Antibodies Transcript (PDF)

PROPRIETARY. DO NOT SHARE. Transcript: Antibodies Section 1: Antibodies Overview Welcome Welcome to Biotech Primer’s online course entitled “Antibodies.” Antibodies are elegant Y-shaped proteins that are naturally made by your immune system to protect you against disease. Scientists have adapted th...

PROPRIETARY. DO NOT SHARE. Transcript: Antibodies Section 1: Antibodies Overview Welcome Welcome to Biotech Primer’s online course entitled “Antibodies.” Antibodies are elegant Y-shaped proteins that are naturally made by your immune system to protect you against disease. Scientists have adapted these same antibodies to be used in diagnostics and to treat disease. This course introduces you to the wonderful world of antibodies. This Antibodies course is divided into 3 sections: Antibodies Overview, Antibodies as Therapeutics, and Antibodies as Diagnostics. Let’s get started. Section 1 - Antibodies Overview Objectives At the end of this section, you should be able to: • Describe the structure and function of an antibody. • Explain the difference between polyclonal and monoclonal antibodies. • Discuss antibodies’ role in the human immune system. • Describe how antibodies are identified for biotech applications. What is An Antibody? Let’s begin by asking the question, what is an antibody? Antibodies are proteins. Antibodies are used in healthcare as both a therapeutic and a diagnostic. What Do Antibodies Do? The simple answer―antibodies fight disease. Antibodies help your body recover from infection caused by pathogens such as viruses, bacteria, and parasites. Antibodies even go after cancer, a disease caused when cells divide uncontrollably and spread into surrounding tissues. As you may know, cancer is caused by changes in your DNA. Where Do Antibodies Come From? Antibodies are naturally produced by your immune system. More specifically antibodies are made by a type of immune cell called a B-cell. B-cells are discussed later in this course. 1 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. How Do Antibodies Recognize Foreign Proteins? Foreign proteins, which are present on the surface of bacteria, virus particles, parasites, and cancer cells, are most often referred to as antigens and sometimes referred to as immunogens. Frequently, immunologists use those two terms, immunogen and antigen interchangeably. An antigen is the official word for any protein on the surface of a cell that an antibody can recognize and bind to. Antigen is a more general term. An immunogen is an antigen capable of inducing an immune response. What is the difference between an antigen and an immunogen? An immunogen refers to a protein that can elicit an immune response by an organism's immune system, whereas an antigen refers to a protein that is capable of binding to the product of that immune response. So, an immunogen is necessarily an antigen, but an antigen may not necessarily be an immunogen. Again, the term immunogen is used interchangeably with the term antigen. But only an immunogen can evoke an immune response. Even though it is more scientifically correct to use the term immunogen in this specific course, we will use the more common term antigen because we believe you will encounter the term antigen more often in your work. Antibody Structure An antibody is a special protein because an antibody is designed to recognize and bind, or stick to, other proteins. Antibodies have, as you can see on the slide, a characteristic Y shape. The antibody is made up of two types of chains: a smaller chain, which is called the light chain, and a longer chain, which is called the heavy chain. Within these chains, there are different sequences of amino acids. Recall that proteins are made up of building blocks called amino acids. Each amino acid sequence combination confers unique properties on the antibody. At the top of this Y-shaped molecule, on both the heavy and the light chain, there is a region that is known as the variable region. The variable region, as you might guess, changes from antibody to antibody. It is the variable region that recognizes and binds to antigens, those foreign proteins on the surface of pathogens and cancer. Let me repeat this important concept; the variable region of an antibody is a unique sequence of amino acids. It is the variable region that allows an antibody to recognize and bind to a specific antigen. The rest of the antibody molecule, especially in the heavy chain, is what is known as the 2 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. constant region. As you might guess, the constant region doesn't change. The constant region is constant—it is how your body knows the protein is an antibody. The constant region is also specific to species. Humans have a constant region that is recognized as “human” and mice have a constant region that is recognized as “mouse” and so on. What Are the Different Kinds of Antibodies? Human antibodies are classified into five isotypes: IgM, IgD, IgG, IgA, and IgE. Ig stands for immunoglobulin. Antibodies are also called immunoglobulins. Immunoglobulins are a class of proteins present in the serum and cells of the immune system. The type of immunoglobulin, or antibody, is determined according to their heavy chains which you will also see written as H chains. Each H chain provides each isotype with distinct characteristics and roles. IgG is the most abundant antibody isotype in the blood, accounting for 70-75% of human antibodies! The 5 Types of Antibodies Let’s take a closer look at the five isotypes of antibodies. IgG is the antibody isotype that most people think of when they're talking about antibodies. It is the antibody that is built by immunization. It activates an immune cascade that eliminates infection. IgG can also neutralize certain toxins. Multiple forms of IgG circulate through the body and respond to infection. IgG is made up of only a single Ig subunit. It is the only type of antibody that can cross the placenta during pregnancy. That is how a fetus takes advantage of their mother's immune response. IgG can attack a fetus’ infection. IgA is the antibody isotype found in mucosal areas, such as the mouth and vagina. It is also found in saliva, tears, and breast milk. IgA is formed by two Ig subunits bound together. When IgA binds to a target, it can stimulate inflammation. In mucosal areas, IgA can also keep pathogens from sticking to epithelial cells. The production of IgA against inappropriate targets is associated with certain autoimmune diseases, such as celiac disease. IgM is one of the first types of antibodies to be produced after a pathogen has entered the body. Since it is made up of five Ig subunits bound together, it binds very strongly to its target. IgM is very important in the early stages of an infection. IgM sometimes appears when an infection 3 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. becomes reactivated, such as with a herpes outbreak. It can also appear when someone is reexposed to a type of disease that they've previously gotten rid of. IgE is the antibody that is responsible for the allergic response. It is mostly found in the lungs, skin, and mucous membranes. When IgE binds to an allergen, it starts the histamine reaction. It's the histamine reaction that causes the symptoms of an allergy attack. This single subunit antibody also helps to protect the body from parasitic worms. IgD is important in the early stages of the immune response. Bound to B-cells, it does not circulate. Instead, it signals B-cells to become active. This helps stimulate inflammation. IgD is the least understood type of antibody, and its functions are still being discovered. Epitopes Let's dig a little deeper and look at what properties of an antigen allow it to be recognized by an antibody. Once again, antigens are surface proteins that are present in all cells including pathogens and cancer cells. A protein is composed of amino acids, which can be hundreds of amino acids long. Different proteins have different amino acid compositions. Within each protein, some amino acid sequences are unique only to that protein. It’s the only protein that has that particular sequence. A unique sequence of amino acids that can be recognized by an antibody is called an epitope. Let me repeat that slowly: A unique sequence of amino acids recognized by an antibody is called an epitope. The epitope is the part of the protein, the part of the antigen, that the antibody recognizes. Large antigen molecules may have as many as 400 amino acids. Of those 400 amino acids, an antibody will recognize 5 to 8 of those amino acids―that is the epitope. An epitope is a very small sub-piece of the antigen that your antibody recognizes. When the antibody interacts with the epitope it is called an “antibody-antigen interaction.” That interaction is said to be a binding event. The antibody binds and attaches to, the antigen. This attachment isn't permanent. This attachment can be undone, but it does stick one to the other. Antigens may have several different epitopes on the same protein. 4 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Different Antibodies Recognize Different Epitopes on The Same Antigen There is one antibody for each epitope. Think of it as “antibody number 1” will recognize “epitope number 1”. “Antibody number 2” will recognize “epitope number 2” and so on. Where Do Antibodies Come From? It's time to learn the secret of your immune system. Antibodies are produced by a specific type of white blood cell called a B-cell. B-cells originate and mature in your bone marrow. They come from what is called bone marrow stem cells or hematopoietic stem cells. Hematopoietic stem cells give rise to all blood cells in your body, but only one particular subclass of cells differentiates and becomes B-cells. Look at the B-cell, do you see a Y-shaped antibody? Antibodies are embedded in the surface of the B-cell. In this case, this particular embedded antibody acts as a receptor. It receives information. What's the information that it receives? It receives the epitope from the virus, bacteria, parasite, or cancer cell. There is one unique B-cell receptor for each epitope that a B-cell may encounter. Let me clarify what that means; you have billions of individual B-cells. Each B-cell with a different receptor can recognize a different epitope. It is said you have B-cells in your body that recognize epitopes that aren’t even present on Earth! Generation Of Antibody Diversity It is important that the immune system must be able to adapt to new and changing pathogens such as viruses which rapidly mutate. How are we able to generate hundreds of millions of different antibodies, specific for evolving pathogens when we only have a single antibody gene? The answer is genetic shuffling. Antibodies are produced by B-cells which originate and mature in the bone marrow; hence the name B-cell. Antibodies are composed of four segments, each coded by a different region of the antibody gene. The four gene segments that make up the final antibody are V, D, J, and C. 5 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. The antibody gene is located on chromosome number 14. The antibody gene contains multiple segments that code for the V region, the D region, the J region, and the C region of the antibody. When the newly created B-cell matures, the antibody gene is shuffled, randomly selecting one V, D, and J region. Initially, all the C or constant regions are retained which will allow the B-cell to later undergo class switching. The mature B-cell then tries to produce the antibody from the randomly shuffled gene. The shuffled gene does not always successfully code for an antibody and many B-cells undergo apoptosis or cell suicide because they cannot produce an antibody from their shuffled gene. Because the antibody has been randomly generated, there is a possibility that it might bind to a “self-antigen”, such as a protein on the outside of a liver cell. This antibody binding to a healthy cell may lead to autoimmune disease. To prevent these potential autoimmune-provoking B-cells from being released from the bone marrow, a second white blood cell, called a dendritic cell, puts the newly created B-cell through a kind of gauntlet. The dendritic cell presents “self-antigens” to the B-cell. If the B-cell antibody binds to the “self-antigen”, the dendritic cell triggers apoptosis or cell suicide and both the dendritic cell and the B-cell die. This prevents most B-cells that bind to “self-antigens” from leaving the bone marrow. If the B-cell does not bind to any of the “self-antigens” presented by the dendritic cell, the B-cell is released from the bone marrow and travels to the lymph system. B-Cell Activation After Exposure to A Pathogen Let’s stop here and put all this information together. Imagine someone sneezing near you. You inhale the flu virus from that sneeze. The flu virus starts to divide within your throat. Some of your immune cells called macrophages and neutrophils come by and try to combat this flu virus. They pick up the flu virus antigen. They carry that antigen to your lymph nodes where your B-cells have been transported from your bone marrow. In your lymph nodes, these macrophages or neutrophils deliver the antigen to that B-cell receptor which is also known as the BCR. BCR stands for B-cell receptor. Within that flu virus antigen, there is a unique viral epitope. That epitope binds to the BCR. Once that epitope binds to the BCR or B-cell receptor, it triggers a whole series of events inside the B-cell. This entire interaction is called a signaling process―it’s the way that cells communicate. In this signaling process, the macrophages and neutrophils are communicating to the B-cell that the body has been invaded and that the B-cell needs to take action. This communication is accomplished by the handoff of the flu virus epitope. 6 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. B-Cells Are Activated by Antigens Ultimately what happens, when the flu virus antigen binds to the B-cell receptor, the B-cell becomes activated. In this case, activation means the B-cell is going to do one of two things: The B-cell will differentiate to become one of two cells, either a plasma cell or a memory B-cell. For activated B-cells, about 90% of them become plasma cells. Plasma cells are the cells that secrete antibodies specific to that flu virus epitope. Memory B-cells, as you might guess, are the cells that stay behind. They don't do anything. They just find another lymph node to sit back and relax in, and there is kept the memory of your flu virus exposure just in case it comes back again. And if that specific flu virus re-infects your body, your body will be able to recognize that flu virus immediately and mount a fight quickly, most likely keeping you from getting that specific type of flu a second time. How Do Antibodies Fight Disease? If you remember, way back in the beginning, when we talked about the structure of the antibody, we said that it has a constant region. Well, it turns out that a part of that constant region, the “FC region” is recognized by macrophages, another type of white blood cell. Macrophages engulf or eat, diseased cells. Macrophages have what's called the “FC receptor.” Pretty clever, huh? The “FC region” of the antibody is recognized by the “FC receptor” of the macrophage. This recognition allows these two cells to “communicate.” The FC receptor on the macrophage binds to the FC region antibody. But what is neat about this process is when the antibody is bound to the virus, the macrophage can come along and bind to the FC region of the antibody. When this interaction occurs, the macrophage engulfs the entire “antibody/virus complex”, and then breaks it down into its molecules that will be reused or eliminated from the body. So, the macrophage digests the virus, destroying it. Ultimately, your viral load is decreased and you recover. This is how the antibody fights disease. Polyclonal Antibodies When we think about using antibodies for research, diagnostic, or therapeutic purposes, we tend to put antibodies into two categories. 7 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Polyclonal antibodies are produced by a collection of different B-cells. As a result, they recognize multiple epitopes on the same antigen, which makes them ideal for some types of diagnostics, where the only requirement is detecting a specific antigen. Traditionally, polyclonal antibodies were made in rabbits using a straightforward procedure. First, a specific antigen was taken from a pathogen. That antigen was injected into a rabbit, which triggered an immune response. This immune response caused the rabbit to produce multiple types of antibodies in their blood serum. A very simple, painless ear bleed on the rabbit was performed and the antibodies were collected and purified. This antibody serum is polyclonal because it has different antibodies that recognize different epitopes on the same antigen. Remember, antigens have more than one epitope. The advantage of polyclonal antibodies is they have a better chance of detecting an antigen. The disadvantage of polyclonal antibodies is they may detect similar, not necessarily identical, antigens. That can lead to a false positive. A false positive is when a screen might indicate an antigen is present, when really what is being seen is just a weak response to a different, but very similar protein. Polyclonal antibodies are fairly easy and inexpensive to produce when compared to monoclonal antibodies. Polyclonal antibodies are ideal for research and diagnostics. Monoclonal Antibodies (mAbs) The second type of antibody we want to talk about is monoclonal antibodies. Mono, as you know, means one or single. This is a single antibody that recognizes a single epitope. As you might guess, this type of antibody is highly, highly, specific. Monoclonal antibodies all derive from the same B-cell or its clones. A clone is a descendant. This “sameness” enables monoclonal antibodies to recognize the same epitope. Therapeutic antibodies are always monoclonal, which ensures consistent treatment. Rarely are false positives given with monoclonal antibodies, so accuracy is assured. When using monoclonal antibodies as therapeutics, off-target effects are limited. The first monoclonal antibodies were made in 1975 in a mouse. The production was the same as that used 8 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. to make polyclonal antibodies. The mouse was injected with a viral antigen which led the mouse to raise an immune response that produced many different types of antibodies. Now you are probably thinking, "But wait. That antigen has different epitopes." And you are correct. However, it turns out that B-cells, when they get close to expiring, collect in the spleen. Researchers went into the spleen and isolated thousands of B-cells. Remember each B-cell produces a different, but specific antibody. The challenge at this point in the process was B-cells do not have a long lifespan. Researchers only had 5 days to find the correct B-cell that produced the antibody of interest. Researchers needed more time to sort through the B-cells. So, in a genius move, B-cells were fused with myeloma cells. Myeloma cells are immortal cancer cells. Now researchers had as much time as they needed to search for and find the B-cell of interest. Believe it or not, you can buy myeloma cells from the American Type Culture Collection. The resulting B-cell/myeloma cell combo is a hybrid. These are called hybridoma cells and have the properties of both cell types. Today this procedure is no longer being done in the animal. This is done in cell culture outside of the animal and each B-cell is grown individually in a little multi-well plate. When that one B-cell that produces the antibody of interest is found, that B-cell is replicated. Injecting A Mouse Antibody Into Human Elicits An Immune Response Initially, in the late 1970s, and what now seems to be naive, scientists thought, "We can make monoclonal antibodies in mice to fight disease.” Remember earlier, I said the constant region of antibodies is the same. That is true, it is constant for a given species, but it differs from one species to another. When a mouse-made monoclonal antibody was injected into a human, the human's immune system knew it was a foreign antibody and rejected it. Humanizing Mouse Antibodies Scientists realized that maybe they could manipulate the sequence of the mouse antibody and make it appears to be more human. Think about this- what antibody sequence would you manipulate? You are correct, you would manipulate the constant region. The variable region must be retained because the variable region recognizes the epitope on the diseased cell’s antigen. You eliminate the mouse constant region 9 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. and replace it with a human constant region. Problem solved! This modified antibody is mostly human, but still part mouse. Because DNA sequences code for both the variable and constant regions, these sequences can be produced in a mammalian cell line. Therefore, an unlimited amount of very specific humanized monoclonal antibodies can be produced and this is exactly what the biopharma industry does today. Fully Human mAbs: Genetically Engineered Mice Transgenic mice that produce monoclonal antibodies can also be made. “Trans” means “across”, so we are moving genes across species. In this case, a gene from a human B-cell that codes for the antibody of interest is isolated and injected into a mouse. That mouse now carries a human gene. Anytime a genetically engineered mouse is injected with a foreign antigen, that B-cell will become activated and produce a fully human antibody. We can then screen for that particular Bcell, selecting the B-cell that created the antibody for our antigen, and then transfer that gene to CHO cells to create a large number of these antibodies. Fully Human mAbs: Phage Display Probably the most common way to identify useful antibodies, whether it's for research, diagnostics, or therapeutics, is by using a technique called phage display. A phage is a virus that infects bacteria. Technically, these are called bacteriophages, but you will also hear them referred to as just phages. Phages are viruses that infect bacteria; they don't kill the bacteria, they are parasitic. Once a bacteriophage infects a bacteria cell, the bacteria is directed to make copies of the phage. The copies are eventually released from the bacteria cell and go on to infect other nearby bacteria cells. This produces millions of virus particles in a short time. After observing this natural process, scientists created a man-made process called phage display. Researchers can take a piece of DNA, for example, from a human B-cell and isolate a specific gene that codes for antibodies. That antibody gene is spliced into, or put into, the bacteriophage, and now that virus is used to infect bacteria cells. Once inside the bacteria cell, the phage is replicated, as well as, the antibody that is coded for by the antibody gene the researcher spliced into the virus in the first place. 10 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. In other words, what's happening is the bacteriophage is now using the information from the antibody gene to produce a fragment of the antibody. That antibody fragment is incorporated into the page, and these antibody fragments are presented on, or displayed on, the surface of the phage. And so, in a relatively short time, there are many antibody fragments present on the surface of the phage. If many different antibody genes are mixed in with the phage, then each phage will present a different antibody fragment. This mixture of different antibody fragments can now be used as a screen. The easy way to make a screen is simply to attach the antigen to a stationary surface. Then pour the phage mixture, with the different antibody fragments displayed, over that stationary surface. Then rinse the stationary surface. Any phage that remains bound to the antigen is the phage that displays the antibody you are after. The DNA is recovered from the remaining phage post-rinse. That recovered DNA can be placed into a mammalian cell line, such as a CHO cell, and that mammalian cell line will produce unlimited copies of the monoclonal antibody. This is, believe it or not, a much faster and much simpler process than making monoclonal antibodies by hybridomas. Section 1: Antibodies Overview Summary In summary of section 1, we learned that: • Antibodies are Y-shaped proteins with a variable and constant region. Antibodies recognize pathogens and cancer with their variable region. • Polyclonal antibodies are a collection of different antibodies recognizing different epitopes on the same antigen. Monoclonal antibodies are one type of antibody that recognizes one epitope on an antigen. • Antibodies are produced naturally by B-cells to flag foreign proteins for destruction. • Biotech companies have developed methods for identifying antibodies including phage display and hybridomas. 11 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Section 2: Antibodies as Therapeutics Welcome Monoclonal antibody therapeutics burst onto the healthcare scene more than twenty years ago. They remain one of the most versatile and effective therapies available for a whole range of diseases including different types of cancers, autoimmune diseases, infectious diseases, and more recently, high cholesterol. Tried and true monoclonal antibodies, such as Herceptin and Rituxan, remain in high demand. Let’s take a closer look at therapeutic antibodies. Section 2 - Antibodies as Therapeutics Objectives At the end of this section, you should be able to: • List the advantages of antibody therapeutics. • Explain the various antibody mechanisms of action used to mitigate disease. • Give examples of therapeutic antibodies available to patients today. Antibodies Mechanism of Actions Let’s begin this section by asking the question: why are monoclonal antibodies so useful as therapeutics? Monoclonal antibodies have two desirable characteristics: They are highly selective and versatile. Monoclonal antibodies are highly selective for a specific antigen. Antigens are drug targets. A drug target is a naturally existing molecule involved in a disease that a drug is meant to act on. Therefore, monoclonal antibodies are target specific. Note the term “target” is used interchangeably with the term “drug target.” The follow-on benefit of selectivity is patients experience fewer adverse reactions. Monoclonal antibodies are versatile. A wide range of monoclonal antibodies can be created to bind to a wide range of different targets found on all cells throughout the body. Some of these targets are well known, while many more are still left to be discovered. Antibodies Mechanism of Actions Monoclonal antibodies have 4 main mechanisms of action. • 12 They bind to receptors to block signaling molecules. Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. • They bind to the receptor and flag the cell for destruction by the immune system. • They bind to the signaling molecule and block the signal from binding to the receptor. • They bind directly to the pathogen and prevent the pathogen from causing disease. Let’s look at examples of each. mAbs Block Receptors Monoclonal antibodies bind to receptors to block signaling molecules. Recall that signaling molecules tell a cell to perform a task. If that signaling molecule is blocked, then no task will be performed. Vectibix is indicated for colorectal cancer. Vectibix binds to the epidermal growth factor receptors or EGFR and blocks the epidermal growth factor signals. What task do you think the epidermal growth factor signal tells the cell to perform? You are correct, the epidermal growth factor signal tells the cell to multiply and grow. When a cell gets too many of these signals the cell grows and divides uncontrollably. Vertibix keeps this from happening. mAbs Trigger Immune Response Monoclonal antibodies bind to the receptor AND flag the cell for destruction by the immune system. The drug Herceptin is a classic example. In a percentage of breast cancer patients, the breast cancer cells overexpress a gene known as HER2. The overexpression of HER2 leads to the creation of a large number of HER2 receptors on the surface of the breast cancer cell. Because of this large number of receptors, the cell receives too many signals. Signals known as growth factors bind to this abundance of HER2 receptors, causing uncontrolled cell growth and cell division leading to cancer. Herceptin is a monoclonal antibody that works by binding to the HER2 receptors on the outside of these breast cells. Once bound to the HER2 receptor, that monoclonal antibody blocks the receptor so that the growth factor signal cannot bind to the HER2 receptor. 13 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Herceptin also flags the breast cancer cell as foreign. When a cell has most of its HER2 receptors bound by a large number of monoclonal antibodies, the immune system is alerted and cells like the macrophage are deployed to the site to destroy the flagged cells. Another therapeutic monoclonal antibody example is Rituxan, which treats cancers such as lymphoma and autoimmune diseases such as rheumatoid arthritis. In both diseases, Rituxan binds to the CD20 antigen on the cell surface and tags the cells for destruction by the immune system. mAbs Capture Signals Monoclonal antibodies bind to the signaling molecule and block the signal from binding to the receptor. Avastin is a monoclonal antibody indicated for certain types of cancers of the kidney, lung, colon, rectum, cervix, ovary, or fallopian tube. Avastin binds to a signaling molecule known as vascular endothelial growth factor or VEGF. VEGF is a potent growth factor that stimulates the growth of cells. Which cells do you think VEGF stimulates? You are correct, the cells Avastin targets are known as endothelial cells found in the vascular system. Avastin binds to VEGF and stops the vascular system from growing. Similarly, Humira which is indicated for a host of conditions including some autoimmune diseases, works by binding to a signaling molecule known as tumor necrosis factor or TNF. TNF is a molecule that is naturally produced by our immune system and in many disease states, leads to inflammation. Humira blocks TNF signaling. mAbs Neutralize Pathogens Monoclonal antibodies bind directly to the pathogen and prevent the pathogen from causing disease. Synagis is used to prevent serious lung disease in premature infants. Synagis binds directly to the respiratory syncytial virus also known as RSV. Synagis stops the virus from fusing with and infecting respiratory epithelium cells found in the lungs, thus halting any infection. Currently, therapeutic antibodies are being developed for many pathogens, including the Ebola virus. mAbs Deliver Toxic Drug A game-changing innovation in antibody technology is antibody-drug conjugates or ADCs. These highly-potent, targeted therapeutics combine the targeting power of monoclonal antibodies with 14 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. the cancer-killing ability of toxic drugs. This potent combo can destroy cancer cells with less impact on healthy cells. ADCs have three key components: • A monoclonal antibody that is highly specific for a tumor-associated antigen with little to no expression on healthy cells. • A highly toxic small-molecule drug kills the cancer cell once internalized. • A chemical linker that connects the small molecule drug to the antibody. The linker is stable in a person's bloodstream, releasing the drug once inside the tumor. How does an ADC work? The antibody binds to its target antigen on the cancer cell surface. The antibody-drug conjugate is then taken up – internalized – by the cell. Once inside the malignant cell, the linker degrades and the active drug is released. The ability to target only cancer cells allows doctors to administer medicines higher in toxicity than traditional chemotherapy. This is because the ADC's precision means it avoids healthy tissue that chemotherapy often damages or destroys. There are a few antibody-drug conjugates on the market including Adcetris for Hodgkin's lymphoma, Kadcyla for HER2-positive breast cancer, Besponsa for acute lymphoblastic leukemia, and Mylotarg for acute myeloid leukemia. Targeting Cholesterol: PCSK9 The body naturally keeps bad cholesterol in check with low-density lipoprotein or LDL receptors. These receptors bind to excess LDL, which the liver cell absorbs. The liver breaks down the cholesterol and recycles the receptor back to the cell surface, where the LDL receptor can bind to and remove more LDL. PCSK9 is a protein that also binds to the LDL receptor, which also triggers the liver cell to absorb the pair. However, the entire complex is degraded and the receptor is not recycled—sort of like a murder-suicide. This results in fewer LDL receptors, impeding the process of LDL removal. 15 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Repatha and Praluent, two types of monoclonal antibodies, work by attaching to PCSK9, which prevents the protein's interaction with low-density lipoprotein receptors on the surface of liver cells. By preventing the degradation of these critical receptors, PCSK9 inhibitors lower LDL levels and lessen the risk of a cardiovascular event. Clinical results report a lowering of LDL levels by as much as 60%. One key advantage of the PCSK9 inhibitors is their safety profile—adverse events observed were equivalent to that of the placebo. Bispecific Antibody Construct Antibodies are Y-shaped. The two “arms” of the Y are identical and recognize only one target in all naturally produced antibodies and most man-made therapeutic antibodies. In contrast, bispecific antibodies have been genetically engineered by splicing genes for two different monoclonal antibodies to make a new Y. This way, the bispecific antibody is able to recognize two different targets and bring them in contact with one another. Imagine that one arm of the Y recognizes a cancer cell. Meanwhile, the other arm recognizes and binds to a killer T-cell. Remember, killer T's are white cells that inject toxins directly into cells. By bringing the malignant cell it “caught” into contact with a killer T-cell, the first arm of the Y essentially forces the killer T-cell into action — killing the cancer cell. The cool thing is that it's not even necessary to imagine this scenario — it's the mechanism of action for Blincyto which is indicated for leukemia. Immune System Checkpoint Therapies James Allison and Tasuku Honjo won the 2018 Nobel Prize in Physiology or Medicine for pioneering basic work for today's hugely successful immune checkpoint inhibitor therapies. In the early 1990s, Allison and Honjo independently discovered two proteins, CTLA-4 and PD-1, that serve similar functions in our immune system. They both shut down killer T-cells, a type of white blood cell. This biological safety feature prevents overactive T-cells from attacking our own organs and tissues. Thus, CTLA-4 and PD-1 are known as immune system checkpoints. By inhibiting the CTLA-4 protein on T-cells with a monoclonal antibody, those T-cells become more fully activated in their attacks on tumor cells. Allison developed a monoclonal antibody to bind CTLA-4, blocking its ability to put the brakes on our immune system. Honjo performed similar 16 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. work with the PD-1 checkpoint protein. Checkpoint inhibitors are monoclonal antibodies that bind to CTLA-4, PD-1, or PD-L1 and block the interaction between these three molecules. This allows the T-cell to stay activated and destroy tumor cells. Yervoy As a Check Point Inhibitor The monoclonal antibody, Yervoy, is an example of a checkpoint inhibitor indicated for melanoma. Cytotoxic T-cells would normally recognize and destroy melanoma. However, melanoma cells release a molecule that binds to the antigen CTLA-4 on the cytotoxic T-cell that prevents the Tcell from recognizing the melanoma as foreign. When the CTLA-4 is blocked the T-cell is “turned off.” Yervoy works by binding to CTLA-4 forming a “Yervoy antibody-CTLA-4 complex” that stops the signaling molecule from binding and turning off the cytotoxic T-cell. This allows the T-cells to remain activated, thus allowing the cytotoxic T-cells to seek out and destroy the skin tumor cells. Select PD-1 & PDL-1 Inhibitors in The Clinic PD-1 or PD-L1 inhibitors may have one crucial advantage over CTLA-4 inhibitors: fewer of the potential autoimmune-like side effects seen in CTLA-4 inhibitor drugs. PD-1 and PD-L1 inhibitors' decreased side effects may result from the fact that they seem to primarily activate T-cells already present in tumors' tissues, rather than those present in healthy tissues that would be damaged by a T-cell attack. Oncologists are excited about these new therapies for two main reasons: First, they are proving to be longer-lasting than other treatments, even when compared to the highly effective, targeted monoclonal antibodies. Once activated by checkpoint therapy, the immune system can evolve and change with cancer, unlike static therapies. Second, they promise to fight a range of cancers, unlike most monoclonal antibody therapies, which target one specific type of cancer. However, their efficacy will vary among different subcategories of patients. Researchers are working on identifying the next round of immune system checkpoint inhibitors by deciphering proteins such as LAG-3, TIM-3, and VISTA. This table shows a few examples of 17 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. the checkpoint inhibitors that have been developed and FDA-approved for the treatment of different forms of cancer. Section 2: Antibodies as Therapeutics Summary In summary of section 2, we learned that: • Monoclonal antibodies are highly selective for their drug target allowing for fewer adverse events and are extremely versatile. • Antibody mechanisms of action include blocking a receptor; blocking a receptor and flagging the cell for destruction by the immune system; blocking a signaling molecule; blocking a pathogen. • Antibodies can be used as checkpoint inhibitors to stop cancer cells from evading the immune system. • Some examples of monoclonal antibodies on the market include Humira to treat autoimmune/inflammatory disorders, Avastin to treat various cancers, Synagis to prevent/treat the respiratory syncytial virus, Praluent or Repatha to treat high cholesterol, and many more. 18 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Section 3: Antibodies as Diagnostics Welcome Antibodies as diagnostics. Section 3: Antibodies as Diagnostics Objectives At the end of this section, you will be able to: • Describe the use of antibodies in ELISA assays. • Explain the steps involved in using antibodies in bead immunoassays. • Describe the use of antibodies in home pregnancy tests. Enzyme-Linked Immunosorbant Assay (ELISA) ELISA stands for enzyme-linked immunosorbent assay. ELISAs are quantitative when done correctly and can tell us how much of a particular protein is present. Usually, this is a very small amount, picograms, femtograms, nanograms, so a very small amount of protein. ELISAs are performed in multi-well plates. These are plastic plates with lots of little wells in them. In each well, we put our antibodies, our antigen, and so forth. The antigen is just another term for the protein that the antibody will recognize. On the antigens, there are the epitopes, the specific part of the antigen that an antibody recognizes. ELISA Steps When we start off, we're looking here at a single well in a multi-well plate and just want to see what happens in this particular well. Click each step on the tabs to learn more. Step 1 We start by binding the antibody to the bottom of the well, and you can buy plates where the well is specially designed chemically so the antibody will attach to it. This is a nonspecific interaction. Any protein will stick to the well, but we're attaching an antibody to this particular plate. Step 2 We then add a patient's blood sample, which will likely contain multiple antigens designated here by different colors. Only the purple antigen binds to the antibody. The other antigens do not bind 19 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. because they do not have the epitope that the antibody recognizes. So, the antibody has captured the antigen that we're interested in. Step 3 At this point, though there is no label, there's no way to visualize that any of this has occurred. What we need to do is add something, that allows us to detect this interaction between the antigen and the antibody. We add in a second antibody, which has a label or a dye attached to it. This binds to a different epitope, usually on the antigen, and the second antibody has a fluorescent dye or some other marker on it that allows us to visualize this. Step 4 In this case, we've attached an enzyme. Remember, an enzyme is a protein that carries out a chemical reaction. So, we have chemically attached an enzyme to the antibody. This is called an enzyme antibody conjugate. We have conjugated the enzyme to the antibody, then we add in a substrate, a chemical that the enzyme acts on, and the substrate changes color. ELISA Results The whole process here is we have our first antibody, which we've attached to the well. The first antibody captures the antigen. The second antibody then binds to the captured antigen and generates a color. The more captured antigen we have, the darker the color. That's what's reflected in the plate here on that right side of the screen. Click on the multi-well plate to learn about the differences we see in color for each well. Wells The wells with the darker color have more antigens because they have bound more second antibodies which have the label on it so we can see it. The wells that are colorless or virtually colorless have little or no antigen in them, so no color is generated. There's no secondary antibody binding to the antigen, and even when we add the substrate because there's no secondary antibody, there's nothing to make the color. So that's how an ELISA works. So again, the more intense the color, the more antigen is present. Instead of just looking at color intensity by eye, there are machines called plate readers that quantify or assign a numerical value to the intensity 20 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. of the color in each well. And then you can get a printout of that, and you can analyze it. From that information, you can calculate the absolute amount of antigen that's present. Multiplexed ELISA Now in a multiplexed ELISA, instead of detecting one antigen at a time, we're going to detect multiple antigens at the same time. You can see this saves a lot of time. Instead of doing each thing individually, we're going to do it all at once. We're going to attach the antibody to a bead. So instead of attaching our antibody to the well, we're going to attach it to a bead. The way it's been designed, the multiplex assays can generate beads that have 100 different colors to them. By color, we really mean the wavelength of light. Each bead will interact with a different wavelength of light, which we can generate by a laser beam. We then attach an antibody to these beads to each particular color. So, we're going to have groups of colors. Initially, in one vial, we're going to have these pink beads, and in another vial, we'll have the blue beads. The pink beads get an antibody that recognizes one particular epitope. The blue beads have a different antibody attached to them that recognizes a different epitope. We can do this 100 times with 100 different antibodies, which allows us to recognize 100 different antigens simultaneously. So, we can then use multi-well plates. The next slide will show us the different types of multi-well plates. These are multiples of each other. 96, 384, 1536 Well Plates We started out with 96 well plates, then 384, then 1536 wells. You notice that each plate is the same size and therefore has a footprint. These fit basically in the palm of your hand, just maybe a little bit bigger depending on your hand size. But they're all the same size because each plate can fit into a plate reader, which can be purchased. If you buy a higher quality plate reader, it is configured so that it can actually analyze the different plate types with different numbers of wells. You program into the plate reader that it is reading a 96 well plate, 384 well plates, or 1536 well plate. But in each of those, in each well of that 96-well plate, we can put 100 different beads, which means that we can then analyze 100 different samples or 100 different antigens. The same thing can be done in each well 1536 well plate; the only thing that will change is the total volume of liquid in each well. 21 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. 96, 384, 1536 Well Plate Volumes As we increase the number of wells, the size of the wells decreases, so there's less volume to deal with. That becomes a little bit problematic as we move into the 1536 well plate. It becomes a little bit more problematic because the smaller the well, the less volume we have. We have to begin to worry about evaporation issues. As the liquid evaporates, that changes the concentration, and the beads won't be suspended in the liquid. Therefore, their ability to interact with the antigen may also be altered. We just need to keep the chamber that this occurs in well-humidified so that it minimizes evaporation. Again, these are specially made plates that allow us to pass light through them but also allow the reactions to occur properly within the plate. To learn about how this process has now become automated, click on the "Look Closer" button, otherwise click next to continue. Rapid Multiplexed Analyzers This process is now becoming automated because it is very difficult to accurately pipette into a 1536 well plate a specific volume manually. So, you actually now purchase robots that will pick up the samples, add them to the plate, and the robot adds the same amount of liquid into each well of the plate. They can even pick up the different samples and so forth. Therefore, essentially, all you need is a human being to be able to monitor the process, make sure the plates don't get jammed, make sure all the liquids are filled and in the proper location, and the machine will add all of the liquids to the wells. These machines are fairly large, but they save a lot of time. And they make fewer mistakes in theory than a human does. Bead Immunoassay Let's look at an individual bead then in terms of our multiplexed assay. Click on each tab to learn more. Number 1 We have some sort of inert material, and this inert material is filled with a dye that can be excited by a laser to give us a particular color at a particular wavelength of light. 2 22 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Number 2 Attached to that bead is an antibody, which is specific for a particular epitope on an antigen. This is our capture antibody. This is the antibody that was on the bottom of the well on the ELISA plate. We've now simply attached it to a bead. Number 3 When we add in the antigen, the antigen binds to the antibody and captures it on the bead; it's now attached to the antibody and, by default, to the bead. The color of the bead tells us what epitope the antibody is specific for; that is, we know that if it's a blue bead, it's going to capture epitope number one. Number 4 We still, at this point, just know that we have a blue bead, but we don't know if we've captured the antigen or not; we will only capture that antigen if it's present in the sample. We need to add our second antibody, which has our detection label on it as well. Once again, we've now completed a sandwich. Number 5 And then, we add in our substrate, and we get our color formation. We said, though, we can do this with up to 100 different beads. What changes is the color of the bead, and then the color of the bead is going to tell us what antibody we have attached to it. Multiplexed Assays Here we have simply two beads, a pink bead, and a blue bead, and the capture antibodies are slightly different colors. These will capture different epitopes, epitope one on one protein, epitope two on a different protein. These get captured by the beads. These beads can be placed in the same well so that we can measure both proteins in the same well. Click each number chronologically to learn more. 23 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Number 1 We've captured two different antigens, but we still aren't able to demonstrate they are captured, so we add the second antibody, which will bind to the antigen. This now has the substrate on it that gives us the color. Number 2 Here's the tricky part. The color of the bead tells us which antibody is attached to it, and that tells us which antigen we've captured. The second antibody all have the same substrate on them, so they give us the same color. That tells us how much of each antigen we have present. So that's what we call the analyte signal. The nice part about this is we can detect multiple samples, multiple different antigens in a very small volume. Therefore, we don't need to acquire as much fluid from the patient. Number 3 There is a second signal as well called the discriminator signal. This signal tells us whether we're epitope one or epitope two on the antibody. We can read each bead individually by essentially using a method called flow cytometry, which sorts the beads based on color. Cell Sorter “Reads” Beads In a flow cytometric device, we have a very narrow tube, which only allows one bead at a time to pass through the tube. You see that on the right side of the screen. The tube is so narrow that only a single bead can drop through the tube. As it drops through the tube, it's illuminated by a laser, and the wavelength of light that is the color is detected on the other side. The bead is then counted so we know how many of that color of the bead is in the sample. Home Pregnancy Test Let's look at some real-life examples of our immunoassays. We'll start with a home pregnancy test. We're using a single antibody to detect a single protein. The home pregnancy test is really a sandwich of ELISA on a strip, and we are measuring the beta subunit of human chorionic gonadotropin or hCG. This is a protein that's produced in pregnant women, and it appears in their 24 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. urine. We call this a lateral flow immunochromatographic assay because this is flowing in one direction. Lateral Flow Immunochromatographic Assay There are a couple of details that we need to discuss before we can illustrate the test. Click each number chronologically to learn more. Number 1 First, we have our hCG, the protein that we're measuring. On that protein, remember, there are epitopes, and we've made antibodies to detect the epitopes on this protein. These are anti-hCG antibodies. Notice that we have two different antibodies here to capture the hCG. On one of these antibodies, we've conjugated or attached an enzyme. Enzymes, again, carry out chemical reactions, and the enzyme will interact with the dye molecule to create a color that we can observe. We then need to be able to detect this process. So, we have a second antibody to detect the antibody that has the dye molecule on it. These antibodies were originally made in mice, but this is all packaged into the home pregnancy test kit. Number 2 The way this works is if you think you're pregnant, you obtain a urine sample, you dip the test strip in the urine sample, and by capillary action, just like a Bounty paper towel sucking up a liquid spill, the urine moves up the test strep. Within the urine is our hCG protein, which is then recognized by the initial antibody, which is in the lower part of the strip. This antibody captures the hCG to form an antibody-hCG complex. Number 3 After it's captured, this antibody complex then continues up this test strep, carrying the hCG and the antibody. Our second antibody then recognizes the initial antibody and the hCG complex, and then we have our chemical reaction where it reacts with a dye molecule. If hCG is present, this turns red. 25 Copyright 2023 Biotech Primer, Inc. PROPRIETARY. DO NOT SHARE. Number 4 But we also want to just make sure that this works. Therefore, we have what's called positive control. We also have dye molecules and capture antibodies up there, up at a second strip, a second panel, which will always turn color no matter what. This is called a control. This just ensures that the antibody is working properly and that we're able to detect color. Pregnancy Test Assay Results If you have a positive test, you'll see both panels appear; the color change will appear on both panels. But if you have a negative test, only the control zone will change color. This shows us that, indeed, this particular test is working. If there was no color appearing in a control zone, then it tells us that your test kit is defective and that the test is inconclusive because we don't know if it worked or not. So, we always like to include what's called a positive control, that is, a sample that we have created that gives us a positive result just to know if the test is working correctly. In the positive test, we have the actual patient sample reacting, and we have a positive control that we have artificially included to see if things are working or not. In the negative test, the patient sample did not have hCG, so the bottom panel doesn't change color, but the top panel changes red, indicating that everything is working properly. Section 3: Antibody Diagnostics Summary To summarize section 3, in this section we learned that: • ELISA assays use one antibody to bind to a target protein and another antibody that allows us to visualize and quantify the target protein. • Bead immunoassays allow for the detection of many different targets in a single well of a microarray plate. • ● Home pregnancy tests use a modified ELISA technique known as a lateral flow immunochromatographic assay which detects human chorionic gonadotropin (HCG) in the urine of a pregnant woman. 26 Copyright 2023 Biotech Primer, Inc.