From Cancer Genomics to Cancer Treatment : from hope to reality Yusuke Nakamura Human Genome Center Institute of Medical Science The University of Tokyo International HapMap Consortium
October 27, 2005 Construction of public database for genetic variations in human Country Genotyping Center %Genome Chromosome Platform
Japan RIKEN 24.3% 5, 11, 14, 15, 16, 17, 19 Third Wave Invader
Wellcome Trust Sanger UK 23.7% 1, 6, 10, 13, 20 Illumina BeadArray Institute McGill Univ. / Genome Canada 10.1% 2, 4p Illumina BeadArray QuebecWe Innovation (The Centre SNP Research Center in RIKEN) Sequenom MassExtend, China Chinesecontributed HapMap Consortium the largest9.5% 3, SNP8p, 21 data (24.3%) contributed the largest SNP data (24.3%)Illumina BeadArray
Illumina among the16.1% SNP 8q,typing 9, 18q, 22, centers X Illumina BeadArray
Broad Institute of Harvard and in this paper Sequenom MassExtend, 9.7% 4q, 7q, 18p, Y, mtDNA MIT Illumina BeadArray
USA Baylor College of Medicine 4.6% 12 ParAllele MIP
PerkinElmer AcycloPrime- UCSF / Washington Univ. 2.0% 7p FP High-denstity Perlegen Sciences All oligonucleotide array Biobank Japan Sample Collection at February 29, 2008
Hyperlipidemia 42,354 Prostate cancer 5,839 Leukemia 1,597 Diabetes 39,982 Periodontitis 5,652 Esophageal cancer 1,461 Cataract 19,070 Pollinosis 5,572 Cervical caner 1,405 Cerebral infarction 16,012 Glaucoma 5,257 Hepatitis B 1,391 Arrhythmia 15,440 Lung cancer 4,845 Uterine corpus cancer 1,189 Stable angina pectoris 14,855 Unstable angina pectoris 4,161 Nephro tic syndrome 1,038 MyocardialWe Infarction have 12,956 genotyped Rheumatoid arthritis more than 4,155 Ovarian15,000 cancer patients 976 BronchialWe asthma have 8,657genotyped Atopic dermatitis more than2,967 Tuberculosis15,000 patients 894 Cardiac failure 7,438 COPDat 250K-550K 2,797SNPs Keloid 814 Breast cancer 7,349 Cerebral aneurysm 2,735 ALS 788 Colorectal cancer 6,957 Arteriosclerotic obliterans 2,609 ILD 780 Gastric cancerA 6,869 total Liver cirrhosisof 6-billion data-points.2,494 Drug-induced hypersensitivity 598 Urinary stone 6,605 Liver cancer 2,452 Pancreatic cancer 531 Osteoporosis 6,412 Hyperthyroidism 2,320 CCC 503 Myoma uteri 5,988 Epilepsy 2,250 Febrile seizures 475 Hepatitis C 5,962 Endometriosis 1,827 Total 295,278
ICIC askedasked 235,841 235,841 patientspatients TotalTotal Cases Cases 295,278 295,278 cases cases ICIC obtainedobtained 201,805 201,805 patientspatients WithdrawnWithdrawn 172 172 individuals individuals ((85.6%)85.6%) Genes isolated through genome-wide association studies in RIKEN and University of Tokyo Myocardial Infarction LTA Nature Genetics 2002 LGALS2 Nature 2004 PSMA6 Nature Genetics 2006 Rheumatoid Arthritis PADI4 Nature Genetics 2003 SLC22A4 Nature Genetics 2003 FcRH3 Nature Genetics 2005 Diabetic nephropathySLC12A3 Diabetes 2003 ELMO1 Diabetes 2005 (Diabetes) KCNQ1 Nature Genetics 2008 IgA nephropathy SEL-L,-E Am J Hum Genet 2002 Osteoarthritis Asporin Nature Genetics 2005 Calmodulin 1 Human Mol. Gen 2005 GDF5 Nature Genetics 2007 DVWA Nature Genetics 2008 Disc herniation CILP Nature Genetics 2005 Brain Infarction PRKCH1 Nature Genetics 2007 Kawasaki disease ITPKC Nature Genetics 2008 Crohn disease TNSF15 Human Mol. Gen 2005 Colon cancer multipel genes Nature Genetics 2008 Lung fibrosis TERT JMG 2008 Hapmap Nature 2003 Nature 2005 Nature 2007 From molecular targets to antianti-cancer-cancer drugs
SMC Antibody
Dominant-negative Target Peptide Molecules peptide Molecules vaccine
Anti-sense DNA Cell Gene siRNA Therapy Therapy From molecular targets to antianti-cancer-cancer drugs
SMC Antibody
Dominant-negative Target Peptide Molecules peptide Molecules vaccine
Anti-sense DNA Cell Gene siRNA Therapy Therapy IsolationIsolation ofof molecularmolecular targetstargets forfor cancercancer treatmenttreatment usingusing clinicalclinical materialsmaterials cDNA microarray consisting of 32,000 genes
ComparisonComparison ofof expressionexpression ExpressionExpression profilesprofiles ofof profilesprofiles ofof cancercancer andand 3030 normalnormal humanhuman correspondingcorresponding normalnormal tissuestissues tissuestissues
Selection of novel molecular targets
DiagnosisDiagnosis TreatmentTreatment TumorTumor MarkerMarker SmallSmall molecularmolecular compoundcompound PredictionPrediction toto MonoclonalMonoclonal AntibodyAntibody chemosensitivitychemosensitivity PeptidePeptide VaccineVaccine siRNAsiRNA cDNAcDNA MicroarrayMicroarray systemsystem High-density spotting Cancer 20000 spots/glass Normal tissue tissue
LMM LMM
Cancer cells Normal ductal cells
T7-based RNA Amplification T7-based RNA Amplification (2-rounds) (2-rounds) aRNA aRNA labeling labeling Cy5 Cy3 co-hybridization Number of clinical samples analyzed by cDNA microarray
Tissue Number Tissue Number Lung 126 Bile duct 45 Breast 135 Uterus 44 Soft Tissue 101 ALL 25 AML 87 Kidney 25 Colon 78 Endometriosis 23 CML 84 Liver 20 Ovary 59 Pancreas 20 Malignant lymphoma 54 Melanoma 20 Prostate 54 Thyroid 20 Stomach 51 Neuroblastoma 16 Bladder 55 Testis 13 Eshophagus 45 TOTAL 1200 Criteria for selection of candidate targets for drug development (S) Small molecular compound (P) Peptide vaccine (R) siRNA (A) Antibody 1. Genes which were highly over-expressed in a large proportion of clinical cancer samples examined (S, P, R, A)
2. Genes which were expressed in none of important vital organs; ideally not expressed in any organs (S, P, R, A)
3. Genes whose expressions are essential for cell survival (S, R, A?)
4. Cytoplasmic membrane protein, Secreted protein (A) Development of anti-FZD10 antibody therapy for Synovial Sarcoma
Chikako Fukukawa Satoshi Nagayama Toyomasa Katagiri Frizzled Homologue 10 (FZD10)
RT-PCR C S SS S MFH LMS L M Synovial Sarcoma MPNST cell lines 123456789101112131514 16 17 18 19 20 21 22 23 24 25 26 27 28 FZD10
b2MG
Northern Blotting SS CL Srug. Frizzled family (Wnt signal) Lung Kidney Brain Liver Heart Pancreas SS582 SS487 Placenta HS-SY-2 YaFuSS Bone marrow Bone Wnt? 9.5 7.5 SS cell 4.4 2.37 1.35
SS ; Synovial Sarcoma FZD10 03-24640 04-25950 M Pr N M Pr SS r NNT Pr NP Pr NM C, O/N o Protein expression colon of in cancer FZD10 Pr NM 04-26192 g/mL, 4 g/mL, Pr NM NTNTNTNTNTNTNTNTN Pr 34 41 59 69 123 124 128 129 141 146 147 149 150 151 153 154 155 201 NT NM expression in colon cancer colon in expression 2MG 2MG
β β FZD10 FZD10 FZD10 C, 30sec C, o -FZD10 mAb 92-13 20 μ 92-13 mAb -FZD10 α Paraffin slides EDTA buffer (pH9.0) 125 Celler Immunization Monoclonal antibody recognizing a complex structure Cells (COS-7 etc.)
transfection
Ag
pCAGGS-FZD10 (FL)-myc・His
Immunization to mouse Monoclonal Antibody
Hybrydoma
Antibody stayed at tumor lesion at 5 days after injection InternalizationInternalization ofof anti-FDZ10anti-FZD10 antibodyantibody
FZD10(+) FZD10(-) SYO-1 YaFuSS LoVo No Tx No 92-13 93-22 In vivo effect of Y90-anti-FZD10 Antibody
Day 0 Day 5 Day 9 Day 40 Day 54
90Y-anti-FZD10
Day 0 Day 9 Day 33 Day 40 Day 54
90Y-anti-FZD10
Day 0 Day 9
90Y-CD20-Ab In vivo effect of Y90-anti-FZD10 Antibody Balb-c/nu (male) / SYO-1 tumor 90Y - DTPA - antibody 100uCi Intraveneous injection Single injection on Day0 7 Non-Labeled 92-13 (n=5) Non-treated (n=5) 6 ) 3 5
4
3 Tumor Volume(cm 2 90Y - 92-13 (n=11) 1
0 0 5 10 15 20 25 30 35 40 Days Injection 11 / 11 4 / 11 DevelopmentDevelopment ofof cancercancer peptidepeptide vaccinevaccine andand ConstructionConstruction ofof TRTR networknetwork inin JapanJapan Types of Cancer Vaccines
• Antigen/adjuvant vaccines • Whole cell cancer vaccines • Dendritic cell (DC) vaccines • Idiotype vaccines RecentRecent advancesadvances forfor cancercancer vaccinevaccine • 1991 Discovery of Tumor specific antigen (T. Boon, Science) • 1995 Clinical Trial against melanoma (Int J Cancer) • 1998 IL-2 + Peptides (Rosenberg, Nature Med) DC + Peptide (Nestle, Nature Med) • 2004 Less than 3% response rate for advanced cancer (Rosenberg, Nature Med)
• 2006 33%33% reductionreduction ofof recurrencerecurrence forfor lunglung cancercancer afterafter surgerysurgery (GSK,(GSK, ASCO)ASCO) 50%50% reductionreduction ofof recurrencerecurrence forfor breastbreast cancercancer afterafter surgerysurgery (Peoples,(Peoples, SanSan AntonioAntonio Int.Int. BreastBreast CancerCancer Meeting)Meeting) Expectation to Cancer Vaccine Treatment
100 1998 Promising Results on 2015 Approval melanonma of 15-20 Vac.
2007 Effect on reduction 0 of recurrence 1991 Tumor antigen
2003 Rosenberg report -100 1990 2000 2010 2020 AppropriateCancer Vaccine Cancer Treatment Vaccine atTreatment present 10-cm tumor = 1011-12 cells
CTL CTL CTL CTL<<<<<Cancer<<<<<CancerCancer CellsCells
10000 cells Peripheral Lym 1010 cells
CTL=106-7 cells
CTL Vaccine Treatment at an earlier stage of cancer
Canc er Canc CTL er
CTL Canc Canc er er CTL
Canc CTL Canc Canc er CTL er er
Canc CTL er CTL Canc er CTL
CTL CTL Canc er Canc er CTL CancerFurther Vaccine enhancement Treatment of CTL at activity present
CTL CTL CTL CTL CTL<<<<<Cancer<<<<<Cancer Cells 10000 cells
CTL FurtherTo overcom enhancement the present of CTL status activity
CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL 1.Improvement of CTL inductionCTL CTL CTL CTL CTL 2.BestCTL selection of antigen CTL CTL CTL CTL UnevenCTL precursor T cell population CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL 3.Regulation of Treg CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL
CTL CTL CTL CTL CTL CTL CTL DevelopmentDevelopment ofof cancercancer vaccinevaccine
Development of cancer vaccines using novel tumortumor-specific-specific oncoantigensoncoantigens identifiedidentified through genome-widegenome-wide cDNAcDNA microarraymicroarray analysisanalysis andand subsequent“Oncoantigen” functional analysis is defined as a protein that functions as an oncogene product and can enhance an immunogenic DevelopmentDevelopment ofof cancercancer vaccinesvaccines Development of cancerreaction. vaccines targetingtargeting tumortumor angiogenesisangiogenesis Expression pattern of MPHOSPH1 (Bladder Cancer)
Growth suppressive effect of MPHOSPH1 siRNA From moreSinceMPHOSPH1 than their 50 oncoantigens expressions is defined as identified, are restrictedan “oncoantigen” in cancer and testis we have(or screened fetal tissues 9- or 10- or amino-acidplacenta), peptides (HLA-A02 Low orit is chance HLA-A24very unlikely of loss restricted) ofthat that can stimulate CTLantigenactivated (cytotoxic expression CTLs T-lymphocytes) cause in , and identified ~60cancer peptides cells covering most of adverse reactions like cancer types. autoimmune diseases. Cancer Peptide Vaccine Translational Network (Captivation Network)
Organ Num of Hospitals Esophagus 7 (2) Stomach24Colon Universities Cancer4 PhaseColon II Clinical Research6 (1) Liver 48 protocols3 (1) Bile duct 2 Adjuvant setting Pancreas for 5 (1) KidneyAdvanced cancer 2 (2) Bladder 11 organs1 Lung 16(2008.08) Hospitals2 Breast 1 (2) Head and Neck 0 (2) Total 33 (11) Organizer: Takuya Tsunoda, Yusuke Nakamura Early Phase Development of Cancer Vaccines
• Study objective: Identification of a pharmacologically effective dose or optimal biologic dose rather than MTD
• Rationale Biologically active doses may occur well below the MTD Probably not feasible to examine MTD Immunological endpoints The Aims of Captivation Network
1.Evaluation of Safety
2.Evaluation of immunological responses
3.Searching the most appropriate condition that can induce the maximum immunological response
4.Clinical Effect 50 The45 number of the patients enrolled 40
35
30
25
20
15 Phase 1 10 50 5 45 0 Aug-Oct Nov-Jan Feb-Apr May-Jul Aug-Oct Nov-Jan Feb-Apr May-Jul 40
Cases 35
30
25 20 Phase 2 15
10 5
0 Aug-Oct Nov-Jan Feb-Apr May-Jul Aug-Oct Nov-Jan Feb-Apr May-Jul 2006 2007 2008
Month (Total 212 cases) (Aug 31, 2008) CancerCancer VaccineVaccine MonotherapyMonotherapy forfor AdvancedAdvanced esophagealesophageal cancercancer ((Phase(PhasePhase II)I)) • URLC10, TTK, KOC1 (each 1mg) • IFA as an adjuvant • HLA-A*2402-restricted • Weekly s.c. injection
1 Vac 2 Vac 3 Vac 4 Vac 5 Vac
Vaccination day 1 day 8 day 15 day 22 day29 day43
Yamanashi University, First Department of Surgery Evaluation Drs. Koji Kono Yoshiki Mizukami Hideki Fujii Case 5 Lung Metastasis
S1 S1+2
S1 S1
After 1 course (07.5.2)
S1+2 Before (07.3.7) S1+2 After 2 months (07.7.9) Case 5 Liver Metastasis
About 1X1011-12 cells
Total number of peripheral The number oflymphocytes=~1 caner cells >>> X 10CTLs10 cells
High level of Treg
Loss of HLA or antigen
Before After 1 course After 2 months (07.3.7) (07.5.2) (07.7.9) Case 5Specific T Specific Spots ratio Case 5Specific T 0.2 0.4 0.6 0.8 1.2 1.4 1.6 1.8 Specific Spotsratio: target molecule HLA transfectant/HLA alonetransfectant + 0 1 2 RC0TK6 KOC1 TTK567 URLC10 * * * - - cell response cell response * * *p<0.05 R/S:1 After 1 co inj. 2 After Before Pre-vaccine
Complete regression was observedPost-vaccine 76th day after 2 courses of the treatment.
A total number of tumor cells 108
Post-vaccine 104th day CaseCase 77 Clinical development of cancer vaccine ---patient population
• Metastatic disease • Minimal or no evidence of diseases • Advantages • Disadvantages
Shorter and faster Longer trial period enrollment (short evaluation period)
Large sample size Small sample size
Trial endpoints may be Allows tumor response Allows tumor response challenging evaluation Clinical development of cancer vaccine ---patient population • Metastatic disease • Minimal or no evidence of diseases • Disadvantages • Advantages Often multiple prior cancer Fewer prior therapies treatment Better in immune status Incompetent immune status (from prior Adequate time for evaluation therapies) before disease progression
Inadequate evaluation due Optimal for the proposed to rapid disease MOA of cancer vaccines progression