Juyoun Jin, D.V.M., Ph.D. Institute for Refractory Cancer Research, Samsung Medical Center
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1 Juyoun Jin, D.V.M., Ph.D. Institute for Refractory Cancer Research, Samsung Medical Center
2 Animal Models for Translational Research How to apply preclinical translational research New trial concept of personalized drug development
3 Overview of Anticancer Drug Development Discovery Non-clinical development Clinical Trial Target Identification & Validation Lead Optimization IND Synthesis and Formulation Development PHASE I PHASE II Animal Models for Efficacy PHASE III NDA Assay Development Animal PK and PD Dose Escalation and Initial PK Proof of Concept and Dose Finding Large Efficacy Trials with PK Screen PK/PD Studies in Special Populations Non-Clinical Development Clinical Development PHASE IV
4
5 Ideal Animal Model for Cancer Therapy There is no perfect tumor model Validity Selectivity/Specificity Predictability Reproducibility Similarity
6 Animal Model in Cancer 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)
7 Syngeneic vs xenograft model Human cancer cell Immunedeficient animals Tumor growth
8 Human Tumor Xenografts 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 SC
9 Immune-deficient animals Athymic nude mice NOD-SCID (NOD.CB17- Prkdc scid /NCrCrl) mouse 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 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-2Rγ null ) 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
10 Subcutaneous Xenograft Study Endpoints 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
11 Subcutaneous Xenograft Tumor Weight Change Caliper Tumor weight change ratio (used by the NCI in xenograft evaluation) Defined as: treated/control x 100% Tumor mass volume= (a x b 2 )/2 a = tumor length b = tumor width T/C < 40-50% is considered significant length Width
12 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
13 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
14
15 Unmet Need for Translational Research in Cancer Therapeutics 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 교모세포종 / 뇌전이암 유방암 대장암 폐암방광암전립선암
16 Are subcutaneous models adequate? Subcutaneous Model Is this representative the actual tumor - Microenvironment? - Heterogeneity?
17 The effect of paclitaxel and p-gp inhibitor combination therapy on tumor growth in subcutaneous tissue and brain metastasis tumor model. In subcutaneous model vs. In brain metastasis model Even though the tumors are originated from same cell, the therapeutic responses are different according to the tumor bearing organ.
18 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
19 Types of murine model for studying human cancers. ADVANTAGES 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 Appropriately mimics human tumor microenvironment Can predict the drug response of a tumor in a human patient Provides realistic heterogeneity of tumor cells 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 DISADVANTAGES Mice are immuno-compromised, providing a less realistic tumor microenvironment Expensive Technically complicated 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, (2008)
20 Oncology Tools: Dose Calculator (Human vs Mouse)
21
22 Brain Tumor Orthotopic Model- Intracranial injection 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
23 Experimental Design For In Vivo Study ex. Brain tumor Orthotopic Model 2 X 10 5 U-87MG cells I.C. implantation Tumor volume measurement (B) 0 W 1 W 2 W 3 W 4 W 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 Survival length IHC (PCNA/TUNEL) IHC (Target vali.) Distribution
24 Ex. Anti-tumor effects of TMZ in U-87MG Human GBM Orthotopic Mice Model
25 Ex. Anti-tumor effects of Radiotherapy in U-87MG Human GBM Orthotopic Mice Model
26 Brain Metastases Model- Internal Carotid Artery injection
27 Lung Cancer Brain Metastases Model (Ideal Cell Dose) Cancer Cell line Type of cancer EGFR K-ras Cell dose Survival A549 NSCLC, Adenocarcinoma w/t Mut (G12S) 5X10 5 8W Lung Cancer Brain Metastases H460 NSCLC, LCC w/t Mut (G61H) 5X10 3 3W PC14PE6 NSCLC, Adenocarcinoma E746-A750del w/t 1X10 4 3W H23 NSCLC, Adenocarcinoma w/t Mut (G12C) 5X10 5 5W H1299 NSCLC, LCC w/t w/t 5X10 5 6W H460 PC14PE6 5X10 3 5X10 4 5X105 Group Median Survival Day I H460 (5X10 3 ) 150 II H460 (5X10 4 ) 51 III H460 (5X10 5 ) 36 1X10 4 1X10 5 1X10 6 Group Median Survival Day I 1X II 1X III 1X
28 Whole Brain Radiotherapy for Brain Metastases
29 Brain Brain Metastases Model- Left ventricle injection CANCER RESEARCH , April 15, 1992 Cancer Cell line Type of cancer Site of Implantation Cell dose Survival Lung cancer RFP-labeled A549 NSCLC Left Ventricle 1 X weeks After 8 weeks
30 Breast Cancer Orthotopic Model Mammary Fat Pad Tumor mass inoculate to 4th MFP Tumor mass Cell injection into 2 nd MFP Mammosphere
31 Lung Cancer Orthotopic Model Left lung parenchyma Cell injection into Lung Single cell suspension Cell implantation Left lung : One single lobe Right lung: Cranial, middle, caudal and accessory lobes.
32 Colon Cancer Orthotopic Model Cecal wall Cell injection Mass implantation
33 Gastric Cancer Orthotopic Model Procedure Incision : edge of the rib cage near the chest Draw out the stomach and injection or implantation into the stomach wall Cancer Cell line Type of cancer Site of Implantation Cell dose Survival Gastric cancer SNU-16 Human gastric carcinoma Stomach wall 2X weeks
34 Prostate Cancer Orthotopic Model Procedure Cancer Cell line Type of cancer Prostate cancer PC-3 Human prostate adenocarcinoma Site of Implantation Right dorsal lobe Cell dose Survival 5 X weeks Histopathology (H&E)
35 Pancreatic Cancer Orthotopic Model
36 Colorectal Cancer Liver Metastasis Model Cancer Cell line Type of cancer Colorectal cancer HCT116 Human colorectal carcinoma Site of Implantation Cell dose Survival Spleen 2X weeks Liver metastasis Spleen Head 50 mm 100 mm T Tail T T: Tumor region
37 Bone metastatic model by Intracardiac injection CANCER RESEARCH , April 15, 1992 Cancer Cell line Type of cancer Site of Implantation Cell dose Survival Lung cancer RFP-labeled A549 NSCLC Left Ventricle 1 X weeks After 8 weeks Hip joint Pelvis Knee joint Hind leg paralysis
38 Spontaneous Breast Cancer Lung Meta Model 1 X 10 6 MDA-MB-435 LvBr1 cells M.F.P. implantation 0W 1W 2W 3W 4W 5W 6W 7W 8W 9W 10W 11W 12W Pulmonary metastases mesurement After primary tumor formation (1.3~1.5 cm), tumor resection perform
39 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
40 Disseminated Lymphoma model Intravenous injection Mouse: NOD/Shi-scid/IL-2Rγnull (NOG) Single cell suspension I.V. injection of B cell lymphoma cells
41 Ex. In vivo preclinical efficacy of Rituximab in Disseminated model
42 Subcutanous Xenograft Model 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..
43 Experimental Design: Xenograft model (S.C.) Athymic nude mice S.C. injection of Human cancer cells Several days (After tumor formation) Control Test Treatment of test agents Extract Protein/RNA - Target Validation Make Tissue Slides - H&E, IHC - Target Validation Measure Tumor size Measure Body weight Tumors Measure Tumor weight Distribution study - Optical imaging
44
45 In vivo optical imaging and PET imaging In vivo optical imaging PET imaging
46 In vivo optical imaging and PET imaging
47
48 Example #1 Metronomic (low dose, multiple times) temozolomide chemotherapy for GBM
49 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, HOLLAND, E.C. Proc. Natl. Acad. Sci. U.S.A. 2000, 97,
50 Metronomic (low dose, multiple times) temozolomide chemotherapy 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
51 Microvessel Density Preclinical Data Metronomic chemotherapy is More effective in tumor volume reduction Anti-tumorous via anti-angiogenic and proapoptotic activities
52 Phase II clinical trial Depletion of MGMT by continuous TMZ Anti-angiogenic treatment Increment of total dose of TMZ
53 Example #2 Anti-tumor activities of human cytokine-induced killer cells against glioblastoma
54 Immunotherapy for the treatment of cancer 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) NK cell CIK Cytokine-induced killer (CIK) cells (Thorne et al., 2006) Lymphokine-activated killer (LAK) cells (Takashima et al., 2006) CD56 Dendritic cells (DC) (Kalinski et al., 2006) Natural killer (NK) cells (Raja Gabaglia et al., 2007) LAK CIK cells CD3 Characterized by the expression of CD3 and CD56 molecules Most potent cytolytic activity (Baker et al., 2001; Lu and Negrin, 1994)
55 Human CIK cell Production hpbmc After 14 days Ex vivo expansion Characterization Lymphocyte Culture with IL-2, CD3 hcik; Activated T cell Pre-clinical efficacy test hcik; Activated T cell U-87MG GBM orthotopic xenograft model
56 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 <
57 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.
58 Efficacy study of hcik in GBM xenograft model 2 X 10 5 U-87MG cells I.C. implantation 0W 1 W 2 W 3 W 4 w Intravenous injection of Human CIK cell Tumor Volume Measurement 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.
59 Efficacy study of hcik in GBM xenograft model 2 X 10 5 U-87MG cells I.C. implantation 0W 1 W 2 W 3 W 4 w Intravenous injection of Human CIK cell Tumor Volume Measurement hcik cells inhibit GBM tumor growth in an orthotopic xenograft model. hcik cells were traced by immunohistochemistry using a human CD3 specific antibody.
60 Combinational treatment of hcik cell and TMZ 2 X 10 5 U-87MG cells I.C. implantation 0W 1 W 2 W 3 W 4 w Intravenous injection of Human CIK cell Intraperitoneal injection of TMZ (2.5 mg/kg) 21~25d Tumor Volume Measurement 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.
61 Percentage of PCNA-positive cells per tumor section Decreased tumor cell proliferation (PCNA) 90.0 * P< 0.01 Control Control CIK * P< 0.05 CIK 1X10^ P< 0.05 TMZ 2.5 TMZ + CIK 10^ TMZ CIK+TMZ 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
62 Number of TUNEL-positive cells per tumor section Increased tumor cell apoptosis (TUNEL) 7.0 * P< P< 0.05 Control Control CIK * P< 0.05 CIK 1X10^7 TMZ 2.5 TMZ + CIK 10^ TMZ CIK+TMZ 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
63 Example #3 Schedule Dependent Synergistic Effect of Rituximab on the Methotrexate Chemotherapy against CNS Lymphoma
64 Primary CNS Lymphoma (PCNSL) 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 Incidence of CNS lymphoma Patients who are having biopsies
65 Treatment of Primary CNS Lymphoma 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)
66 Rituximab(RTX) 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!
67 Research Overview
68 Primary CNS Lymphoma Animal Model Intracranial injection of Raji cells Establishment of Group Ⅰ Ⅱ Ⅲ Cell dose 5*10 4 /5 ul 5*10 5 /5 ul 5*10 6 /5 ul I.C. injection
69 Primary CNS Lymphoma Animal Model Establishment of Features of histopathology Perivascular cuffing Leptomeningeal seeding IHC(anti-CD20)
70 Quantification of Penetration of RTX into CNS Lymphoma In vivo optical imaging RTX-AF680
71 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.
72 Evaluation of Antitumor Activity RTX treatment followed by MTX administration showed significantly reduced tumor volume.
73 Example #4 Bioequivalent Efficacy Study of Similar-Rituximab in Lymphoma model
74 In vivo Bioequivalent Efficacy Evaluation System for Rituximab in Human Lymphoma Xenograft Model Genentech Our Lab Mouse: Female Balb/c-nu, 6wks 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 = mm 2 ) Volume (width 2 x length x 0.5 = mm 3 )
75 Efficacy study of Original Rituximab and Similar Rituximab in Ramos Lymphoma Model Tumor volume (mm^3) 1 X 10 7 Ramos cells S.C. implantation 0W 1W 2W 3W 4W 5W 6W Intravenous injection of Rituximab After tumor formation (100mm 3 ) CT (PBS) O-RTX S-RTX
76 Decrease of Proliferation in Ramos Tumors (PCNA Staining) PCNA-positive cells per tumor section Control O-RTX S-RTX P< Cont. S-RTX G-RTX O-RTX S-RTX
77 Increase of Apoptosis in Ramos Tumors (TUNEL Staining) TUNEL-positive cells per tumor section Control O-RTX S-RTX P< S-RTX O-RTX Cont. G-RTX RTX S-RTX
78 Example #5 How to discover therapeutic target Development of Radio-sensitizer for Brain Metastasis
79 Metastatic Brain Tumors Most common intracranial tumor MRI scan of brain metastatic patient 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) 79
80 Limitations of Current Treatments Chemotherapy Whole Brain Radiotherapy Dementia Ischemic stroke Brain atrophy Blood Brain Barrier 80
81 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
82 Development of Radiosensitizer for Brain Metastasis Brain Metastasis Animal Models Relevance of DNA damage checkpoint signaling in prognosis of patient In vivo RT O Gy 5 Gy 10 Gy RT-resistant clone cdna MicroArray Target molecule
83 Development of Radio-sensitizer for Brain Metastasis Radiosensitizer : Chk1 inhibitor?? AZD7762 (ATP-competitive checkpoint kinase inhibitor) Chk1 Knock- down
Juyoun Jin, D.V.M., Ph.D. Institute for Refractory Cancer Research, Samsung Medical Center
Juyoun Jin, D.V.M., Ph.D. Institute for Refractory Cancer Research, Samsung Medical Center Overview of Anticancer Drug Development Discovery Non-clinical development Clinical Trial Target Identification
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