PHAR 504: Antibodies - Structure, Function, and Therapeutic Applications

LECTURE 3: Antibodies - Foundational Knowledge

Introduction to Monoclonal Antibody (mAb) Therapy

This lecture aims to explain the basis of monoclonal antibody (mAb) therapy. After this lecture, you will be able to:

Describe the structure and function of mAbs.
Explain how mAbs interact with their targets (antigens / epitopes).
Explain how mAbs are made.
Compare methods for engineering mAbs for improved therapeutic performance.

Therapeutic Applications of Monoclonal Antibodies

Monoclonal antibodies have a wide range of therapeutic applications, including:

Cancer Therapy
Autoimmune Diseases
Infectious Diseases
Allergic Conditions
Ophthalmology
Immunotherapy
Bone Disorders
Transplantation
Cardiovascular Diseases
Neurological Disorders

Antibody (Ig/Ab) Structure and Function

Structure

Total Size: Approximately $150 ext{ kDa}$ (really large compared to other therapeutic agents).
Heavy (H) Chains: Two identical heavy chains, each approximately $50 ext{ kDa}$ .
- Classes/Isotypes: Five possible classes: IgM, IgA, IgD, IgG, IgE. These are determined by the heavy-chain constant (C) regions.

Light (L) Chains: Two identical light chains, each approximately 25 ext{ kDa}$.

Types: Either \kappa $(kappa) or$ \lambda $(lambda).</li></ul></li><li>Variable (V) Region (Orange): Responsible for antigen binding.<ul><li>Present on both Heavy chain ($ VH $) and Light chain ($ VL $).</li></ul></li><li>Constant (C) Region (Blue): Determines the effector functions of the antibody.<ul><li>Heavy Chain: In many examples, it has three domains:$ CH1 $-$ CH3 $. The number of domains varies between classes.</li><li>Light Chain: Just one domain:$ C_L $.</li></ul></li></ul><h5 id="761589c6-b699-42a5-b50b-04da175e14aa" data-toc-id="761589c6-b699-42a5-b50b-04da175e14aa" collapsed="false" seolevelmigrated="true">Functional Features</h5><ul><li>Antigen (Ag)-binding site: Composed of both heavy and light chains, providing specificity.</li><li>Fab (Fragment antigen binding) region:<ul><li>Contains the Ag-binding domain.</li><li>Composed of both heavy and light chains.</li><li>Composed of both variable (orange) and constant (blue) regions.</li></ul></li><li>Fc (Fragment crystallizable) region:<ul><li>Only consists of the heavy chain constant region.</li><li>Specifies biological activity (e.g., the Fc of IgG is bound by Fc receptors on phagocytes to mediate phagocytosis of opsonized particles).</li></ul></li><li>Hinge region: A flexible segment providing conformational flexibility to the antibody, allowing it to bind to epitopes at various distances and angles.</li></ul><h4 id="db2c74ab-d3ef-427e-a89e-2bd172b7f5fb" data-toc-id="db2c74ab-d3ef-427e-a89e-2bd172b7f5fb" collapsed="false" seolevelmigrated="true">Post-translational Modifications</h4>These modifications can significantly impact antibody function and efficacy, especially relevant for biosimilars.<ul><li>Glycosylation (CHO):<ul><li>Attachment of carbohydrates to the protein.</li><li>Not templated or programmed by DNA; may differ depending on growth conditions during production.</li></ul></li><li>Disulfide linkages (S-S):<ul><li>Programmed by the amino acid sequence and proper protein folding.</li><li>Significantly influence the overall structure of the immunoglobulin (Ig).</li><li>Chemically labile, meaning proper conditions for use and storage must be maintained to preserve their integrity and function.</li></ul></li></ul><h4 id="fca94650-c759-4abc-898d-1e4f682d5543" data-toc-id="fca94650-c759-4abc-898d-1e4f682d5543" collapsed="false" seolevelmigrated="true">How B Cells Produce Antibodies</h4><h5 id="c59c9aff-8b4d-4767-960d-1fc2fa124813" data-toc-id="c59c9aff-8b4d-4767-960d-1fc2fa124813" collapsed="false" seolevelmigrated="true">Producing Antibody Diversity: V(D)J Joining</h5><ul><li>Susumu Tonegawa: Awarded the 1987 Nobel Prize in Physiology or Medicine for his discovery of somatic rearrangement of Ig genes.</li><li>Somatic Rearrangement: Ig gene segments are organized into multigene families and assembled through recombination.</li><li>Heavy Chain Variable Region Encoding: The variable region of the human Ig heavy chain is encoded by combining V, D, and J regions.<ul><li>Human genome encodes:<ul><li>$ 123 $V regions</li><li>$ 27 $D regions</li><li>$ 9 $J regions</li></ul></li><li>Total possible heavy chain variable ($ V_H $) sequences:$ 123 \text{ V regions} \times 27 \text{ D regions} \times 9 \text{ J regions} = 29,889 $</li></ul></li><li>Diversity of Variable Regions and Antigen Binding:<ul><li>Total possible light chains:$ 200 $</li><li>Total possible heavy chains:$ 29,889 $</li><li>Total possible Ig (L+H) combinations:$ 200 \times 29,889 = 5,977,800 $</li><li>In addition to combinatorial diversity from heavy and light chain pairing, junctional diversity is also generated during VDJ recombination, leading to an estimated$ 3,586,680 $additional combinations due to imprecise joining. The total possible combinations considering all factors are approximated as$ 9,564,480 $.</li></ul></li></ul><h5 id="6f4916da-fd95-4c8a-b58e-02fb93b344c3" data-toc-id="6f4916da-fd95-4c8a-b58e-02fb93b344c3" collapsed="false" seolevelmigrated="true">Antigen-Driven Affinity Maturation</h5><ul><li>Complementarity Determining Regions (CDRs):<ul><li>These physically interact with epitopes (specific antigen sites).</li><li>Composed of the variable regions of both heavy and light chains.</li></ul></li><li>Process: After V(D)J joining and before Class-Switch Recombination, antigen stimulation triggers affinity maturation, which alters the DNA sequence of gene segments encoding the CDRs and selects for high-affinity binders.<ul><li>Somatic Hypermutation:<ul><li>Changes the DNA sequence of CDR genes.</li><li>Induces point mutations in the CDRs at a rate$ 10,000 $times the normal rate.</li><li>Resulting CDRs may have better, same, or worse affinity for the antigen compared to the original Ig.</li></ul></li><li>Clonal Selection:<ul><li>B cells producing Igs with high affinity for the antigen will bind better and be stimulated to reproduce, becoming a dominant B cell population; their high-affinity Ig will be a major component of the total Ig.</li><li>B cells producing Igs with low affinity will not be stimulated to reproduce, and their low-affinity Igs will remain a minor component.</li></ul></li></ul></li><li>Outcome: Affinity maturation increases both the variety and specificity of the Ig response.</li><li>Immunization in Action:<ul><li>Primary immune response (initial exposure/vaccination): Slow, low-grade response with low-affinity antibodies.</li><li>Secondary immune response (secondary exposure): Faster, high-grade response with high-affinity antibodies due to affinity maturation and clonal selection.</li></ul></li></ul><h4 id="05b6f004-9eb7-4d25-9945-794cbaff7a45" data-toc-id="05b6f004-9eb7-4d25-9945-794cbaff7a45" collapsed="false" seolevelmigrated="true">Ig Function: Ig Classes and Class-Switch Recombination</h4><h5 id="66ac4039-c026-4687-ba92-6f020d20d62a" data-toc-id="66ac4039-c026-4687-ba92-6f020d20d62a" collapsed="false" seolevelmigrated="true">Ig Classes (Isotypes)</h5><ul><li>Classes: IgM, IgD, IgG, IgA, and IgE.</li><li>Determination: Determined by the heavy-chain constant (C) regions, encoded by heavy chain genes:$ \gamma $(G),$ \alpha $(A),$ \mu $(M),$ \delta $(D), and$ \epsilon $(E).</li><li>Specific Biological Functions: Each class/isotype is associated with specific functions (e.g., IgG is abundant in blood, IgA in mucus membranes; IgM is typically a pentamer; IgA is a dimer).</li></ul><h5 id="50e7a4da-eb5e-4b4a-a190-fa259233c952" data-toc-id="50e7a4da-eb5e-4b4a-a190-fa259233c952" collapsed="false" seolevelmigrated="true">Switching from One Class to Another</h5><ul><li>IgM and IgD Production: Produced first by alternative splicing of the primary RNA transcript.</li><li>IgA, IgG, and IgE Production (with same Ag specificity): The B cell undergoes Class Switch Recombination (similar to V(D)J joining) to produce these classes.<ul><li>The$ C_H $region CHANGES.</li><li>The$ V_H $region stays the same (maintaining antigen specificity).</li><li>The light chain stays the same.</li><li>Class switch recombination is irreversible.</li></ul></li><li>Immune Response: Earlier responses often involve IgM, while higher, more mature responses involve IgG, IgA, or IgE after class switching.</li></ul><h4 id="ef431886-556e-42b8-b230-258e71973f71" data-toc-id="ef431886-556e-42b8-b230-258e71973f71" collapsed="false" seolevelmigrated="true">Role of Fc in mAb Mechanisms of Action</h4>The Fc region of an antibody dictates its effector functions:<ul><li>(A) Blocking of signaling pathways: The Fab region binds to a target, preventing it from interacting with its receptor or other molecules, thereby blocking downstream signaling.</li><li>(B) Antibody-dependent cellular cytotoxicity (ADCC): The Fab region binds to a target cell, and the Fc region is recognized by Fc receptors on immune effector cells (e.g., NK cells), leading to the lysis of the target cell.</li><li>(C) Complement-dependent cytotoxicity (CDC): The Fc region can activate the complement system, leading to the formation of the membrane attack complex and lysis of the target cell.</li><li>(D) Antibody-dependent cellular phagocytosis (ADCP): The Fab region binds to a target particle or cell, and the Fc region is recognized by Fc receptors on phagocytes (e.g., macrophages, neutrophils), leading to engulfment and destruction of the target.</li></ul><h4 id="c0eb76b5-ff9e-42b9-853b-89dec038622a" data-toc-id="c0eb76b5-ff9e-42b9-853b-89dec038622a" collapsed="false" seolevelmigrated="true">Characteristics of IgG (aka Gamma Globulin)</h4>IgG is the primary class for therapeutic, industrial, and research uses due to its favorable characteristics:<ul><li>Soluble: Easy to produce, store, and use.</li><li>Gamma ($ \gamma $) Heavy Chain with Subclasses: IgG1, IgG2, IgG3, IgG4. Therapeutic Igs may be labeled accordingly.<ul><li>IgG1: Most abundant; responds to protein antigens; key to viral and cellular pathogen response; highly effective at activating complement.</li><li>IgG2: Responds well to bacterial capsular polysaccharides.</li><li>IgG3: Best activator of complement; highly effective at promoting phagocytosis.</li><li>IgG4: Least abundant (2-3% of IgG); relatively weak effector functions; often considered "anti-inflammatory."</li></ul></li><li>Abundant: Predominant Ig in blood and lymph; comprises ~$ 15\% $of the total protein in serum.</li><li>Stable: Has a long half-life in serum of ~$ 23 days, providing good pharmacokinetic properties.
Glycosylated (carbohydrate modification): May influence function and therapeutic efficacy, significant for biosimilars.

Summary of Ig Production in B Cells

V(D)J Joining: Generates diverse variable regions that bind antigens.
Affinity Maturation & Clonal Selection: Selects for B cells producing antibodies with better binding affinity.
Class-Switch Recombination: Changes the antibody class, leading to different functions while retaining antigen specificity.

Producing Igs: Polyclonals, Hybridomas, and mAbs

Polyclonal Igs / Antiserum

Product of Many B Cells: Stimulated to produce Ig against the same antigen.
Complex Mixtures: Contain hundreds to thousands of Igs, each with a different affinity and epitope specificity.
Production: Produced by a single animal host after immunization.
Reproducibility: Not reproducible from one host to another, leading to lot-to-lot variability.
Role: Important in many early-phase diagnostic tests and therapeutic applications.
Making Polyclonal Igs: Immunization of an animal (e.g., rabbit, mouse, goat) with an antigen, followed by collecting blood ("bleeds") and purification of polyclonal antibodies (pAb).

Hybridomas and Monoclonal Antibodies (mAbs)

Monoclonal Antibodies (mAbs): Produced by hybrid cells called hybridomas.
Hybridoma Composition: A hybrid between an immortal tumor cell and plasma B-cells from the spleen of an immunized mouse.
Clonal Expansion: An individual hybridoma cell is isolated and clonally expanded to produce a clonal cell line (a population of identical cells) that produces the same mAb.
- This ensures uniformity in production.
Selection: To produce a commercially successful mAb, multiple clonal lineages are isolated, expanded, and evaluated (screened) to identify clones that produce mAbs with the desired affinity and specificity.
Immortality: Hybridomas are immortal and can be grown in culture indefinitely or frozen for storage, providing an indefinite potential for mAb production and a long-term source.
Natural Processes: V(D)J joining, antigen-driven affinity maturation, and class switch recombination all occur naturally in vivo (in the host animal) during the immune response to the antigen. Once established in the host, there is no further potential for changes to the Ig sequence or functional capabilities; they are fixed.

Polyclonal vs. Monoclonal Antibodies

Feature	Polyclonal (Pros)	Polyclonal (Cons)	Monoclonal (Pros)	Monoclonal (Cons)
Versatility	Greater reagent versatility	Potential for reduced specificity	High specificity	Longer development
Cost	Inexpensive	High lot-to-lot variability	Consistent performance	Expensive
Development	Shorter development; available first	Finite supply of each lot	Constant supply

Therapeutic mAbs: Classes and Nomenclature

Engineering of Therapeutic mAbs for Reduced Immunogenicity

Therapeutic mAbs are engineered to reduce immunogenicity (the chance of triggering an immune response in the patient).

Murine (0% human): Mouse-derived antibodies. High potential for immunogenicity (human anti-mouse antibody/HAMA response).
Chimeric (65\% $human): Mouse variable regions fused to human constant regions. Reduced potential for immunogenicity.</li><li>Humanized ($ >90\% $human): Only the CDRs are from mouse, grafted onto a human antibody framework. Further reduced potential for immunogenicity.</li><li>Human ($ 100\% $human, aka "fully human"): Derived from human genes. Lowest potential for immunogenicity.</li></ul><h5 id="f7706736-a368-4041-92b3-f54f88fa1c43" data-toc-id="f7706736-a368-4041-92b3-f54f88fa1c43" collapsed="false" seolevelmigrated="true">Pre-2022 mAb Nomenclature</h5><ul><li>Chosen by the company that developed the drug.</li><li>Utilized infixes to indicate source (e.g., -xi- for chimeric, -zu- for humanized, -u- for human).</li><li>Example: Rituximab (a chimeric mAb).</li></ul><h5 id="e71e0c4b-9d0f-4929-b10c-111ba05b5b0e" data-toc-id="e71e0c4b-9d0f-4929-b10c-111ba05b5b0e" collapsed="false" seolevelmigrated="true">2022 mAb Nomenclature (for drugs developed after January 1, 2022)</h5><ul><li>This is a recent update, so uptake is slow, but it provides a good structure for explaining Ig technologies.</li><li>"-tug" (unmodified Igs):<ul><li>Closely resemble naturally occurring Igs in the human immune system.</li><li>Have not undergone significant engineering or modifications beyond what might occur naturally.</li></ul></li><li>"-bart" (artificial antibodies):<ul><li>Monospecific full-length Igs with engineered amino acid changes in the constant regions.</li><li>Examples of modifications:<ul><li>Replacing parts of mouse/rat Ig with human components to reduce immunogenicity (e.g., Eculizumab, trade name Soliris, a humanized terminal complement inhibitor, uses CDR grafting).</li><li>Mutations in the Fc region to reduce complement activation and thus complement-dependent cytotoxicity (CDC).</li><li>Glycoengineering to increase Ig-dependent cell-mediated cytotoxicity (ADCC) for anti-cancer therapeutic mAbs.</li></ul></li></ul></li><li>"-ment" (fragment):<ul><li>Monospecific agents derived from the variable domain of antibodies, such as scFv (single-chain variable fragment).</li><li>Advantages over full-sized antibodies:<ul><li>Smaller size: Better tissue penetration.</li><li>Reduced nonspecific binding: No Fc region.</li><li>Faster clearance from the body.</li><li>Simpler production: No glycosylation required.</li><li>Modular nature.</li><li>No Fc-mediated effects (can be an advantage or disadvantage depending on the desired mechanism).</li></ul></li></ul></li><li>"-mig" (multi-Igs):<ul><li>Bi-specific antibodies: Bind two different antigens. Improve specificity to a particular cell type or bring two molecules together to trigger biological functions.</li><li>Bi-paratopic antibodies: Bind to two different epitopes on the same antigen. Can achieve high-affinity binding without crosslinking.</li><li>Bi-valent antibodies: Bind two of the same target (epitope) (e.g., natural Igs).</li><li>Multi-specific antibodies: Bind three or more different targets. Can increase the affinity or specificity of bi-specifics.</li><li>Example: Blinatumomab (Blincyto®): A Bi-specific T cell Engager (BiTE).<ul><li>It is a single-chain variable fragment (scFv) from two different mAbs, one binding CD3 (a T cell co-receptor that activates cytotoxic and helper T cells) and the other binding CD19 (expressed on all B cells, identifying B cell lymphoma).</li><li>Used for relapsed/refractory B-cell precursor acute lymphoblastic leukemia (ALL) and B-cell precursor ALL with minimal residual disease.</li><li>Functions by redirecting T cells to cancer cells, leading to cancer cell death.</li></ul></li></ul></li></ul><h5 id="7fac1a00-5083-4902-a15f-6a0a28e9a153" data-toc-id="7fac1a00-5083-4902-a15f-6a0a28e9a153" collapsed="false" seolevelmigrated="true">Antibody-Drug Conjugates (ADCs)</h5><ul><li>Definition: A class of biopharmaceutical drugs that combine a monoclonal antibody (or fragment) with a cytotoxic payload via a chemical linker.</li><li>Aim: To deliver a potent cytotoxic agent specifically to cancer cells while sparing healthy cells, thereby reducing systemic toxicity.</li><li>Payload Examples: Toxins, radionuclides (radioactive elements), enzymes, or other cytotoxic drugs.</li><li>Components:<ul><li>Antibody: Targets a specific antigen on the surface of cancer cells.</li><li>Payload: A potent cytotoxic agent that kills the cancer cell upon release.</li><li>Linker: Chemically connects the antibody and the payload; some types of linkers are designed to control the release of the payload only once inside the target cell or in the tumor microenvironment.</li></ul></li></ul><h4 id="1f3a991d-58d5-4f72-a359-19baa848c6b9" data-toc-id="1f3a991d-58d5-4f72-a359-19baa848c6b9" collapsed="false" seolevelmigrated="true">Biologics and Biosimilars</h4><h5 id="8259a3ad-f444-4816-8e5a-b36001d3bed3" data-toc-id="8259a3ad-f444-4816-8e5a-b36001d3bed3" collapsed="false" seolevelmigrated="true">Biologics</h5><ul><li>Definition (aka Biological Products): Derived from living organisms or contain components of living organisms. They are produced using biotechnology from human, animal, or microorganism sources.</li><li>Range of Products: Includes vaccines, blood and blood components, allergenics, somatic cells, gene therapy products, tissues, and recombinant therapeutic proteins (like mAbs).</li><li>Therapeutic Use: Used to treat numerous diseases and conditions, including autoimmune disorders, cancers, diabetes, and more. They have revolutionized treatment for many conditions.</li><li>Characteristics: Typically larger, more complex molecules, or complex mixtures, compared to traditional small-molecule drugs.</li></ul><h5 id="e869c741-a367-4746-8d69-e9355b5cf586" data-toc-id="e869c741-a367-4746-8d69-e9355b5cf586" collapsed="false" seolevelmigrated="true">Biosimilars</h5><ul><li>FDA Approval Process: Undergo an abbreviated FDA approval process compared to the reference biologic.</li><li>Emphasis: More emphasis is placed on establishing similarity with the reference biologic through analytical, preclinical, and clinical data.</li><li>Purpose: Intended to increase access to important biologic medicines and potentially reduce healthcare costs by introducing competition.</li><li>Differences from Generics: Unlike generic small-molecule drugs (which are exact chemical copies), biosimilars are not exact copies of their reference products due to the inherent complexity and manufacturing variability of biologics.</li></ul><h5 id="ae410b7d-e8c9-4053-835b-9ef720f4c86a" data-toc-id="ae410b7d-e8c9-4053-835b-9ef720f4c86a" collapsed="false" seolevelmigrated="true">Interchangeability and Substitution</h5><ul><li>Substitution by Pharmacists: Substitution of a biosimilar or interchangeable product for a reference product is governed by state pharmacy law.</li><li>Illinois Pharmacy Practice Act: Allows pharmacists to substitute an interchangeable biologic under certain conditions:<ul><li>Notification of the patient.</li><li>Prescriber has not prohibited substitution.</li><li>Communication of the change to the prescriber.</li></ul></li><li>Interchangeable Biosimilars: Have met additional regulatory requirements beyond those for biosimilar approval.<ul><li>In many U.S. states, interchangeable biosimilars may be substituted for the reference biologic at the pharmacy level without consulting the prescriber, similar to generic drug substitution.</li><li>Important: Regular biosimilars (without the interchangeability designation) cannot be automatically substituted without prescriber consultation.</li></ul></li><li>Humira Biosimilar Landscape: Humira (adalimumab) is a fully human anti-TNF$ \alpha$$ IgG1 mAb used to treat several autoimmune conditions. Its biosimilars (e.g., Amjevita (adalimumab-atto), Cyltezo (adalimumab-adbm)) are distinguished by a unique four-letter suffix added to the end of the nonproprietary name, as established by the FDA to differentiate them from the reference product and from each other.