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Page 1: Preclinical)evalua,on)and)biomarker…€¦ · CT3$LDA$Tumor$Volume$/$10$Cells PBS Irinotecan a/LGR5$ a/LGR5$Irinotecan 0 50 100 150 200 250 300 Prior$Treatment$Group m ³ 1 Preclinical)evalua,on)and)biomarker

CT3$LDA$Tumor$Volume$/$10$Cells

PBS Irinotecan a/LGR5$ a/LGR5$Irinotecan0

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Prior$Treatment$Group

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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)

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GPR49 neg 1000

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Sorted&LGR5+&Cells&from&Pa@ent&Derived&Tumor&

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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&

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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"

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uency"of"Can

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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

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BNC101: 15mg/kg BIW

BNC101 MDA-MB231 Tumor Efficacy Study

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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'

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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$Tumor$

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LGR5 Expression in Origene Tumor Tissue Array

0" 1" 2" 3" 4" 5" 6" 7" 8" 9"

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CT1 LDA Tumor Volumes

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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

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D. Colorectal Cancer Patient-Derived Xenograft Study #3: Patient CT3: BNC101 FOLFIRI Standard of Care (SOC) combination

CT3 LDA Tumor Regrowth - 30 cells

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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    

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Figure 7. BNC101 Activity in Small-cell Lung Cancer (SCLC) PDX Models

Tumor Growth of BLG293 Tumors Treated with BNC101

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