FGFR-Selective Tyrosine Kinase Inhibitors, Such As Infigratinib, Show Potency and Selectivity for FGFR3 at Pharmacologically Re

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FGFR-selective tyrosine kinase inhibitors, such as infigratinib, show potency and selectivity for FGFR3 at pharmacologically relevant doses for the potential treatment of achondroplasia Katherine Dobscha, MBA1, Ge Wei, PhD1, Carl L Dambkowski, MD1, Daniela Rogoff, MD, PhD1 1QED Therapeutics, Inc., San Francisco, CA, USA #5126

Figure 1. Literature review flow chart ■ Efficacy and toxicity findings were concentration- or dose-dependent Figure 3. Dose response with infigratinib in an achondroplasia Figure 5. Ex-vivo improvement in tibia growth and Col10a3 Background (illustrative subset shown in Figure 2). mouse model 9 Growth improvement of ex vivo Restoration of normal growth ■ Germline mutations in fibroblast growth factor receptor genes 1–3 (FGFR1–3) tibia cultures with ARQ 087 architecture with ARQ 0870 Papers, manuscripts, abstracts other full texts ■ FGFR-selectivity of TKIs included in this review varied, with FGFR3 IC50 can cause skeletal dysplasias and craniosynostoses. Control FGF2 [50 ng/ml] FGF2 + ARQ087 ARQ087 [1 µM] Identiﬁcation identiﬁed through database searching ranging from 1.0 nM (infigratinib2) to 4.5 nM (ARQ 0873) for FGFR-selective HE

■ Mutations in FGFR2 (e.g. S351C, Y375C, S252W) and FGFR3 (e.g. G380R, (n=683) e Axial skeleton Appendicular skeleton 4 5 i c

compounds, and from 5 nM (PD173074 ) to 190 nM (A31 ) to 500 nM m 20% + K650E, N540K, Y373C) are known to cause skeletal dysplasias including / *** ***

a 6 67 C 3 (NF449 , ) for non-selective compounds. Y 3 *** craniosynostoses, short-stature syndromes such as achondroplasia and r 16% g f F *** HE hypochondroplasia, and thanatophoric dysplasia. d *** e t

■ Infigratinib was the most-commonly identified TKI, with eight publications on a 12% *** e r

t *** ■ Over the past decade, several FGFR1–3 tyrosine kinase inhibitors (TKIs), such - ** e Records eligible after screening Records screened out for date l preclinical data in models of skeletal dysplasias. Key results for infigratinib show: i c *** as infigratinib (BGJ398), AZD4547, and PD173074 have been studied in a h Screening for date and duplicates and duplicates e 8%

v **

s ** * *** Cj 2 v (n=357) variety of preclinical models of FGFR-driven skeletal dysplasias. (n=326) *** *** E/Co/10 – FGFR3 IC 1.0 nM, FGFR3-K650E IC 4.9 nM. e *** * *** 50 50 s ** * * a 4% *** e r

■ Achondroplasia is the most common form of disproportionate short stature c i n

– In-vitro data: inhibition of FGFR1–3 activity at concentrations ranging from t driven by an FGFR genetic alteration. It is most commonly caused by an n 0% rc e Foramen L4-L6 height Skull length Tail length Femur Tibia Humerus Ulna Radius 1 e autosomal dominant G380R substitution in FGFR3. 5 to 500 nM, including reversal of established growth arrest in chondrocytes P magnum Cj Infigratinib at 7 nM and an ‘optimal concentration’ of 5–10 nM (Figure 2).7 length ■ Achondroplasia is the most frequently studied FGFR-driven skeletal dysplaisa Records assessed for full text after Records screened out based 0.2 mg/kg/day 1 mg/kg/3 days 0.5 mg/kg/day *p<0.05, **p<0.01, ***p<0.001 although, to date, no study has comprehensively examined the literature Eligibility excluding irrelevant titles and abstracts on title and abstract – Ex-vivo data: overgrowth of ACH mouse femurs vs. WT at 1x103 nM.8 regarding the potential therapeutic usage of FGFR1–3 TKIs in achondroplasia (n=16) (n=310) or other FGFR-driven skeletal dysplasias. – In-vivo studies: dose-dependent improvements in foramen magnum and long Additional publications bone length in Fgfr3Y367C/+ mice at SC doses of 0.2–2 mg/kg/day (Figure 3).8,9 included for review – In-vitro data show rescue of RCS cells from FGF2-mediated inhibition of Conclusions (n=2) – No preclinical studies reported a survival disadvantage and one showed proliferation at concentrations ranging from 10 nM for AZD4547 to 15 nM for Purpose: ■ a significant survival advantage for infigratinib-treated ACH mice (Figure 4).9 PD173074.7 While two studies suggest toxicity with FGFR-selective TKIs; this was ■ Explore the publicly available literature to evaluate the dose Records screened out after full text review produced at doses significantly higher than pharmacologically relevant dependency and toxicity profiles of FGFR-selective TKIs in (n=4) ■ In relation to other FGFR TKIs besides infigratinib: – One study of PD173074 (100 nM) showed significant rescue of primary cilia for the treatment of achondroplasia or other skeletal dysplasias. (PC) length in chondrocytes from FGFR3Y367C/+ mice to 96% of the length preclinical skeletal dysplasia models. – Off-target biochemical effects on other RTKs were reported for most of Records included for review ■ In-vivo studies in an achondroplasia mouse model treated with low Included observed in control chondrocytes, and in human fetal TD chondrocytes to ■ Evaluate, based on the comprehensive non-clinical evidence of (n=14) these agents across studies, with the exception of ARQ 087, which was 94% of that observed in control chondrocytes.10 doses of infigratinib showed increase in growth of long bones and foramen safety and efficacy of FGFR-selective TKIs, the potential for a reported to only slightly inhibit KIT, FLT4, and TYRO3 at 500 nM magnum with a good dose-response relationship. No toxic effects were 3 therapeutic option in FGFR-driven skeletal dysplasias. (FGFR1–3 IC50 1.8–4.5 nM). – Ex-vivo data in one study demonstrated restoration of normal growth plate observed at these low but efficacious doses. architecture and ~50% growth improvement compared with controls in ■ Y367C/+ mouse tibia cultures treated with 1x103 nM ARQ 087, both in the presence One study demonstrated a survival advantage in Fgfr3 mice treated Figure 2. Representative data: concentrations of FGFR inhibitors explored preclinically in models of skeletal dysplasia and absence of FGF2 (Figure 5).3 with infigratinib. Methods – In-vivo data: one study showed that AZD4547 decreased survival in ■ A systematic literature review was performed to investigate non-clinical data newborn wild-type mice (CD1) treated at doses of 1x106 to 2x106 nM.7 from studies of infigratinib and other FGFR-selective TKIs relevant to FGFR- driven skeletal dysplasias. Another study showed limb malformation in chicken embryos treated with Clinical relevance: PD173074 or PD161570 at doses of 1x106 to 50x106 nM, and increased ■ 7 100 1000 3x10E4 2x10E6 Given the totality of evidence, low-dose infigratinib appears to be a Two major types of sources were searched on October 22/23 2019: 5 6 11 500 50x10E6 embryo mortality above 1x10 nM. potentially safe option for further development in children with – Major databases (e.g., PubMed, Medline [NLM Catalog]) were searched for relevant articles from the past 10 years. Log(nM) achondroplasia. – Conference archives (e.g., ENDO, ESPE, ISDS, ASHG, ASBMR) were 1 10 100 1,000 10,000 100,000 1,000,000 10,000,000 100,000,000 searched for relevant abstracts from the past 5 years. Figure 4. Survival benefit with infigratinib in an achondroplasia mouse model 9 ■ Full text was included where possible. nM 5 7 100 500 1000 6000 – 3x104 1x106 – 2x106 1x106 – 50x106 (drug) (infigratinib) (infigratinib) (infigratinib) (infigratinib) (infigratinib) (NF449a) (AZD4547) (PD173074, PD161570) ■ Key words used in the searches included, but were not limited to, the following: Acknowledgements 100 – Achondroplasia. 100 Vehicle Vehicle The authors would like to acknowledge the following: – Skeletal dysplasia. Rescue of FGF2- Rescue of FGF2 growth- Complete Complete 0.5 mg/kg/day mediated inhibition inhibited phenotype; Bone overgrowth 0.5 mg/kg/day – FGFR inhibition. reversal of fully- rescue of bone Significant rescue of None; significant None; shortening of right 8800 0.2 mg/kg/da■y Lee Miller (Miller Medical Communications) for provision of medical writing/ y of chondrocyte restoration of normal vs. wt of 0.2 mg/kg/day c

a established growth defect growth arrest shortening of long bones wing or mandible in editing support for this poster.

Y367C/+ ) c – Infigratinib. proliferation; Y367C/+ growth plate architecture Fgfr3 ) 1 mg/kg/13 m dag/yskg/3 days

ﬃ growth arrest of of Fgfr3 phenotype in RCS and skull bones in chicken embryos (HH25- % %

E optimal active including Col10a1 embryonic ( ( 60

– BGJ398. RCS embryonic chondrocytes newborn wt mice 29) ■ concentration noted expression in embryonic femurs ex vivo This work was funded by QED Therapeutics. chondrocytes femurs ex vivo

– AZD4547. at 5-10 nM tibia cultures va l i va l i v 40 – PD173074. v 40 u r S u r Significance p<0.001 – Tyrosine kinase inhibitor. S Significance p<0.001

y 20

t No apparent cellular

i References

c Limb, mandible ■ Eligibility criteria for inclusion was determined in advance to exclude content i None noted None noted None noted None noted None noted toxicity throughout Lethality 20 x malformation o

not relevant to the purpose of this literature review. T range 1. Horton WA, et al. Lancet 2007;370:162–72. 0 0 0 10 15 20 25 2. Guagnano V, et al. J Med Chem 2011;54:7066–83. y

d b u In vitro In vitro Ex vivo Ex vivo Ex vivo In vitro In vivo Ex vivo 0 10 15 20 25 t Days 3. Balek L, et al. Bone 2017;105:57–66. Results S NumberD oaf myisce Number of mice 4. Krejci P, et al. J Biol Chem 2010;285:20644–53.

■ 14 publications were included in this review (Figure 1). . f Gudernova et al. Gudernova et al. Komla-Ebri et Gudernova et al. Komla-Ebri et al. Krejci et al. 7 Horakova et al.

e at P1 at P16 7 7 8 7 8 4 Gudernova et al. 2016 11 R 2016 2016 al. 2016 2016 2016 2010 2014 5. Jonquoy A, et al. Hum Mol Genet 2012;21:841–51. – 683 publications were identified through the initial search, with 326 remaining 0.5 mg/kg/day Num1b2er of mice N1u0mber of mice after screening for date and duplicates. 0.2 mg/kg/day 12at P1 10 at P16 6. El Mdawar M-B, et al. Sci Rep 2019;9:5159. – 310 publications were excluded based on title and abstract, leaving 16 0.5 mg/kg/day 12 10 1 mg/kg/3 days 14 13 7. Gudernova I, et al. Hum Mol Genet 2016;25:9–23. remaining to assess full text. Representa ve data reﬂected. 0.V2e mhicg/lekg/day 23 12 11 10 aDespite being referenced as an FGFR inhibitor in multiple papers, NF449 has more recently been characterized as a selective P2X1 receptor (P2RX1) antagonist.6 Concentrations with reported eﬃcacy 8. Komla-Ebri D, et al. J Clin Invest 2016;126;1871–84. – Of the 16 publications reviewed as full texts, four were excluded given focus bIn this study, AZD4547 was injected intraperitoneally. Concentrations with reported toxicity 1 mg/kg/3 days 14 13 outside the scope of this literature review (e.g., focus on targets other than FGFR, Vehicle 23 11 9. Demuynck et al. ASHG 2019. discussion of therapeutic space without provision of new non-clinical data). 10. Martin L, et al. Hum Mol Genet 2018;27:1–13. – Two additional publications found through reference review of identified publications were included due to direct relevance. 11. Horakova et al. Dev Growth Differ 2014.

The data shown in this poster were accepted but not presented at ENDO 2020 as the conference was cancelled amid concerns about COVID-19