Juyoun Jin, D.V.M., Ph.D.

Institute for Refractory Cancer Research, Samsung Medical Center

Animal Models for Translational Research

How to apply preclinical translational research

New trial concept of personalized drug

development

Overview of Anticancer Drug Development

IND NDA

Synthesis and Formulation Development

Animal Models

for Efficacy

Assay

Development

Animal PK and

PD

Dose

Escalation

and Initial PK

Proof of

Concept and

Dose Finding

Large Efficacy

Trials with

PK Screen

PHASE I

Non-Clinical Development Clinical Development

PK/PD Studies in Special

Populations

PHASE IV

Discovery Non-clinical development Clinical Trial

Target

Identification

& Validation

Lead

Optimization

PHASE II PHASE III

Ideal Animal Model for Cancer Therapy

Validity

Selectivity/Specificity

Predictability

Reproducibility

Similarity

“There is no perfect tumor model”

Spontaneous tumors

Idiopathic

Carcinogen-induced

Transgenic/gene knockout animals: p53, RB, etc

Transplanted tumors

Animal tumors syngenic: Lewis lung, S180 sarcoma, etc

Human tumor xenografts:

Human tumor lines implanted in immunodeficient mice

(current NCI standard in vivo efficacy testing system)

Animal Model in Cancer

Syngeneic vs xenograft model

Human cancer cell

Immunedeficient animals

Tumor growth

Athymic “nude”mice developed in 1960’s

Human cancers grown in immune-deficient animals.

First human tumor xenograft of colon adenocarcinoma by

Rygaard & Poulson, 1969

Subcutaneous Xenograft model

Human Tumor Xenografts

SC

Immune-deficient animals

Athymic “nude” mice

Developed in 1960’s

Mutation in nu gene on chromosome 11

Lack thymus gland, T-cell immunity, Macrophage and NK cells are active

NOD-SCID (NOD.CB17-

Prkdcscid/NCrCrl) mouse

NOD scid Spontaneous mutant model was developed by the Fox Chase Cancer Center by transferring the scid mutation from a C.B-17 congenic background to a diabetes-susceptible non-obese diabetic background

T cell, B cell deficiency and depressed NK cell activity

NOG (NOD/Shi-scid/ IL-2Rγnull) mouse

New generation of severely immunodeficient mouse, Developed in 2000

No activity of T cell, B cell and NK cell, Dysfunction of macrophage, DC

Lack of NK cells, dendritic cell dysfunctions, and other unknown deficiencies due to inactivation of the IL-2Rγ gene

Efficacy Endpoints

Clonogenic assay

Tumor growth assay (corrected for tumor doubling time)

Treated/control survival ratio

Tumor weight change

Toxicity Endpoints

Drug related death

Net animal weight loss

Subcutaneous Xenograft Study Endpoints

Subcutaneous Xenograft Tumor Weight Change

Tumor weight change ratio

(used by the NCI in xenograft evaluation)

Defined as: treated/control x 100%

Tumor mass volume= (a x b2)/2

a = tumor length

b = tumor width

T/C < 40-50% is considered significant

length

Width

Caliper

Subcutaneous Xenograft Advantages

Many different human tumor cell lines transplantable

Wide representation of most human solid tumors

Allows for evaluation of therapeutic index

Good correlation with drug regimens active in human lung,

colon, breast, and melanoma cancers

Subcutaneous Xenograft Disadvantages

Different biological behavior, metastases rare

Survival not an ideal endpoint:

death from bulk of tumor, not invasion

Shorter doubling times than original growth in human

Difficult to maintain animals due to infection risks

Increasing unmet medical need of developing cancer therapeutics

Increasing new anticancer drugs under R&D projects in pharmaceutical companies

Current subcutaneous xenograft models do not translate the clinical outcome

Need to develop clinically relevant organ-specific orthotopic tumor models to

develop effective targeted therapies

Unmet Need for Translational Research in Cancer Therapeutics

교모세포종/뇌전이암

대장암

유방암

폐암 방광암 전립선암

Are subcutaneous models adequate?

Subcutaneous Model

Is this representative the actual tumor

- Microenvironment? - Heterogeneity ?

The effect of paclitaxel and p-GP inhibitor combination therapy on tumor growth in subcutaneous tissue and brain metastasis tumor model.

Even though the tumors are originated from same cell,

the therapeutic responses are different according to the

tumor bearing organ.

In subcutaneous model vs. In brain metastasis model

Advantages and Disadvantages of Orthotopic Model

Advantages

Resembles the original tumors morphologically, biologically and

biochemically

Important for the research of cancer metastasis

Short-term screening of variable cancer therapy strategy

Disadvantages

Necessary to have skillful technique

Wide variation

Types of murine model for studying human cancers.

ADVANTAGES DISADVANTAGES

Allows for rapid analysis of human tumor response to a therapeutic regime

Can predict the drug response of a tumor in a human patient

Provides realistic heterogeneity of tumor cells

Mice are immuno-compromised, providing a less realistic tumor microenvironment

Appropriately mimics human tumor microenvironment

Can predict the drug response of a tumor in a human patient

Provides realistic heterogeneity of tumor cells

Expensive Technically complicated

Tumor exists in the presence of competent immune system (realistic microenvironment)

Defined mutations can mimic those identified in human tumors

Can follow tumor development from early time points

Targets a limited number of genes which is usually not reflective of the complex heterogeneity of human tumor cells

Development is costly and time consuming, often requiring years of work before validation

Tumor development in animals is slow and variable

Disease Models & Mechanisms 1, 78-82 (2008)

http://www.accessdata.fda.gov/scripts/cder/onctools/animalquery.cfm

Oncology Tools: Dose Calculator (Human vs Mouse)

Novel device for the translational research

7 mice injection/30min 1 mice injection/30 min

VS.

• Device invented for the translational research for the brain tumor orthotopic model • Cells with same condition were injected into seven mice simultaneously

Brain Tumor Orthotopic Model- Intracranial injection

Experimental Design For In Vivo Study ex. Brain tumor Orthotopic Model

2 X 105 U-87MG cells I.C. implantation

1 W 2 W 3 W 4 W 0 W

Tumor volume measurement (B)

21~25d Survival length (C)

I.C.injection of Human GBM cells

Treatment of

test agents

1. Measurement of tumor volume 2. Survival length 3. IHC study (PCNA, TUNEL) 4. IHC (Target validation) 5. Distribution study 6. Measurement of body weight

Tumor volume IHC (PCNA/TUNEL) IHC

(Target vali.) Distribution Survival length

Ex. Anti-tumor effects of TMZ in U-87MG Human GBM Orthotopic Mice Model

Ex. Anti-tumor effects of Radiotherapy in U-87MG Human GBM Orthotopic Mice Model

Brain Metastases Model- Internal Carotid Artery injection

Lung Cancer Brain Metastases Model (Ideal Cell Dose)

Cancer Cell line Type of cancer EGFR K-ras Cell dose Survival

Lung Cancer Brain

Metastases

A549 NSCLC, Adenocarcinoma w/t Mut (G12S) 5X105 8W

H460 NSCLC, LCC w/t Mut (G61H) 5X103 3W

PC14PE6 NSCLC, Adenocarcinoma E746-A750del w/t 1X104 3W

H23 NSCLC, Adenocarcinoma w/t Mut (G12C) 5X105 5W

H1299 NSCLC, LCC w/t w/t 5X105 6W

H460

Group Median

Survival Day

I H460 (5X103) 150

II H460 (5X104) 51

III H460 (5X105) 36

5X103

5X104

5X105

1X104

1X105

1X106

PC14PE6

Group Median

Survival Day

I 1X104 21

II 1X105 14

III 1X106 10

Whole Brain Radiotherapy for Brain Metastases

Brain Metastases Model- Left ventricle injection

Bra

in After 8 weeks…

Cancer Cell line Type of cancer Site of Implantation Cell dose Survival

Lung cancer RFP-labeled A549 NSCLC Left Ventricle 1 X 106 8 weeks

CANCER RESEARCH 52. 2304-2309, April 15, 1992

Breast Cancer Orthotopic Model – Mammary Fat Pad

Tumor mass inoculate to 4th MFP Cell injection into 2nd MFP

Tumor mass Mammosphere

Lung Cancer Orthotopic Model – Left lung parenchyma

Single cell suspension

Cell implantation

Cell injection into Lung

Left lung : One single lobe Right lung: Cranial, middle, caudal and accessory lobes.

Colon Cancer Orthotopic Model – Cecal wall

Cell injection Mass implantation

Gastric Cancer Orthotopic Model

Incision : edge of the rib cage near the chest Draw out the stomach and injection or implantation into the stomach wall

Procedure

Cancer Cell line Type of cancer Site of

Implantation Cell dose Survival

Gastric cancer SNU-16 Human gastric carcinoma Stomach wall 2X106 5 weeks

Prostate Cancer Orthotopic Model

Procedure

Histopathology (H&E)

Cancer Cell line Type of cancer Site of

Implantation Cell dose Survival

Prostate cancer

PC-3 Human prostate adenocarcinoma

Right dorsal lobe

5 X 105 10 weeks

Pancreatic Cancer Orthotopic Model

Colorectal Cancer Liver Metastasis Model

Cancer Cell line Type of cancer Site of

Implantation Cell dose Survival

Colorectal cancer

HCT116 Human colorectal

carcinoma Spleen 2X106 7 weeks

Liver metastasis

50 mm

Spleen

100 mm

Head

Tail

T

T

T: Tumor region

Bone metastatic model by Intracardiac injection

CANCER RESEARCH 52. 2304-2309, April 15, 1992

After 8 weeks…

Cancer Cell line Type of cancer Site of Implantation Cell dose Survival

Lung cancer RFP-labeled A549 NSCLC Left Ventricle 1 X 106 8 weeks

Hind leg paralysis

Knee joint

Hip joint Pelvis

0W 1W 2W 3W 4W 5W 6W 7W 8W 9W 10W 11W 12W

1 X 106 MDA-MB-435 LvBr1 cells M.F.P. implantation

After primary tumor formation (1.3~1.5 cm), tumor resection perform

Pulmonary metastases mesurement

Spontaneous Breast Cancer Lung Meta Model

Ex. Effects of Erlotinib in Spontaneous Breast Cancer Lung Meta Model

Tumor pathology:

Gross and histological observation for measurement of tumor progression,

metastasis, and target modulation.

YJ Choi et al, Oncology Report 16:119122, 2006

Single cell suspension I.V. injection of

B cell lymphoma cells

Mouse: NOD/Shi-scid/IL-2Rγnull (NOG)

Disseminated Lymphoma model – Intravenous injection

Ex. In vivo preclinical efficacy of Rituximab in Disseminated model

50-60 human derived cancer cell line Brain tumor (U87-MG, U373-MG, U251- MG….) Breast cancer cell line (MDA-MB-435, MDA-MB-231, MCF-7….) Colon Cancer (Lovo, SW480, Colo205, HT29, HCT116…..) Lung Cancer (PC14-PE6, A549, H23, H460…….) Lymphoma (Raji, Ramos, Daudi, BJAB, Toledo, SKW 6.3…….) Other Cancer Cell lines..

Subcutanous Xenograft Model

Experimental Design: Xenograft model (S.C.)

Athymic nude mice

S.C. injection of

Human cancer cells

Control Test

Several days

(After tumor formation)

Treatment of test agents

• Measure Tumor size • Measure Body weight

Tumors

Extract Protein/RNA - Target Validation

Make Tissue Slides - H&E, IHC - Target Validation

Distribution study - Optical imaging • Measure Tumor

weight

In vivo optical imaging and PET imaging

In vivo optical imaging PET imaging

In vivo optical imaging and PET imaging

Metronomic (low dose, multiple times)

temozolomide chemotherapy for GBM

Example #1

Glioblastoma multiforme (GBM)

Grade 4 astrocytoma (WHO classification) Most common primary brain tumors High degree of morbidity and mortality Combination of surgery, radiation therapy, and chemotherapy Poor prognosis and GBM patients die within 1 year Novel therapeutic approaches to treat gliomas are needed

1. DAVIS, F.G et. al, Neurooncol. 2001, 3, 152–158. 2. HOLLAND, E.C. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 6242–6244.

• Preclinical study (2004) – published

• Pilot clinical trial (2005) – published

• Phase II clinical trial (2006~2008) – Neuro-Oncology

• Example of successfully translating from preclinical to clinical trials

• First to show benefit of metronomic in vitro, in vivo, and clinically

Metronomic (low dose, multiple times) temozolomide chemotherapy

Metronomic chemotherapy is…

• More effective in tumor volume reduction

• Anti-tumorous via anti-angiogenic and pro-apoptotic activities

Mic

rove

ssel D

ensity

Preclinical Data

Phase II clinical trial

• Depletion of MGMT by continuous TMZ

• Anti-angiogenic treatment

• Increment of total dose of TMZ

Anti-tumor activities of

human cytokine-induced killer cells

against glioblastoma

Example #2

Immunotherapy for the treatment of cancer

CD3

CD56

NK cell CIK

LAK

Immune cell-based cancer therapy

: Eliminate cancer cells through the transfer of ex vivo expanded and activated immune cells.

Active immune cell for the cancer therapy • Cytotoxic T lymphocyte (CTL) (Wang et al., 2006b)

• Cytokine-induced killer (CIK) cells (Thorne et al., 2006)

• Lymphokine-activated killer (LAK) cells (Takashima et al., 2006)

• Dendritic cells (DC) (Kalinski et al., 2006)

• Natural killer (NK) cells (Raja Gabaglia et al., 2007)

CIK cells

• Characterized by the expression of CD3 and CD56 molecules

• Most potent cytolytic activity (Baker et al., 2001; Lu and Negrin, 1994)

Human CIK cell Production

hPBMC

Lymphocyte Culture with IL-2, CD3

hCIK; Activated T cell

After 14 days

Pre-clinical efficacy test

Ex vivo expansion

hCIK; Activated T cell U-87MG GBM orthotopic xenograft model

Characterization

The phenotypic characterization of hCIK cells

Molecular characteristics of hCIK cells

(A) CD3, CD8 and CD56 expression of cultured hCIK cells were analyzed by flow cytometry.

(B) Phenotypes of fresh PBMC and CIK cells were compared.

Data are expressed as the mean ± SE of eight separate experiments. *** P < 0.001.

Cytotoxicity of hCIK against U-87MG

hCIK cells are cytotoxic against human GBM cells in vitro.

U-87MG human GBM cells were incubated for 4 h with fresh PBMC or

14-day cultured hCIK cells (effector-to-target ratios = 10:1 or 30:1).

Cytotoxicities of PBMC and hCIK cells were compared by the LDH

assay. Data are expressed as the mean ± SE. ***P<0.001.

Efficacy study of hCIK in GBM xenograft model

2 X 105 U-87MG cells I.C. implantation

1 W 2 W 3 W 4 w 0W

Tumor Volume Measurement Intravenous injection of Human CIK cell

hCIK cells inhibit GBM tumor growth in an orthotopic xenograft model.

The timeline for the assessment of in vivo anti-tumor activities of hCIK cells in an orthotopic xenograft model.

hCIK cells reduced U-87MG tumor volumes in a dose-dependent manner.

Efficacy study of hCIK in GBM xenograft model

2 X 105 U-87MG cells I.C. implantation

1 W 2 W 3 W 4 w 0W

Tumor Volume Measurement Intravenous injection of Human CIK cell

hCIK cells inhibit GBM tumor growth in an orthotopic xenograft model.

hCIK cells were traced by immunohistochemistry using a human CD3 specific antibody.

Combinational treatment of hCIK cell and TMZ

2 X 105 U-87MG cells I.C. implantation

21~25d Intraperitoneal injection of TMZ (2.5 mg/kg)

1 W 2 W 3 W 4 w 0W

Tumor Volume Measurement Intravenous injection of Human CIK cell

Immunotherapy using hCIK cells potentiates anti-tumor

effect of TMZ.

The timeline for the assessment of in vivo therapeutic effects of

combinational treatment of hCIK cells and TMZ in an

orthotopic xenograft model.

hCIK cells and TMZ created addictive or synergistic therapeutic

effects in the U-87MG human GBM animal model.

Decreased tumor cell proliferation (PCNA)

Control CIK

TMZ CIK+TMZ

40.0

50.0

60.0

70.0

80.0

90.0 Control

CIK 1X10^7

TMZ 2.5

TMZ + CIK 10^7

* P< 0.05

Pe

rce

nta

ge o

f P

CN

A-p

osit

ive

ce

lls

per

tum

or

se

cti

on

* P< 0.01

+ P< 0.05

Tumor cell proliferation (PCNA) is altered by either hCIK, TMZ, or hCIK + TMZ treatment in vivo.

Proliferating cells was analyzed by anti-PCNA antibody in tumor masses.

Numbers of PCNA- positive cells was calculated and compared

Increased tumor cell apoptosis (TUNEL)

Control CIK

TMZ CIK+TMZ

Nu

mb

er

of

TU

NE

L-p

osit

ive

ce

lls

per

tum

or

se

cti

on

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0 Control

CIK 1X10^7

TMZ 2.5

TMZ + CIK 10^7

* P< 0.05

* P< 0.01

+ P< 0.05

Tumor cell apoptosis (TUNEL) is altered by either hCIK, TMZ, or hCIK + TMZ treatment in vivo.

Apoptotic cells was analyzed by TUNEL assay in tumor masses.

Numbers of TUNEL-positive cells was calculated and compared

Schedule Dependent Synergistic Effect of

Rituximab on the Methotrexate

Chemotherapy against CNS Lymphoma

Example #3

• Malignant lymphoid neoplasm restricted

at presentation to the brain, spinal cord or meninges

• Histopathology • Aggressive non-Hodgkin’s lymphoma (NHL)

• Infiltrate walls of cerebral vessels

Patients with compromised immune systems

Patients who are receiving immune suppressive therapies

Patients who are having biopsies

Incidence of CNS lymphoma

Primary CNS Lymphoma (PCNSL)

• Radiotherapy (RT)

• Chemotherapy; high dose (HD)-methotrexate (MTX)

• Combinational treatment of RT and HD-MTX

• Immunotherapy; Rituximab, Zevalin, Bexxar, etc.

Treatment failure • Frequently multifocal infiltration of CNS proper

• Leptomeningeal dissemination (25 ~ 30% of patients)

Treatment of Primary CNS Lymphoma

• Monoclonal anti CD20 antibody

• CD20 is a cell surface receptor present on all B lymphocytes

• Rituximab (Rituxan) binds to CD20 and eventually leads to cell lysis

• Very well tolerated drug, infusion reactions are possible

Application for the treatment of CNS lymphoma is still controversial because of the BBB!

Rituximab(RTX)

Research Overview

Establishment of

Group Cell dose

Ⅰ 5*104/5 ul

Ⅱ 5*105/5 ul

Ⅲ 5*106/5 ul

I.C. injection

Primary CNS Lymphoma Animal Model

Intracranial injection of Raji cells

Establishment of

Features of histopathology Perivascular cuffing Leptomeningeal seeding

IHC(anti-CD20)

Primary CNS Lymphoma Animal Model

RTX-AF680

In vivo optical imaging

Quantification of Penetration of RTX into CNS Lymphoma

Penetration of RTX across the BBB or the blood tumor barrier was significantly increased by changing the treatment order from MTX + RTX to RTX + MTX.

Quantification of Penetration of RTX into CNS Lymphoma

RTX treatment followed by MTX administration showed significantly reduced tumor volume.

Evaluation of Antitumor Activity

Bioequivalent Efficacy Study of

Similar-Rituximab in Lymphoma model

Example #4

Genentech Our Lab

Compare parameter Genentech (Patent No. 5,843,439) Our Lab

Animal model Ramos Lymphoma Xenograft Ramos Lymphoma Xenograft

RTX Conc. 200 ug/mouse 200 ug/mouse

TX schedule & Route Once a week, IV Once a week, IV

Mass size Size (width x length = mm2) Volume (width2 x length x 0.5 = mm3)

Mouse: Female Balb/c-nu, 6wks

In vivo Bioequivalent Efficacy Evaluation System for Rituximab in Human Lymphoma Xenograft Model

Efficacy study of Original Rituximab and Similar Rituximab in Ramos Lymphoma Model

0

500

1000

1500

2000

2500

3000

3500

4000

4500

24 26 28 30 32 34 36 38 40 42 44 46

CT (PBS)

O-RTX

S-RTX

Tum

or

volu

me (m

m^

3)

0W

1 X 107 Ramos cells S.C. implantation

1W 2W 3W 4W 5W

Intravenous injection of Rituximab After tumor formation (100mm3)

6W

Decrease of Proliferation in Ramos Tumors (PCNA Staining)

Control O-RTX S-RTX

50

70

90

110

130

150

170

190

210

230

250

Cont. G-RTX RTX S-RTX S-RTX

PC

NA

-po

sit

ive

cell

s p

er

tum

or

sec

tio

n

P<0.05

O-RTX

Increase of Apoptosis in Ramos Tumors (TUNEL Staining)

Control O-RTX S-RTX

0

5

10

15

20

25

30

Cont. G-RTX RTX S-RTX

TU

NE

L-p

os

itiv

e c

ell

s p

er

tum

or

sec

tio

n

P<0.05

O-RTX S-RTX

How to discover therapeutic target

Development of Radio-sensitizer for Brain Metastasis

Example #5

Metastatic Brain Tumors

Most common intracranial tumor

Incidence

3 ~ 11/ 100,000 person-year

Probability

20 ~ 50 % of all malignancy

Treatment

Supportive care with steroid

Surgical resection

Chemotherapy

Radiosurgery

Whole brain radiation

Importance

Important cause of death

Worst factor for quality of life

Mean survival without Tx: 1 month

Mean survival with Tx: extend only 4-6 months

Ref. Neuropathology (2004)

MRI scan of brain metastatic patient

79

Limitations of Current Treatments

Chemotherapy

https://rad.usuhs.mil

Blood Brain Barrier

Whole Brain Radiotherapy

80

• Dementia

• Ischemic stroke

• Brain atrophy

Sensitizing tumor cells to radiation

Radiosensitizers (chemicals or biological agents)

- increase the lethal effects of radiation on the tumor

- without causing additional damage to normal tissue

Ref. Nat Rev Cancer (2011) 81

Development of Radiosensitizer for Brain Metastasis

Brain Metastasis Animal Models

RT-resistant clone cDNA MicroArray Target molecule

In vivo

RT 5 Gy 10 Gy O Gy

Relevance of DNA damage checkpoint

signaling in prognosis of patient

Development of Radio-sensitizer for Brain Metastasis

Radiosensitizer : Chk1 inhibitor ??

Chk1 Knock- down

AZD7762

(ATP-competitive checkpoint

kinase inhibitor)