Covalent FGFR Inhibitors - Principia Biopharma · Poster Print Size: This poster template is 30”...
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• Tailored Covalency TM technology delivered potent and selective FGFR1-4 inhibitors • Irreversible covalent binding conveyed high potency against cell lines harboring FGFR amplifications, mutations, and translocations • Tumor regression in xenograft models was observed with daily or intermittent oral dosing • Selected patient-derived tumor explant models showed durable tumor regression • FGFR mediated hyperphosphatemia potentially may be managed with a dosing schedule that maintains strong anti-tumor activity • Molecules from this series are advancing into clinical development for the treatment of cancers Conclusions RT4 tumor sections stained with hematoxylin and eosin or nuclear Ki-67 showed tumor necrosis and reduced tumor cell proliferation (Figure 9). An increase in plasma phosphate is an on-target toxicity effect associated with FGFR inhibition. In mice, 2-day dose holiday allows plasma phosphate levels to return to baseline while tumor growth inhibition is uncompromised (Figure 10). Covalent inhibitors show superior inhibition of cell proliferation when compared to reversible inhibitors (Table 2). Cellular activity is retained against common activating mutations. Screening of 100+ cell lines reveals sensitivity for FGFR aberrations (Figure 5). In a SNU16 gastric cancer xenograft model, oral dosing inhibits FGFR autophosphorylation and shows dose dependent tumor growth inhibition (Figure 6). In RT4 (FGFR3:TACC3 fusion) bladder cancer (Figure 7a) and SNU16 (FGFR2-amplified) gastric cancer (Figure 7b) models, PRN1371 induces rapid tumor regression following daily oral dosing. In the SNU16 tumor model, 5 days of dosing or an intermittent dosing schedule resulted in prolonged tumor regression (Figure 7c-d). Potent tumor regression was also observed in several PDX models (Figure 8). Representative molecules from the lead series, PRN1109 and PRN1371, are irreversible FGFR1-4 inhibitors designed to bind a conserved cysteine residue in the glycine rich loop (Figure 3). These molecules are potent FGFR1-4 inhibitors that do not appreciably inhibit other kinases sharing the same conserved cysteine residue (Table 1) or other kinases including VEGFR2 (100-1000x). Results Irreversible Covalent Pan-FGFR Inhibitors are Highly Efficacious Against FGFR-dependent Cancers Vernon T. Phan, Erik Verner, Mary Gerritsen, J. Michael Bradshaw, David M. Goldstein, Ronald J. Hill, Dane Karr, Jacob LaStant, Philip Nunn, Danny Tam, Jin Shu, Jens Oliver Funk, Ken A. Brameld Principia Biopharma, South San Francisco, CA Principia Biopharma (www.principiabio.com) Irreversible covalent pan-FGFR inhibitors that target a conserved cysteine residue within the kinase domain of FGFR1-4 were discovered to be potent and highly selective. Covalency imparted long lasting PD inhibition and tumor regression in multiple xenograft models. Preclinical dosing schedules demonstrated complete tumor regression while significantly sparing FGFR on-target plasma phosphate changes. Abstract Introduction Deregulation of the FGF/FGFR signaling pathway (Figure 2) has been associated with tumor progression in a variety of solid and hematological tumors. Recently, Phase 1 studies have demonstrated an encouraging utility of FGFR inhibitors for the treatment of FGFR-driven cancers. Challenges remain to identify FGFR inhibitors that do not have off-target inhibition, e.g. of VEGFR2, and that allow for strategies that translate into significant reduction of FGFR activity to improve clinical responses in patients while minimizing on-target toxicity. Assay PRN1109 PRN1371 BGJ398 AZD4547 Transfected Ba/F3 IC50 (nM) FGFR1 WT 8 < 1 6 27 FGFR2 WT 1 < 1 7 11 FGFR2 (K660E) 2 < 1 14 10 FGFR2 (K660N) 2 < 1 7 11 FGFR2 (N550K) 23 < 5 87 120 FGFR3 WT 13 < 5 7 37 FGFR3 (K650M) 18 < 5 45 130 FGFR4 WT 120 50 260 290 Cell proliferation IC50 (nM) SNU16 (FGFR2 amp) 2.5 < 5 5 7 RT4 (FGFR3:TACC3) 55 < 5 180 230 RT112 (FGFR3:TACC3) 21 < 5 19 36 Hep3B (FGFR4; FGF19) - 6 96 116 Activated FGFR Kinase FGF ligand overproduction FGFR gene over- expression FGFR mutation Gene translocations and fusions Covalent pan-FGFR Inhibitor Figure 2. Irreversible inhibitors targeting the kinase function block all aberrant FGFR signaling while sparing off-targets. Table 2. Cellular potency against wild-type (WT) and mutant FGFR bearing cell lines. Figure 6. A SNU16 xenograft model was used to establish tumor PD by monitoring the blockade of pFGFR in tumor tissue post dosing and tumor growth inhibition. Days on treatment Tumor volume (mm 3 ) RT4 xenograft model Figure 8. PRN1371 inhibits patient derived tumor models. Figure 3. Modeled bound conformation of PRN1109 (green) in the ATP binding pocket of FGFR1. Covalent binding to Cys 488 is highlighted in yellow. Cys488 Kinase PRN1109 PRN1371 FGFR1 1 < 1 FGFR2 2 < 1 FGFR3 6 < 5 FGFR4 40 < 20 FGR 3% 280 YES 15% 380 SRC 21% 440 TNK1 10% 580 LIMK1 not tested 540 PINK1 not tested not tested Table 1. Biochemical enzymatic inhibition for kinases that have a cysteine residue homologous to Cys488 of FGFR1. Values reported are IC50 (nM) or % inhibition at 1 uM. 0 400 800 1200 1600 2000 2400 0 3 6 9 12 15 18 21 24 27 Tumor size(mm 3 ) Days post treatment 0 200 400 600 800 1000 1200 0 3 6 9 12 15 18 21 24 Tumor size(mm 3 ) Days post treatment Glioblastoma NSCLC Vehicle control PRN1371 15mg/kg BID Figure 1. Chemical structure of PRN1109. Cysteine targeting irreversible binding element highlighted in red. Figure 7. Oral daily dosing of PRN1371 inhibits tumor growth in FGFR-driven xenograft models (A-B). Durable tumor regression is achieved after 5 days of dosing near MTD or with an intermittent schedule (C-D). Figure 9. Representative RT4 tumor sections stained with hematoxylin and eosin shows PRN1371 increased tumor necrosis, while Ki-67 staining confirms significant inhibition of tumor cell proliferation. Figure 10. Continuous dosing of PRN1371 increases plasma phosphate. A 2-day dose holiday is sufficient for phosphate levels to return to baseline while tumor regression is maintained. A. B. C. D. 0 200 400 600 800 0 3 6 9 12 15 18 21 24 27 Tumor volume (mm 3 ) Days post treatment Vehicle control PRN1371 20mg/kg BID 6 2 3 3 Dosing days 0 200 400 600 800 1000 0 3 6 9 12 15 18 21 24 27 30 Vehicle control PRN1371 40mg/kg BID 5 Days post treatment Tumor volume (mm 3 ) Dosing days 0 200 400 600 800 1000 0 3 6 9 12 15 18 21 24 27 30 Tumor volume (mm 3 ) Days post treatment Vehicle control PRN1371 5.0 mg/kg BID PRN1371 10 mg/kg BID PRN1371 15 mg/kg BID 0 200 400 600 800 0 3 6 9 12 15 18 21 24 27 Vehicle PRN1371 2.5mg/kg BID PRN1371 12.5mg/kg BID Tumor Volume (mm 3 ) Days post treatment Figure 5. Cell proliferation screening of PRN1109 . Most cell lines are insensitive to 10uM inhibitor (not shown). Sensitive cell lines are consistent with an FGFR alteration. PRN1109 Cell Line Screening #483 EORTC-NCI-AACR 2014 – Barcelona November 18-21, 2014 Plasma Phosphate (mg/DL) Vehicle 12.5 mpk/BID continuous 50mpk/QD 5d on/2d off Phosphate on day 7 RT4 SNU16 SNU16 SNU16 Figure 4. Kinase selectivity profile of lead molecules screened against 250 kinases. Dots represent individual kinase with > 90% inhibition at 1 uM.
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• Tailored CovalencyTM technology delivered potent and selective FGFR1-4 inhibitors
• Irreversible covalent binding conveyed high potency against cell lines harboring FGFR amplifications, mutations, and translocations
• Tumor regression in xenograft models was observed with daily or intermittent oral dosing
• Selected patient-derived tumor explant models showed durable tumor regression
• FGFR mediated hyperphosphatemia potentially may be managed with a dosing schedule that maintains strong anti-tumor activity
• Molecules from this series are advancing into clinical development for the treatment of cancers
Conclusions
RT4 tumor sections stained with hematoxylin and eosin or nuclear Ki-67 showed tumor necrosis and reduced tumor cell proliferation (Figure 9). An increase in plasma phosphate is an on-target toxicity effect associated with FGFR inhibition. In mice, 2-day dose holiday allows plasma phosphate levels to return to baseline while tumor growth inhibition is uncompromised (Figure 10).
Covalent inhibitors show superior inhibition of cell proliferation when compared to reversible inhibitors (Table 2). Cellular activity is retained against common activating mutations. Screening of 100+ cell lines reveals sensitivity for FGFR aberrations (Figure 5). In a SNU16 gastric cancer xenograft model, oral dosing inhibits FGFR autophosphorylation and shows dose dependent tumor growth inhibition (Figure 6).
In RT4 (FGFR3:TACC3 fusion) bladder cancer (Figure 7a) and SNU16 (FGFR2-amplified) gastric cancer (Figure 7b) models, PRN1371 induces rapid tumor regression following daily oral dosing. In the SNU16 tumor model, 5 days of dosing or an intermittent dosing schedule resulted in prolonged tumor regression (Figure 7c-d). Potent tumor regression was also observed in several PDX models (Figure 8).
Representative molecules from the lead series, PRN1109 and PRN1371, are irreversible FGFR1-4 inhibitors designed to bind a conserved cysteine residue in the glycine rich loop (Figure 3). These molecules are potent FGFR1-4 inhibitors that do not appreciably inhibit other kinases sharing the same conserved cysteine residue (Table 1) or other kinases including VEGFR2 (100-1000x).
Results
Irreversible Covalent Pan-FGFR Inhibitors are Highly Efficacious Against FGFR-dependent Cancers Vernon T. Phan, Erik Verner, Mary Gerritsen, J. Michael Bradshaw, David M. Goldstein, Ronald J. Hill, Dane Karr,
Jacob LaStant, Philip Nunn, Danny Tam, Jin Shu, Jens Oliver Funk, Ken A. Brameld Principia Biopharma, South San Francisco, CA
Principia Biopharma (www.principiabio.com)
Irreversible covalent pan-FGFR inhibitors that target a conserved cysteine residue within the kinase domain of FGFR1-4 were discovered to be potent and highly selective. Covalency imparted long lasting PD inhibition and tumor regression in multiple xenograft models. Preclinical dosing schedules demonstrated complete tumor regression while significantly sparing FGFR on-target plasma phosphate changes.
Abstract
Introduction Deregulation of the FGF/FGFR signaling pathway (Figure 2) has been associated with tumor progression in a variety of solid and hematological tumors. Recently, Phase 1 studies have demonstrated an encouraging utility of FGFR inhibitors for the treatment of FGFR-driven cancers. Challenges remain to identify FGFR inhibitors that do not have off-target inhibition, e.g. of VEGFR2, and that allow for strategies that translate into significant reduction of FGFR activity to improve clinical responses in patients while minimizing on-target toxicity.
Assay PRN1109 PRN1371 BGJ398 AZD4547
Transfected Ba/F3 IC50 (nM)
FGFR1 WT 8 < 1 6 27
FGFR2 WT 1 < 1 7 11
FGFR2 (K660E) 2 < 1 14 10
FGFR2 (K660N) 2 < 1 7 11
FGFR2 (N550K) 23 < 5 87 120
FGFR3 WT 13 < 5 7 37
FGFR3 (K650M) 18 < 5 45 130
FGFR4 WT 120 50 260 290
Cell proliferation IC50 (nM)
SNU16 (FGFR2 amp) 2.5 < 5 5 7
RT4 (FGFR3:TACC3) 55 < 5 180 230
RT112 (FGFR3:TACC3) 21 < 5 19 36
Hep3B (FGFR4; FGF19) - 6 96 116
Activated FGFR Kinase
FGF ligand overproduction
FGFR gene over-
expression FGFR mutation
Gene translocations and fusions
Covalent
pan-FGFR
Inhibitor
Figure 2. Irreversible inhibitors targeting the kinase function block all aberrant FGFR signaling while sparing off-targets.
Table 2. Cellular potency against wild-type (WT) and mutant FGFR bearing cell lines.
Figure 6. A SNU16 xenograft model was used to establish tumor PD by monitoring the blockade of pFGFR in tumor tissue post dosing and tumor growth inhibition.
Days on treatment
Tu
mo
r vo
lum
e (
mm
3)
RT4 xenograft model
Figure 8. PRN1371 inhibits patient derived tumor models.
Figure 3. Modeled bound conformation of PRN1109 (green) in the ATP binding pocket of FGFR1. Covalent binding to Cys 488 is highlighted in yellow.
Cys488
Kinase PRN1109 PRN1371
FGFR1 1 < 1
FGFR2 2 < 1
FGFR3 6 < 5
FGFR4 40 < 20
FGR 3% 280
YES 15% 380
SRC 21% 440
TNK1 10% 580
LIMK1 not tested 540
PINK1 not tested not tested
Table 1. Biochemical enzymatic inhibition for kinases that have a cysteine residue homologous to Cys488 of FGFR1. Values reported are IC50 (nM) or % inhibition at 1 uM.
0
400
800
1200
1600
2000
2400
0 3 6 9 12 15 18 21 24 27
Tu
mo
r s
ize
(mm
3)
Days post treatment
Group1 Vehicle 0.5% Methyl cellulose p.o. 10μl/g BIDx21
Group2 PRN-1371 15mg/kg p.o. 10μl/g BIDx21
0
200
400
600
800
1000
1200
0 3 6 9 12 15 18 21 24
Tu
mo
r s
ize
(mm
3)
Days post treatment
Glioblastoma NSCLC
Vehicle control
PRN1371 15mg/kg BID
Figure 1. Chemical structure of PRN1109. Cysteine targeting irreversible binding element highlighted in red.
Figure 7. Oral daily dosing of PRN1371 inhibits tumor growth in FGFR-driven xenograft models (A-B). Durable tumor regression is achieved after 5 days of dosing near MTD or with an intermittent schedule (C-D).
Figure 9. Representative RT4 tumor sections stained with hematoxylin and eosin shows PRN1371 increased tumor necrosis, while Ki-67 staining confirms significant inhibition of tumor cell proliferation.
Figure 10. Continuous dosing of PRN1371 increases plasma phosphate. A 2-day dose holiday is sufficient for phosphate levels to return to baseline while tumor regression is maintained.
A. B.
C. D.
0
200
400
600
800
0 3 6 9 12 15 18 21 24 27
Tu
mo
r vo
lum
e (
mm
3)
Days post treatment
Vehicle control
PRN1371 20mg/kg BID
6 2 3 3
Dosing days
0
200
400
600
800
1000
0 3 6 9 12 15 18 21 24 27 30
Vehicle control
PRN1371 40mg/kg BID
5
Days post treatment
Tu
mo
r vo
lum
e (
mm
3) Dosing days
0
200
400
600
800
1000
0 3 6 9 12 15 18 21 24 27 30
Tu
mo
r vo
lum
e (
mm
3)
Days post treatment
Vehicle control
PRN1371 5.0 mg/kg BID
PRN1371 10 mg/kg BID
PRN1371 15 mg/kg BID
0
200
400
600
800
0 3 6 9 12 15 18 21 24 27
Vehicle
PRN1371 2.5mg/kg BID
PRN1371 12.5mg/kg BID
Tu
mo
r V
olu
me
(m
m3)
Days post treatment
Figure 5. Cell proliferation screening of PRN1109 . Most cell lines are insensitive to 10uM inhibitor (not shown). Sensitive cell lines are consistent with an FGFR alteration.
PRN1109 Cell Line Screening
#483
EORTC-NCI-AACR 2014 – Barcelona November 18-21, 2014
Pla
sma
Ph
osp
hat
e (m
g/D
L)
Vehicle 12.5 mpk/BID continuous
50mpk/QD 5d on/2d off
Phosphate on day 7
RT4 SNU16
SNU16 SNU16
Figure 4. Kinase selectivity profile of lead molecules screened against 250 kinases. Dots represent individual kinase with > 90% inhibition at 1 uM.
