Introduction of Monoclonal Antibody

Definition and History of Monoclonal Antibody

Monoclonal antibodies (mAbs), sometimes referred to as moAbs, are antibodies produced from a cloned unique white blood cell lineage. All subsequent antibodies derived through this process trace back to a single parent cell. Monoclonal antibodies possess monovalent affinity, binding exclusively to the same epitope (the antigen part recognized by the antibody). These antibodies differ from polyclonal antibodies, which are produced by different B cells and can bind to multiple epitopes on the same antigen.

The concept of creating monoclonal antibodies stemmed from the observation that certain cancerous B cells, known as myelomas, could secrete large quantities of a single type of antibody. However, the antigen recognized by the myeloma antibody was unknown and uncontrollable. In 1975, Georges Köhler and César Milstein invented hybridoma technology, enabling the production of mouse monoclonal antibodies targeting specific known antigens. This breakthrough involved fusing mouse spleen cells, immunized with a desired antigen, with mouse myeloma cells, which were immortal and capable of proliferating in culture. The resultant hybrid cells, termed hybridomas, inherited both the ability to produce specific antibodies and the capacity to proliferate indefinitely. Köhler and Milstein were jointly awarded the Nobel Prize in Physiology or Medicine in 1984 for this pioneering discovery.

Generation of Monoclonal Antibodies

Monoclonal antibodies possess the unique ability to recognize specific antigens and are produced by identical clone cells. The primary methods for generating monoclonal antibodies are as follows:

• Hybridoma Technology: This is the most common method, involving the fusion of lymphocytes from animals (typically mice) immunized with a specific antigen, with malignant myeloma cells. This fusion forms hybridoma cells capable of continuously dividing and secreting monoclonal antibodies. However, due to their origin from mice, these monoclonal antibodies may cause immune rejection in humans.
• Mouse-Human Chimeric Antibody Technology: This improved method connects the variable regions (V regions) of mouse monoclonal antibodies with the constant regions (C regions) of human monoclonal antibodies, resulting in mouse-human chimeric antibodies. This approach reduces the immunogenicity of monoclonal antibodies while maintaining their high affinity and specificity.
• Humanized Monoclonal Antibody Technology: This more advanced method involves transplanting a few antigen-binding related amino acid residues from the V regions of mouse monoclonal antibodies to the V regions of human monoclonal antibodies, forming humanized monoclonal antibodies. This method technique further minimizes the immunogenicity of monoclonal antibodies while preserving their high affinity and specificity to the greatest extent.
• Human Monoclonal Antibody Technology: This is the most desirable method, directly obtaining or creating monoclonal antibodies with specific antigen recognition ability from human B cell.

Structure and Function of Monoclonal Antibodies

Monoclonal antibodies predominantly belong to the IgG class, consisting of two heavy chains and two light chains, each with a constant region and a variable region. The variable regions form the Fab (fragment antigen-binding) segments, which specifically recognize and bind to the same epitope. On the other hand, the constant regions form the Fc (fragment crystallizable) segment and the FcR-binding site, facilitating interactions with effector cells or complement components, thereby mediating biological functions. The IgG class further divides into four subclasses: IgG1, IgG2, IgG3, and IgG4, based on the amino acid sequence differences in their heavy chain constant regions. These subclasses differ in their ability to activate complement and bind to Fc receptors, which impact their effector functions. The structure of a typical IgG1 monoclonal antibody is shown in Fig.1.

Fig.1 A Diagram of a Typical IgG1 Antibody Along With Its Constituent Parts (Mabry III, 2005)

The effectiveness of monoclonal antibodies primarily relies on the combined action of their Fab and Fc segments. The Fab segments are instrumental in interfering with the signal transduction or functional expression of target molecules by blocking, antagonizing, activating, or cross-linking them. For example, trastuzumab (Herceptin) hinders the binding of human epidermal growth factor receptor 2 (HER2) to its ligands, thereby inhibiting its downstream signaling pathways in breast cancer cells. Similarly, ipilimumab (Yervoy) activates cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) on T cells, augmenting their anti-tumor activity. Furthermore, rituximab (Rituxan) cross-links CD20 on B cells, leading to apoptosis induction. On the other hand, the Fc segments initiate cytotoxicity, phagocytosis, inflammation, immune regulation, and other effects by binding to effector cells or complement components. Notably, omalizumab (Xolair) binds to IgE on mast cells and basophils, restraining their release of inflammatory mediators through inhibition of FcεRI signaling. Likewise, eculizumab (Soliris) binds to C5 on the complement cascade, blocking its cleavage into C5a and C5b, and thereby inhibiting C5 convertase activity.

Types, Pharmacology, and Applications of Monoclonal Antibodies

Monoclonal antibodies can be classified into four types based on their source and engineering degree: murine, chimeric, humanized, and fully human. Murine monoclonal antibodies are derived from mouse B cells and possess high immunogenicity when administered to human patients. Chimeric monoclonal antibodies comprise mouse variable regions and human constant regions, reducing their mouse content and immunogenicity. Humanized monoclonal antibodies, composed of mouse complementarity-determining regions (CDRs) and human framework regions, further reduce the mouse content and immunogenicity. Fully human monoclonal antibodies originate from human B cells or transgenic mice carrying human antibody genes, eliminating the mouse content and immunogenicity. Monoclonal antibodies can also be categorized based on their structure and function, monoclonal antibodies can be divided into several forms, such as naked antibodies, conjugated antibodies, bispecific antibodies, and fragment antibodies. Naked antibodies are unmodified monoclonal antibodies without any attachments. They exert therapeutic effects by binding to their targets and modulating their functions or activating immune responses. Conjugated antibodies are monoclonal antibodies linked to cytotoxic agents or radioactive isotopes, enhancing their therapeutic effects by delivering the payload directly to target cells. Bispecific antibodies, in contrast, are capable of binding to two different antigens simultaneously, thereby increasing their specificity and efficacy by targeting multiple pathways or recruiting immune cells. Lastly, fragment antibodies represent smaller pieces of monoclonal antibodies that retain their antigen-binding ability, thereby improving their penetration and clearance through size reduction.

Monoclonal antibodies possess distinct pharmacological properties compared to small molecule drugs. Their high specificity and affinity for targets result in fewer off-target effects and reduced drug-drug interactions. However, due to their large molecular weight and hydrophilicity, they have limited oral bioavailability and tissue distribution. Additionally, monoclonal antibodies have extended plasma half-life and low clearance rate, necessitating less frequent dosing but potentially increasing the risk of accumulation and toxicity. Their complex biophysical and biochemical characteristics can also affect stability and solubility. Moreover, their pharmacokinetics and pharmacodynamics are nonlinear, influenced by target-mediated drug disposition (TMDD) and immune-mediated drug disposition (IMDD). TMDD refers to the process in which monoclonal antibodies binding to their targets alters the concentration and elimination of both the antibody and the target. IMDD, on the other hand, describes the immune system's recognition and elimination of the monoclonal antibodies as foreign substances. These phenomena can lead to nonlinear dose-response relationships, saturation effects, hysteresis, time-dependent changes, interindividual variability, and other complexities.

Monoclonal antibodies offer numerous advantages, including strong specificity, high titer, homogeneity, minimal or no serum cross-reactivity, ease of mass production, and cost-effectiveness. These features make them highly promising for disease treatment and clinical practice. Monoclonal antibodies find extensive applications in clinical treatment and disease diagnosis. In disease treatment, play a pivotal role in various conditions. For instance, antibodies targeting PD-1/PD-L1, HER2, CD20, and other specific targets have been employed to treat cancer. They can block tumor cell  escape mechanisms, enhance the immune system's ability to eliminate cancer cells, or conjugate with drugs or radioactive substances for targeted tumor localization and treatment. Also, monoclonal antibodies have proven effective in treating autoimmune diseases. Antibodies targeting tumor necrosis factor (TNF) are used to manage diseases like rheumatoid arthritis and Crohn's disease. By inhibiting the pro-inflammatory effects of TNF, they help reduce tissue damage and functional impairment. Furthermore, monoclonal antibodies have been instrumental in combating infectious diseases. Antibodies directed against the novel coronavirus have been utilized to prevent and treat COVID-19 by neutralizing the virus's spike protein, preventing it from binding to host cell receptors and thereby reducing the risk and severity of infection. These examples illustrate only a fraction of the potential applications of monoclonal antibodies, as researchers continuously explore and develop new possibilities in various fields. Monoclonal antibodies represent efficient, specific and safe biological agents, making them some of the best-selling drugs in today's pharmaceutical market.

Challenges and Future Prospects of Monoclonal Antibodies

Despite the remarkable success of monoclonal antibodies, certain challenges and limitations remain to be addressed. One of the challenges is the potential immunogenicity of these antibodies, leading to the formation of anti-drug antibodies (ADAs) in patients. ADAs can decrease drug efficacy, increase clearance, or even cause adverse reactions. Various factors influence immunogenicity, such as the antibody's source, structure, modification, dose, route, frequency, and duration of administration, along with the patient's genetic background, disease state, and concomitant medications. To mitigate immunogenicity, researchers have developed strategies such as engineering human or humanized antibodies, removing or masking immunodominant epitopes, modifying glycosylation patterns, optimizing formulation and delivery systems, and co-administering immunosuppressive agents. Another challenge involves the potential toxicity of monoclonal antibodies. They may bind to their targets or off-targets in normal tissues or cells, causing undesirable effects on physiological functions or homeostasis. Fc-mediated effector functions of monoclonal antibodies, such as cytokine release syndrome (CRS), infusion reactions, or hypersensitivity reactions, can also contribute to toxicity. Conjugated payloads or radiolabels of monoclonal antibodies may induce toxicity, leading to issues like bone marrow suppression, hepatotoxicity, or nephrotoxicity. The nature of toxicity can be dose-dependent or dose-independent, acute or chronic, and reversible or irreversible. While preclinical studies using animal models or in vitro systems can help predict toxicity, some toxicities may only manifest in humans due to species differences or complex interactions. Therefore, careful monitoring and management of toxicity are essential for ensuring the safe and effective use of monoclonal antibodies.

In summary, monoclonal antibodies represent a highly effective immunotherapy method, specifically recognizing and binding to pathological antigens to exert therapeutic effects. As molecular-targeting medicine continues to develop, monoclonal antibodies hold vast potential for various applications. Future research directions involve discovering new targets, enhancing antibody performance, developing multifunctional or bispecific antibodies, creating immunotoxins or immunocytokines, and exploring intrabodies capable of acting inside cells. These ongoing studies will open up new possibilities and choices for monoclonal antibody therapy.

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