Lecture 1: History and Terminology of Monoclonal Antibodies PDF

Course: Monoclonal antibodies Program: Medical Biotechnology Level: 4 Date 3/10/2024 Lect. 1: History and terminologies of Monoclonal antibodies (mabs) Assoc. Prof. Reda M. Mansour  Course: Monoclonal antibodies...

Course: Monoclonal antibodies Program: Medical Biotechnology Level: 4 Date 3/10/2024 Lect. 1: History and terminologies of Monoclonal antibodies (mabs) Assoc. Prof. Reda M. Mansour  Course: Monoclonal antibodies (PhB-4108) Theoretical Part 4th level (Medical Biotechnology) Lecture 1: History and terminologies of Monoclonal antibodies 3/10/2024 Assoc. Prof. / Reda M. Mansour E-mail: [email protected] Office : 237 School of Biotechnology Office Hours: Wednesday 2:00 pm to 4:00 pm Text Books: 1- Micheel, B. (2006). Monoclonal Antibodies. In Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine (pp. 1174-1181). Berlin, Heidelberg: Springer Berlin Heidelberg. 2- An, Z. (2009): Therapeutic Monoclonal Antibodies: From Bench to Clinic. John Wiley & Sons, Inc. 3- Waldmann, H. and Steinitz, M. (2014): Human Monoclonal Antibodies: Methods and Protocols. Humana Press.  Course Objectives: 1- Understand the principles and methods used to produce monoclonal antibodies (mAbs). 2- Gain hands-on experience in the techniques used for producing and characterizing mAbs. 3- Learn about the applications of mAbs in diagnostics, therapeutics, and research. 4- Understand the regulatory and ethical issues surrounding mAbs production. 3  Genetics Course Syllabus: 1- Week 1 History and terminologies of Monoclonal antibodies 2- Week 2 Introduction to Monoclonal Antibodies 3- Week 3 Hybridoma Technology 4- Week 4 IN VITRO DISPLAY TECHNOLOGY: Antibody Phage Display 5- Week 5 Recombinant DNA Technology for Monoclonal Antibodies 6- Week 6 Diagnostic Applications of Monoclonal Antibodies 7- Week 7 Therapeutic Applications of Monoclonal Antibodies 8- Week 8 Mid-term 9- Week 9 Advances in Monoclonal Antibody Engineering 10- Week 10 Case Studies and Clinical Trials 11- Week 11 Final Project and Presentations  Course degrees Exams & activities Marks weighting 1- Quizzes & Assignments 15 % 2- Practical exam & attendance 20 % 3- Mid-term exam 15 % 4- Oral exam 10 % 5- Final written exam 40 % Regulations Quizzes Attendance 6  Antibodies are one of the main defense mechanisms of vertebrate animals, able to neutralize and destroy pathogens with help of other components of the immune system. Until the development of monoclonal antibodies (mAbs) in 1975 by Köhler and Milstein, antisera containing a mixture of several types of antibodies (polyclonal antibodies) were used for diagnostic or therapeutic purposes. The first evidence that mAbs with therapeutic potential came in the early 1980s, when a patient (Philip Karr) suffering from a lymphoma showed a clear response to treatment with mouse antibodies. Soon after, several pharmaceutical companies started to develop antibodies for the treatment of cancer, autoimmune diseases, as well as against transplant rejection processes. After several immunizations, patients developed hypersensitivity reactions and anti-murine antibodies were produced (HAMA: human anti-mouse antibodies or HARA: human anti-rat antibodies), thereby reducing the potential of antibodies in human therapy. Although the use of fully human mAbs could have several advantages compared with the use of murine antibodies (lower immunogenicity, better interaction with human effector systems such as opsonization, binding to Fc receptors, or the same glycosylation pattern), the production of human mAbs has resulted to be more tedious than first thought. The use of phage libraries carrying human Ig genes was one of the techniques that allowed the production of fully human mAbs. The cloning of human Ig coding regions in phage genes (single chain Fv fragments, scFv) allowed the production of phages libraries with different Ig specificities.  In the 1990s, some researchers tried to engineer mouse strains by the introduction of human immunoglobulin genes codifying for heavy and light chains into the mouse germ line. These transgenic mice carrying human immunoglobulin genes could potentially now produce B cells expressing human Igs. The development of the “final” transgenic mouse producing fully human Igs in the absence of mouse endogenous antibodies is a very complex process, which has required and continues to require, the effort and time of several research groups and companies. Prosperities & nature of Antigens  An antigen isa molecule that is capable of binding to a component immune response (antibodies or T-cell receptor). An immunogen is a molecule that is capable of eliciting an immune response. Immunogenicity is the extent to which an antigen is Nature of capable of initiating an immune response. “All immunogens are antigenic, but not all antigens are Antigens immunogenic” In order to be immunogenic, some antigens must be coupled to macromolecular 'carriers', usually proteins. This is especially the case for small molecules such as drugs or other compounds of molecular weight less than a few thousands. Such small molecules are termed haptens.  Haptens are small molecules that elicit an immune response (It is not immunogenic by itself) only when attached to a large carrier such as a protein. Nature of Antigens Epitope: An epitope or antigenic determinant, is the part of an antigen that is recognized by an antibody, B-cell receptor or T-cell receptor. Generally an antigen has several or many different epitopes and reacts with many different antibodies. an epitope is approximately five or six amino acids in length.  Adjuvant An adjuvant is a pharmacological or immunological agent that modifies the effect of other agents. Directly most antigens will lead to a poor immune response and rapid removal of the antigen from the body. To prevent this, the antigen is first combined with an adjuvant, which is a material that helps stimulate and enhance the immune response against the antigen.  Structure of Antibodies/immunoglobulins (Ig) Antibodies, or immunoglobulins (Ig), are proteins produced by the immune system to recognize and neutralize pathogens like bacteria and viruses. B and T lymphocytes express different receptors that recognize antigens: membrane-bound antibodies on B cells and T cell receptors (TCRs) on T lymphocytes. The principal function of cellular receptors in the immune system, as in other biologic systems, is to detect external stimuli (antigens, for the antigen receptors of the adaptive immune system) and trigger responses of the cells. Antigen receptors are clonally distributed, meaning that each lymphocyte clone is specific for a distinct antigen and has a unique receptor, different from the receptors of all other clones. (Recall that a clone consists of a parent cell and its progeny.) The total number of distinct lymphocyte clones is very large, and this entire collection makes up the immune repertoire. Antibodies are a central component of the adaptive immune response. They are produced by B cells and exist in two forms: 1- Membrane-bound antibodies: These serve as B cell receptors (BCRs). 2- Secreted antibodies: These circulate freely in the blood and lymphatic system to recognize and neutralize antigens. Each antibody is specific to a particular antigen, allowing the immune system to target a wide variety of pathogens. Fig.1: Properties of antibodies. Antibodies (immunoglobulins) may be expressed as membrane receptors or secreted proteins; When immunoglobulin (Ig) signals are delivered to the lymphocytes by proteins associated with the antigen receptors. The antigen receptors and attached signaling proteins form the B cell receptor (BCR).  Structure of Antibodies Antibodies have a characteristic Y-shaped structure, composed of four polypeptide chains: 1. Two heavy chains (H chains): These are larger and more complex. 2. Two light chains (L chains): These are smaller. Key Structural Components: A- Variable (V) and Constant (C) Regions: - The Variable Region is responsible for binding to specific antigens. This region varies greatly between antibodies, allowing for antigen specificity. - The Constant Region determines the class or isotype of the antibody and dictates how the immune system will respond after binding to an antigen. - There are five types of heavy chains, called μ, δ, γ, ε, and α, which differ in their C regions; in humans, there are four subtypes of γ chain and two of the α chain. Antibodies that contain different heavy chains belong to different classes, or isotypes, and are named according to their heavy chains (IgM, IgD, IgG, IgE, and IgA). B- Fab and Fc Fragments: - Fab Fragment (Fragment antigen-binding): The two arms of the Y. Each Fab contains one variable region from both the heavy and light chains. This fragment binds to the antigen. - Fc Fragment (Fragment crystallizable): The stem of the Y. It is made only from heavy chain constant regions. This fragment interacts with immune cells and mediates effector functions like opsonization, complement activation, and antibody-dependent cell-mediated cytotoxicity (ADCC). C- Hinge Region: This flexible region allows the two arms of the antibody to move and bind to antigens at different angles, enhancing the antibody’s ability to interact with antigens on cell surfaces or in soluble form. The antigen-binding site of an antibody is composed of the V regions of both the heavy chain and the light chain, and the core antibody structure contains two identical antigen binding sites. Each variable region of the heavy chain (called VH) or of the light chain (called VL) contains three hypervariable regions, or CDRs (complementarity-determining regions ). Of these three, the greatest variability is in CDR3, which is located at the junction of the V and C regions. As may be predicted from this variability, CDR3 is also the portion of the Ig molecule that contributes most to antigen binding. FIGURE 4-2 Structure of antibodies. Schematic diagrams of A, a secreted immunoglobulin G (IgG) molecule, and B, a molecule of a membrane-bound form of IgM, illustrating the domains of the heavy and light chains and the regions of the proteins that participate in antigen recognition and effector functions. N and C refer to the amino-terminal and carboxy-terminal ends of the polypeptide chains, respectively. C, The crystal structure of a secreted IgG molecule illustrates the domains and their spatial orientation; the heavy chains are colored blue and red, the light chains are green, and carbohydrates are gray. D, The ribbon diagram of the Ig V domain shows the basic β-pleated sheet structure and the projecting loops that form the three CDRs. CDR, Complementarity determining region. (C, Courtesy of Dr. Alex McPherson, University of California, Irvine.) The "upper" part (Fab region) of an antibody. The complementarity-determining regions of the heavy chain are shown in red. CDRs are where these molecules bind to their specific antigen and their structure/sequence determines the binding activity of the respective antibody. A set of CDRs constitutes a paratope, or the antigen-binding site. As the most variable parts of the molecules, CDRs are crucial to the diversity of antigen specificities generated by lymphocytes.