indianapolis

Presenting author – Joshua Ballard

I have the following financial relationships to disclose:Employee of Loxo Oncology at Lilly and share holder of Eli Lilly and Company

I will not discuss off label use and/or investigational use in my presentation

Background

• Activating FGFR3 gene alterations are found in ~15-20% of advanced bladder cancers.

• All approved and investigational FGFR small molecule inhibitors are similarly potent against FGFR1-3. As a result,

their efficacy is potentially limited by toxicities driven by inhibition of both FGFR1 and FGFR2. Additionally, existing

drugs lose potency in the setting of FGFR3 gatekeeper mutations, which have been reported as mechanisms of

acquired resistance to existing pan-FGFR inhibitors.

• Here we describe the preclinical profile of LOXO-435, a potent and highly isoform-selective FGFR3 inhibitor with

preserved potency against FGFR3 gatekeeper resistance mutations.

FGFR1 and FGFR2 inhibition drive important, dose-limiting toxicities

• FGFR1-mediated hyperphosphatemia is a dose-limiting toxicity of pan-FGFR inhibitors1-4

• FGFR2-mediated cutaneous/nail, ocular, and perioral toxicities drive chronic intolerance of pan-FGFR inhibitors5

Erdafitinib1 Pemigatinib2 Infigratinib3 Bemarituzumab5

Inhibitor classification Pan-FGFR Pan-FGFR Pan-FGFR FGFR2b mAbIndication FGFR2/3-altered UC FGFR2-altered CCA FGFR2-altered CCA Investigational

Dose and schedule8+1 mg/day(dose changes based on phosphate levels)

13.5 mg/day14 days on, 7 days off

125 mg/day21 days on, 7 days off

15 mg/kg every 2 weeks (plus 7.5 mg/kg on day 8 of first cycle only)

ORR 32.2% 36% 30.8%4 –Hyperphosphatemia 76% 60% 46%4 –

Common adverse eventsHyperphosphatemia,

ocular disorders, embryofetal toxicity

Hyperphosphatemia, ocular disorders,

embryofetal toxicity

Hyperphosphatemia, ocular disorders,

embryofetal toxicity

Common adverse events: stomatitis, ocular disorders

Cell IC50 (nM)1.7 (FGFR1)1.0 (FGFR2)

3.3 (FGFR3 S249C)

0.9 (FGFR1)1.5 (FGFR2)

2.0 (FGFR3 S249C)

4.3 (FGFR1)4.9 (FGFR2)

8.4 (FGFR3 S249C)–

Selectivity (target) vs FGFR3 S249C

3.3x (FGFR2),1.0x (FGFR3) 1.4x (FGFR2) 1.7x (FGFR2) –

1Balversa. Prescribing information. Janssen Pharmaceutical Companies; 2020. 2Pemazyre. Prescribing information. Incyte corporation; 2021. 3Truseltiq. Prescribing information. QED Therapeutics; 2021. 4Lyou Y et al. 2020 JCO, 38:15_suppl, 5038. 5Catenacci DVT et al. 2021 JCO, 39:15_suppl (May 20, 2021) 4010.

LOXO-435 is a potent and highly isoform-selective FGFR3 inhibitor in enzyme assays, with gatekeeper activity

LOXO-435 108.2 19.7 0.3 1.1 361x 66x

Enzyme Inhibition Fold Selectivity

FGFR1IC50 (nM)

FGFR2IC50 (nM)

FGFR3 IC50 (nM)

FGFR3 V555M

IC50 (nM)

FGFR3 over FGFR1

FGFR3over FGFR2

Erdafitinib 0.3 0.6 0.2 1218.0 1.5x 2.0xPemigatinib 0.5 0.3 1.0 752.0 0.5x 0.3xInfigratinib 0.4 0.7 0.3 579.8 1.3x 0.6xFutibatinib 0.7 0.4 0.4 14.4 1.8x 1.8x

• LOXO-435 is potent against and highly selective for FGFR3 and FGFR3 V555M enzymes while sparing FGFR1 and FGFR2

Enzyme assays were performed using a PerkinElmer commercial HTRF fluorescence assay (KinEASE)

Proliferation assays were performed in 3D cell culture allowing compound to be exposed to the spheroids for 4 days. After exposure, cells were lysed, and total cellular ATP concentration was measured via homogeneous luminescent CellTiter-Glo (Promega) assay

LOXO-435 preserves its potency and selectivity in cellular proliferation assays

0.01 0.1 1 10 100 1000 10000 1000000

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LOXO-435 (nM)

Cel

lula

r ATP

Dec

reas

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Proliferation in Cancer Cell Lines Fold selectivity

FGFR1 (DMS114) IC50 (nM)

FGFR3 S249C (UMUC-14) IC50 (nM)

FGFR3-TACC3 (RT112) IC50 (nM)

UMUC-14 over DMS114

Erdafitinib 32.0 2.5 1.3 13xPemigatinib 22.1 12.9 5.2 2xInfigratinib 109.7 31.5 6.7 4xFutibatinib 8.1 3.7 8.8 2x

LOXO-435 4712.6 12.6 15.1 374x

FGFR3-TACC3 (RT112)

FGFR3 S249C (UMUC14)

FGFR1 (DMS114)

Cell based phosphorylation assays were conducted using HEK293 cell lines engineered to express FGFR1, FGFR2, FGFR3 (S249C) and FGFR3 (S249C, V555M). Assays were developed utilizing In-Cell Western (LICOR) by monitoring FGFR activation loop phosphorylation normalized to GAPDH antibody signal

Cellular Phospho Target Inhibition (HEK293) Fold Selectivity

pFGFR1 IC50 (nM)

pFGFR2 IC50 (nM)

pFGFR3 S249C

IC50 (nM)

pFGFR3 S249C, V555M

IC50 (nM)

pFGFR3 S249Cover

pFGFR1

pFGFR3 S249Cover

pFGFR2Erdafitinib 1.7 1.0 3.3 132.4 0.5x 0.3x

Pemigatinib 0.9 1.5 2.0 1451.7 0.5x 0.8xInfigratinib 4.3 4.9 8.4 244.9 0.5x 0.6xFutibatinib 1.0 1.0 2.7 63.4 0.4x 0.4x

Cellular signaling assays best exemplify LOXO-435’s potential to avoid FGFR1/2-mediated toxicities

LOXO-435 207.8 112.2 3.4 9.7 61x 33x

Biochemical kinase selectivity panel: Reaction Biology Corp, panel of 362 WT kinases tested at Km ATP, percent inhibition at 250 nM. Illustration reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com), FGFR3 Cellular IC50 derived from S249C

LOXO-435 has no clinically relevant off-target kinase activity

Enzyme IC50 (nM) Cell IC50 (nM)FGFR4 78 331

ROS/ROS1 0.5 NDCDK18 ND >10000

Aurora A 61 >10000MUSK 20 7670

FGFR3 0.3 3.4

• In a panel of 362 kinases, LOXO-435 inhibited 5 non-FGFR3 kinases by >70% at 250 nM test concentration

• Follow-up cellular assays ruled out potential off-target toxicity concerns

• LOXO-435 hERG ion channel IC50 >10 µM

Kinome selectivityHighly selective for FGFR3

Rodent efficacy and target inhibition studies were performed by subcutaneous implantation of mouse NIH3T3 cells engineered to express either FGFR3 (S249C) or FGFR3 (S249C, V555M) double mutant in immunocompromised mice. Treatment started when the average tumor size was between 200-400 mm3. **p≤0.001, ***p≤0.0001 compared to vehicle

LOXO-435 inhibits FGFR3 (S249C) and gatekeeper mutantsin vivo without FGFR1 engagement

Vehicle 1h 4h 8h 24h 1h 4h 8h 24h 4h0

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p/t F

GFR

3 S

igna

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LOXO-435

***

ErdaMTD30 mpk 90 mpk

******

****** *** ***

Vehicle 1h 4h 8h 1h 4h 8h 4h0

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p/t F

GFR

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gnal

(%)

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LOXO-435

ErdaMTD

** *****

**

30 mpk 90 mpk

In vivo pFGFR3 (S249C) inhibition In vivo pFGFR3 (S249C,V555M) inhibition

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Unb

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FG

FR1

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90 mpk30 mpk ErdaMTD

LOXO-435

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FGFR1 target engagement in lungs

Rodent efficacy and target inhibition studies were performed by subcutaneous implantation of human urothelial cancer line UMUC-14 in immunocompromised mice. Treatment started when the average tumor size was between 150-200 mm3. Start of treatment. Total serum phosphate changes determined by measuring total serum phosphate pre and post treatment, then compared to vehicle control. ***p≤0.0001 compared to vehicle. †Erdafitinib MTD in rats is 20 mpk. Note: LOXO-435, 90 mpk rat hyperphosphatemia study in progress

LOXO-435 displays a wide therapeutic index in vivo

• LOXO-435 causes tumor regressions without hyperphosphatemia

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LOXO-435 Erda

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LOXO-435 in UMUC-14 (FGFR3 S249C)

VehicleLOXO-435, 10 mpkLOXO-435, 30 mpkLOXO-435, 90 mpkErdafitinib, 30 mpk

↑

Conclusions

• LOXO-435 is a potent and highly isoform-selective FGFR3 inhibitor with potency against FGFR3

gatekeeper resistance mutations

• LOXO-435 spares FGFR1 and FGFR2, thus avoiding dose limiting hyperphosphatemia and other

adverse events that drive chronic intolerance to pan-FGFR inhibitors

• LOXO-435 causes significant tumor regression in FGFR3-mutant in vivo models

• An IND submission is planned for 2022

Presenter: Josh Ballard, jballard@loxooncology.com