From Pharmacology to Drug Development~Preclinical Oncology Animal models~

1. Copyright 2018 1Hsiang-Wen Tseng 1Copyright 2018 Hsiang-Wen Tseng
From Pharmacology to Drug Development
~Preclinical Oncology Animal models~
Hsiang-Wen Tseng (曾湘文), Pharmacist, PhD.
January 11th, 2018

2. Copyright 2018 2Hsiang-Wen Tseng
Outlines
Preclinical study in Pharmaceutical
Industry
Animal models for oncology drug
development
 Immunodeficient mice and Humanized mice
 Human xenograft / Murine allograft tumor
models
 Human PDX xenograft
Subcutaneous/Orthotopic models
Other animal models for drug discovery
Pharmacology/Toxicology approaches
in Drug Discovery and Development

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https://www.slideshare.net/rahul_pharma/drug-discovery-and-development-10698574
Timeline of Drug Discovery and Development

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https://www.embodi3d.com/blogs/entry/340-how-3d-printing-is-changing-drug-discovery-and-testing/
Drug Discovery Process

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Pharmaceutical Discovery & Development
– Target selection

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Animal Study in Lead Optimization

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Animal Study in Non-Clinical Development and Toxicology Study

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Onco Targets Ther. 2016; 9:545-55.
Mouse models for Drug Discovery
Cell Mol Immunol. 2012; 9(3):208-14.

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The road to humanized SCID mice
(Brehm 2014)
1983
1995

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Immunodeficient mice for humanized models
Three strains of immunodeficient IL2rgnull mice are widely used:
 NOD.Cg-PrkdcscidIl2rgtm1Wjl(NSG) mice
completely lack the gamma chain
 NODShi.Cg-PrkdcscidIl2rgtm1Sug (NOG) mice
truncated cytoplasmic domain of the gamma chain
 C;129S4- Rag2tm1FlvIl2rgtm1Flv (BALB/c-Rag2null IL2rgnull mice or BRG) mice
completely lack the gamma chain
NSG™ Mouse Model Variants Model in Jackson Lab.
 NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (005557)
 NOD.Cg-PrkdcscidIl2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (013062)
 NOD.Cg-Rag1tm1MomIl2rgtm1Wjl/SzJ (007799)
 NSG-HLA-A2.1 (009617)/NSG-HLA-A2/HHD (014570)
 DR1 (012479)DR4 (017637)
 NSG B2m (010636)/NSG-(KbDb)null (023848)
Annu Rev Pathol. 2017 Jan 24;12:187-215
https://www.jax.org/jax-mice-and-services/find-and-order-jax-mice/nsg-portfolio

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Applications of the human immune system in the NSG mice
(http://jaxservices.jax.org/invivo/humanized-nsg.html)

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(Brehm 2014)
Humanized
mice
models

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Humanized mice in Jackson Lab
ONCO-HU® MODELS
 Onco-Hu® Models are a robust immuno-
oncology platform for efficacy testing of
novel immunotherapies targeting T cells and
myeloid cells to help destroy cancers in
vivo.
PDX LIVE™ TUMOR MODELS
 Get experimental data up to 80% faster with
PDX Live™ tumors. This valuable off-the-
shelf resource can save your project more
than 6-12 weeks time.
NSG™ VARIANTS PORTFOLIO
 Our study-ready NSG™ and NSG™-SGM3
mice are the most versatile immunodeficient
strains. Start your research today with our
readily-available models.
https://www.jax.org/jax-mice-and-services/in-vivo-pharmacology/humanized-mice

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Comparison of NSG™ Mouse Model Variants in Jackson Lab. (1)
Name & Stock Number
NOD.Cg-Prkdcscid
Il2rgtm1Wjl/SzJ (005557)
NOD.Cg-PrkdcscidIl2rgtm1Wjl Tg(CMV-
IL3,CSF2,KITLG)1Eav/MloySzJ (013062)
NOD.Cg-Rag1tm1Mom
Il2rgtm1Wjl/SzJ (007799)
Branded or common
name
NSG™(branded name), NOD scid gamma
NSGS, NOD scid gamma Il3- GM-SF
(NSG-SGM3)
NRG, NOD Rag gamma
Leakiness Very Low Absent Absent
Irradiation tolerance Low Low High
Lymphoma incidence Low Low Low
Benefits
•Engrafts the widest range of solid and
hematological cancers, including ALL
and AML
•Most sensitive host for cancer stem cells
when compared to NOD scid or nude mice
•Longer lifespan than NOD scid; supports
long-term engraftment studies and
capabilities; >89 weeks median
•Increased CD4+ FoxP3+ regulatory T
cell population
•Enhances human myelopoiesis and
terminal differentiation
•Increased efficiency of engrafting
human acute myeloid leukemia (AML)
•Long-term multilineage
hematopoeitic stem cell
repopulation similar to NSG mice
•Engrafts human PBMC without
irradiation similar to NSG
•Engrafts a wide range if solid and
hematological cancers
•Considerations
•No thymic lymphomas, can be used for
long & short-term experiments
•Sensitive to irradiation
•Compromised human stem cell
regeneration
•Suppression of human erythropoiesis
•Reduction of human B-lymphopoiesis
•No thymic lymphomas, can be
used for long & short-term
experiments
•Requires higher dose of irradiation
to obtain human HSC engraftment
Mature B cells: absent ; Mature T cells: Absent; Dendritic cells: Defective; Macrophages: Defective; Natural killer cells: Absent;
Complement: Absent
https://www.jax.org/jax-mice-and-services/find-and-order-jax-mice/nsg-portfolio

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Comparison of NSG™ Mouse Model Variants in Jackson Lab. (2)
Name & Stock
Number
NSG-HLA-A2.1 (009617)
NSG-HLA-A2/HHD (014570)
DR1 (012479)
DR4 (017637)
NSG B2m (010636)
NSG-(KbDb)null (023848)
Branded or common
name
HLA Class I-A2 Transgenics HLA Class II Transgenics MHC Class I-null NSG™
Leakiness Low Low Low
Irradiation tolerance Low Low Low
Lymphoma incidence Low Low Low
Benefits
•High engraftment of HLA-A2-
restricted immune cells
•Enable functional CD4+ T cell
responses to viral infection
(014570)
•Useful for transplantation
studies in the absence of xeno-
GVHD
•Develop allo-GVHD post-
engraftmentof DR4-negative
CD4+ T cells (017637)
•Resistant to xeno-GVHD (010636)
•Attenuated xeno-GVHD development
post-hPBMC transplantation (023848)
•High hCD45+ cell engraftment (023848)
•Useful for studying mechanisms for
xeno-GVHD
Considerations
•No thymic lymphomas - can be
used for long-term experiments
•Sensitive to irradiation
•Reduced CD45+ cell
engraftment compared to NSG
•Higher proportion of mice with
no CD45+ cell engraftment as
compared to NSG (017637)
•Reduced survival post hCD34+ cell
transplantation compared to NSG mice
(023848)
Mature B cells: absent ; Mature T cells: Absent; Dendritic cells: Defective; Macrophages: Defective; Natural killer cells: Absent;
Complement: Absent
https://www.jax.org/jax-mice-and-services/find-and-order-jax-mice/nsg-portfolio

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ONCO-HU® MOUSE MODELS
NSG™-SGM3 mice: human IL3, GM-CSF and SCF
https://www.jax.org/jax-mice-and-services/in-vivo-pharmacology/oncology-services/onco-hu

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PDX LIVE™
Type Model ID Tumor Markers
Bladder
TM00015 PIK3CA H1047R
TM00020
TP53 E336*; KDR Q472H; PTEN
T321fs
Breast
TM00089 TNBC ER-/PR-/HER2-; BRCA1 V757fs
TM00096 TNBC ER-/PR-/HER2-
TM00098 TNBC ER-/PR-/HER2-
TM00386 ER+/PR+/HER2-
Colon TM00170 BRAF V600E/PIK3CA G1049R
Lung
TM00302 KRAS G12D/KDR Q472H/TP53 R158L
TM00199 EGFR L858R
TM00206 EML4-ALK fusion
Ovary
TM00335 CA125 & MUC16 mRNA elevated
TM00916 ER+/PR+
Prostate
TM00298 TP53 R273C/PTEN R233*/PTEN
L265fs, AR+
Skin
TM00702
TM01149 POLB P242R, NF1 R1362

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Human Tumorgraft Model 價值及趨勢
•取代細胞株平台，正確判定
藥效減少新藥開發成本
•腫瘤分子特徵研究
•個人化藥物治療
•BC Cancer Research centre / Living Tumor Lab.
•The Jackson Lab.
•Crownbio. (liver cancer)
•ONCOTEST.
•GeneScript (liver cancer)
•Biopharmaceutical Research Inc. (BRI)
•XenTech
•Deshpande Lab.
http://www.livingtumo
rcentre.com/About_Us
1.html
Tumor line

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Oncology Databases of CrownBio
HuBase™
 A unique asset, to easily search for models that meet your criteria. An online
database containing the complete genomic annotation of our HuPrime PDX
models:
MuBase®
 Online database containing information for CrownBio’s immuno-oncology murine
efficacy platforms
XenoBase®
 The world’s first online database for cell lines and Cell Line Derived Xenograft
models, collating data from over 1,000 tumor cell lines.
OncoExpress™
 OncoExpress is newly launched search engine allowing clients to explore all
CrownBio models contained in HuBase, XenoBase, and MuBase, in just one
search. OncoExpress also includes model information such as growth curves,
standard of care pharmacological data, mutation, and copy number analysis.

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HuPrime®

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MiXeno™

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Patient-derived human hepatocellular carcinoma xenograft of Subcutaneous and
Orthotopic models for pharmacodynamic effects of sorafenib

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Immunotherapy:
Using the Immune System to Treat Cancer
Immune Checkpoint Modulators
Adoptive Cell Transfer
CAR (chimeric antigen receptor) T-Cell Therapy
TCR (T-cell receptor) Therapy
TIL (tumor-infiltration lymphocytes) Therapy
Therapeutic Antibodies & ADCs (antibody-drug conjugates)
Cancer Treatment Vaccines
Immune System Modulators: eg. interleukins and interferons
https://www.cancer.gov/research/areas/treatment/immunotherapy-using-immune-system#1

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Immune Checkpoint Inhibitors approved by FDA
Anti-CTLA4 Ab:
Ipilimumab (Yervoy; BMS, 2011)
Anti-PD-1 Abs:
Pembrolizumab (Keytruda; Merck, 2014/9/4）
Nivolumab (Opdivo; BMS, 2014/12/22）
Anti-PD-L1 Abs:
Atezolizumab (Tecentriq; Roche, 2016/5/18）
Durvalumab (Imfinzi; AstraZeneca, 2017/5/1）
Avelumab (Bavencio)（Merck / Pfizer，
2017/5/9）
Journal of Immunology Research Volume 2017 (2017)

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In vivo PD study of Ipilimumab (Yervoy)
BMS-734016, BMS-663513_Peripheral blood long term immunophenotyping in
cynomolgus monkey
Ipi: Monoclonal antibody adjuvants for the improvement of DNA vaccines
Ipi: Unstaged MC38 tumors in hCTLA-4 Tg mice
9D9 and dexamethansone in a therapeutic SAI/N tumor model
9D9, MDC-1106 (anti-mouse PD-1 Ab) in murine MC38 colon model
9D9, MDC-1106 in murine CT26 colon model
9D9, MDC-1106 in murine SA1/N fibrosarcoma model
9D9, MDC-1106 in murine B16-F10 melanoma and J558 myeloma models
BMS-863019, anti-CD137 Ab in murine tumor models
 P815 in DBA/2
 SA1/N in A/J mice
 EMT-6 in BALB/c mice
 B16-F10-Lu in B6 mice
From: FDA NDA Documents of Pharmacology Review

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In vivo PD study of Atezolizumab
(Tecentriq; Roche, 2016/5/18）
Syngeneic MC38.OVA colorectal model in C57BL/6 mice
Syngeneic CT26 colorectal cancer model in B/c
Syngeneic Cloudman S91 melanoma model in DBA/2 mice
Syngeneic MC38 colorectal model in C57BL/6 mice
Chronic LCMV CL-13 infection in mice
MC38.OVA CT26 Cloudman
S19
MC38
From: FDA NDA Documents of Pharmacology Review

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Bispecific antibody (BsAb)
Bi-specific T-cell engagers (BiTEs)
Catumaxomab (Removab® , EMA 2009, Trion Pharm):
EpCAM-CD3
Blinatumomab (BLINCYTO® , AMG103, MT103, FDA 2014):
CD3-CD19
Ertumaxomab (Rexomun): HER2/neu-CD3
FBTA05 (Lymphomun): CD20-CD3
TRBS07 (Ektomab): GD2-CD3
Emicizumab (Hemlibra, factor Ⅸa-Ⅹ, FDA 2017,
Roche)_haemophilia A
Other drugs: IL-17°-IL-23、IL-1°-IL-1β、CD19-CD3

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Blinatumomab (BLINCYTO® , AMGEN)
Bi-specific T-cell engagers
(BiTEs), CD3-CD19, that exert
action selectively and direct
the human immune system to
act against tumor cells.
Blinatumomab specifically
targets the CD19 antigen
present on B cells.
In December 2014 it was
approved by the US Food and
Drug Administration under the
accelerated approval program;
marketing authorization
depended on the outcome of
clinical trials that were ongoing
at the time of approval.

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J Immunol. 2014 Nov 1;193(9):4739-47
blinatumomab-expanded T cells (BET)

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Antibody-Drug Conjugates
Gemtuzumab ozogamicin (Mylotarg® , Pfizer/Wyeth, FDA 2001)
 Anti-CD33-calicheamicin
 acute myelogenous leukemia
 2010 withdraw
Brentuximab vedotin (Adcetris® , Seattle Genetics and Millennium/Takeda, FDA
2011, EMA 2012)
 Anit-CD30-valine-citrulline-MMAE
 relapsed HL and relapsed sALCL
Trastuzumab emtansine (Kadcyla/T-DM1, Genentech and Roche, FDA 2013)
 Anti-Her2-mertansine
 HER2-positive metastatic breast cancer (mBC)
Inotuzumab ozogamicin (Besponsa, Celtech/Wyeth, FDA, EMA 2017)
 Anti-CD22-ozogamicin
 refractory CD22-positive B-cell precursor acute lymphoblastic leukemia (ALL)

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Developmental Cancer Animal Models in ITRI
Head and Neck:
RPMI 2650, HSC-3, FaDu, Detroit 562
Hepatocellular carcinoma:
Huh-7, SK-Hep1, HA22T/VGH, and
PLC/PRF/5
4 PDXs and BNL 1MA (murine)
Leukemia:
MV4-11, RS4-11 and MOLM-13 Lung cancer:
NCI-H460, A549, NCI-H226, NCI-H520
Breast cancer:
BT-474, MDA-MB-453, MCF-7, MDA-
MB-231
Gastric cancer:
SNU-1, NCI-N87, TMC-1, TSGH and
TSGH-S3
Colon cancer:
COLO 205 and HT-29
Pancreatic cancer:
PANC1 and MIA-PaCa-2
Thyroid carcinoma: TT
Melanoma (murine):
B16, B16-F10, and B16-F10-Luc2

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Other disease animal models
糖尿病動物模式(Diabetes animal models): db/db第二型糖尿病小
鼠(db/db mice)
非酒精性脂肪肝動物模式 (Non-Alcoholic Fatty Liver Disease,
NAFLD)
慢性傷口癒合動物模式(Chronic wound healing models)
馬兜鈴酸誘發腎臟傷害/腎病變動物模式(Renal injury
/nephropathy models (aristolochic acid induction)
血管新生動物模式 (In vivo angiogenesis models)
肌少症動物模式 (Sarcopenia/muscle wasting models)

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DMPK/non-GLP Tox study for Drug Discovery
DMPK study
Drug formulation
Dosing frequency
PK/PD correlation study of biomarker
Non-GLP toxicology study
Acute toxicity
Subacute toxicity
Targeting organ toxicity

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