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Issues in Biotechnology: The Way We Work With Life Dr. Albert P. Kausch life edu.us Medical Biotechnology Lecture 31 Part IVb. Cancer Biology and Emergent Treatments: Applications of Biotechnology in Cancer Treatment © life_edu Issues in Biotechnology: The Way We Work With Life Dr. Albert P. Kausch Kimberly Nelson OnCampus Live BCH 190, MIC 190, AFS 190, NRS 190, PLS 190 OnLine BCH 190 A Sweeping General Survey on Life and Biotechnology A Public Access College Course The University of Rhode Island Issues in Biotechnology: Biotechnology, Our Society and Our Future life edu.us Issues in Biotechnology: The Way We Work With Life Dr. Albert P. Kausch life edu.us BCH 190 Section II. The Applications of Biotechnology A Sweeping General Survey on Life and Biotechnology © life_edu The University of Rhode Island Issues in Biotechnology: The Way We Work With Life Dr. Albert P. Kausch life edu.us Medical Biotechnology Lecture 31 Part IVb. Cancer Biology and Emergent Treatments: Applications of Biotechnology in Cancer Treatment © life_edu Emergent Technologies in Cancer Treatment Cancer Basics, and the Application of Biotechnology to Treat Disease Cancer: Presentation Overview - Part 2 Fundamentals of Oncology • Socio-economic burden of cancer • Origins of cancer (carcinogenesis) • Pathophysiology and Molecular pathology • Contemporary target discovery/validation Applications of Biotechnology in Cancer Treatment • A brief history of cancer chemotherapy • Three Case Studies - New Drugs Making a Difference • Issues and opportunities for doing even more DNA Microarray A technology that is reshaping molecular biology Microarrays consist of ordered sets of DNA fixed to solid surfaces provide pharmaceutical firms with a means to identify drug targets. In the future, the emerging technology promises to help physicians decide the most effective drug treatments for individual patients. The complex interaction between a microbial pathogen and a host is the underlying basis of infectious disease. By understanding the molecular details of this interaction, we can identify virulence-associated microbial genes and host-defense strategies and characterize the cues to which they respond and mechanisms by which they are regulated. This information will guide the design of a new generation of medical tools. How Does Chip Technology Work? RNA expression Disease free Metastases Nature 415: 530-536 2002 Pharmacogenomics • Genetic causes of interpatient variability in phenotype • Relationship between patient phenotype and genotype Phenotype: Genotype: Responders Clinical symptoms Pharmacokinetic variability Response to drug - efficacy - side effects A genetic marker(s) distinguishing specific variations within a DNA sequence Non-responders Pharmacogenomics Human Genetics • SNPs • Haplotypes • Sequencing Expression Profiling • Specific transcript levels • Total RNA profiling Phenotype Proteomics • Specific biochemical markers • Protein profiling • Drug response • Disease Prediction Whan that Aprill, with her shoures soote The droghte of March is perced to the roote And bathed every veyne in swich licour, Of which vertu engendred is the flour; Whan Zephirus eek with his sweete breeth Inspired hath in every holt and heeth The tendre croppes, and the yonge sonne Hath in the Ram his halfe cours yronne, And smale foweles maken melodye, And slepen al the nyght with open eye(So priketh hem Nature in hir corages); Thanne longen folk to goon on pilgrimages And especially from every shires ende to room 10, in CBLS they wende, Whan that Aprill, with her shoures soote The droghte of March is perced to the roote And bathed every veyne in swich licour, Of which vertu engendred is the flour; Whan Zephirus eek with his sweete breeth Inspired hath in every holt and heeth The tendre croppes, and the yonge sonne Hath in the Ram his halfe cours yronne, And smale foweles maken melodye, And slepen al the nyght with open eye(So priketh hem Nature in hir corages); Thanne longen folk to goon on pilgrimages And especially from every shires ende to room 10, in CBLS they wende, When in April the sweet showers fall That pierce March’s drought to the root And bathed every vein in sweet liquor That has power to generate therein and sire the flower; When Zephyr also has with his sweet breath Filled again, in every holt and heath, The tender shoots and leaves, and the young sun His half-course in the sign of the Ram has run, And many little birds make melody That sleep through all the night with open eye (So Nature pricks them on to ramp and rage) Then folk do long to go on pilgrimages And especially from every shire’s end Of URI they to 271 Chaffee they went Variation in Phenotype is Variation in Genetics X Variation in Environment Vp = Vg X Ve Complex (Multifactorial) Phenotypes: What can we expect? Genei I. Few Genes each contributing large risk • ls> 4.0 Genen Expression of Phenotype = Gi + Gn + E Environment II. Many genes each contributing small risk • ls< 2.0 Gi Gi Gi Gi E Gi Phenotype Genetics X Environment What Causes Cancer? Genetics Environment What Causes Cancer? Genetics Mutations Oncogenes Anueploidy Chromosomal Rearrangements Human Cancer Genes: Genes Causally Linked to Oncogenesis About 290 genes have been linked to cancer by identification of mutation in primary human tumors. Changes include… Point mutations Activating (ex. Ras in pancreatic cancer) Inactivating (ex. p53 in ~50% of all cancers) Translocations Aberrant fusion proteins (ex. bcr/abl in CML) High Expression (ex. erbB2 in breast cancer) Deletions/insertions/frame shifts (ex. APC in colon cancer) Point mutations Frame shift mutations Deletions Insertion Translocations Additional gene copies Anueploidy Chromosomal rearrangements Oncogene activations Viruses (e.g. HPV) Step-wise (ordered) Genetic Changes in Colon Tumorigenesis “Full Blown” Tumorigenesis requires 4-17 events (mutations) Kinzler and Vogelstein Cell 1996 What Causes Cancer? Environment Carcinogens (Too many to name) Toxic compounds (everywhere) Sunlight (UVb) Tobacco smoking Radiation Emergent Technologies in Cancer Treatment: Lecture Overview Fundamentals of Oncology • Socio-economic burden of cancer • Origins of cancer (carcinogenesis) • Pathophysiology and Molecular pathology • Contemporary target discovery/validation Emergent Technologies in Cancer Treatment Applications of Biotechnology in Cancer Treatment • A brief history of cancer chemotherapy • Three Case Studies - New Drugs Making a Difference • Issues and opportunities for doing even more Emergent Technologies in Cancer Treatment: Lecture Overview Fundamentals of Oncology • Socio-economic burden of cancer • Origins of cancer (carcinogenesis) • Pathophysiology and Molecular pathology • Contemporary target discovery/validation Applications of Biotechnology in Cancer Treatment • A brief history of cancer chemotherapy • Three Case Studies - New Drugs Making a Difference • Issues and opportunities for doing even more The Bari Myth • USS John Harvey, an American ship in Bari Harbor, carried a highly classified load of 2,000 100-lb mustard bombs • Dec 2, 1943 - A German raid damaged 17 ships, including the Harvey • Fire on the Harvey caused a mustard-laden smoke that spread quickly • 617 Mustard gas poisoning cases among troops and merchant marine seamen, with a 14% fatality rate (3-fold higher that of World War I) • Commonly cited as the key observation that led to Goodman and Gilman’s pioneering work on the use of nitrogen mustards in cancer chemotherapy R. Joy, HISTORICAL ASPECTS OF MEDICAL DEFENSE AGAINST CHEMICAL WARFARE The Bari Myth (cont’d) After World War I, researchers were highly aware of the human effects of mustard gas—it destroyed lymphatic tissue and bone marrow. It was broadly reasoned that mustards could also kill cancer cells in the lymph nodes. It was not until 1942, more than a year before the Bari raid, however, that two young assistant professors in Yale’s new Department of Pharmacology, Louis S. Goodman, M.D., and Alfred Gilman, Ph.D., were commissioned by the Defense Dept. to study nitrogen mustard and its derivatives. Their pre-clinical studies in mice produced dramatic regressions of lymphomas. In December of 1942, a 48-year-old patient with terminal lymphosarcoma was given 10 consecutive doses of nitrogen mustard, a 10th of a milligram to a milligram per kilogram of body weight, roughly 2.5 times what became the standard dose. Within two days a softening of the tumor masses was noted. By the end of treatment the tumors disappeared. A month later, however, the patient relapsed, and subsequent courses of treatment were less effective. Nevertheless the scientists were encouraged. The clinical trials remained a classified military secret, even from caregivers, until 1946—the first Yale patients’ charts said only, “0.1 mg. per kg. compound X given intravenously.” From Humble Beginnings… R Ruddon, Anticancer Drugs Goodman and Gilman usher in 60 years of cytotoxic chemotherapy R Ruddon, Anticancer Drugs Cytotoxic Drugs in 2012 - Still Among Standards of Care in Cancer Chemotherapy 1st Line 2nd Line Breast Ca Adriamycin+ Cytoxan +/- Taxol Cytoxan + 5FU+methotrexate Cytoxan + Adriamycin + 5FU Taxol + Herceptin (erbB2+ only) Tamoxifen Taxol Xeloda Herceptin Taxotere NSCLC Taxol and carboplatin Gemcitabine + CDDP Tarceva, Taxotere Etoposide, Navelbine Colorectal Camptosar + 5FU+ leucovorin Oxaliplatin + 5FU+leucovorin Xeloda Irinotecan Prostate LHRH analog (Lupron/Zoladex) +/- antiandrogen Casodex+mitoxantrone+pred -nisone Hodgkins Lymphoma ABVD: doxorubicin+bleomycin+ vinblastine+ dacarbazine The key limitation of cytotoxic chemotherapy… How fast is the tumor growing? Tumor Type Burkitt's lymphoma Testicular cancer Ewings' sarcoma Small cell lung cancer Colon cancer Breast cancer Lung cancer Doubling time (days) 2 - 5 days 21 22 81 96 129 134 Populations of committed stem cells in normal tissues (bone marrow; epithelium of GI tract) have doubling times < 1 day Cancer: Presentation Overview Fundamentals of Oncology • Socio-economic burden of cancer • Origins of cancer (carcinogenesis) • Pathophysiology and Molecular pathology • Contemporary target discovery/validation Applications of Biotechnology in Cancer Treatment • A brief history of cancer chemotherapy • Three Case Studies - New Drugs Making a Difference • Issues and opportunities for doing even more Inhibition of Angiogenesis as a Target Required for tumor metastasis & growth beyond 1-2 mm3 Limited role in adults Broad spectrum of activity May circumvent acquired drug resistance Folkman: Angiogenesis is Necessary Angiogenesis and Tumors Blood vessels grow toward and into tumors VEGF Vascular Epithelial Growth Factor DNA Technology And Small Molecule Drug Design Precise treatments for: Cancers Cardiovascular diseases Inflammation Behavior Obesity Depression Schizophrenia Small Molecule Cancer Drug Design Small Molecule Cancer Drug Design Gleevec: Imatinib, is a drug used to treat certain cancers. It is marketed by Novartis as Gleevec (USA) or Glivec (Europe/Australia/Latin America) as its mesylate salt, imatinib mesilate (INN). Imatinib is the first of a new class of drugs that act by specifically inhibiting a certain enzyme – a receptor tyrosine kinase – that is characteristic of a particular cancer cell, rather than non-specifically inhibiting and killing all rapidly dividing cells. Recombinant DNA And New Cancer Drugs DNA Technology and Pharmaceuticals Allows precise treatments for: Cancers Cardiovascular diseases Inflammation Behavior Obesity Depression Schizophrenia Genetic Constructs Now Make Proteins That Are New Cancer Drugs Promoter Coding Sequence Terminator Your favorite gene Controlled expression “making protein” Erbitux Herceptin Avastin Rituxan TM TM TM TM Stop transcription Message stability Advance 1: AvastinTM (Bevisizumab) Why an Antibody to VEGF? Ferrara: VEGF is Necessary for Angiogenesis Antibodies for targeted therapy Advantages Ideal for extracellular targets Small molecule approaches successful for “beautiful” binding sites, but generally limited for all others – Non-enzymes – Non-receptors – Shallow pocket enzymes Opportunity for exquisite selectivity – Enzymatic selectivity for targets with promiscuous binding sites IGF-1R & Insulin receptor – Eliminates potential for chemotype-based toxicities associated with small molecules Relatively long duration of coverage over target – T 1/2 for human antibodies (weeks vs. hours for drugs) Antibodies for targeted therapy Potential Issues/Risks Distribution of Abs may limit activity relative to small molecules – Distribution: potential for limited tumor penetration Single chain variable fragments may improve penetration – Potential for immunogenicity Fully human antibodies may mitigate risk Manufacturing more complicated; high COG Long T1/2: Little opportunity to limit exposure if toxic – Points to advantage for small molecule, or Ab fragments Human Abs rarely x-react with rodents: preclinical modeling is challenging – Efficacy modeling for anti-angiogenesis targets – Estimates of TI in the efficacy species Therapeutic Antibodies for Cancer Antibodies already a key weapon in the Cancer arsenal Regulatory approvals for Abs in solid tumor indications are on the rise Product Target Company Indication Approval date RituxanTM CD20 IDEC/Roche/ Genentech NHL 11/97 HerceptinTM erbB2 Genentech/ Roche Metastatic BC 9/98 AvastinTM VEGF Genentech 1st line CRC 2/2004 ErbituxTM EGFR ImClone/BMS 2nd line CRC 2/2004 Additional Benefit from Avastin Breast Cancer: Phase III study of Avastin plus paclitaxel in first-line metastatic breast doubled the duration of surviving without cancer progression compared to chemotherapy alone Median progression-free survival was 11 months for patients treated with Avastin plus chemotherapy, compared to six months for patients treated with chemotherapy alone In patients with measurable disease, the overall response rate was 28 percent (93/330) in the Avastin plus chemotherapy arm, vs. 14 percent (45/316) observed in the chemotherapy alone arm KD Miller, ASCO 2005 Lung Cancer: Phase III study of Avastin plus paclitaxel and carboplatin chemotherapies in first-line non-small cell lung cancer (NSCLC) patients showed a 30 percent improvement in overall survival (12.5 months vs. 10.2 months)compared to patients who received chemotherapy alone AB Sandler, ASCO 2005 Advance 2: HerceptinTM (traztuzumab) EGF TGF- Amphiregulin Betacellulin HB-EGF Heregulins NRG2 NRG3 Heregulins Betacellulin AEV Extracellular Ligandbinding Domain Tyrosine Kinase Domain ErbB-1 (EGFR) 5’ v-erbB v-erbA ErbB-2 ErbB-3 3’ ErbB-4 erbB2 as a Cancer Target • erbB2 (HER2) is a member of the epidermal growth factor receptor (EGFR) family of transmembrane tyrosine kinases • Amplification (an excess number of gene copies) or over-expression (excess production of protein) confers on the affected cancer cell aggressive behavioral traits, including enhanced growth and proliferation, increased invasive and metastatic capability, and stimulation of angiogenesis • Patients with breast cancer in which HER2 is amplified (FISH-positive tumors) or HER2 is over-expressed are likely to have poorly differentiated tumors with a high proliferative rate, positive axillary lymph nodes, and decreased expression of estrogen and progesterone receptors NE J Med 353: 1734 (2005) Gabriel N. Hortobagyi, M.D. erbB2 as a Target: Checklist HerceptinTM (traztuzumab) 1. 2. 3. 4. 5. 6. Is the target a ‘gain-of-function’ gene that provides a key advantage to the tumor: • Progression and expression are linked? • Inhibition of gene function restores normal homeostasis? Is the target gene differentially expressed in tumor vs. normal cells (plausible basis for a TI?) Can the target be measured in biopsy material to select patients for clinical treatment? Can modulation of the target after Rx be monitored by a relatively non-invasive technique (biomarker)? Does target structure/function indicate a plausible basis for therapeutic attach (Is it an enzyme? A receptor?) Is it plausible that inhibition of the target would combine with other known Rx? /? /? Herceptin (anti-erbB2) Monoclonal Antibody: Biological Effects ErbB-2 LA Emens, Amer J Therapeutics 12: 243 (2005) Herceptin: Clinical Activity in Advanced Breast Cancer LA Emens, Amer J Therapeutics 12: 243 (2005) Herceptin – Activity in Improving Survival (D + C, TX =/- trastuzumab) Romond et al., NE J Med 353: 1673 (2005) “On the basis of these results, our care of patients with HER2-positive breast cancer must change today. Certainly, patients with lymph-node–positive, HER2-positive breast cancer should receive trastuzumab as part of optimal adjuvant systemic therapy… Since most HER2-positive tumors have other adverse prognostic factors, this risk–benefit scenario is likely to apply to many patients with node-negative breast cancer.” NE J Med 353: 1734 (2005) Gabriel N. Hortobagyi, M.D. Issues in Biotechnology One of the standards of care in cancer treatments include the use of chemotherapy. Many of the drugs used in modern chemotherapy are: (A) carcinogenic (B) cytotoxic (C) not developed by large pharmaceutical companies (D) free of any side-effects (E) deadly Therapeutic Antibodies for Cancer Antibodies already a key weapon in the Cancer arsenal Regulatory approvals for Abs in solid tumor indications are on the rise Product Target Company Indication Approval date RituxanTM CD20 IDEC/Roche/ Genentech NHL 11/97 HerceptinTM erbB2 Genentech/ Roche Metastatic BC 9/98 AvastinTM VEGF Genentech 1st line CRC 2/2004 ErbituxTM EGFR ImClone/BMS 2nd line CRC 2/2004 Issues in Biotechnology Antibodies have some distinct advantages over old school chemotherapies for treating cancer because they: (A) all of these reasons (B) are ideal for extracellular targets involved with tumor growth (C) eliminate potential for chemotype-based toxicities associated with small molecules (D) have a relatively long duration of coverage over the tumor target Advance 3: TarcevaTM (erlotinib) EGFR (erbB1) Why Target EGFR for Anti-tumor Drug Discovery ? • Over-expression of EGFR can transform cells in a liganddependent manner • EGFR or TGFa are frequently over-expressed in carcinomas, for example 42% of Lung Cancers over-express EGFR and many express EGF or TGFa • EGFR “Knockout” Mice are viable and hematopoietic cells lack EGFR The Use of Mouse As A Human Cancer Model The Use of Mouse Genomics As A Human Cancer Model OncoMouse OncoMouse Pharmacological Selectivity - Among close oncogene kindreds O HN O O O O HN O N N O CP-358774 erbB2 kinase: 1714 nM EGFR kinase: 2 nM 857x selective for EGFR N N erbB2 kinase: 43 nM EGFR kinase: 18 nM represents >500000x shift in selectivity! O O N OMe H N H N O HN HN N N N CP-724714 erbB2 kinase: 8 nM EGFR kinase: 6173 nM 772x selective for erbB2 N erbB2 kinase: 40 nM EGFR kinase: 5998 nM N Tarceva Antibodies Advantages Extracellular targets Small molecules successful for “beautiful” binding sites Opportunity for exquisite selectivity Relatively long duration of coverage over target – T 1/2 for human antibodies (weeks vs. hours for drugs) Intracellular targets Tyrosine Kinases active sites Exquisite selectivity (500,000-fold vs. erbB2) Relatively long duration of coverage over target – T 1/2 in humans ~12 hours… q.d. oral dosing (strong patient preference) High tumor penetration Low COG New Frontiers - Tumor Stem Cells BCRP/ABCG2 (breast cancer resistance protein) Telomerase Oct4 Nanog Stat3 Aldehyde Dehydrogenase CXCR4/SDF-1 Cancer: Summary for Part 2 • Targeted therapies, those abrogating the key molecular advantages of tumor cells, are adding importantly to patient survival • These approaches offer the promise of less morbidity and higher quality of life • Monoclonal antibodies have ‘credentialed’ targeted therapies; small molecule drugs are now emerging as well • The future is likely to be focused on understanding ‘systems biology’ vs. individual gene targets, on tumor stem cells vs. their diverse (and somewhat less talented) progeny, and on novel combinations of targeted agents where patient survival will be measured in decades, not months No Walls The Clear bead at the center changes everything There are no edges to my being now I have heard it said that there is a window That opens from one mind to another But where there are no walls There is no need for a window, or fitting a latch. Rumi 1279 AD 11. There have been genes that have been identified to be associated with certain types of cancer. Microarrays or biochips have been used to examine differences in RNA population in cancer vs. non-cancerous cells. Human cancer genes of special interest are: (A) genes that are causally linked to cancer development or oncogenesis (B) those that have been introduced by genetic engineering (C) now being investigated in cloned humans who have had these genes removed from their genome (D) only simple somatic cell mutations (E) currently only those involved with breast cancer 12. One of the standards of care in cancer treatments include the use of chemotherapy. Many of the small molecule chemical drugs still used in modern chemotherapy are: (A) carcinogenic (B) cytotoxic (kill fast growing cells) (C) not currently developed by large pharmaceutical companies (D) free of all side-effects (E) only placebos 13. Biopsy and histology has been an effective tool for the staging of colon cancer using Dukes’ staging. This type of staging works well only for very good / very poor prognosis patients (Dukes’ stage A and D), but it is not very informative when predicting long-term outcomes of intermediate prognosis patients (Dukes’ stage B and C). A bigger problem is that a biopsy and a Dukes’ classification provides a only single snapshot in time… within the long natural history of a colon tumor (until the day of biopsy). Molecular staging in colon cancer may have the following implications: (A) there will be an increase of misdiagnosis (B) molecular staging will be in the control of the big pharmaceutical companies who will prefer to keep it secret because it will decrease the sales of their most effective chemotherapies and decrease sales (C) molecular staging itself may cause cancer (D) molecular staging may provide more accurate and early predictions of patient outcome than is currently possible with clinical staging (E) directly cause an increase in health care costs and therefore be prohibitive 14. Progression of cancer development is thought to be due to both genetic and non-genetic changes at the molecular level. The result of clonal progression in carcinogenesis is: (A) a fortunate event because it signals programmed cell death of the tumor (B) a clone of cells that is unresponsive to normal growth controls, and is able to expand indefinitely (C) means that the cancer is untreatable (D) a result of a gain in function of the tumor suppressor genes (E) what renders some cancers as not dangerous 15. The USS John Harvey, an American ship in Bari Harbor, carried a highly classified load of 2,000 100-lb mustard bombs on Dec 2, 1943 when a German raid damaged 17 ships, including the Harvey. Fire on the Harvey caused a mustard gas-laden smoke that spread quickly. 617 mustard gas poisoning cases among troops and merchant marine seamen occurred. Studies of mustard gas by two young assistant professors in Yale’s new Department of Pharmacology, Louis S. Goodman, M.D., and Alfred Gilman, Ph.D had already shown interesting biological effects of mustard gas that eventually lead to the development of: (A) (B) (C) (D) (E) radiation therapy chemotherapy molecular staging gene therapy stem cell therapy 16. Going from a microscopic view of the histological appearance of tumors in biopsies to a molecular view of analyzing the genes that are expressed or not expressed in tumors can be done now by: (A) generating a molecular profile on a GeneChip (microarray) (B) sequencing all of the genes in the patient (C) comparing all the genes of a patient to a healthy individual (D) doing a ‘Southern blot’ on the patient’s DNA (E) electron microscopy 17. The growth of blood vessels is known to have a strong influence on tumor growth. AVASTIN is a very effect anticancer recombinant DNA drug that as an antibody reacts with VEGF to inhibit tumor: (A) (B) (C) (D) (E) angiogenesis DNA synthesis transcription protein production origins 18. Herceptin is an antibody based drug that is produced from cloned DNA that has been introduced into cells so that it can be produced in large quantities. Herceptin binds to erbB2 and is very effective against what type of cancer? (A) (B) (C) (D) (E) brain cancer herceptin treats all of these cancer types effectively breast cancer prostate cancer lung cancer 19. Antibodies have some distinct advantages over old school small molecule chemotherapies (such as mustard gas) for treating cancer because they: (A) are highly specific (B) are ideal for extracellular targets involved with tumor growth (C) eliminate potential for chemotype-based toxicities associated with small molecules (D) have a relatively long duration of coverage over the tumor target (E) all of these reasons 20. Avastin is a humanized antibody that binds the peptide factor VEGF with a half-life in humans of 17-21 days. This drug is produced from a cloned segment of DNA that has been introduced into cell culture to make a stable transgenic cell line. These cells are then grown in large fermenters that are then harvested and the antibody is then purified through biomanufacturing processes. This purified antibody is sold as the drug Avastin which when introduced into the patient will cause: (A) the neutralization of VEGF that reduces or eliminates the ongoing angiogenesis in tumors (B) tumors to stop replicating their DNA (C) metastasis (D) debilitating side effects, such as paralysis (E) a shortened death process