preclinical)evalua,on)and)biomarker…€¦ · ct3$lda$tumor$volume$/$10$cells pbs irinotecan...
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CT3$LDA$Tumor$Volume$/$10$Cells
PBS Irinotecan a/LGR5$ a/LGR5$Irinotecan0
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Preclinical evalua,on and biomarker iden,fica,on for BNC101 an,-‐LGR5 mAb in K-‐Ras Mutant CRC and Solid Tumors Peter Chu, Kristen Smith, Farbod Shoajei, Colin Walsh, John Norton, Jose Iglesias and Christopher Reyes.
Oncology Discovery, Bionomics, Inc., San Diego, California.
• LGR5 is a cancer stem cell receptor overexpressed in colorectal cancer (CRC), pancrea,c, breast and most other solid tumors
• LGR5 is a co-‐receptor in the Wnt signaling pathway that focuses and enhances stem cell signaling to LGR5+ normal and cancer stem cells
• LGR5 therapeu,c mAb candidates for preclinical development were selected based on biochemical characteris,cs, and in vitro and in vivo func,onal ac,vity against cancer stem cells (CSCs) from primary pa,ent-‐derived CRC xenograW (PDX)
• BNC101 is a humanized an,-‐LGR5 monoclonal an,body that has successfully completed IND studies for IND-‐filing and Phase I clinical studies in 2015
BNC101 therapeu,c strategy is to delay recurrence and/or metasta,c disease by preven,ng CSCs from re-‐seeding cancer following front-‐line surgery and standard of care chemotherapy
Introduc,on Results
Background Fig 1. LGR5 Marks Normal and CSCs in the Colon
Day 1 Day 5 Day 60
Barker et al., Nature 2007
Lineage Tracing of Lgr5-‐LacZ Stem Cells LGR5 expression is highly specific to normal intes,nal stem cells
Wnt ac,va,on in non-‐stem cells No progress to cancer (284 days)
Wnt ac,va,on in LGR5+ stem cells Massive tumors, mice die ~ day 24
LGR5 cells are cancer stem cells at the root of colon cancer
Barker et al., Nature 2009
LGR5+ stem cells
LGR5+ cancer stem cells
Normal Crypt
Tumor
LGR5+&CSCs&are&important&targets&for&the&treatment&of&cancer&&
Tumor Volume (Mean)
0 20 40 600
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Days Post Tumor Inoculation
GPR49 neg 1000
GPR49 pos 1000
Tum
or V
olum
e (m
m3 )+
SE
M
N=10&mice/group&
Sorted&LGR5+&Cells&from&Pa@ent&Derived&Tumor&
LGR5+&
LGR5neg&
Reyes,&Chu,&2009&& Source:&Merlos4Suarez&et&al.,&Cell&Stem&Cell&2011&
LGR5hi&ISC&Gene&Signature&Predicts&10Efold&Higher&Disease&Relapse&in&CRC&Pa@ents&
LGR5+&High&
LGR5+&Med&
LGR5+&Low&
Fig 4. LGR5 Marks Highly Tumorigenic CRC Cells and Predicts Relapse in CRC Patients
mBNC101 Treatment Prevents Tumor Re-Growth in Serially Transplanted Hosts*
* No additional treatment in this experiment
C. Colorectal Cancer Patient-Derived Xenograft Study #2: Patient CT3: Stage IIIb CRC, with mutations in K/H-Ras, PI3K, PTEN, APC, STK11, RB1, TP53, FGFR2
BNC101 Summary and Conclusions ● BNC101: First-‐in-‐Class mAb targe,ng LGR5 CSCs, Ac,ve in: K-‐Ras mutant CRC, Triple-‐nega,ve breast and Lung cancer Pa,ent-‐derived XenograWs
● Targets and Reduces CSCs in vivo, Prevents Tumor Re-‐Growth in Re-‐implant Assays
● BNC101 combined with FOLFIRI significantly Delays Tumor Recurrence in CRC PDX re-‐implanta,on models
● BNC101 treatment of CRC and SCLC PDX tumors Modulates Wnt Gene Expression
BNC101 LGR5 mAb treatment may significantly extend pa,ent survival in CRC, TNB, SCLC and also Pancrea,c Cancer
Wnt genes and LGR5+ CTCs iden,fied as BNC101 biomarkers for upcoming Phase I trial
* P < 0.001 vs Control ** P < 0.001 vs Irinotecan Ini?al tumor size ~ 200 mm3
Outsized Tumor Inhibi,on Ac,vity • ~10% Lgr5+ cells in CT1 • 43% an,-‐tumor ac,vity
LDA: Tumor Growth Inhibi,on of 30 cell Re-‐implant aWer Primary Treatment ¥
¥ No additional treatment in this experiment
Re-‐implant tumor cells
aWer treatment
Figure 5. BNC101 Targets CSCs in K-Ras Mutant CRC That Seed New Tumors
0"
0.002"
0.004"
0.006"
0.008"
0.01"
Freq
uency"of"Can
cer"S
tem"Cells"
Treatment"BNC101 BNC101/
Chemo Chemo CON
mBNC101 Reduces CSC Frequency (Limiting Dilution Analysis)
B. Limiting Dilution Assay (LDA) on CT1
A. Colorectal Cancer Patient-Derived Xenograft Study #1: Patient CT1: Stage IV, mCRC (liver) with mutations in K-Ras, PI3K, PTEN, p53
1/100
1/300
Secondary Cancer Stem Cell LDA re-implant assay
BNC101: 15mg/kg BIW
BNC101 MDA-MB231 Tumor Efficacy Study
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Tum
or V
olum
e (m
m3) MOPC
BNC101
49% T/C
MDA-231 LDA Time to Progression
20 30 40 50 60 70 80 900
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Perc
enta
ge o
f Tum
orFr
ee A
nim
als
(%)
Study Day
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BNC101
MDA-231 LDA Tumor Volume
MOPC BNC101 0
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Tum
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olum
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m3)
Figure 6. Single Agent Activity of BNC101 in Triple-Negative Breast PDX Models
Fig 3. LGR5 RNAi Knockdown Inhibits Growth of Colorectal Tumor Lines
siRNA treatment of HCT116
Control'siRNA'
LGR5'siRNA'
Soft Agar
0
20
40
60
80
HCT116 SW620 DLD-1 LS174T
% in
hibi
tion
In Vivo CRC Tumor Xenografts
Fig 2. LGR5 is Highly Expressed in Primary and Metastatic Cancer Indication Assay %-LGR5-
Expression-(n)Fold;increase Reference
Colorectal- Microarray 87%-(77) 6.7 Biogen-Idec
Metastatic-Colon Microarray NA 16 Bionomics
Ovarian qRT;PCR 53%-(33) >3 McClanahan,-Cancer-Bio-Ther-2006;-Bionomics
Microarray 83%-(36) 8.9 Biogen-Idec
Pancreatic Microarray 27%-(11) 3;4 Bionomics
IHC 100%-(17) 2 Simon,-PlosOne-2012
-Metastatic-Breast FACS 3;6%-of-cells NA Bionomics
-Lung Microarray 20%-(15) 2;4 Bionomics;-Jackson-Labs
-Metastatic-Prostate FACS 1%-of-cells NA Bionomics
-Gastric IHC 91%-(32) 2.5 Simon,-PlosOne-2012
--Esophageal- IHC/PCR 85%-(60) 3.5 von-Rahden,-J-Exp-Clin-Cancer-Res-2011
--Liver-(HCC) qRT;PCR 53%-(38) >3 Yamamoto,-Hepatology-2003;-Zucman;Rossi,-
-----β;cat-mutant-HCC qRT;PCR 100%-(11) >2Bioulac;Sage-,-Hepatology-2007
CT1$CRC$PDX$Tumor$
JH109$PANC$PDX$
CRC$Grade$I$
CRC$Grade$II$ CRC$Grade$III$
CRC$Metasta;c$
LGR5 IHC Expression
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Day post-implantion
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LGR5 RNAiControl RNAi
20,000 cells/mouse
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Day post-implantion
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LGR5 RNAi
Control RNAi
LGR5 Expression in Origene Tumor Tissue Array
0" 1" 2" 3" 4" 5" 6" 7" 8" 9"
Rectum"
R"Ovary"
R"Frontal"Brain"
L"Ovary"
Right"adnexa"
R"Ovary"
Pleural"Fluid"
Liver"
Ovary"
R"Ovary"
Pelvic"Tumor"
R"Ascending"Colon"
Liver"
Liver"
Liver"
R"Ovary"
R"Ovary"
Bowel"Tumor"
Brain"Tumor"
mRNA"(Log2)"
LGR5"Expression"in"MetastaOc"CRC"Panel""
CT1 LDA Tumor Volumes
Irinotecan mBNC101 mBNC101 Chemo0
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Tum
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@ 8
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Time to Progression
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Tum
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ree
Ani
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s (%
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Study Day
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BNC101
BNC101 Irinotecan
CT1 LDA Tumor Growth
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Study Day
Tum
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Irinotecan mBNC101mBNC101 Chemo
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CT1 Colorectal PDX Tumor Efficacy Study
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Study Day
Tum
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m3) Irinotecan
Control
mBNC101
mBNC101 + Irino
CT1 LDA Tumor Growth
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Study Day
Tum
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m3)
Irinotecan mBNC101mBNC101 Chemo
Tumor-Free Mice After Re-Implantation of mBNC101 Treated CRC Tumors (8 weeks)
Results
Fig 8. Modulation of Wnt proteins and target genes in CRC tumors treated with BNC101
Study Design: 1) cDNA from n=10 BNC101 or MOPC treated CT1 or CT3 tumors was pooled. 2) QPCR in Wnt Plates Containing Primers for 84 genes. 3) Data Analyses
BNC101 modulates the expression of a panel of Wnt genes
BNC101 Mediated Changes in Wnt Expression Consistent Among Different CRC PDX Tumors Downregulated vs Upregulated
Genes Post-‐BNC101 treatment
LGR5 Expression in Metasta,c CRC Panel
CT3 LDA Time to Progression - 30 Cells
Study Day
Perc
enta
ge o
f Tum
orFr
ee A
nim
als
(%)
30 40 50 60 70 800
50
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150 BNC101 FOLFIRI 30 cells
FOLFIRI 30 cells
CT3 LDA Tumor Volume 30 cells
FOLFIRI 30 BNC101 FOLFIRI 30
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Tum
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m3)
@ d
ay 6
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CT3 LDA Tumor Growth - 30 cells
30 40 50 60 70 800
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Study Day
Tum
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olum
e (m
m3)
BNC101 FOLFIRI 30 cells
FOLFIRI 30 cells
D. Colorectal Cancer Patient-Derived Xenograft Study #3: Patient CT3: BNC101 FOLFIRI Standard of Care (SOC) combination
CT3 LDA Tumor Regrowth - 30 cells
0 30 60 90 120 1500
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Study Day
Tum
or V
olum
e (m
m3) mBNC101
Irinotecan
mBNC101 Irinotecan
Fig 8. Average Inhibition of LRP6 phosphorylation by BNC101 (murine 18G7) without added RSPO or Wnt, and with R-spo and Wnt (CM-conditioned media) ligands
BNC101 reduc,on of pLRP protein
Adre
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0.250.51.52.53.54.5
Fold
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T1)
Nor
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to G
APD
H
* P < 0.05 vs normal** P < 0.01 vs normal
*
**
**
*
Figure 7. BNC101 Activity in Small-cell Lung Cancer (SCLC) PDX Models
Tumor Growth of BLG293 Tumors Treated with BNC101
64 66 68 70 72 74 76 78 80 82 840
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Study Day
Tum
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Fold
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MOPCBNC101
Etoposide/Cisplatin10mpk/5mpk
Normal Colon
LoVo cells
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phos
pho-
LRP
6 : p
an-L
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6(%
R-s
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R-Spo +Wnt CM +BNC101
R-Spo +Wnt CM
R-Spo
PD Post-Study Gene Exp Analysis
Con Ab1 Ab2 mBNC101
BLG222 SCLC PDX Treated with BNC101
Study Day
Fold
Sta
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Vol
ume
43 45 47 49 51 53 55 57 59 61 63 65 67 69 710
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PBSMOPCBNC101Chemo