English Transcript Drug Discovery Of Biologics_edited version.pdf

PROPRIETARY. DO NOT SHARE. Transcript: Drug Discovery of Biologics Section 1: Setting the Stage Welcome Welcome to the Biotech Primer’s Drug Discovery of Biologics. This class begins with a brief overview of the drug development process, followed by a deep dive into the drug discovery process for b...

PROPRIETARY. DO NOT SHARE. Transcript: Drug Discovery of Biologics Section 1: Setting the Stage Welcome Welcome to the Biotech Primer’s Drug Discovery of Biologics. This class begins with a brief overview of the drug development process, followed by a deep dive into the drug discovery process for biologics. Let’s get started. Section 1- Setting the Stage Objectives By the end of this section, you should be able to: • Define the terms drug and drug target. • Compare the characteristics of small molecule drugs and biologics. • List the steps of the drug development process. • State the length of time and number of candidate drugs and the cost of the average drug discovery process. What Is a Drug? Before we explain how biologics are discovered, let’s start by defining the word “drug.” Based on the FDA's definition, a drug is a substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease. The keyword is ‘intended.’ Intended to affect the structure or function of the body. Intended for use as a medicine component, but not a device. The Food and Drug Administration or FDA regulates drugs in the United States. And finally, drugs are recognized by official pharmacopoeia or formulary. Pharmacopeia is volumes of information regarding medications approved across any indication and are excellent references for molecules in development. Drug Size and Complexity Now that you know the definition of a drug let’s look at two types of drugs--- small molecule drugs and large molecule drugs, also known as biologics. As you might guess, the names given to each are based on their size as determined by a unit of measurement called a dalton. Small molecule drugs have a relatively low molecular weight, below 900 daltons. Aspirin is an example of a small molecule drug. A large molecule drug or biologic is typically a high molecular weight compound of more than 4000 daltons. An example of a biologic is a therapeutic antibody. The differences in size are shown here. 1 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Size Determines Drug Target Interaction Size significantly affects a drug’s ability to interact with a drug target. A drug target is a molecule in the body, usually a protein, associated with a particular disease. Small-molecule drugs can enter and exit cells more freely than biologics, enabling a small-molecule drug to reach intracellular and intranuclear drug targets. Intracellular refers to drug targets inside the cell. Intranuclear refers to drug targets in the nucleus, where DNA is housed. Whereas many biologics, such as therapeutic antibodies, tend to target extracellular drug targets. Extracellular refers to the outside of the cell. Drug targets on the outside of the cell are the receptors. The receptors are proteins that receive signals from other areas of the body. However, there are known rare monoclonal antibodies, such as RAD52 important in cancer, that appear to be able to enter certain cell types. Criteria Used in the Discovery Process This table compares specific criteria used in the discovery process to understand the differences between small molecule drugs and biologics. As we stated in the last screen, small molecule drugs target intracellular and intranuclear spaces. Biologics target the extracellular areas. Moving on to half-life, the half-life of small molecules is typically in the range of 2 to 6 hours, versus a monoclonal antibody, which as a result of its design, can be 4 to 28 days. The half-life of a drug is the time taken for the plasma concentration of a drug to reduce to half its original value. Half-life estimates how long it takes for a drug to be removed from your body. Half-life influences the medication schedule, which includes the appropriate dose and the correct times to take them. Another critical topic is safety; small molecule drugs can access several tissues and cells that may be off-target, resulting in toxicities and adverse reactions, whereas monoclonal antibodies, limited to interacting with highly specific extracellular protein receptors, typically have far fewer side effects. A small molecule drug’s route of administration is often oral, whereas a biologic is most often intravenous. Focus On Antibodies: Their Roles According to Market Data Forecast, the size of the 2022 global next-generation antibody therapeutics market is $4.33 billion US dollars. It is expected to grow by 13% to reach $7.97 billion US dollars by 2027. Therapeutic antibodies are based on naturally occurring antibodies that our immune system produces; both the naturally occurring and the biopharma-produced antibodies fight diseases such as cancer, infectious disease, and autoimmune disease. 2 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Naturally occurring antibodies and biopharmaceutical antibodies have four primary roles: 1. Antibodies bind to and inactivate pathogens and toxins. 2. Antibodies attach to receptors, blocking chemical signals from reaching a cell. 3. Antibodies activate the complement system to destroy bacterial cells by punching holes in their cell wall 4. Antibodies facilitate the clean-up of foreign material by flagging the debris to be eaten by other immune cells called macrophages. Focus on Antibodies: Human Immunoglobulin Functions Currently, all marketed therapeutic antibodies are derived from an immunoglobulin, specifically, an IgG. Immunoglobulin is a class of proteins present in the body’s serum (blood) and immune cells. Immunoglobulin functions as antibodies. This table lists each immunoglobulin class with its associated function. IgG makes up roughly 75% of all known immunoglobulin. Please pause here to review, on your own, the different types of immunoglobulins. Focus on Antibodies: Human Immunoglobulin Structures Human immunoglobulins differ in their structure, hinge regions, the number of heavy chains, and the distribution of carbohydrate groups. Each one of these features can be manipulated to alter the antibody shape. Since structure influence function, an antibody’s structure plays an outsized role in its ability to bind to a drug target. By changing the angle of a hinge region, the size of the heavy chains, or the number of carbohydrate groups, an antibody’s shape, and function can be altered to become a more potent drug candidate. The Big Picture It’s essential to view the entire drug development process, as seen here, to understand where the drug discovery process fits into the overall scheme of drug development. Drug development comprises all the activities involved in transforming a compound from a drug candidate (the endproduct of the discovery phase) to a drug product approved for marketing by the appropriate regulatory authorities (the end of the clinical stage). This process is efficient. Efficiency in drug development is critical for commercial success for two main reasons. First, drug development accounts for roughly 75 percent of the total R&D costs. The cost per project increases sharply as the project moves into the later stages of clinical 3 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. development. Keeping costs under control is a primary concern for management, so the failure of a compound late in development represents a significant amount of wasted money. Second, speed in development is essential in determining sales revenues. Time spent in development shortens the 20-year patent production once the drug goes to market. As soon as the patent expires, competition sharply reduces sales revenue. The Big Picture in Numbers Here we list the average time, cost, and number of candidates associated with each critical development stage. These numbers represent the industry totals based on treating a variety of indications with large molecules. The discovery process includes the early phases of research, which are designed to identify potential drug candidates. According to the non-profit organization PhRMA, the drug discovery process takes three to six years to complete and costs up to $200 million US dollars. Of the multiple candidates screened in the drug discovery phase, on average, 250 are worthy of being sent to preclinical development. Preclinical development tests the 250 candidates in animal models. The goal of preclinical development is to assess which candidates are safe, to establish a starting dose for clinical trials, and to a lesser extent, to show efficacy potential. On average, it takes four years to complete the preclinical stage and to generate the data necessary to select roughly seven candidates to enter clinical development. Clinical development rigorously tests drugs in humans to determine the compound’s safety and efficacy. On average, it takes seven years to test seven biologics and, assuming safety and efficacy are proven, to produce one biologic worthy of a BLA or biologic license agreement submission. If the BLA receives FDA approval, that biologic can be marketed to patients. In Silico, In Vivo, and In Vitro Testing Funneling these numbers down from 250 to one relies on large databases compiled from screening, pharmacology testing, and toxicology testing using in silico (computers), in vitro (cells), and in vivo (live animals) systems. Let’s stop here briefly to discuss animal testing. The biopharma industry and the FDA back the “Three Rs”—replacement, reduction, and refinement—which are guiding principles for more ethical use of animals in drug testing and scientific research. 4 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. • Replacement: Use non-vertebrates in place of vertebrates because nonvertebrates have a lower potential for pain reception. • Reduction: Researchers must reduce the number of animals used in each experiment, relying more on in silico and in vitro testing. • Refinement: Researchers must refine their procedures to minimize adverse conditions for animals. As always, safety is a crucial endpoint at all stages of development; thus, the discovery stage has several methods to screen for compounds most likely to succeed in clinical trials. We will explore these methods in the following sections. Section 1: Setting the Stage Summary In this section, you learned the following: • Based on the FDA's definition, a drug is a substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease. • Biologics bind to extracellular drug targets in a highly specific manner. They are limited to blood and extracellular space, so they have a low volume of distribution in the body. • Biologics have a medium half-life at low doses and a long half-life at high doses. Off-target toxicities are rare. The route of administration is typically intravenous. Small molecule drugs bind to intracellular and intranuclear drug targets in a less specific manner. They have a high volume of distribution in the body and a short half-life. Off-target toxicities are more common, and the route of administration is often oral. • The drug development process begins with drug discovery, preclinical development, and clinical development, ending with the drug either receiving regulatory approval or not being marketed to patients. • The average drug discovery process takes 3-6 years, produces 250 drug candidates to move on to preclinical development, and costs as much as $200 million. 5 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Section 2: Drug Target Identification and Validation Welcome This section begins with drug target identification, identifying a possible drug target and its role in the disease. It ends with drug target validation, verifying that the predicted drug target plays a role in the disease. We will explore several methods used to identify and validate targets. However, please note that many other techniques are used in drug discovery and are too complex for this introductory course. Section 2: Drug Target Identification and Validation Objectives By the end of this section, you should be able to: • List the steps of drug discovery. • State the purpose of drug target identification. • State the purpose of drug target validation. • Name the methodologies used in drug target identification. • Name the methodologies used in drug target validation. Drug Discovery Workflow Let’s begin by examining the drug discovery workflow. • Drug target identification. • Drug target validation • Drug candidate identification • Lead candidate selection • Lead candidate optimization We will focus on the first two steps. Drug target identification aims to identify a possible drug target and its role in the disease. Drug target validation endeavors to verify that the predicted drug target plays a role in the disease. Target Identification: In Silico Many drug targets are initially identified using in silico methods such as reading scientific literature and combining online public databases. Two popular databases are DrugBank and Therapeutic Target Database. 6 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. DrugBank contains information on drugs and drug targets. As both a bioinformatics and a cheminformatics resource, DrugBank combines detailed chemical, pharmacological, and pharmaceutical data with the drug target sequence, structure, and signaling pathway information. The Therapeutic Target Database (TTD) provides information about known protein, DNA, and RNA drug targets, disease conditions, signaling pathways, and corresponding drugs for each drug target. TDD cross-links to other databases that include information about the target sequence, 3D structures, function, drug binding properties, drug usage and effects, and related literature. DrugBank and the Therapeutic Target Database are used by the biopharma industry, medicinal chemists, pharmacists, and physicians. Target Identification: In Vitro Methods Drug targets can be identified by performing in vitro mechanistic studies and studying mRNA expression profiles. In vitro mechanistic studies provide information on the molecular, cellular, and physiological disease mechanisms that affect the cells. mRNA expression profiles indicate what a cell is doing at a point in time. Recall from first-year biology class that a gene codes for mRNA, and in turn, mRNA codes for a protein. If mRNA is produced, the associated gene that codes for that mRNA is said to be "on“—the gene is working. If no mRNA is made, the related gene is turned "off“ and not working. Altered levels of a specific mRNA may indicate a changed need for that protein, perhaps indicating disease. For example, suppose breast cancer cells express higher levels of mRNA associated with a particular receptor, such as the HER2 receptor, compared to healthy cells. In that case, it might be that the associated HER2 receptor plays a role in breast cancer. HER2-positive breast cancer is one type of cancer that is mitigated with a monoclonal antibody called Herceptin. Drug Target Identification: CRISPR Today, the primary method used for target identification is CRISPR, a DNA editing tool. The ability to edit specific genes by “knocking them out” or “in” can reveal if a gene or its associated proteins are responsible for a particular disease. Although it may sound counterintuitive, one of the most direct ways to determine what a gene does is to see what happens to the cell when it is knocked out. Simply put, change the expression of a gene, and observe what happens. Early work using genome-wide CRISPR loss of function screens showed their promise for target identification. For example, Vemurafenib treats melanoma patients. However, after months of treatment, patients often develop resistance to vemurafenib leading to melanoma progression. Using 7 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. CRISPR, researchers identified 33 genes that regulate resistance to vemurafenib. Any of these 33 genes or their associated proteins may be drug targets that, if appropriately manipulated, may allow vemurafenib to continue working in melanoma in patients. Drug Target Validation Once drug target identification has been established, the drug target must be confirmed. Drug target validation verifies that the predicted drug target plays a role in the disease. The questions researchers ask are: Is the drug target druggable? Does the drug target play a key role in the disease process? Is binding a drug to that target likely to be safe and effective? Drug Target Validation: In Silico Drug target validation begins in silico with databases such as the Therapeutic Target Database (TTD), which houses three types of target validation data: • The level of drug potency against the drug target. • The effects of drug candidates against the drug target. • The effects of drug target knockout via CRISPR and RNA interference. Target validation is crucial to help scientists identify the most promising approaches before going into the laboratory to develop potential drug candidates, increasing the efficiency and effectiveness of the R&D process. Drug Target Validation: RNAi RNAi is a family of non-coding RNAs synthetically made to bind to mRNA. A double-stranded RNA (dsRNA) is formed when RNAi and mRNA are bound together. Since naturally occurring RNA is single-stranded, enzymes patrolling the cell will degrade the dsRNA, thus preventing the associated protein from being made. The drug target can be validated by creating a synthetic RNAi to bind to the mRNA that codes for the identified drug target. Drug target validation is a crucial step in the drug discovery process because, as the development process continues, it gets progressively more expensive―if a drug is unlikely to be successful, it is better to find out sooner rather than later. 8 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Section 2: Drug Target Identification and Validation Summary In this section, you learned: • The steps of drug discovery are drug target identification, drug target validation, drug candidate identification, lead candidate selection, and lead candidate optimization. • The purpose of drug target identification is to identify a possible drug target and its role in the disease. • The purpose of drug target validation is to verify that the predicted drug target plays a role in the disease. • The methodologies used in drug target identification include searching databases for drug target information, performing in vitro mechanistic studies, studying mRNA expression profiles, and using CRISPR technology. • The methodologies used in drug target validation include searching databases for drug 9 validation information and performing in vitro RNAi tests. Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Section 3: Drug Candidate Identification, Selection, and Optimization Welcome In this section, we will look at the final three discovery steps, drug candidate identification, lead candidate selection, and lead candidate optimization. Let’s get started. Section 3: Drug Candidate Identification, Selection, and Optimization Objectives By the end of this section, you should be able to: • State the purpose of drug candidate identification. • State the purpose of lead candidate selection. • State the purpose of lead candidate optimization. • Name the methodologies used in drug candidate identification. • Name the methodologies used in lead candidate selection. • Name the methodologies used in lead candidate optimization. Drug Discovery Workflow You are familiar with the drug discovery workflow as seen on the screen. In the last section, we discussed the first two stages, now our focus is on selecting a lead candidate to move into preclinical development. Steps three, four, and five include drug candidate identification, lead candidate selection, and lead candidate optimization. Drug candidate identification screen libraries to determine the best biologics for the drug target of interest. Lead candidate selection chooses the best drug candidates to become the lead candidates by assessing toxicology and pharmacology findings. Lead candidate optimization enhances the lead candidate’s physiochemical properties to best stop disease progression. Drug Candidates The drug candidate is any compound, small or large molecule, that can bind to the drug target to stop the progression of the disease. Drug candidate identification is difficult and expensive and is accomplished in a variety of ways. These include identifying compounds found in nature, creating a molecule from living or synthetic material, genetically engineering living systems to produce disease-fighting molecules, and using compound libraries to select a few promising possibilities from among thousands of potential candidates. 10 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Living Systems Produce Drug Candidates Let’s explore two of the four methods of obtaining drug candidates in more detail, starting with genetically engineered living systems that produce monoclonal antibodies. Researchers use genetically engineered mice to produce human monoclonal antibodies. To create such mice, the antibody gene in a mouse embryo is first silenced. Next, a human immunoglobulin gene is inserted into the embryo. The embryo is implanted in a surrogate mouse, which gives birth to the genetically engineered mouse that produces human antibodies. This mouse is bred to create a line of mice with the human antibody gene. When these genetically engineered mice are injected with a foreign substance known as an antigen, every antibody the mice produce is fully human. Meaning if the fully human antibody were given to a person, there would be no rejection of the antibody as the person’s immune system will recognize it as human---as self. If it had remained a mouse antibody, the human immune system would reject it before it could treat the disease for which it is intended. The mice B-cells, which produce the antibodies, are screened to select the antibodies that best bind to the drug target. The corresponding gene for the desired antibody is elucidated and transferred to a manufacturing cell line for production. Since the antibody is human, the cell line most likely to be chosen is the Chinese hamster ovary or CHO cell line. This mammalian cell line produces very large and complex monoclonal antibodies, as CHO cells are capable of various cellular modifications that must occur before the monoclonal antibody is functional. These mammalian cell structural modifications are only performed by a mammalian cell line. A disadvantage of this technology is the mouse’s immune system does not easily recognize the human antibody. As a result, the mouse has a less robust immune response and will create fewer B-cells that recognize the antigen of interest. This may decrease the likelihood of finding an antibody that binds the drug target with high affinity. Libraries Produce Drug Candidate Instead of making the drug candidates from scratch, companies most often use a library. A library is a collection of compounds that have a natural grouping. There are multiple compound libraries, including natural product libraries, FDA-approved drug libraries, and target-specific libraries. Taking a closer look at target-specific libraries, they are used when the drug target is known. Some examples are RNA libraries, DNA libraries, and specific receptor libraries. These libraries may be 11 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. further grouped. For example, the specific receptor libraries include a specific GPCR library or a specific calcium ion channel library. It’s worth noting that ‘Hits’ detected in the initial screen often turn out to be compounded with undesirable features, such as excessive polarity or possession of molecules known to be associated with toxicity. Computer-based molecular modeling uses computational prescreening of compound libraries to eliminate undesirable biologics. Screening Libraries Once a library is accessed, a collection of potential candidates is chosen by screening. High throughput screening (HTS) is an integral part of drug discovery that uses robotics, data processing/control software, liquid handling devices, and sensitive detectors to quickly conduct thousands of chemical, genetic, and pharmacological tests to identify active biologic compounds, such as antibodies, that modulate a particular disease pathway. High Throughput Screening To find the best possible drug candidate for a drug target, it is necessary to screen hundreds to thousands of compounds using HTS. Assays are performed in the wells of a microtiter plate containing 96 or 384 wells. Assay choices are many and include cell-free enzyme assay, membrane-based binding assay, and cellular response assay. Let’s use the example of the EGF receptor (EGFR) to understand the power of HTS. Suppose we conclude that the drug target is the EGF receptor. We also know that to stop disease progression, EGFR must be blocked. To screen for an EGFR inhibitor, genetically modified cells with EGF receptors are used to make the cellular response assay. This assay is loaded into the wells. Next, a variety of EGFR inhibitor candidates obtained from the EGFR-specific receptor library are added, one to each well. A growth factor signaling molecule is added to activate each EGFR inhibitor drug candidate. All the wells in which the EGFR is activated (not inhibited) fluoresce green. Any wells in which EGFR is inhibited will not fluoresce. To see which wells are fluoresced or not, the microtiter plate is read by a plate reader. Any “blank” wells represent potential EGFR inhibitors, which are taken for further studies. These potentially promising compounds are referred to as “Hits.” You may have heard the term “Hit to Lead.” This references the drug candidate “hit” from the screening process, that moves on to become a lead candidate. 12 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Lead Candidate Selection Lead compounds undergo a series of tests to provide a preliminary safety assessment. Normally performed via computational models (in silico), in living cells (in vitro), and in animals (in vivo), these studies help researchers prioritize lead compounds early in the discovery process. During lead candidate selection, scientists assess how the body processes the drug candidate—what the body does to the drug—also referred to as pharmacokinetics. And they evaluate the impact the candidate drug has on various functions within the body—what the drug does to the body—or the pharmacodynamics. Successful drugs must be: • Absorbed into the bloodstream. • Distributed to the proper site of the body meaning the drug target is in the correct tissue. • Metabolized efficiently and effectively. • Successfully excreted from the body. Demonstrated to be not toxic in the tests performed. Focus On Pk and PD Pharmacokinetics (PK) measures the absorption, half-life, and elimination of the drug. It is used to optimize dosing. Pharmacodynamics (PD) measures the effects of the drug at increasing concentrations until adverse effects are seen. Animals used in PK and PD drug discovery studies may include fruit flies, zebrafish, and rodents. Interspecies scaling allows for the prediction of in vivo drug absorption, distribution, and clearance in humans from the experimental observations made in one or more animal models. These tests are used to optimize dosing. Lead Candidate Optimization Lead Candidate Optimization is the last step before preclinical development. It is the starting point for detailed chemical modifications to further improve target specificity, selectivity, pharmacokinetic profile, and safety profile while maintaining the favorable properties of the lead compounds. Hundreds of variations or “analogs” of the initial leads are produced and tested. By slightly changing their structures, scientists can give them different properties. For example, they can make a compound less likely to interact with other chemical pathways in the body, thus reducing the potential for side effects. 13 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Other assays used in optimization include: • hERG assay determines lead candidate potency. • Ames assay determines if the lead candidate can mutate DNA. • Protein binding assay determines how the lead candidate interacts with the drug target. The resulting optimized biologic is the candidate drug which is sent forward to preclinical development, where it undergoes years of further testing and analysis before potentially being reviewed and assessed for approval by a regulatory agency such as the Food and Drug Administration (FDA). Section 3: Drug Candidate Identification, Selection, and Optimization Summary In this section, you learned: • The purpose of drug candidate identification is to screen libraries to determine the best biologics for the drug target of interest. • The purpose of lead candidate selection is to choose the best candidates to become the lead by assessing toxicology and pharmacology findings. • The purpose of lead candidate optimization is to enhance the lead candidate’s • physiochemical properties to best stop disease progression. • The methodologies used in drug candidate identification include identifying compounds found in nature, creating a molecule from living or synthetic material, genetically engineering living systems to produce disease-fighting molecules, and using high-throughput screening techniques. • The methodologies used in lead candidate selection include computer-based molecular modeling, X-ray crystallography, and high throughput screening. • The methodologies used in lead candidate optimization include pharmacokinetic and pharmacodynamic studies, interspecies scaling, hERG assay, Ames assay, and protein binding assay. 14 Copyright 2023 Biotech Primer, Inc.