Foundations in Biotechnology PDF Module 1-7

Foundations in Biotechnology Welcome - Introduction to Biotechnology BIOT 5120 Module 1 Diaa Alabed, Ph.D. 9/10/2024 Agenda for Today Welcome and Opening Remarks Instructor and Students’ Introductions Syllabus Review Introductio...

Foundations in Biotechnology Welcome - Introduction to Biotechnology BIOT 5120 Module 1 Diaa Alabed, Ph.D. 9/10/2024 Agenda for Today Welcome and Opening Remarks Instructor and Students’ Introductions Syllabus Review Introduction to Biotechnology Break Biopharmaceutical Industry/Biotechnology and Drug development Final Group Project Topic Review Items for Next Week Opening remarks This class is designed to provide you with an introduction to biotechnology: o Covers examples of the foundations in biotechnology, molecular biotechnology, receptor pharmacology, drug development process, scale-up bio-products, regulatory affairs, genomics, proteomics etc… o Helps your understanding and application of biotechnological concepts which can be useful in real life professions Integrate knowledge from multiple disciplines (including process engineering, chemistry, biology, and business practices) to formulate comprehensive views of the workings of the biotechnology industry for the production of biopharmaceuticals from discovery to product approval Apply such views to understand the process of value creation and the role of protein manufacturing in the biotechnology industry Implement common and best practices and techniques to plan and apply tasks for the development of process and analytical procedures in the development and biomanufacturing of pharmaceuticals Apply acquired skills to work as an individual or team contributor in research and commercial biotechnology organizations About the instructor/students Background Ph.D. (Molecular Genetics and Biotechnology) Toledo Rockets! Scientist and Educator (Biotechnologist) Has worked in academy, government, and biotech industries Associate Teaching Professor/Faculty Lead in Biotechnology at Northeastern University Syllabus and additional notes Please read Syllabus Carefully Attendance (follow the rules) You are expected to show up to every class in person Notify the instructor if you miss a class before class starts Attendance may affect your grade Assignments Use accurate and concise language Due at start of following class. Late submission will be graded up to half credit. Submit online (Canvas) Solutions will be posted in Canvas Grade system See syllabus Grades may be adjusted at instructor’s discretion Emphasize on ability of communication Logical reasoning (which reflects your level of understanding) Express your opinion (tell your story) by connecting key points (ideas) Our Goals for The Class Respect Problem Solving and Critical Thinking Leadership Ethics Working together Project management Advanced communication skills 6  Canvas website Navigate Canvas website https://canvas.northeastern.edu/ What is the difference between Biotech and Bioscience companies?  Biotech: Develops or produces products and processes from living organisms and/or biological processes. Agriculture (or even aquaculture! – uses genetic, cellular and molecular techniques technologies to allow fish farmers to produce more abundant, resilient, and healthier supply of seafood!), Food production and medicine Genomics, recombinant gene technology, immunology, pharmaceutical and diagnostics (like Puritan Medical Products, Puritan Medical Products | Contact Us (puritanmedproducts.com)) Bioscience: life science, consists of all scientific disciplines that study life (animals, plants and humans) Growing and specialized fields Northeastern University and others (Council of State Bioscience Associations https://www.bio.org/csba)  Biotechnology clusters in the US Biotechnology Bay San Francisco, Northern California Biotechnology Beach Southern California Biotechnology Capital Delaware, Maryland, Virginia, Washington, D.C. Biotechnology Midwest Illinois, Indiana, Iowa, Michigan, Minnesota, Missouri, Nebraska, Ohio, and Wisconsin Biotechnology Indiana Biotechnology NC Research Triangle Park, North Carolina Biotechnology Forest Idaho, Montana, Oregon, and Washington, Genetown Massachusetts Pharm Country Connecticut, New York, New Jersey, Pennsylvania, and Rhode Island BioSpace has documented the areas and companies within Massachusetts where the biotech industry, along with other hotbed areas of the US. Learn more about the biotech industry in MA, at the Genetown (Links to an external site.) website. Massachusetts’s cluster Predominantly located Cambridge and Boston, Massachusetts The metro area is home to almost 1,000 biotechnology companies —from the earliest startups, to $50 billion companies — academic centers, and life science organizations. “Genetown” Useful link: GenetownLinks to an external site. Genetown | BioSpace Cambridge and Kendall Square Cambridge has ~250 biotech companies Biotech hub of experienced researchers Linked to the city’s 1977 rDNA research law NIH guidelines to working with rDNA attracted numerous researchers to the area MIT professor Phillip Sharp founded Biogen in Kendall developed a novel rDNA technology Biogen was the first industry to start Cambridge, Mass. is densely populated with life science companies. Here's where some of the largest biotechs and pharmas are located. Map: Karissa Waddick/PharmaVoice Source: Cambridge, Mass Embed Created with Datawrapper The next era of Greater Boston’s biotech boom | PharmaVoice  2022 Industry snapshot 2021, biopharma employment grew by 13.2% The life sciences industry employs 106,704 with an average annual wage of $201,549 for a total of $21.5 billion in total Massachusetts- based wages Top NIH-funded state per capita, with $3.3 billion constituting over 9% of all NIH funding Biopharma companies have received 26% of all venture capital investment nationally The drug pipelines of MA headquartered companies make up 7.2% of the global pipeline and 15.6% of the US pipeline 2022 Industry Snapshot (2022). MassBio. https://www.massbio.org/industry-snapshot/  Life Sciences sees large employment growth 2022 Massachusetts Life Sciences Workforce Analysis Report (2022). MassBio. https://www.massbio.org/2022-workforce-analysis-report/ Useful links Genetown | BioSpace MassBio News Archive – MassBio Dr. Robin Scheffler - Genetown: Biotechnology and Inequality in Greater Boston - MIT Events The next era of Greater Boston’s biotech boom | PharmaVoice Biopharmaceutical Industry – From Startups to Global Operations BIOT 5120 Module 2 Biotechnology in statistics 40 regional hubs that observe high activity in developing technology-driven solutions across the industry like novel drugs, biopolymers, and sustainable chemicals to name a few. 1219 startups employing technology-driven solutions to innovate in the industry. Silicon Valley, Boston, London, New York City, and San Diego are home to 431 startups and account for 35% of global activity in biotech. Boston: 114 Startups The biotech ecosystem in Boston is primarily focused on pharma research and development (R&D), thanks to the city’s position as a leader in pharmaceuticals and medicine industries. Major drug makers have moved to the city and initiatives such as the Massachusetts Life Sciences Center provide ideal conditions for biotech startups. Startus-insights.com Top Performing Countries in Biotechnology Labiotech.eu Biotechnology in statistics VENTURE CAPITAL FINANCING IN EUROPEAN AND U.S. BIOTECH INDUSTRY IN 2021 26.2 bn USD TOP GLOBAL COMPANY BY BIOTECH REVENUE IN 2021 Pfizer COUNTRY WITH THE HIGHEST NUMBER OF BIOTECH PATENTS IN 2018 Germany Statosta.com Impact of Mergers on Pharmaceutical R&D Major companies' reduction (Last 15 yrs.) R&D productivity concerns Research momentum slowing Pipeline reduction Social consequences Uncertainty about future Jobs lost Employees survive merger (Motivation) R&D future and mergers Patients, doctors and investors Therapeutic Choices, Small Molecules vs Biologics and how they work Effect of drugs on targets Drug Actions Direct receptor (stimulate or depress a process) Blocking mechanism Stabilizing action (receptor action) Direct beneficial chemical Direct harmful chemical to destroy the cells Pre-Discovery Manufacturing Patent Protection Marketing, Sales, Ethics, and Continuous patients monitoring and Drug related improvements Drug Development Pre-discovery: Understand the disease and understand the underlying conditions Drug discovery: High throughput screening (HTS) of large compounds libraries Target identification and validation Drug targets (GPCR): G protein-coupled receptors (GPCRs), largest family of membrane receptors that are targeted by approved drugs, and the number of such drugs that target GPCRs Protein Kinases add phosphate groups from ATP to proteins and activate them (Signal transduction) Early safety testing (absorbed, distribution, metabolized, excreted and nontoxic) Optimization (more effective and safer) Drug Discovery Choosing a drug target and understanding the impacts Established Targets New Targets Determining the AKT1 AKT2 TSC-1 roadmap to virus AKT3 TSC-2 PIP2 mTORC1 infection using GPCR-5HT2 PIP3 mTORC2 PI3K Nucleus Cytoplasm bioinformatics RAS Activates Numerous Transcription factors BRAF CRAF MSK1 MSK2 MNK1 MNK2 MAPKAP2 MAPKAP3 TRAF Human MEK1 MEK2 ERK1 P38-α P38-γ P38-δ JNK3 DLK ZAK disease ERK2 P38-β JNK2 ASK-1 MTK1 Q: What genes are “turned on” or “turnedMEK6 off” during viral infection? MEK3 JNK1 MEK4 A: Computer software and coding, can TAK1 determine MLK3 what genes (i.e. MEK7 tools) the virus use to infect the cell and ultimately cause disease RAC1  What do the COVID-19 vaccines target? Spike (S) protein Why this protein? (instead of the M, E (E) Envelope or N protein?) (M) Membrane Why was the development of the (N) vaccine so quick? Nucleocapsid Previous research SARS-CoV-2 Faster ways to manufacture vaccines A LOT of funding “The lightning - fast quest for COVID vaccines - and what it means for other diseases” The lightning-fast quest for COVID vaccines — and what it means for other diseases (nature.com)  Vector vs mRNA vaccines Vector vaccines mRNA vaccines Drug Development Preclinical testing: Is the drug safe for human testing? Lab and animal testing to determine the safety of a drug for human (Food and Drug Administration (FDA) requirement) In vitro and In vivo Lab in test tubes Cell cultures and animals How to make large quantity for clinical trials Narrow down the number of potential compounds (1-5) Drug Development Developmental process: File Investigational new drug application (IND) with the FDA Preclinical work, drug's chemical structure, how it works in the body, side effects, clinical trials plan (how & where) and manufacturing information Institutional Review Board (IRB) must review and approve clinical trials Companies must provide regular clinical trials update reports to FDA and IRB Clinical trail phases Design of each trial Validate results (Placebo control, randomize with double-blinded) Regular clinical trials update reports to FDA and IRB New drug application (NDA) and approval Drug approval process, benefit vs risk Manufacturing From small-scale to large-scale Sustain high quality with the large production Drug Development Pharmacogenomics: Influence of genetic variation on drug response in patients (gene expression with drug’s efficacy or toxicity) Optimize drug with respect to patients’ genotype and ensure max. efficacy with min. adverse effects Patent protection: Intellectual property (IP) is important during innovation cycle Early stage of drug development to encourages researchers to innovate and find new treatments Encourages further development of the product Financial benefits Generic companies may compete after IP right expiration Patent protection period Preclinical (6-10 yrs.) Patent protection (20 yrs.) Marketing and drug sales Pharmacokinetics (PK) vs. Pharmacodynamics (PD) PK: Absorption and distribution of an administrated drug. The chemical changes of the substance in the body and effects and routes of excretion of the metabolites of the drug What the body does to the drug Alpha phase (Initial phase with rapid concentration decrease due to distribution) Beta phase (Gradual decrease after alpha phase and due to metabolism and excretion) PD: Biochemical and physiological effects of a drug on the body or any organisms Mechanism of a drug, its’ concentration and effect What the drug does to the body Mimic or inhibit normal physiological/biochemical process Inhibit vital processes in microbial organisms Protein therapeutics “Proteins are large molecules that contain the fundamental biological and chemical components of all living organisms.” Recombinant DNA technology has made this area of biotech successful (module 2, lesson 4) Recombinant protein therapeutic (Human Insulin) Increasingly steady in the market (>130 protein therapeutics) Hormones, Growth factors, Recombinant vaccines Largest group of product sales in Biologics: monoclonal antibodies (Mabs) Treat cancer (Herceptin, Avastin, Erbitux) and immune disorders (Mabs… Rituximab, natalizumab) mAbs can directly attack cell toxicity, activate/modify immune response, bind to certain ligands /receptors on surface of strange cells (cancer, pathogens..), blocking cell growth etc.. mAbs are becoming attractive options for many biotech companies Protein therapeutics Monoclonal antibody therapy emerging tool for chronic diseases (Cancer) Significant investment in developing novel therapeutics Rising in chronic diseases Numerous DNA recombinant technologies increasing the range of Biotech potential Therapeutic products: Vaccines, hormones, recombinant proteins Genetically modified products Fruits, GM crops and animals Diagnosis Gene therapy, monitoring devices Energy applications Biohydrogen, Bioethanol Biotherapeutic process of proteins Before the invention of recombinant DNA technology, it was very difficult to produce enough protein to be used as a therapeutic agent. Proteins were usually extracted from tissues, organs of animals or human Now they are produced in labs using micro-organisms  Process overview 1. DNA isolation 2. Cloning into vector (plasmid) 3. Transformation (into bacteria) 4. Fermentation (DNA multiply) and protein production 5. Purification of the protein 6. Formulation (medicine) Advantages vs. Challenges Can be produced in large amounts and short time Engineering aspects of all equipments used to make protein Biochemistry of reagents and products proteins Physical nature of some proteins can hinder therapeutic functionality Protein molecules’ susceptible to structural changes and modifications in many areas. One or two of the amino acid messengers Multiple proteins can form aggregates Chemical, thermal and conditions instability Acidic and high temperature conditions Protein development process errors Analytical methods Use of analytical methods to monitor the production process Check final protein product for quality and Required to make sure safety and quality is maintained Next Week Week/Module Date Topic Assessment Due Group selection by Instructor Individual Assignment 1 (Module 1 Discussion Board, due 9/15 and 9/16) Week 3/Module 3 9/17/24 Drug Target/Transcription and Translation Module 2 Quiz (Take home due by 9/15) Read lessons files in Canvas Quiz Module 3 (Next week in class)  Mass. Biotechnology Council https://www.massbio.org/ https://readymag.com/MassBio/2022IndustrySnapshot/ Mass. Biotechnology Council, Module 1 Discussion Board Explore this site, more specifically, explore the “Career Center” page and see what jobs may interest you. You also can utilize other sites for Biotechnology jobs such as Indeed Write about it in the discussion board (on Canvas) What are your plans after graduations? Think about what you need to know now to be an attractive candidate for when you would apply! Post a self discussion (make sure to utilize class readings as well as at least 2 external references) Give your feedback to 2 other colleagues Action items (FIXED DATES) Quiz Module 2 Take home Respondus LockDown Browser Due: Sunday 9/15 by midnight Module 1 discussion board Biotechnology job of interest provide own response and feedback to two colleagues Due: Sunday, 9/15 by midnight Due: Monday, 9/16 by midnight Module 3 lessons Canvas pre-reads (coming soon!) Module 3 Quiz in class 9/17/24 Drug Target / Transcription and Translation Module 3 BIOT 5120 Diaa Alabed Ph.D. 9/17/2024 Agenda Questions about Module 2 Quiz Group Assigning Final Group Project Overview Protein Therapeutics Transcription/Translation Break Module 3 Quiz Group activity  Final Group Project Proposal Outline Disease/condition overview Your drug development process Incidence, prevalence, etiology, Overview of your drug/device how many patients are affected candidate Biological mechanism (MoA) Structure What gene(s)/organs are affected? Mechanism of action What proteins are malfunctioning? IP landscape (patents Why have you selected this condition? filed/issued) Discovery research What drugs have been Pre-clinical studies developed/approved by the Clinical trials FDA for this condition? Manufacturing Drugs in development Pricing Approved drugs Ethical issues Efficacy of the drugs Timeline Cost of the drugs Is there an unmet need and why? Summary References Final Group Project Timeline Final Project Topic due: 10/15: Final Project Proposal Outline draft (Not graded) 11/12: Final Project Proposal Outline due (Graded) 11/19: Final Project Presentation submission all groups 11/26: Final Project Group Presentations 12/3: Final Project Group Presentations  Numerous DNA recombinant technologies increasing the range of Biotech potential Therapeutic products: Vaccines, hormones, recombinant proteins Genetically modified products Fruits, GM crops and animals Diagnosis Gene therapy, monitoring devices Energy applications Biohydrogen, Bioethanol The use of DNA in biotechnology Genomic DNA (natural) Contain introns and exons Other regulatory elements Anything natural is not patentable unless modified by human Complementary DNA (cDNA) is not natural Reverse-transcribed from mRNA Introns are eliminated Patentable potential Patients and industry benefits Increase competition among industry (genetic testing) Reduce treatment pricing  Protein therapeutics “Proteins are large molecules that contain the fundamental biological and chemical components of all living organisms.” Recombinant DNA technology has made this area of biotech successful (module 2, lesson 4) Recombinant protein therapeutic (Human Insulin) Increasingly steady in the market (>130 protein therapeutics) Hormones, Growth factors, Recombinant vaccines Largest group of product sales in Biologics: monoclonal antibodies (mAbs) Treat cancer (Herceptin, Avastin, Erbitux) and immune disorders (Mabs… Rituximab, natalizumab) mAbs can directly attack cell toxicity, activate/modify immune response, bind to certain ligands /receptors on surface of strange cells (cancer, pathogens..), blocking cell growth etc.. mAbs are becoming attractive options for many biotech companies Protein therapeutics Monoclonal antibody therapy emerging tool for chronic diseases (Cancer) Significant investment in developing novel therapeutics Rising in chronic diseases Biotherapeutic process of proteins Before the invention of recombinant DNA technology, it was very difficult to produce enough protein to be used as a therapeutic agent. Proteins were usually extracted from tissues, organs of animals or human Now they are produced in labs using micro-organisms  Process overview 1. DNA isolation 2. Cloning into vector (plasmid) 3. Transformation (into bacteria) 4. Fermentation (DNA multiply) and protein production 5. Purification of the protein 6. Formulation (medicine) Advantages vs. Challenges Can be produced in large amounts and short time Engineering aspects of all equipments used to make protein Biochemistry of reagents and products proteins Physical nature of some proteins can hinder therapeutic functionality Protein molecules’ susceptible to structural changes and modifications in many areas. One or two of the amino acid messengers Multiple proteins can form aggregates Chemical, thermal and conditions instability Acidic and high temperature conditions Protein development process errors Analytical methods Use of analytical methods to monitor the production process Check final protein product for quality and Required to make sure safety and quality is maintained Begin to think about the mechanism of disease and how drugs can be designed to target it Transcription and Translation Central Dogma of Molecular Biology DNA/Transcription and Translation DNA backbone made of sugar (deoxyribose) and phosphate group (outside) Double helix two chains held by hydrogen One of four bases attached to sugar via Glycosidic bonds between basis from the inside bond: Adenine (A) Cytosine (C) 5’ 3’ Guanine (G) Thymine (T) Double strands (sequences): A-T 3’ 5’ C-G Nucleotides are linked by phosphodiester bond RNA backbone made sugar (ribose) and phosphate group One of four bases attached to sugar via Glycosidic bond: Adenine (A) tRNA attaches A.As. Uracil (U) to the ribosome Cytosine (C) rRNA links A.As. to Guanine (G) form protein A-U G-C Types of Mutations and their Effects MUTATIONS  Any mistakes in the DNA code can result in a “broken” (non-functional) protein  A mutation affecting only a few somatic cells (body cells) might not have any effect, unless the mutation turns the cell cancerous  A mutation affecting a sex cell can be passed on to the offspring TYPES OF MUTATIONS  Point mutation: base substitution that may or may not code for a different amino acid  Insertion mutation: one or more bases is inserted into the DNA strand  Deletion: one or more bases is deleted from the DNA strand Eukaryotes vs. Prokaryotes Overall process of protein synthesis is similar in all living cells Significant differences between bacteria and eukaryotes: Eukaryotic cells contain mitochondria and chloroplasts, which have their own DNA and their own ribosomes The ribosomes made up of rRNA of these organelles operate similarly to those of bacteria The ribosomes of eukaryotic cells are larger, contain more rRNA and protein molecules than those of prokaryotes In eukaryotic protein synthesis, it is usually the cytoplasmic ribosomes that translate nuclear genes Several aspects of eukaryotic protein synthesis are more complex Eukaryotes have more initiation factors and a more complex initiation procedure Eukaryotic vs. Prokaryotic DNA DNA in chromosomes and in nucleus. Primary mRNA transcript is processed to remove introns, cap the 5’ end, and add a poly A tail on the 3’ end. The processed mRNA is transported out of the nucleus to the cytoplasm where it is then translated on cytoplasmic ribosomes or on DNA in nucleoid within cytoplasm. ribosomes in the rough Translation of the mRNA transcript to endoplasmic reticulum. produce protein begins on ribosomes Post-translation, Proteins are that are present in the cytoplasm modified (glycosylated) even before the transcription process - Addition of carbohydrate is completed. Post-translation (sugar) Proteins are not modified (not glycosylated). Recombinant protein expression systems CHO cells as recombinant DNA hosts and therapeutic proteins Provides consistency and reproducibility of results from a batch of clonal cells CHO cells can produce proteins with gylcoforms that are both compatible and bioactive in humans Protein folding and post-translational modifications -> pharmacodynamics and pharmacokinetic properties Adaptability and ease of genetic manipulation as well as grow in suspension Cell cultures can either be in suspension or be adherent Transcription/Translation The genes in DNA encode protein molecules ("workhorses" of the cell) Enzymes, including those that metabolize nutrients and synthesize new cellular constituents, as well as DNA polymerases and other enzymes that make copies of DNA during cell division, are all proteins Expressing a gene means manufacturing its corresponding protein The process has two major steps In the first step, the information in DNA has transferred to a messenger RNA (mRNA) molecule by way of a process called transcription During transcription, the DNA of a gene serves as a template for complementary base- pairing, and an enzyme called RNA polymerase II catalyzes the formation of a pre-mRNA molecule, which is then processed to form mature mRNA The resulting mRNA is a single-stranded copy of the gene, tRNA carries specific amino acids to the ribosome follow by mRNA read at the ribosomes in the rough ER (Translation) Next must be translated into a protein molecule  Transcription/Translation Genes in DNA contain information to make proteins The cell makes mRNA copies of genes that are needed The mRNA is read at the ribosomes in the rough ER Protein is produced What is transcription? DNA to RNA Nucleus Pre-initiation complex (PIC) -> Initiation -> Elongation -> Termination mRNA is shuttled to the Ribosomes in the rough ER for protein to be produced All starts with transcription factors  =A =C Transcription Review =U =G =T Coding sequence 3’ 5’ 3’ 5’ Template sequence Newly transcribed mRNA sequence 5’ RNA polymerase 3’ 3’ Template sequence 5’ Why was it thought that targeting RNA molecules and transcription factors was “undruggable”? Targeting transcription (and even translation) may affect multiple pathways Side effects to healthy cells that require these processes Answer? Develop transcription-targeted therapeutic targets Anti-cancer therapeutics Single-gene targeted agents may not be needed because cancer cells are often driven by a group of genes coregulated by transcription factors, like p53 But.. Off-target effects have not been extensively explored…  What are the transcriptional targets? A simplified overview of the four stages of transcription and where the inhibitors are targeting. Pol II, RNA polymerase II; TFIID, transcription factor IID; CDK, cyclin dependent kinase. Laham-Karam N, Pinto GP, Poso A and Kokkonen P (2020) Transcription and Translation Inhibitors in Cancer Treatment. Front. Chem. 8:276. doi: 10.3389/fchem.2020.00276 Merck pill, Molnupiravir SARS-CoV-2 Positive-strand RNA virus During viral replication: The drug (MTP) +RNA competes with CTP (cytosine -RNA (to be used as a template) triphosphate) and to UTP (lesser extent) GTP or ATP gets +RNA (RdRp reads it and incorporated when creates new +RNA the RNA polymerase viral genome) reads the MTP Error catastrophe Kabinger, F., Stiller, C., Schmitzová, J. et al. Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis. Nat Struct Mol Biol 28, 740–746 (2021). Translation The completed mRNA molecule then moves from the nucleus to the rough ER for translation. Translation  Initiation begins with a tRNA bearing methionine (met) attaching to one of the ribosomal units.  The codon for methionine is a universal “start” codon for “reading” the mRNA strand. Translation  The ribosomal unit binds to mRNA where the code for met is located (AUG). The anticodon (UAC) of the tRNA matches the “start” codon on mRNA (AUG) 5’-3’ direction Translation  Larger ribosomal subunit binds to the smaller unit, forming a ribosomal complex. The tRNA binds to the first active site on the ribosome and Translation begins  The second codon in mRNA (GUU) matches the anticodon of a tRNA carrying the amino acid valine (CAA). The second tRNA binds to the second active site on the large subunit  A new peptide bond forms between val. and his. on the catalytic site. The tRNA that carried val. will detach and find another amino acid in the cytoplasm. The mRNA strand will then shift over one more codon  The process continues until the ribosome finds a “stop” codon. The subunits detach from one another, the mRNA is released, and the polypeptide chain moves down the ER for further processing. The initial met is removed and the chain is folded into its final shape How RNA is read to Protein GGG: Gly UGC: Cys UAA: Stop Gly-Cys-Stop GCS  Targeting Translation Translation initiation -> translation elongation -> translation termination -> recycling of translation machinery Laham-Karam N, Pinto GP, Poso A and Kokkonen P (2020) Transcription and Translation Inhibitors in Cancer Treatment. Front. Chem. 8:276. doi: 10.3389/fchem.2020.00276 Targeting transcription and translation conclusions Targeting these central cellular processes is a more directed approach to cancer cells compared to non-specific chemotherapeutic agents (i.e., cisplatin) BUT all cells require transcription and translation Need to continue to target the individual proteins involved*** RNA activation of transcription (like transcription factors) and oncogenes are now becoming druggable targets Targeting -> specified binding sites to transcription factors, oncogenes and RNA molecules Next Week Week/Module Date Topic Assessment Due Module 3 Discussion due 9/21 and 9/22 Week 4/Module 4 9/24/24 Proteins and the Omics in Biotechnology Read lessons files in Canvas Study for Module 4 Quiz Action items (FIXED DATES) Module 3 Discussion board due (9/21 and 9/22) Module 4 pre-reads Prepare for Quiz module 4 Work with your group to organize the team Start to think about what disease you want to develop a drug against Final Group Project Proposal Outline Proteins and Omics in Biotechnology Module 4 BIOT5120 Diaa Alabed, Ph.D. 9/24/2024 Agenda for Today Questions about Module 3 Final Group Project Overview Proteins and Omics in Biotechnology Break Quiz module 4 Group activity (explore proteins) Review Items for Next Week Introduction to Omics Omics’ technologies adopt a holistic view of the molecules that make up a cell, tissue, or organism Universal detection of genes (genomics), mRNA (transcriptomics), proteins (proteomics), and metabolites (metabolomics) in a specific biological sample in a non‐targeted and non‐biased manner The integration of these techniques is called systems biology Introduction to Omics Traditional studies, which are largely hypothesis‐driven or reductionist Systems biology experiments are hypothesis‐generating, using holistic approaches where no hypothesis is known or prescribed but all data are acquired and analyzed to define a hypothesis that can be further tested Omics Genomics: Study of organisms set of genetic information, functions, structure etc.. vs. Genetics: study of heredity and how genes are inherited Metagenomics: Genetic material recovered directly from environmental samples Pharmacogenomics: Study of genetic variation on drug response Proteomics: Study of proteins, the structure and functions Bioinformatics: Study big biological data as well as the storing and retrieving the data Protein and Amino Acid Structures and Abbreviations 20 different L-α-amino acids by cells for protein construction Contain basic amino group and an acidic carboxyl group How RNA is read to Protein GGG: Gly UGC: Cys UAA: Stop Gly-Cys-Stop GCS  Amino Acids What are the two groups that R group you recognize on this structure? H CH3 H CH3 O O + N C C H N C C H H OH H H O Amino Alpha (α) Carboxylic acid group carbon group Amino acid (accepts H+) (donates H+) Zwitterion (dipolar ion)  Peptides and Proteins Alanine N-terminal C-terminal end end (Plus water) Glycylalanine Glycine Protein structure and Functionality Proteins begin as a chain but can form a very specific and complicated structure (e.g. primary, secondary, tertiary or, quaternary) Protein structures are based on the thermodynamic properties of the amino acid chains and how they interact with the surrounding environment Protein structure and functionality Primary: Refers to its sequence of amino acids, which amino acid comes 1st, 2nd, 3rd and 4th etc. The linear structure is referred to as a polypeptide Secondary: Functional protein requires more than a primary structure. Secondary structures may contain additional disulfide bonds that are folded into αlpha helices (light blue cylinders) and βeta strands (light green arrows) Hemaglutinin protein Protein structure and Functionality Tertiary: Caused by folding the helices and strands as they fold into this complicated yet compact three-dimensional structure Quaternary: More complicated final structures of multiple peptides. When several peptides with tertiary structures come together, they form the quaternary structure  Levels of Proteins protein organization Primary proteins Sequence of a chain of amino acids Alpha helix Secondary Pleated Sequence of amino acids are proteins sheet linked by hydrogen bonds Tertiary proteins Attractions with alpha helices and pleated sheets Quaternary proteins Protein consisting of multiple polypeptides chains  Ionization of Amino Acids H3O+ OH- Zwitterionic Concentration form Both groups Both groups protonated deprotonated 0 2 4 6 8 1 12 1 pH 0 4 What are enzymes made of? Enzymes are proteins Proteins are made up of amino acids How these proteins fold (i.e. their structure) determines their function What are enzymes and why are they useful in biological reactions? Energy need to form products (i.e. Activation energy) Energy need to form products with an enzyme Energy CO2 + H2O Energy release by reaction HCO3 - + H+ Time of Reaction Denaturation of proteins Disruption of the interactions between the R groups Covalent amide bonds stay connected Increase in temperature pH becomes very acidic or basic  Enzyme Kinetics: Temperature? Optimum Too high and temperature the enzyme denatures The active site Reaction Rate Too low and the changes shape enzyme works and the enzyme slow can’t break down the substrate Temperature (C°)  Enzyme Kinetics: pH? Maximum enzyme activity Optimum pH Reaction Rate Too acidic Too basic pH Enzyme Kinetics and Protein-Protein Interactions (PPIs) Enzyme Substrate Product (i.e., cellular process) Competitive Inhibitors Slows down the breaking of product Noncompetitive (Allosteric) Inhibitors Competes with the substrate for the enzymes active site Binds to another spot of the enzyme but can also provide protein- changing the shape of the active site protein stabilization Protein-Protein Interaction and interfaces (PPI) Selective Binding AA with a protein Two proteins Beta sheets & beta-sheet with have different complementary surfaces when surfaces complementary surface-surfaces interaction  PPIs = new potential therapeutic targets PPIs important in cell signal transduction, cell proliferation, growth, apoptosis, etc. Classic drug target: enzymes, ion channels, or receptors Recent advances in the development of protein–protein interactions modulators: mechanisms and clinical trials | Signal Transduction and Targeted Therapy (nature.com) Table 1 Summary of some PPI modulators in clinical trails Table 1 Summary of some PPI modulators in clinical trials (nature.com)  Inhibitors of PD-1/PD-L1 interactions Programmed Cell Death PD-L1 PD-1 PD-1 Tumor cell Immunosuppressive receptor Expressed in T cell activated immune cells PD-1/PD-L1 signaling pathway inhibitors: Overactivation -> Monoclonal antibodies negative regulation Peptides of T cells Small molecule inhibitors Five monoclonal antibody drugs: Pembrolizumab (Keytruda) -> melanoma, non-small cell lung cancer Opdivo (Nivolumab) -> melanoma, head and neck cancer Tecentriq (Atezolizumab) -> non-small cell lung cancer, bladder cancer Bavencio (Avelumab) -> Merkel cell carcinoma Imfinzi (Durvalumab) -> Urothelial carcinoma “OMICS : Genomics, Proteomics, Bioinformatics ” Tools for Drug Development Genetic Engineering Direct manipulation of an organism's genome using biotechnology Genome sequencing has been critical for advancing DNA manipulation technologies Viral genome was the first to be sequenced (Bacteriophage fX174, in 1970s) Yeast Chromosome III was then sequenced (1992) Bacterium was the first organism to have a complete genome sequenced (1995) Human genome sequenced followed (2001) New DNA maybe inserted, a gene maybe removed or editing of a native gene etc.. Plants, vegetables, crops, animals and other organisms…… Bacterium was the first organism to be genetically modified (1973) Genetic manipulation in produce and poultry Method dependent on restriction enzymes Gene cloning used in a vector Locating single nucleotide polymorphisms (SNPs) Restriction digests Genetic Engineering Genetic engineering does not include the techniques that do not use recombinant nucleic acids or a genetically modified organism in the process: Traditional animal and plant breeding In vitro fertilization Induction of polyploidy Mutagenesis Cell fusion techniques Genetic Engineering Cloning and stem cell research, although not considered genetic engineering, are closely related and genetic engineering can be used within them Synthetic biology is an emerging discipline that takes genetic engineering a step further by introducing artificially synthesized genetic material from raw materials into an organism  Genetic Engineering Mass-production of insulin, monoclonal antibodies, and vaccines Genetic engineered animal models (i.e., study and model cancer, obesity, diabetes, ageing) Bioluminescent tumors in mice Transparent zebrafish to track Transgenic axolotls to fight aging immune cell trafficking Applications of Genetic Engineering: Gene Therapy Gene therapy is the genetic engineering of humans by replacing defective human genes with functional copies. This can occur in somatic tissue or germline tissue If the gene is inserted into the germline tissue it can be passed down to that person's descendants Gene therapy has been used to treat patients suffering from immune deficiencies (notably Severe combined immunodeficiency) and trials have been carried out on other genetic disorders The success of gene therapy so far has been limited and a patient (Jesse Gelsinger) has died during a clinical trial testing a new treatment There are also ethical concerns should the technology be used not just for treatment, but for enhancement, modification or alteration of a human beings' appearance, adaptability, intelligence, character or behavior The distinction between cure and enhancement can also be difficult to establish Transhumanists consider the enhancement of humans is desirable  Gene therapy It can cure or treat diseases Advancements in science and technology today are changing the way we define disease, develop drugs, and prescribe treatments Cells are the basic building blocks of all living things; the human body is composed of trillions of them Thousands of genes in our cells that provide the information to produce specific proteins that help FDA.gov make up the cells The genes provide the information that makes different cells do different things Gene therapy Sometimes the whole or part of a gene is defective or missing from birth. This is typically referred to as a genetically inherited mutation Healthy genes can change (mutate) over the course of our lives. These acquired mutations can be caused by environmental exposures Scientists can do one of several things in gene therapy: They can replace a gene that is missing or is causing a problem They can add genes to the body to help treat disease Or they can turn off genes that are causing problems Vectors need to be able to efficiently deliver genetic material into cells Gene therapy can be used to modify cells inside or outside the body The product must be tested in clinical studies for safety and effectiveness so FDA scientists can consider whether the risks of the therapy are acceptable considering the potential benefits FDA.gov Genomics, rapid growth and development Genetic Engineering in the United States: Regulations The USA is the largest commercial grower of genetically modified crops in the world For a genetically modified organism to be approved for release, it is assessed by the USDA, the FDA and the EPA USDA evaluates the plants potential to become weeds, the FDA reviews plants that could enter or alter the food supply and the EPA regulates the genetically modified plants with pesticide properties Most developed genetically modified plants are reviewed by at least two of the agencies Final approval can still be denied by individual counties within each state. In 2004, Mendocino County, California became the first and only county to impose a ban on the "Propagation, Cultivation, Raising, and Growing of Genetically Modified Organisms", the measure passing with a 57% majority Each transgenic event is regulated separately as the transgene insertion locus varies even when using identical constructs and host genotypes. This could result in different expression patterns or could affect the function of other endogenous genes within the host What is Proteomics? Proteomics is the large-scale study of proteins, particularly their structures and functions The term "proteomics" was first created to make an analogy with genomics, the study of the genes The word "proteome" is a blend of "protein" and "genome", and was coined by Marc Wilkins in 1994 while working on the concept as a PhD student The proteome is the entire complement of proteins, including the modifications made to a particular set of proteins, produced by an organism or system. This will vary with time and distinct requirements, or stresses, that a cell or organism undergoes While proteomics generally refers to the large-scale experimental analysis of proteins, it is often specifically used for protein purification and mass spectrometry Applications of Proteomics One of the most promising developments to come from the study of human genes and proteins has been the identification of potential new drugs for the treatment of disease This relies on genome and proteome information to identify proteins associated with a disease, which computer software can then use as targets for new drugs For example, if a certain protein is implicated in a disease, its 3D structure provides the information to design drugs to interfere with the action of the protein A molecule that fits the active site of an enzyme, but cannot be released by the enzyme, will inactivate the enzyme. This is the basis of new drug-discovery tools, which aim to find new drugs to inactivate proteins involved in disease As genetic differences among individuals are found, researchers expect to use these techniques to develop personalized drugs that are more effective for the individual Biomarkers: The FDA defines a biomarker as, "A characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention" Understanding the proteome, the structure and function of each protein and the complexities of protein–protein interactions will be critical for developing the most effective diagnostic techniques and disease treatments in the future What is Bioinformatics? Bioinformatics is a branch of biological science which studies methods for storing, retrieving and analyzing biological data, such as nucleic acid (DNA/RNA) and protein sequence, structure, function, pathways and genetic interactions It generates new knowledge useful in such fields as drug design and development of new software tools Bioinformatics also deals with Algorithms Databases and information systems Web technologies Artificial intelligence and soft computing Information and computation theory Structural biology What is Bioinformatics? Bioinformatics also deals with Software engineering Data mining Image processing Modeling and simulation Discrete mathematics Control and system theory Circuit theory Statistics Modeling Biological Systems Systems biology involve the use of computer simulations of cellular subsystems: The networks of metabolites and enzymes which comprise metabolism Signal transduction pathways Gene regulatory networks to both analyze and visualize the complex connections of these cellular processes Artificial life or virtual evolution attempts to understand evolutionary processes via the computer simulation of simple (artificial) life forms Explore protein structures and the interactions they have with specific drugs Therapeutic Target Database Protein Data Bank (PDB) Protein Data Bank – 101 What complex are you looking at? Is the structure of a human protein, what is the protein called? What are a few drug targets and the amino acid sequences for them? Examine a ligand interaction with the drug target, what is the specific interaction is has? Quiz Reminders Taken in class Requires Respondus LockDown Browser Please don’t talk during quiz even if you finished No open notes or web browser Don’t leave quiz window, RLB will exist you from the quiz Using any type of notes or web browser will lead to points deductions and potential for failing the class Only one machine (laptop, tablet etc..) is allowed on the desk No phones allowed on desk You can keep a backup machine in your back Good Luck! Next Week Week/Module Date Topic Assessment Due Discussion board Module 4 Read lessons files in Canvas Week 5/Module 5 10/1/24 Drug Development Process, A Global Approach Class discussion activity papers (Molupiravir) Quiz Module 5 Action items (FIXED DATES) Module 5 pre-reads Week 5/Module 5 (10/1): Prepare for Class Discussion Activity (Read Molnupiravir papers (Merck)) Module 5 Quiz Module 4 Discussion Board Saturday 9/27 by 11:59 p.m. (Part #, Own Post) Sunday 9/28 by 11:59 p.m. (Part #2, Reply Post) Work on your Final Group Project Proposal Outline Optional draft not graded (Due 10/15/24) Graded proposal (Due 11/12/24) Drug Development Process – Proteins as Therapeutics Module 5 BIOT 5120 Diaa Alabed Ph.D. 10/1/2024 Agenda today Final group project questions Drug development process Proteins as therapeutics Drug development challenges Ethical considerations mAbs example Quiz Merck activity Next class items  Final Group Project Proposal Outline Disease/condition overview Your drug development process Incidence, prevalence, etiology, Overview of your drug/device how many patients are affected candidate Biological mechanism (MoA) Structure What gene(s)/organs are affected? Mechanism of action What proteins are malfunctioning? IP landscape (patents Why have you selected this condition? filed/issued) Discovery research What drugs have been Pre-clinical studies developed/approved by the Clinical trials FDA for this condition? Manufacturing Drugs in development Pricing Approved drugs Ethical issues Efficacy of the drugs Timeline Cost of the drugs Is there an unmet need and why? Summary References Small molecules vs. Biologics There are over 80 autoimmune disorders identified as of today, affecting almost 50 million Americans, mainly females Therapies target activated T/B lymphocytes, pro-inflammatory cytokines, inflammatory mediators May treat several autoimmune conditions due to shared mechanisms Monotherapy (single agent) Combination therapy (two or more agents) Case Study Biologics Small molecules Drug development Progress in drug development contributes to human health Involves many steps from basic research to commercial launch of the drug More specifically, it refers only to the clinical parts of this process and the discovery nonclinical research components The process involves tremendous investments from the industry and the National Health Institutes (NIH) & Companies $230 million for list of targets against Alzheimer’s disease, type II diabetes, arthritis and Lupus Protein Structure and Target Potential Target protein Involvement in a relevant pathway Functional and structural characterization Drug ability A good target protein has a structure that favorites its’ interaction with a drug 3D structures screening Can be achieved by sequence homology of a protein to drug targets Lahti et. al. 2012 Protein Targets Found in multiple locations in the body Many are secreted (Plasminogen) Transmembrane (P2Y receptor) Subcellular locations (mTORC1) Different functions Transmitting signals from outside the cell to the inside Catalyzing biochemical reactions Controlling ion influx across membrane Regulating gene expression There are 22,000 protein coding genes in human genome Only 6000-8000 are druggable In the US, 1400 small molecule drugs are marketed to target less than 450 unique human proteins Lahti et. al. 2012 Drug development Aspects of drug discovery include identifying a disease, discovering drug targets, identifying and optimizing lead compounds, preclinical studies and phase 1 through phase 4 clinical studies Quick view (Clinical trials phases) Pharmacokinetics (PK): A study of what the body does to the drug Pharmacodynamics (PD): A study of what the drug does to the body File Investigational new drug application (IND) with the FDA Institutional Review Board (IRB) must review and approve clinical trials Phas e 0: No safety or efficacy testing Sub-therapeutic dose A small number of subjects Phas e 1: Usually, healthy volunteer Establishing PK and PD A small number of volunteers 100 subjects) Subjects randomly assigned to a treatment group Continued safety assessments May include genetic testing often determines if a compound performs as planned Phase II – IIa and IIb Phase Ila – specifically targets dosing Phase lIb – targets efficacy of dose Phase 3 Usually large multicenter trials (>300) Sometimes called Studies effectiveness of treatment against the current standard of care Long duration Expensive Difficult Follow up periods of at least a week Sometimes called pre-marketing Can be used for “label” expansion Some studies continue “compassionate use” while an application is being reviewed Especially big in the world market Results are used to submit for regulatory filing, New Drug Application (NDA) Can be repeated if regulatory authorities are not satisfied with results or believe more data is needed Phase 4 & Phase 5 Post marketing Observes safety after treatment has been sold Designed to detect long-term effects of the treatment not yet observed in the other phases Can you think of any drugs that were taken off the market due to observed safety issues? Treatment into the population…the real clinical study, no? What are the current challenges we face in the drug development field? Discovery dilemma Pipelines are running empty Blockbuster model is no longer viable Current R&D model is too expensive Discovery research will be greatly impacted Budget cutbacks, FTE reductions, site closures These realities will force changes in discovery in Biotech and Pharma Greater reliance on Academia to fuel early pipeline Increased reliance on CROs (Contract Research Organizations) to support R&D Hybrid research discovery model will emerge Challenges of drug discovery Long Development cycles (> 10 years) High Attrition Rates (Less than 1 in 10 clinical drug candidates succeed to market) Cost of drug development is increasing Constant pressure to increase R&D productivity Constant pressure to innovate Biotechnology landscape is constantly changing  Expensive and drug pipelines (drug candidates have pharmaceutical companies running empty) Published data on potential drug targets > ~80% of data does not match with in-house findings Phase I ~ 30% of drugs fail safety Lack of reproducibility Phase II and Phase III failure rate (20% fail due to safety) PubMed Central, Figure 1: Clin Transl Sci. 2018 Nov; 11(6): 597–606. Published online 2018 Jul 30. doi: 10.1111/cts.12577 (nih.gov) Challenges of In-licensed drug targets During the project, transfer focus changes from “interesting” to “feasible/marketable” $$ Investments in HTS in early programs are substantial Most companies run-in house validation programs The general impression that published results are hard to reproduce The best chance for success with a dedicated target validation group that can confirm and extend published data FF. Prinz et al (Bayer HealthCare), Nature R eviews Drug Discovery 10, 712 (September 2011)  What are the solutions that address the problems in the drug development field? Drug Development The National Institutes of Health (NIH), a part of the U.S. Department of Health and Human Services, is the nation’s medical research agency — making important discoveries that improve health and save lives NIH is the largest public funder of biomedical research in the world (>$45 bill. 2022) NIH is made up of 27 Institutes and Centers, each with a specific research agenda, often focusing on particular diseases or body systems. NIH initiative Dec 2010 - NIH proposes new National Center for Advancing Translational Sciences to accelerate drug development The new center will be a hub and driver for NIH efforts to push promising discoveries further down the drug discovery pipeline The hope is that NIH would eventually license the most promising compounds to pharma and biotech companies, which would then take the drugs the rest of the way through the FDA approval process and to market Pharmaceutical external R&D innovation models Model Description Examples Traditional pharma– A collaboration with an academic investigator by providing funding or academic partnership: other resources in exchange for the investigator’s knowledge and Numerous one company–one contribution to research investigator Creates a contest to encourage external scientists to submit proposals Lilly PD2, TD2; Bayer Grants4Targets, or ideas. Awards are given in the form of grants or access to drug Grants4Leads, Grants4Apps; GSK Discovery Fast Open crowdsourcing discovery expertise, tools and reagents with potential for longer-term Track Competition; AstraZeneca open innovation follow-up collaborations web portal; third-party platforms such as Incentive Numerous, for example, Pfizer CTIs; AstraZeneca – Builds master agreements with one or more universities; in some cases, Academic centers of Karolinska Institute; MedImmune – INSERM; scientists from pharmaceutical laboratories are co-localized to the excellence Janssen – KU Leuven – Wellcome Trust; Sanofi – academic institutions to facilitate collaboration UCSF; Boehringer Ingelheim – Harvard University Numerous, for example, Sanofi – Third Rock Ventures for Warp Drive Bio; GSK – Avalon Biotech co-creation Invests capital funds and/or contributes assets to the biotech start-ups Ventures for multiple start-ups; Lilly – Atlas Ventures – OrbiMed for Arteaus; Celgene – Versant Ventures for Quanticel; Astellas – MPM Capital for Mitokyne Many examples; for example, development of Pharmaceutical Two (or more) pharmaceutical companies co-develop clinical candidates SGLT2 inhibitors: BMS – AstraZeneca on peers risk sharing to share development cost Dapagliflozin, Lilly – Boehringer Ingelheim on Empagliflozin, Pfizer – Merck on Ertugliflozin Creates a regional center in a biomedical hub to facilitate collaborations Innovation centers with academia and biotechs., biotech start-up creation, in-licensing and Bayer, J&J, GSK, and Merck merger activities. Details may vary by each company Wang et al. 2015 SBIR/STTR Small Business Innovation Research/Small Business Technology Transfer The United States Congress created the SBIR program in 1982 and the STTR program in 1992 These programs congressionally require eligible governmental agencies to set aside a percentage of their extramural budget so that domestic small businesses can engage in R&D that has a strong potential for technology commercialization SBIR/STTR Small business grants solicitations can be in the form of SBIR or STTR, but the contract solicitation can only be SBIR (not STTR). A large majority of NIH SBIR awards are in the form of grants, although a small, but growing percentage of SBIR awards are through contract procurement. Grant and contract proposals are both submitted electronically, but have different submission requirements SBIR/STTR The SBIR program includes the following objectives: Stimulate technological innovation Meet federal research and development needs Increase private sector commercialization of innovations developed through federal R&D funding Foster and encourage participation in innovation and entrepreneurship by socially and economically disadvantaged persons and women-owned small businesses. The Small Business Administration (SBA) serves as the coordinating agency for the SBIR and STTR programs.  Reliance/collaboration with Academia to replenish the pipeline PubMed Central, Figure 2: Clin Transl Sci. 2018 Nov; 11(6): 597–606. Published online 2018 Jul 30. doi: 10.1111/cts.12577 (nih.gov) Additional material on Canvas: Racing-to-define-pharmaceutical-R&D(2).pdf: BIOT5120 20405 Foundations in Biotechnology SEC 10 Fall 2021 [PM-2-TR] (instructure.com) Contract Research Organizations Support biotech companies on a contract basis WHO and American Society for Quality (ASQ) create guidelines to follow, and pharmaceutical industry should promote these guidelines to improve R&D productivity  Where should those guidelines be? GxP (Good practices) regulations are in place to ensure the purity, quality, and safety of pharmaceuticals. Fall under Good manufacturing practice (GMP) regulations Safety studies for drugs are regulated by good laboratory practice (GLP) regulations mRNA vaccines: creating mRNA vaccines require in vitro transcription enzymatic reactions… what are the current challenges? Few challenges relate to mRNA vaccine development and cGMP Ex: the components for this reaction must be from certified suppliers that guarantee that all the material is animal component-free and GMP-grade  Regarding the COVID-19 pandemic Biomedical Research COVID-19 Impact Assessment: Lessons Learned and Compelling Needs - National Academy of Medicine (nam.edu) 1. Invest in NIH and NIH-funded researchers to understand foundational knowledge about SARS-CoV-2 and COVID-19 2. Speed innovation in COVID-19 testing technologies through NIH’s recently launched Rapid Acceleration of Diagnostics (RADx) initiative 3. Join public-private partnerships: NIH’s Accelerating COVID-19 Therapeutic Interventions and Vaccines (ACTIV) partnership, and federal partnerships such as Operation Warp Speed (OWS) 4. Invest in preventative treatment and public health measures 5. Ensure that diagnosis, treatment, and prevention options are accessible and available for underserved and vulnerable populations that have greatest risk for the most severe disease threats  Monoclonal antibody (mAbs) classification Antigen binding site variable constant Light chain (light green) constant Heavy chain (dark blue) Murine antibody Chimeric antibody Humanized antibody Human antibody (-momab) (-ximab) (-zumab) (-umab) Monoclonal antibody development Hybridoma technology Hybridomas = short-lived antibody-producing B cell + immortal myeloma cell 1. Develop and optimize an immunogenic antigen 2. Host animal is immunized to elicit an immune response and create B cells 3. Isolate B cells from spleen and fuse to myeloma cells 4. Validate and amplify production Compared with other biologics, mAbs have high affinity towards drug target mAbs became humanized to avoid immune rejection Chimeric mAbs or targeting specific residues that can cause an immune response  Approaches for development of therapeutic antibodies Lu, RM., Hwang, YC., Liu, IJ. et al. Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci 27, 1 (2020). https://doi.org/10.1186/s12929-019-0592-z Timeline from 1975 of successful development of therapeutic antibodies and applications Lu, RM., Hwang, YC., Liu, IJ. et al. Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci 27, 1 (2020). https://doi.org/10.1186/s12929-019-0592-z  mAbs to target SARS-CoV-2 Antibodies in the battle against COVID-19. Mode of action of therapeutic monoclonal antibodies for treatment or prevention of COVID-19. Antibodies that bind to the viral spike protein can block the interactions with the cellular receptor angiotensin-converting enzyme 2 (ACE2), inhibiting viral entry. Some regions of the antibody such as the Fc region can be specifically engineered to improve the characteristics of therapeutic mAbs. mAbs, monoclonal antibodies; CH, constant heavy domain; CL, constant light domain; Fab, fragment antigen-binding; Fc, fragment crystallizable; FcR, Fc receptor; VH, variable heavy domain; V-L, variable light domain. Hunting for antibodies to combat COVID-19 (nature.com) Dealmakers, B. Hunting for antibodies to combat COVID-19. Biopharma Deal (2020) doi:10.1038/d43747-020-01115-y. International Council for Harmonization (ICH) Technical Requirements for Pharmaceuticals for Human Use brings together regulatory authorities and pharmaceutical industry to discuss scientific and technical aspects of pharmaceutical product development and registration Europe, Japan, and the United States without compromising the regulatory requirements for safety and effectiveness Identify and then reduce regional differences in technical regulatory requirements for pharmaceutical products while preserving a consistently high standard for drug efficacy, safety, and quality FDA participates in ICH as a founding member and implements all ICH guidelines as FDA guidance Quality requirements Includes large and small molecules discovery and non-clinical development Good practice (GxP) regulations ensure purity, quality and safety of pharmaceuticals Good manufacturing practice (GMP) regulations ensure manufacturing and quality control testing of approved drugs Good laboratory practice (GLP) regulations oversee safety studies of a drugs Best Quality Practices Management System Technical Requirements Organization Test Equipment Project Management Test methods / Method Validation Quality Management System Sampling and Chain of Custody Documentation Document control / Document approval and Materials issue Legal and Ethical Considerations Document changes Vendor Selection and Qualification Document Storage ICH Guidelines Quality Guidelines Quality area include pivotal milestones such as the conduct of stability studies, defining relevant thresholds for impurities testing, and a more flexible approach to pharmaceutical quality based on Current Good Manufacturing Practices (cGMP) risk management Safety Guidelines ICH has produced a comprehensive set of safety guidelines to uncover potential risks like carcinogenicity, genotoxicity, and reprotoxicity. A recent breakthrough has been a non-clinical testing strategy for assessing the QU interval prolongation liability: the single most important cause of drug withdrawals in recent years Efficacy Guidelines Concerned with the design, conduct, safety, and reporting of clinical trials. It also covers novel types of medicines derived from biotechnological processes and the use of pharmacogenetics/genomics techniques to produce better target medicines Multidisciplinary Guidelines Those are the cross-cutting topics that do not fit uniquely into one of the qualities, safety, and efficacy categories. It includes the ICH medical terminology(MedDRA), the Common Technical Document (CTD), and the development of Electronic Standards for the Transfer of Regulatory Information (ESTRI) cGLP and Ethical Considerations The benefits of GLP is the creation of technically defensible scientific data, by which its quality, reliability, and trustworthiness can be assured The regulations and standards associated with cGLP, the fundamental requirements of cGLP, and the consequences of non-compliance for regulated laboratories Differences between, Good Laboratory Practice, Good Clinical Practice (GCP), and Good Manufacturing Practice (GMP) regulations as they relate to laboratory testing GLPs, GCPs, and GMPs cover lab testing but are very different Scientists and quality control/quality assurance personnel participating in GLP, GCP, and GMP studies play different roles The GLPs are designed to protect scientific data integrity and to provide the EPA or FDA with a clear and auditable record of open-ended research studies Ethical Considerations The GCPs are intended to be an ethical and scientific quality standard for designing, conducting, recording, and reporting studies that involve the participation of human subjects Provides public assurance that the rights, safety, and well-being of clinical study subjects are protected, consistent with the principles that have their origin in the Declaration of Helsinki (WMA), and that the clinical data are credible GMPs are intended to demonstrate to the FDA whether or not individual batches of a regulated product are manufactured according to predefined manufacturing criteria Ethics  Bioethics Ethical guidelines in Belmont Report Created as a result of the National Research Act of 1974 to identify basic ethical principles to conduct biomedical and behavioral research involving human subjects Respect for persons Acknowledgement of autonomy and protection of those with diminished autonomy. Beneficence The duty to act in the best interests of patients or research subjects. The goal of maximizing benefits and minimizing harm. Also called non-maleficence. Justice The duty or obligation to treat patients equally and to distribute, by allocating fairly, what is rightly due in terms of benefits, risks and cost. What about the ethics that surround AI and big data in laboratory medicine?  Examples of Ethical Principles and Risks Ethical Principle Ex amples of Risk Ex amples of mitigation Patients cannot exercise informed Ban block boxes; algorithms need to be consent if they do not understand the made available for independent Autonomy risks and benefits of the individual academic study. algorithms. Healthcare AI models might emerge Prohibit sharing of personal health that primarily serve nonmedical information with technology firms unless Beneficence function such as targeted advertising. the downstream uses are explicitly for medical benefit. Massive data sets create potential for Implement short time limits on retention even de-identified health data sets to of health data by tech companies. Nonmaleficence be used in ways that harm individuals. Prohibition both sharing with third parties and re-identification. AI may exacerbate health disparities Develop reasonable pricing clauses and through exploitative pricing models. adapt intellectual property law to reflect Justice ownership rights by the public whose data were used to train the algorithm. SOURCE: Ethics of Al and Big Data in Laboratory Medicine | AACC.org Bioethics in Different Analytical Stages Pre-Analytical Phase: Responsibility of the laboratory, the health care provider (Consent), researcher, phlebotomist, nurse, or whoever collects the specimen, correct sample labeling Analytical Phase: Sample labels Confidentiality, quality and competence of personnel, patient may decline the analyses, GLP, SOPs and accuracy Post-Analytical Phase: Reporting or interpretation of results, sample storage, confidentiality, archiving results, data retention time, patient consent about who can access information Merck pill, Molnupiravir Merck requested for emergency use authorization (EUA) to the FDA What is an EUA? Provide more timely access to drugs, diagnostic tests, and other critical medical products when no adequate approved drug available They evaluate risk : benefit ratio with information they have IND, clinical phase data Full approval takes longer, and more data is required and reviewed over a longer period  Merck’s antiviral pill, molnupiravir Is this drug a small molecule or is it a biologic? How is it different then other treatments, like remdesivir and mAb treatment? What are the advantages of molnupiravir? What is the target of molnupiravir, the mechanism, and outcome? Thinking ahead questions: Who helped developed the phase I clinical trials and what were they determining in phase I? What was the basis of preliminary data that allowed the phase 1 trial to be performed smoothly? Next Week Week/Module Date Topic Assessment Due Read lessons files Module 6 in Canvas Week 6/Module 6 10/8/24 Introduction to Clinical Research Quiz Module 6 Module 5 discussion due 10/5 and 10/6 Action items (FIXED DATES) Module 5 discussion (due 10/5 and 10/6) Module 6 pre-reads Week 6/Module 6 (10/8): Prepare for Class Discussion Activity Read: FDA Advisory Board Briefing Israel data: 1st vs 2nd dose Consideration for Booster Module 6 Quiz (next week) Work on your Final Group Project Proposal Outline Quiz Reminders Taken in class Requires Respondus LockDown Browser Please don’t talk during quiz even if you finished No open notes or web browser Don’t leave quiz window, RLB will exist you from the quiz Using any type of notes or web browser will lead to points deductions and potential for failing the class Only one machine (laptop, tablet etc..) is allowed on the desk No phones allowed on desk You can keep a backup machine in your back Good Luck! Introduction to Clinical Research Module 6 BIOT 5120 Diaa Alabed, Ph.D. 10/8/2024 Agenda for Today Introduction to Clinical Research Review Items for Next Week Quiz module 6 COVID -19 Booster Decision Class Activity Reminder Midterm exam in two week, 10/22/24 (Modules 1-7) Biostatistics reminder Sample vs. population Main statistics (mean, standard deviation, range, mode etc..) Confidence of intervals (CI) Represents the values that expected to contain the population mean with a certain level of confidence CI = mean +/- (Z) X SD N Z value: How many SDs is the value from the mean 𝑥 − 𝑚𝑒𝑎𝑛 𝑍 𝑆𝑐𝑜𝑟𝑒 = 𝑆𝐷 Example: Testing a new drug for lowering Cholesterol level Clinical Trials Testing a drug to lower Cholesterol level Randomly sample 30 volunteers taking the drug for 3 months Record Cholesterol level mean and standard deviation Calculate CI at 95% (use the z value for CI @ 95%) Construct intervals for the true mean of the population Make a decision on go no go with the drug What is Clinical Research? Research conducted with human subjects (or on the material of human origins such as tissues, specimens, and cognitive phenomena) for which an investigator directly interacts with human subjects Clinical trials, also known as clinical studies, test potential treatments in human volunteers to see whether they should be approved for broader use in the general population. A treatment could be a drug, medical device, or biological, such as a vaccine, blood product, or gene therapy. Potential treatments, however, must be studied in laboratory animals first to determine potential toxicity before they can be tried in people. Treatments having acceptable safety profiles and showing the most promise are then moved into clinical trials, comparing the effect and value of intervention(s) against a control in human beings. Must use one or more active intervention techniques such as a single or combination of diagnostic, preventive, or therapeutic drugs biologics, devices, regimes, or procedures.  Involves a particular person or group of Research with people or uses materials from humans: human subjects 1) mechanisms of human disease, 2) therapeutic interventions, 3) clinical trials, and 4) development of new technologies. Patient-orientated research Outcomes and Mechanisms of disease health services Therapeutic interventions research Clinical Clinical trials Research Development of new techniques Seek to identify the most effective and most efficient interventions, treatments, and services. Examine the distribution of disease, the factors that Epidemiological and affect health, and how behavioral studies people make health-related decisions. Fundamentals of a Clinical Trial A crossover study is administering two or more experimental therapies, one after the other, in a specified or random order to the same group of patients First is the order in which treatments are administered may affect the outcome Second is the carry-over between treatments. In practice, carry-over can be dealt with using a wash-out period between treatments or by making observations sufficiently later after treatment What are clinical trials? Goal: Test potential treatments for use in general population How: Use of humans as subjects Cooperation of clinical centers, like hospitals IRBs enforce safe and ethical studies IRB approval is required before study begins Clinical trials are performed in phases Preclinical, phase I (early stage), phase II, phase III, phase IV Institutional Review Board (IRB) Under FDA regulations, an IRB is a group that has been formally designated to review and monitor biomedical research involving human subjects The purpose of IRB review is to assure, both in advance and by periodic review, that appropriate steps are taken to protect the rights and welfare of humans participating as subjects in the research (protocols, research materials, documentations etc..) IRB What is it? The “guardian” of clinical research Review: protocol, Informed consent, advertising, any other documents What you need: risks minimized, risk: benefit ratio, subjects selected fair, data monitored, privacy protected, vulnerable populations safeguarded Exempt from IRB approval Investigations starting before July 27,1981 under FDA regulations before that time Emergency use of a test article. The use must be reported to the IRB within 5 working days Taste and food quality evaluations and consumer acceptance studies What is its function? Committee that approves, disapprove, modifies, monitors, and reviews biomedical and behavioral research involving humans IRB continued Research can be terminated or suspended: Unexpected serious harm Not being conducted according to requirements of IRB What’s needed: clinical investigator and research, condition under study and/or purpose, list of benefits, time commitment, contact information No promises of cure, safety, or efficacy, or imply it is a better treatment, information- research or investigational purposes, description of criteria needed to participate Tuskegee Study Overview 1932 United States Public Health Services (USPHS) study: “record the natural history of syphilis in Blacks” (i.e. “Tuskegee Study of Untreated Syphilis in the Negro Male”) Treated for “bad blood” (referring to anemia, fatigue and syphilis, etc.) 600 Black men, 399 (syphilis) and 201 uninfected (controls) In exchange for taking part in the study, the men received free medical exams, free meals, and burial insurance. Health monitored over time 1943: Penicillin was the treatment of choice for syphilis and widely available Therapeutic treatments were given instead. What were they? Lumbar puncture/injection  Aim of the Tuskegee Study and moving forward in the study Concern (before the study): The rate of syphilis was higher among African Americans than among whites Social and economic factors BUT also different susceptibility Today: higher rates of diabetes, stroke, high blood pressure (**social factors**) Genetic differences: lower number of methyl groups in people of West African ancestry- more prone to heart disease, diabetes, inflammatory disease, kidney disease, and cancer (1) Ex: P2RY1 gene, causes blood clotting disorders (1) Observe the effect of disease on untreated patients PROBLEM: Deliberately withheld prescribing medicines to patients 1943: Penicillin was the treatment of choice for syphilis and widely available 1947: widely available and used as a treatment Park CS, De T, Xu Y, Zhong Y, Smithberger E, Alarcon C, Gamazon ER, Perera MA. Hepatocyte gene expression and DNA methylation as ancestry-dependent mechanisms in African Americans. NPJ Genom Med. 2019 Nov 25;4:29. doi: 10.1038/s41525-019-0102-y. PMID: 31798965; PMCID: PMC6877651.  Deaths from the Study 28 patients died directly from syphilis 100 died from complications related to syphilis 40 of the patients' wives- infected with syphilis 19 children- born with congenital syphilis https://www.mcgill.ca/oss/article/history/40-years-human-experimentation- america-tuskegee-study The Study Exposed and the Impact Mid- 1960s a PHS investigator, exposed the study as unethical, Peter Buxton Reporter of the Study? Jean Heller, Associated Press 1973, Congress held hearings, and some participants received a $10 million out-of-court settlement 1974: The National Research Act, creating the National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research. Late 1970’s: Ethics Advisory Board: Created the Belmont Report  Biotherapeutic Roadmap NDA (New Drug Application) is an application that is filed to FDA for approval of a chemical entity to be used as a pharmaceutical. Chemical entities usually refer to small molecules. IND (Investigation of New Drugs) is an application to be completed before clinical BLA (Biological Licensure Application) is trials begin. This form is filed with the FDA, an application similar to NDA, but specific who will determine if it is sufficiently to biologic compounds of interest including convincing that the product will have protein biopharmaceuticals potential benefit to the human subject, and is also proven to be safe based on pre- clinical studies. Biotherapeutic development Discovery research Tar