preclinical characterization of loxo-435 (lox-24350), a
TRANSCRIPT
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
20
40
60
80
100
120
LOXO-435 (nM)
Cel
lula
r ATP
Dec
reas
e (%
)
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
40
80
120
160
p/t F
GFR
3 S
igna
l (%
)
LOXO-435
***
ErdaMTD30 mpk 90 mpk
******
****** *** ***
Vehicle 1h 4h 8h 1h 4h 8h 4h0
50
100
150
200
p/t F
GFR
3 Si
gnal
(%)
**
LOXO-435
ErdaMTD
** *****
**
30 mpk 90 mpk
In vivo pFGFR3 (S249C) inhibition In vivo pFGFR3 (S249C,V555M) inhibition
Vehicle 1h 4h 8h 24h 1h 4h 8h 24h 4h0
50
100
150
200
Unb
ound
FG
FR1
(%)
90 mpk30 mpk ErdaMTD
LOXO-435
***
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
Vehicle 30 mpk 20 mpk80
100
120
140
160
Cha
nge
in S
erum
Pho
spha
te (%
)
***
LOXO-435 Erda
Serum phosphate in rat
†5 10 15 20 25
0
400
800
1200
Treatment Days
Tum
or V
olum
e (m
m3 )
↑
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, [email protected]