ChemPartner Oncology May 2017 - CPhI Online Oncology-file076520.pdf · Assays by Target Type GPCR...

ONCOLOGYONCOLOGYMay, 2017

ChemPartner Discovery Biology Expertise

In Vitro Biology/HTS/Assay Technology In Vivo Biology/ Pharmacology

Assay Development HTS

Oncology Panel Screening (>700 cell lines)

HTS Target Classes

GPCRs: (32) Nuclear receptors (22)

Cell line‐derived xenograft models (>160) Patient‐derived xenograft models (>200) Tumor immunology

Neuroscience Kinases (161) Epigenetic enzymes (>100) Metabolic enzymes (>40) Ion channels transporters

Behavioral Neuropharmacology In Vitro Electrophysiology (QPatch, Brain Slice) In Vivo Microdialysis & Receptor Occupancy Primate Cognitive NeuropharmacologyIon channels, transporters

Anti‐infectious assays 150+ clients, 200+ projects, 6.5M+ data points

Metabolic & Cardiovascular Diseases DIO, db/db, ZDF Diabetes & Metabolic Diseases (Diabetic

Nephropathy)

Cell Biology

Target Validation (siRNA, shRNA, CRISPR, cDNA,

Inflammation & Immunology Primary Immune Cell Assays CIA, AIA, IBD, lung fibrosis, PD models

Biomarkerlentivirus)

Target to Hit, Hit to Lead assays Cancer cell line panel screening HCA, ELISA, AlphaScreen, FACS, Luminex

PK/PD correlation PD biomarkers: Western blot, qPCR, ELISA, Luminex,

MSD, LC/MS/MS Pathology service

© 2017 ChemPartner 2

, , p , ,

In Vitro BiologyIn Vitro Biology


Assays by Target Type GPCR Kinase and PDE

Calcium flux (FLIPR)cAMP assay (HTRF, LANCE, cAMP‐Glo)Receptor binding assay

GPCR Kinase and PDE

Caliper mobility shift assay[33P] ATP filter binding assayFRET assayReceptor binding assay

Receptor occupancy on brain homogenateReporter gene (luciferase, b‐gal, bla)β‐arrestin assayIP O HTRF

FRET assayTR‐FRET assayFP transcreener assayLanthaScreen Eu kinase binding assayIMAPIP‐One HTRF assay

IP3 AlphaScreen assay Ion Channel and TransporterNuclear Receptor

TR‐FRET assay

IMAP assay

Membrane Potential assayQ‐PatchTR FRET assay

Luciferase reporter assayFull‐length NRLBD‐GAL4

R t bi di

Q PatchManual PatchFluxOR screening with potassium channelsPredictor hERG FP assayT t t k

Anti‐bacteria testing serviceAnti HBV and HCV testing service

Epigenetic TargetsMicrobiology

Receptor binding assay In‐Cell protein: protein interaction assay

AlphaLISA/ AlphaScreen

Transporter uptake assay

Anti HBV and HCV testing service

Others

AlphaLISA/ AlphaScreenRadioisotopeFluorimetryLance Ultra Assay Development


Mobility shift assayy p

Kinase Panel: 161 Kinase Assays Established Tyrosine Kinase

A F tABLARGBLK BRKBTKCSK

ALKALK (L1196M)AXLcKIT

FGFR2FGFR3FGFR4FLT1/VEGFR1FLT3

Caliper mobility shift assayKinase-glo assay

ADP-Glo assayLanthaScreen assay

LanceUltra assay

Assay Formats

CSKFAKFES FERFGRFYNHCK

cMETECK/EphA2EGFR EGFR (d746‐750)EGFR (L858R)

FLT4/VEGFR3KDR/VEGFR2HER2HER4 IGF1RINSRLTK

MAPKAPK5MARK1 MARK2

AMPKα1BRSK1 BRSK2

AKT1AKT2AKT3MSK1

PKCηPKCθPKCιPKN1

PI3K

HCKITKJAK1 JAK2 JAK3LCK LYNa

(L858R) EGFR (T790M)EGFR (T790M,L858R)EGFR (T790M L746‐750)

LTKMUSKPDGFRa PDGFRb RETRON ROS

MEK1MINKMST1MST2MAP4K2 MAP4K4

MARK2 MARK3 MYLK2PIM1 PIM2PKD1PKD2

BRSK2CAMK2αCAMK2βCAMK2γCAMK2δ CAMK4CHK1

MSK2p70S6KPDK1PKACαPKCαPKCβ1PKCβ2

PKN2PKN3PRKG1ROCK1ROCK2RSK1RSK2 PI3K

PI3KPI3KPI3Kmr

SRCSYKTYK2 YESZAP70

(T790M, L746 750)EphA1 EphB1 EphB2FGFR1

ROS TIE2TRK‐A

MASKPAK2PAK4TAOK2 TNIK

PKD2PKD3 SIK TSSK1TSSK2

CHK1 CHK2 DCAMKL2 MAPKAPK2MAPKAPK3

PKCβ2PKCγPKCδ PKCεPKCζ

RSK2RSK3SGKSGK2SGK3

STECAMK

AGC

Atypical

IRAK1 C K1 K2 CK1δ

STE

CMGC CK1

CAMK

Other

Atypical

TKL

IRAK1IRAK4BRAFBRAF(600E)CRAF

CDK1CDK2 CDK4CDK6 CDK7CDK9DYRK1α

ERK2GSK3α GSK3β JNK2JNK3P38αP38β

CK1δCK1ε

AUR AAUR BAUR CIKKβNEK2


DYRK1αDYRK1βERK1

P38βP38γ P38δ

Epigenetic Panel: 120 Assays Established Assay FormatsAssay Formats

HDAC1HDAC2

NSD1NSD2NSD3PRDM9

DOT1LEHMT1

EZH1EZH2

ATAD2A BAZ1BBAZ2B

BRD2(1,2)BRD2(D1)

CECR2CREBBPEP300FALZPBRM1(D1)

Radiometric assayAlphaLisa assayFluorescence Intensity

HTRF assayAlphaScreen assayMobility Shift assay

Assay FormatsAssay FormatsMulti‐colours stands for multi‐methods

HDAC2HDAC3HDAC4HDAC5HDAC6HDAC7

PRMT1PRMT3

CBPEP300

PRDM9SETD1BSETD7SETD8SETDB1

EZH2EZH2 (Y641F)EZH2 (Y641C)EZH2 (Y641N)EZH2 (Y641S)

JARID1A JARID1BJARID1C

JMJD2DJMJD2EJMJD3

BRD2(D1)BRD2(D2)BRD3(1,2)BRD3(D1)BRD3(D2)BRD4(1 2)

PBRM1(D1)PBRM1(D4)PBRM1(D5)PBRM1(D6)SMARCA2SMARCA4

Fluorescence IntensityLANCE Ultra

Mobility Shift assayMTase-Glo assay

NatDPCAF

HDAC7HDAC8HDAC9

HDAC10HDAC11

SIRT1SIRT2SIRT3

PRMT4PRMT5PRMT6PRMT7PRMT8

GCN5 HAT1KAT5KAT6BKAT7

SMYD2SMYD3SUV39H1SUV39H2SUV420H1

EZH2 (A677G)G9a

MLL1MLL2MLL3

JARID1CJMJD1AJMJD1BJMJD2AJMJD2B

JMJD3LSD1PHF8UTXFBXL10

BRD4(1,2)BRD4(D1)BRD4(D2)

BRD7BRD9

BRDT(D1)

SMARCA4SP140SP140LTAF1(D2)TRIM24

L3MBTL1L3MBTL3

me me ac

HKMT HKDM HDAC&SIRT HATHRMT

Histone Mark

PRMT8 KAT7KAT8

SUV420H1SUV420H2

MLL3MLL4 JMJD2C FBXL11 BRDT(D1)

BRDT(D2)MethylReader Bromodomain

R KKmeDNMT

DNMT1AKT1AKT2

IKKaJAK2

PIM1PKCb1

CpGmDNMT1DNMT3ADNMT3B

AKT3AurB

MSK1MSK2

Histone Mark PKinase

PKN1RSK2


S/T/Y

HDAC: Long-Term SAR Program

Reference IC50 on Target HDAC-X

Historical Reference IC50 on HDAC-X

8 0

8.5

9.0

ef IC

50)

20

40

60

80

100

120

IC50 = 5.6 nM

Inhi

bitio

n %

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4

7.0

7.5

8.0

-Log

(Re

-2 -1 0 1 2 3 4-20

0

20

Log Concentration (nM)

I

1/4/

2012

1/10

/201

21/

17/2

012

2/7/

2012

2/13

/201

22/

21/2

012

2/28

/201

23/

6/20

123/

13/2

012

3/20

/201

23/

27/2

012

4/10

/201

24/

17/2

012

4/24

/201

212

/17/

2012

1/14

/201

31/

22/2

013

1/28

/201

32/

5/20

132/

18/2

013

2/25

/201

33/

11/2

013

3/18

/201

34/

1/20

134/

8/20

134/

15/2

013

4/22

/201

34/

27/2

013

5/6/

2013

5/13

/201

35/

20/2

013

5/27

/201

36/

3/20

136/

17/2

013

6/24

/201

37/

1/20

137/

8/20

137/

15/2

013

7/22

/201

38/

26/2

013

9/2/

2013

9/16

/201

39/

23/2

013

10/8

/201

310

/14/

2013

10/2

2/20

1310

/28/

2013

11/4

/201

311

/11/

2013

11/1

8/20

1311

/25/

2013

12/2

/201

312

/9/2

013

12/2

3/20

1312

/30/

2013

1/6/

2014

Assay Date

Selectivity against HDAC-S1, S2 and S3　IC50 of CPD-1 (nM) Selectivity

HDAC-X 2.1 n/aHDAC-S1 356 169HDAC-S2 380 181HDAC-S3 1785 850


HDAC S3 1785 850

MOA study: Modality of Inhibition

Competitve cmpd Non competitve cmpd Un competitve cmpd

Global fitting for MoICompetitve cmpdMixed mode fitting

10

1541.0024.6014.768.865.313.19

eloc

ity

Non-competitve cmpdMixed mode fitting

10

15804020105el

ocity

Un-competitve cmpdMixed mode fitting

4

6

80.1240.0620.0310.0160.008el

ocity

0 2000 4000 60000

5

0

1.911.150.690.41

Substrate conc. [uM]

Ve

0 500 1000 1500 20000

5 0


Ve

0 50 100 150 200 2500

20.00


Ve

Lineweaver-Burk plotC titi d

Lineweaver-Burk plotN titi d

Lineweaver-Burk plotU titi d

Lineweaver–Burk plot (double reciprocal plot) for MoI

Competitive cmpd

1.0

1.5

2.014.768.865.313.191.910

1/V

Non-competitive cmpd

4

6

88040201050

1/V

Un-competitive cmpd

0.4

0.6

0.80.1240.0620.0310.0160.0080.00

1/V

Competitive cmpd

0.000 0.002 0.004 0.006 0.0080.0

0.50

1/S

Non competitive cmpd

0.00 0.01 0.02 0.03 0.040

20

1/S

Un competitive cmpd

0.00 0.01 0.02 0.03 0.04 0.050.0

0.20.00

1/S


Competitive cmpd Non‐competitive cmpd Un‐competitive cmpd

MOA study: Slow-on/Slow-off Characterization

Kon test for slow‐on cmpds and irreversible cmpds

Kon and Koff determination


Cell BiologyCell Biology


Cell Biology Overview

Target Validation Cancer Cell Line Panel ScreenTarget Validation Cell Line Selection for Target Validation siRNA-Mediated Gene Knockdown shRNA-Mediated Gene Knockdown

Cancer Cell Line Panel Screen 700+ cell line collection Mycoplasma tested and STR verified Various assay formats to choose from

CRISPR-based Gene Knockout Stable Cell Line Generation with Lentivirus Rescue to Prove On-Target Effect In Vivo Target Validation

y Dedicated team with 8 years experience Assay validated by uniformity test and

test/retest Stringent in-study QCg

200+ new targets have been validated in 100+ cell lines by siRNA or shRNA KD, 1000+ stable cell lines generated

g y30+ panel screens conducted and 600,000+ high-quality data points delivered each year

Assay PlatformsCell-based Assays

Cell functional assays Proliferation

Assay Platforms Absorbance-based assays Luminescence-based assays Fluorescence-based assays

Cytotoxicity Migration Apoptosis Cell cycle

y High content analysis Flow cytometry & cell sorting Quantitative PCR Western blot, ELISA, Luminex, y

Cell signaling assays Cell metabolite assays50+ clients, 600+ studies, functional service or part of drug discovery programs to support

, , ,AlphaScreen, etc.

We customized our assays to suit your project needs


p g y p g ppSAR finding

siRNA Transient Gene Knockdown

Knock down target gene by siRNA pool or individual oligos Taqman or Western blot analysis to confirm the knockdown efficiency at mRNA level or

protein level, respectively The effect of tumor cell growth inhibition is monitored by CellTiter Glo cell viability assay

(2D) and/or soft agar colony formation assay (3D)

siRNA Anti‐Proliferation CTG Assay in Cell Line XTaqman KD Evaluation siRNA Anti‐Proliferation CTG Assay in Cell Line XTaqman KD Evaluation

siRNA Soft Agar Screen in Cell Line XWestern Blot KD Evaluation (GAPDH as an example)Day 2 Day 3 Day 4 Day 5 Day 6

GAPDH

Actin


Constitutive and Inducible shRNA Knockdown

pTRIPZ System (Open Biosystems) – tet‐inducible target gene knockdown pTRIPZ System (Open Biosystems) tet inducible target gene knockdown

pSLIK System (ATCC) – tet‐inducible target gene knockdown

pGIPZ System (Open Biosystems) – constitutive target gene knockdownp y ( p y ) g g

pLKO1 System (Open Biosystems) – constitutive target gene knockdownVerify Inducible Target KD in Stable Cell Lines by RT‐TaqmanCommercially

purchased shRNAsClient provided

shRNAs

40%60%80%

100%120%

ativ

e m

RN

A

Leve

l

Dox-D

In‐house made shRNAs

0%20%

shRNA1 shRNA2 shRNA4 shRNA6

Rel

a Dox+

Verify Target KD in Stable Cell Lines by Western blot

Packaging Lentivirus

Generate shRNA‐containing bl ll li

Gene A

Cell Line 1

M WT NT sh5 sh6 M

Gene B

M WT NT 1025 1462 M

Cell Line 2Verify Target KD in Stable Cell Lines by Western blotstable cell lines

Validate stable cell lines by Taqman and Western blot

56 kDa 25 kDa

ActinActin

2D CellTiter Glo Cell Viability Assay

2D Clonogenic Assay

3D Soft Agar Colony Formation Assay

Apoptosis Assay

Downstream pathway/biomarker

In Vivo target lid ti


validation

CRISPR Target Gene Knockout – Generation of KO Stable Cells

M WT GA‐1 GA‐2 GA‐3 M

Detect Target A Expression by Western Blot in Pooled KO Cells (Lentivirus infection)

ells)

Detect Biomarker by LC/MS/MS in Pooled KO Cells

GAPDH

Target A

/million ce

GAPDH

LOW Conc. (ng

Detect Target B Expression by Western Blot in Single KO Clones (Transient Transfection)

Target B

M WT 1‐1 1‐2 1‐3 1‐4 1‐5 1‐6 1‐7 1‐8 2‐1 2‐2 2‐3 2‐4 M

GAPDH


CRISPR Target Gene Knockout – Phenotypic Study

2D Clonogenic Assay in A549 WT/Cas9 cells following sgCopGFP/sgPCNA virus infection

A549‐wt

A549 WT cells A549‐Cas9 cells

sgGFPInfect with pRSG16‐sgGFP or sgPCNA

sgPCNA

Puromycin Selection

Replate into 6 well plates

A549‐Cas9

Replate into 6‐well plates

Colony FormationsgGFP

Knockout of PCNA, an essential gene for cell survival, by Lenti‐sgRNA

y

sgPCNAfor cell survival, by Lenti sgRNA infection, led to scarce colony

formation


Cellular Functional Assays

• Cell proliferation/viability Soft Agar Colony Formation Assay• Cell proliferation/viability‒ CellTiter‐Glo cell viability assay‒ CyQuant assay

MTT/MTS

Apoptosis Analysis

Soft Agar Colony Formation AssayStaurosporine(Rel_IC50=0.00073μM) on A375 cell line by soft agar assay

80

100

DMSO treated‒ MTT/MTS assays‒ Direct cell counting by HCA‒ Clonogenic assay

inhi

bitio

n%

0

20

40

60DMSO treated

‒ 14‐day long‐term proliferation assay

• 3D growth‒ Soft agar colony formation assay

Cell Cycle Analysis

Compound Concentration(μM)1x10-5 0.001 0.1

-20

‒ Matrigel 3D assay‒ Ultra‐low attachment assay

• Cytotoxicity: LDH release assay

Cell Cycle AnalysisDMSO Cisplatin (5

μM) STS treated

y y• Apoptosis

‒ FACS (AnnexinV & PI)‒ Caspase 3/7 Glo PI (FL3‐A)

Num

ber

Caspase 3/7 Glo‒ Cell death detection ELISA

• Cell Cycle: FACST ll i ti T ll

Cell cycle was changed in A549 after cells being treated with Cisplatin for 48h.

PI (FL3 A)


• Tumor cell migration: Transwell assay

14-day Long-Term Proliferation Assay- To Support Epigenetics Program

Cell Line X, Compound Y-day4 Cell Line X, Compound Y-day7120

Day 4 Day 7

row

th In

hibi

tion

20

40

60

80

100

row

th In

hibi

tion

20

40

60

80

100

concentration(uM)0.001 0.1 10

%G

-20

0

20

concentration(uM)0.001 1

%G

-20

0

20

D 14Cell Line X, Compound Y-day11

ition 100

Cell Line X, Compound Y-day14

ition 100

Day 11 Day 14

%G

row

th In

hib

10

40

70

%G

row

th In

hib

10

40

70

A long‐term proliferation assay (time courses on Day4, 7, 11,and 14) was performed to


-20


-20


determine the anti‐proliferation effect by a compound targeting an epigenetic target.

Cell Signaling Assays Protein Phosphorylation

473

p‐Akt detected by AlphaScreen SureFire™Western blot Cell‐based ELISA AlphaScreen SureFire

pAKT473 in Suspension Cells ByAlphaScreen SureFire

100

150

Cpd

ition

High content analysis MSD FACS

-2 0 2-50

0

50

Log[compound] uM

%in

hib

Nanopro

Nuclear Translocation Reporter Gene Assay Afatinib Erlotinib

Afatinib Erlotinibp‐EGFR and p‐HER2 detected by ELISA

p y

C d ( M)

Doxycycline +++ ++-

0 2-- 202 200

Phospho‐Protein detected by Western Blot

%in

hibi

tion

10

30

50

70

90

Erlotinib

%in

hibi

tion

20

40

60

80

100

GFR

(pan

‐Tyr)

Compound (nM)

Total target protein

160kD

110kD-P-target protein

0.2-- 202 200

160kD

Cmpd Concontration (nM)0.01 10

-10

10

Cmpd Concontration (nM)1 1000

0

Afatinib

n

90

Erlotinib

n

90

P‐EG

‐Tyr)

GAPDH40kD

30kD

g p110kD

Cmpd Concontration (nM)1 1000

%in

hibi

tion

10

30

50

70

Cmpd Concontration (nM)10 1000 100000

%in

hibi

tion

10

30

50

70

P‐HER

2 (pan

‐


C pd Co co t at o ( ) C pd Co co t at o ( )

Cell-based Metabolite Assays using LC/MS/MS- To Support Cancer Metabolism Programs

C di i l M di ( d b li )Compounds

Conditional Medium (secreted metabolites)

Cell Extracts (intracellular metabolites)

Assay Optimization‐ Find the best condition that

biologically makes sense and also is

Assay Reproducibility‐ Include a reference compound in each round of the screening as QC

Routine Screening‐Medium throughput, over 5,000 data points per assay per week

robust enough for routine screening

4 cell‐based metabolite assays are being carried out routinely, to guide SAR in various programs The candidate compound from 2 of such programs is now in Phase II trial We newly developed metabolomics platform, in which >100 metabolites can be quantified


simultaneously in one single cell extract

Largest Cancer Cell Line Collection among CROs 700+ well‐selected cancer cell lines ready‐for‐screening700+ well selected cancer cell lines ready for screening

>80% of the cell lines are in CCLE panel with genetic annotations Representative of genomic diversity, tissue and ethnic origins High quality cell panel guaranteed by STR verification and mycoplasma testing

Primary Cancer Lines /PDCs No.liver 14

pancreas 20others 10+

Tumor Type No. Of Cell Lines

Lung 153

ChemPartner proprietary

Asian (JCRB/RIKEN/KCLB/SIBS) No.lung 21brain 18

esophagus 13stomach 12

Brain 50Leukemia 49Breast 48

Lymphoma 45l liver 11

ovary 9pharynx 7leukemia 6

adrenal gland 4

Melanoma 38Colon 37

Pancreas 31Liver 31Ovary 25 tongue 4

kidney 3pancreas 3

endometrial 2skin 2

Ovary 25Esophagus 25Kidney 20Stomach 19Bone 17

Human Normal Cells/Cell Lines No.kidney 2skin 2breast 1

Others 12Bone 17

Bladder 12Tongue 12Myeloma 11Prostate 7


umbilical cord e 1Others 73

State-of-the-Art Facility and Instrumentation

f l l f l7,500 sq.ft. First‐class tissue culture facility Well‐equipped with cutting‐edge technology & instruments

C d H dli A R d tCell Counting & Plating Compound Handling Assay Readout

Flexstation 3Vi‐Cell Cell Viability Analyzer Caliper Zephyr Envision

EnSpire

Hamilton Starlet

Multidrop CombiHP D300

E h 550INCELL 2000

Acumen eX3


Echo 550

Stringent QC at Different LevelsC ll Li Ch i iCell Line Characterization: Growth Property Doubling Time Cell Seeding Density

Cell Lines from Vendor

Master Cell Bank (20 vials) Ce Seed g e s ty

Routine Assays

Master Cell Bank (20 vials)Backup Cell Bank (2 vials)

k ll k ( l )Routine Assays

>2 passages post recovery

Assay and Data QC

Working Cell Bank (20 vials)

Cell Identification

MycoplasmaTest

Cell Morphology Compound Solubility &

(STR Verification)

Historical IC50s of STS Compound Solubility &

Precipitation Max/Min Signals Uniformity and Edge Effect Z factor and SW Replicates Variation Historical IC50s of Reference

Cmpds


Cmpds

PharmacologyPharmacology


Cancer Pharmacology Overview

• 8+ years of experience

• SPF animal facility (former CRL facility): Cell Biology

– AAALAC accreditation, OLAW assurance

• Early Discovery to Clinical Development

(Ph II lik li i l t i l i )

Cell Biology48% In Vivo

Pharmacology46%Ex Vivo– (Phase II‐like clinical trial in mouse)

• Functional service or part of integrated services

46%Ex Vivo6%

• Study Types

– Cell line‐Derived Xenograft (CDX)– Patient‐Derived Xenograft (PDX) Oncology Studies

Conducted

Compounds / Data Points Major Client

Numbersg ( )– Cancer Immunology & Syngeneic models– In Vivo Drug Efficacy

PK/PD (with CP Bioanalytic)

Conducted Screened Numbers

In Vitro (Cell

Biology)~ 700 > 4 million

data points >100– PK/PD (with CP Bioanalytic)– Biomarkers and Pathology – New model development (in collaboration

with Cell Biology & hospitals)

Biology)

In Vivo ~ 2,400> 1,000

compounds > 100


In Vivo Pharmacology Models

Model Type Highlights

Cell line‐derived

Xenograft (CDX)

• 160+ optimized models (growth curves, standard of care treatment)• Compatible with Cancer Cell Panel screen• Experience : Epigenetics, Cancer Metabolism, ADC

Patient

• ~270 optimized models (growth curves, standard of care treatment)• Specialized in cholangiocarcinoma with 17+ PDX modelsPatient

derived‐Xenograft (PDX)

• Facilitating model selection: Whole genome sequencing and RNA‐seq data Ex vivo chemosensitivity assay 40+ PDX derived primary cancer cell lines 40+ PDX‐derived primary cancer cell lines

i

• ~20 in vivo models (sc, iv, orthotopic, resection settings)• Efficacy studies with immunotherapy & combination therapiesSyngeneic

tumor models

Efficacy studies with immunotherapy & combination therapies• Ex vivo analyses

Flow cytometry IHC, soluble factors, genetic profiling


1. Breast cancer (10): MDA-MB-231 (+M); MCF-7(+M); ZR-75-1 (+M); HCC70 (+M); BT-474 (+M); MDA-MB-468 (+M); T-47D(+M);

Cell Line Derived Xenograft Models 160+

Subcutaneous model (136)

( ) ( ); ( ); ( ); ( ); ( ); ( ); ( );MCF-7-Her2(+M); MX-1(+M); DU4475

2. Colorectal cancer (9): HCT-116; SW620; HT-29; SW480; DLD-1; COLO 205; NCI-H716(+M); RKO; SW948(+M)3. Gastric cancer (6): SGC-7901 (+M); BGC-823; HGC-27 (+M); MKN-45; AGS (+M); NCI-N874. Glioblastoma (2): U87MG; U87MG(rat,+M) 5. Kidney cancer (4): Caki-1 (+M); A-498; 786-O(+M); G-401(+M)6. Lung cancer (41): A549; NCI-H460; SPC-A-1; NCI-H1975; NCI-H292 ;NCI-H2009(+M); NCI-H1299(+M); NCI-H2122; NCI-H522(+M);

Calu-3(+M); Calu-6(+M); NCI-H441; NCI-H446(+M); NCI-H1155(+M); NCI-H2171(+M); NCI-H841(+M); SHP-77(+M); DMS 53(+M); NCI-H69(+M); NCI-H209(+M); NCI-H524(+M); NCI-H1048(+M); NCI-H1930(+M); DMS 79(+M); NCI-H2081(+M); NCI-H1092(+M); ChaGo-K-1(+M); NCI-H1770(+M); NCI-H146(+M); DMS 153(+M); NCI-H2029(+M); NCI-H1417 (M); NCI-H2030(+M); NCI-H1703(+M); NCI-H1650(+M); NCI-H596(+M); NCI-H1395;A-427(+M); NCI-H510(+M); NCI-H526(+M); NCI-H838(+M); NCI-H226(+M);

7. Liver cancer (4): BEL-7402; BEL-7404; SMMC-7721; Hep 3B2.1-78. Melanoma (2): A375; A20589 Nasopharyngeal (1): CNE9. Nasopharyngeal (1): CNE10.Neuroblastoma (2): BE(2)-C (+M); SH-SY5Y11. Ovarian cancer (4): OVCAR-3(+M) ; A2780; Caov-3; SKOV-3 (+M)12. Pancreatic cancer (6): MIA PaCa-2; Capan-1; PANC-1; AsPC-1;CFPAC-1; BxPC-3(+M)13. Prostate cancer (3): PC-3; LNCaP (+M); DU14514. Leukemia (13): K562 (+M); HL-60(+M); MV-4-11; MOLT-4 (+M); Kasumi-1(+M); KU812(+M); THP-1(+M); TF-1(+M); HEL 92.1.7(+M);

SKM-1 (+M) ;NOMO-1; ARH-77; Kopn-8SKM 1 (+M) ;NOMO 1; ARH 77; Kopn 815. Lymphoma (14): NAMALWA(+M) ; Daudi; Raji(+M); Mino(+M); DB(+M); Toledo(+M); SU-DHL-6(+M); MC116; OCI-Ly19; WSU-DLCL2; Z-

138; REC-1; Granta-519; RPMI 6666; Pfeiffer 16. Myeloma (4): RPMI 8226 (+M); MM.1S(+M); NCI-H929; U266B1(+M)17. Medulloblastoma (1): Daoy (+M)18. Bladder cancer (2): RT112; HT-1376(+M)19. Fibrosarcoma (1): HT-1080( )20. Medullary thyroid carcinoma (1): TT21. Hypopharyngeal (1): FaDu22. Cervical adenocarcinoma(1): Hela23. Mesothelioma (2): MSTO211H; NCI-H28

Blue font: NSCLC; Red font: SCLC

+M, Matrigel usedBreast cancer (1): MDA-MB-231 (+M)

Orthotopic model (3) Liver cancer (1): Hep 3B-Luc; Glioblastoma (1): U87MG (survival)

Systemic-survival model (12)

Leukemia (6): HL-60; K562; MV-4-11; HEL 92.1.7; KU812; THP-1;Lymphoma (3): NAMALWA; Daudi; RajiMyeloma (1): NCI-H929 Neuroblastoma (1): BE(2)-C


Neuroblastoma (1): BE(2) C Liver cancer (1): Hep G2-Luc

In Vivo Live ImagingXenogen Lumina XR

Human Primary +

Xenogen Lumina XRCancer cell lines (CDX)expressing luciferase

yTumor primary tumor

CellsRFP

Lenti‐virus

Orthotopic Gastric Cancer Model

PDX

Tumor growth80

Vehicle106 p

/s])

Orthotopic Gastric Cancer Model

Vehicle

20

40

60 DDP 5 mg/kg Q7dIrrinotecan 100 mg/kg Q7d

e (T

otal

Flu

x [

DDP

10 20 30-20

0

Days post administration

Tum

or v

olum

Irinotecan


Patient Derived Xenograft Models: Genetic Annotations

Cancer type Number of established models Sequenced

HCC 38 28 (WGS+RNAseq)Cholangiocarcinoma 16 13 (RNAseq)Pancreatic cancer 34 30 (WGS+RNAseq)( q)Colorectal cancer 75 34 (WGS+RNAseq)Gastric cancer 33 0Lung cancer 23 NSCLC +3 SCLC 13+3 (RNAseq)

RNA‐seq will be completed for all models g ( q)

Esophageal cancer 25 0Head & Neck 9 0

Gall bladder cancer 2 1

soon.

Ovarian cancer 4 0Kidney cancer 1 0Breast Cancer 2 0

Endometrial Cancer 2 0Cervical Cancer 3 0


Patient-Derived Cell Lines and Applications SNP

Cell panel screen

2‐3 wksLIX012 LIXC012

SNPs

One of the proprietary PDX related cell line

172914145

sensitive validation biomarkers

Proprietary cell line derived xenograft: PK/PD + efficacy + biomarker

1.5 months

Original PDX model: PK/PD + efficacy + biomarker6‐9 months


Immuno-Oncology Platforms

Immune‐Modulating Target

•Chemical synthesis Compound•Hybridoma•Chemical synthesis

•MedChemCompound generation

•Target specific assays: enzyme cell based Compound

•Phage display•hIg transgenic mice

Ab generation

•Binding assaysLead enzyme, cell‐based• Immune functional assays

ppool

• Syngeneic models•Combination therapies Early leads

g y• Target specific assays

Lead identification

•HumanizationLead•Combination therapies

•Ex vivo analysesEarly leads

•hPBMC assaysA ti ifi it

Lead(s)

•Affinity maturationoptimization

•MLR& other PBMC assays• In vivo efficacy(humanized

Lead characterization •Antigen specificity

( )

Candidate

y(mouse model)

characterization

Candidate


In Vitro Immuno-Oncology Assay Panel

ID Assay Type Cell Type In CP Panel1 Biochemical Assays Cell‐free √

2 Mixed Lymphocyte Reaction (MLR) Primary human DC + T cells √cells

3 T cell activation assay (anti‐CD3/CD28) Primary human T cells √4 T cell activation assay (SEB superantigen) Primary human T cells √

5 T cell activation assay (engineered tumor cells) Primary human T cells + engineered t mor cells √y ( g ) engineered tumor cells

6 CMV antigen recall assay Primary human T cells √7 Target specific reporter gene assay Engineered cell lines √8 ELISpot Primary human PBMC √

9 T cell cytotoxicity assay Primary human T cells + tumor cells In development

10 Antibody Dependent Cellular Cytotoxicity (ADCC) Primary human NK cells √11 Complement Dependent Cytotoxicity (CDC) Complement √11 Complement Dependent Cytotoxicity (CDC) Complement √12 Treg differentiation assay Primary human T cells √13 Treg differentiation assay Primary mouse T cells √14 Th1 polarization assay Primary human T cells √15 Th2 polarization assay Primary human T cells √16 Th17 polarization assay Primary mouse T cells √

17 Macrophage differentiation/ polarization assay Primary mouse macrophages √


p g

Mixed Lymphocyte Reaction (MLR)

MLR IFN Dose-Response60000

MLR IL-2 Dose-Response300

40000

00.01 ug/ml0.03 ug/ml0.1 ug/ml0.3 ug/ml1 ug/ml

Antibody Conc.

pg/m

l200

g/m

l

20000

1 ug/ml3 ug/ml10 ug/ml

IFN

- p

100IL-2

pg

Dendritic cells derived from primary human monocytes are co‐cultured with allogeneic CD3 T cells for 5 days. IFN‐and IL 2 production were measured to evaluate the effect of immune checkpoint inhibitors on MLR

Ab1 Ab2 Ab3 Ab4 Ab5 Ab60

Ab1 Ab2 Ab3 Ab4 Ab5 Ab60


and IL‐2 production were measured to evaluate the effect of immune checkpoint inhibitors on MLR.

Combination with Immune Checkpoint Inhibitors

SyngeneicM d l

Melanoma B16‐F10 (sc, iv, orthotopic, resection), B16‐F1, B16‐F0

Breast cancer 4T1 (orthotopic, resection), EMT6 (orthotopic), EpH4.1424, JC

Models Lung cancers Lewis lung carcinoma (sc, iv) Colon cancer CT26 (sc, orthotopic), MC‐38Renal cancer RENCA , RAG (sc, orthotopic)Leukemia/myeloma A20 (iv, sc), EL4, L1210, P815Liver cancer Hepa 1‐6 (sc, orthotopic), H22Fibrosarcoma WEHI 164, M7Reticulum cell sarcoma J774A.1

Tumor Growth - CT26 Model

2000

2500 IgG control

Compound X

PD-L1mm

3 )

Survival - CT model

100

val

1000

1500aPD-L1+Cpnd X

or v

olum

e (m

50 Vehicle ControlNormal IgG

**

rcen

t sur

viv

0 5 10 15 20 250

500

D t d i i t ti

Tum

o

0 20 400

Normal IgGPD-L1Compound XPD-L1+Compound X

***Pe

r


Days post administration Days post administration

Ex Vivo Immuno-Profiling: Tregs

Tumor Tumor‐Draining Lymph Node

Total Lymphocytes

CD4 T cells CD4 T cells

CD4CD4 CD4

l llTregs

TregsTotal CD4+ cells

FoxP3 F P3


FoxP3 FoxP3

Case 1: an IS Project

1 Early lead CpdKick off

Month 0 Month 1 Month 2 Month 9

Goal• Chemistry• Protein production• Biochemistry Assays

• Chemistry: ~50 cpds• Protein: active protein delivered

• BioChemistry Assays:

• Crystal Structures: Crystal structure with Ref

• Chemistry: ~500 cpds; grams of scale‐ups

• Protein: supply for all biochemical assays• Biochemistry Assays

• Cellular Assays• hPBMC Assays• Syngeneic tumor

on‐line• Cellular Assays: basic assay on‐line, build monoclonal

• BioChemistry Assays: near 1,000 cpds; MOA

• Cellular Assays: near 500 cpds

models• ADME Assays• PK/PD • Crystal Structures

• hPBMC Assays: Optimization

• Efficacy & PK/PD: 1ststudies w/ ref.

• Efficacy & PK/PD: multiple cpds; combination

• ADME Assays: multiple cpds testedCrystal Structures • ADME Assays

• Crystal Structures• Crystal Structures: 3 solved, more crystalized

• Initial safety• Key in vivo assays


Case 2: Pharmacology for a Lead Compound

A l d d

Efficacy in orthotopic 4T1 model,

MOA: immune

Combination efficacy

Immune memory &

A lead cpd w/ good PK, efficacy in 1 CDX model 1st i M i C bi i R h ll

1o tumor, metastasis response Specificity

1st syngeneic model

More syngeneic & nude mice

Combination Re‐challenge

CT26 Tumor growth-118 days after 1st inoculation of CT26Orthotopic 4T1 Model Tumor growth in nude mice

(4T1 cell line)Tumor growth

(CT26) y

500

1000

1500Naive mouseRechallenge mouse

Tum

or v

olum

e (m

m3 )

0

500

1000

1500

2000 VehicleCPD X,50mg/kgCPD X,100mg/kgCPD X,200mg/kg

***

******

Tum

or v

olum

e (m

m3 )

(4T1 cell line)

0

500

1000

1500

2000vehicle controlCPD X, 50mg/kgCPD X, 100mg/kgCPD X, 200mg/kg

Tum

or v

olum

e(m

m3)

( )

500

1000

1500

2000

2500 IgG control

Compound X

PD-L1

PD-L1+Cpnd X

Tum

or v

olum

e (m

m3 )

0 5 10 15 20 250

Days post inoculation

0 10 20 30 400


Lung Weight

1.5

###

****

***

g)

Tumor growth(CT26)

800 Vehicle Control

CPD X 25mg/kgm3 )

0 10 20 300

Days post administration0 5 10 15 20 25

0


CT26 model

100al

A20 Tumor growth-3 months after 2nd challenge of CT26

2000Naive

Rechallenged micem3 )

vehicl

ePD X

, 50m

g/kg

DX, 1

00mg/kg

DX, 2

00mg/k

g

naive

0.0

0.5

1.0

Lung

wei

ght(

g

0 5 10 15 200

200

400

600CPD X, 25mg/kgCPD X, 50mg/kg

CPD X, 100mg/kgCPD X, 200mg/kg

Tum

or v

olum

e (m

m

0 20 400

50 Vehicle ControlNormal IgGPD-L1Compound XPD-L1+Compound X

***

**


Perc

ent s

urvi

va

10 15 20 25 300

500

1000

1500Rechallenged mice

NS

NS, not statistically significant;

Days post inoculation

Tum

or v

olum

e (m

m


CPD

CPD X

CPD X Days post administration Days post administration NS, not statistically significant;Repeated measurement Two-way ANOVA

Thank You!Thank You!


ChemPartner Oncology May 2017 - CPhI Online Oncology-file076520.pdf · Assays by Target Type GPCR...

Documents

Transcript of ChemPartner Oncology May 2017 - CPhI Online Oncology-file076520.pdf · Assays by Target Type GPCR...