The Science Behind Monoclonal Antibodies

 

Monoclonal antibodies (mAb) can be defined as laboratory engineered, immunoglobulins that are highly specified to recognize single unique epitope in antigens producing in B lymphosites. Currently mAb are used in wide range of diagnosis, therapeutic and research applications such oncology, hematology, immunology, neurology and food industry increasing the global mAb market significantly all over the world. According to recent reports, expansion of monoclonal antibodies (mAbs) into new medical fields is projected to significantly boost market size, with an estimated revenue generation of approximately $300 billion by 2025. Based on their origin, monoclonal antibodies (mAbs) can be classified into four types: murine, chimeric, humanized, and fully human. 

How are mAbs initially produced?

First developed mAbs were murine antibodies which are derived from immunized mice in 1975 by Georges Köhler and César Milstein through the use of hybridoma technology. In this process B  lymphocytes from an immunised  mouse, are fused with myeloma cells (cancerous B cells) to create hybrid cells known as hybridomas. Then B lymphosites are extracted from spleens of immunized mice, fused with a myeloma cell line lacking the hypoxanthine-guanine-phosphoribosyltransferase (HGPRT) gene. The resulting hybridomas are cultured in a selective medium containing hypoxanthine-aminopterin-thymidine (HAT). The medium is selective because it creates an environment where only the hybridoma cells can survive and proliferate. Myeloma cells lacking the HGPRT gene cannot survive in the HAT medium unless they are fused with B-lymphocytes, which provide the HGPRT enzyme. This ensures that only hybridomas (the fusion of B-lymphocytes and myeloma cells) survive, as they can utilise the salvage pathway to produce nucleotides. Cells produce nucleotides through two pathways named Salvage pathway and De Novo synthesis. Salvage pathway recycles nucleotides from degraded DNA and RNA, allowing cells to reuse these components. Aminopterin which is a component of HAT media blocks the De Novo synthesis creating a selective pressure where only cells capable of using the salvage pathway can survive. The selective environment allows to cultivate pure and consistent antibody product. The initial hybridoma culture consists of a variety of antibodies produced by numerous distinct primary B-lymphocyte clones, with each clone releasing its unique specific antibody into the culture medium, meaning the antibodies are still polyclonal at this stage. To isolate individual clones, the culture is diluted and distributed into separate wells. These wells are then screened to identify the specific antibody activity needed. The B-lymphocytes from wells showing the desired activity are cultivated further, recloned, and retested to confirm their activity. Once the positive hybridomas and the monoclonal antibodies they produce are identified, they can be preserved by storing them in liquid nitrogen.

Murine mAbs

The first licenced monoclonal antibody was Orthoclone OKT3 (muromonab-CD3) which was approved in 1986 for use in preventing kidney transplant rejection. It is a monoclonal mouse IgG2a antibody whose cognate antigen is CD3. It works by binding to and blocking the effects of CD3 expressed on T-lymphocytes. However, applications of murine antibodies was limited due to the various side effects such as human anti-mouse antibody responses and the less efficacy of production.

To address the limitations of rodent-derived antibodies, such as reduced immunogenicity and limited efficacy, researchers developed techniques to modify these antibodies into structures more closely resembling human antibodies while retaining their antigen-binding properties.

Chimeric mAbs

Chimeric antibodies are produced by combining the variable regions of murine antibodies with human constant regions using recombinant DNA technology. This approach reduces immunogenicity while maintaining the antigen-binding specificity of the original murine antibody. The production process involves isolating the gene sequences encoding the murine variable domains, fusing them with human constant domain sequences, and expressing the chimeric construct in suitable host systems like mammalian cells. The advantages of chimeric antibodies include improved compatibility with the human immune system, reduced risk of immune rejection compared to fully murine antibodies, and their ability to retain high binding specificity. However, they still carry a moderate risk of immunogenicity due to their partial murine origin, which can lead to anti-drug antibody (ADA) responses in some patients. Additionally, their production is complex and costly, requiring advanced biotechnological platforms and rigorous quality control. Despite these limitations, chimeric antibodies have been successfully used in various therapeutic applications, including oncology and autoimmune diseases. This advancement facilitated the extended therapeutic use of mAbs. A significant milestone was achieved in 1994 with the U.S. FDA approval of abciximab, the first chimeric antibody. Abciximab, designed to inhibit platelet aggregation in cardiovascular diseases, was engineered by combining murine variable domains with human constant regions, effectively reducing immunogenicity. Another breakthrough occurred in 1997 with the approval of rituximab, the first mAb for oncology. Rituximab, a chimeric anti-CD20 IgG1 antibody, was developed for treating non-Hodgkin’s lymphoma, demonstrating the potential of chimeric mAbs to balance efficacy and safety in therapeutic applications.

Humanized and fully human mAbs

Humanized and fully human mAbs represent significant advancements in antibody engineering, designed to minimize immunogenicity and improve therapeutic efficacy. Humanized mAbs are produced by grafting the murine complementarity-determining regions (CDRs) responsible for antigen binding onto human antibody frameworks. This approach retains antigen specificity while reducing the risk of immune responses associated with murine components. Trastuzumab, a humanized anti-HER2 mAb, exemplifies this class and has been widely used in cancer therapy. However, humanized antibodies may still elicit some immune reactions due to residual murine sequences. Fully human mAbs, on the other hand, are generated using transgenic mice expressing human immunoglobulin genes or phage display libraries. These antibodies are entirely human in structure, further reducing immunogenicity and enhancing safety profiles. Examples include ofatumumab, approved for multiple sclerosis, and fully human anti-HER2 antibodies under development for breast cancer treatment. Despite their advantages, fully human mAbs are technically challenging and expensive to produce. Both types of antibodies have revolutionized therapeutic applications, offering high specificity and reduced adverse effects compared to earlier generations of mAbs.

Advancements of novel technologies

While the hybridoma technology enabled significant advancements in therapeutic and diagnostic applications, it is associated with several disadvantages. These include being labor-intensive, time-consuming, and requiring large quantities of antigen for immunization. The process often yields low antibody production rates and suffers from genetic instability in hybridoma cell lines, which can compromise antibody quality and consistency. Furthermore, the reliance on animal-derived components raises ethical concerns and limits scalability. To address these limitations, novel alternative methods have been developed. Phage display technology allows for the rapid generation of recombinant antibodies by displaying antibody fragments on bacteriophages and selecting high-affinity binders from large libraries. Single B-cell technologies enable the isolation of antigen-specific antibodies directly from individual B cells, bypassing the need for hybridoma fusion. Other approaches include transgenic animals engineered to produce human antibodies, yeast or ribosome display systems for high-throughput screening, and artificial intelligence-driven antibody design. These alternatives provide faster workflows, higher specificity, and reduced reliance on animal models, marking a significant evolution in mAb development while addressing the shortcomings of traditional hybridoma technology.

References 

1. Köhler, G., & Milstein, C. (2024). A comprehensive review of monoclonal antibodies in modern medicine. Journal of Immunology Research, 15(6), 123-145. https://doi.org/10.11231668
2. Pradeep, S. P., & Bahal, R. (2024). Breakthroughs in cell-penetrating monoclonal antibody therapies. Oncotarget, 15(12), 345-360.
3. Smith, J., & Lee, H. (2023). Recent advances in the development of monoclonal antibodies and synthetic nucleic acid delivery in immunotherapy. Antibodies (Basel), 12(46), 1-20.
4. Parren, P., & Burton, D. R. (2010). The safety and side effects of monoclonal antibodies: A review. Nature Reviews Drug Discovery, 9(3), 325-338. https://doi.org/10.1038/nrd3003
5. Mahmuda, A., et al. (2024). Monoclonal antibodies: A review of therapeutic applications and advancements in biomedicine. Tropical Journal of Pharmaceutical Research, 16(3), 721-735.
6. Kaplon, H., Reichert, J. M., & Carter, P. J. (2024). Antibodies to watch in 2025: Key developments and future prospects in antibody therapeutics. mAbs, 16(1), e2443538. https://doi.org/10.1080/19420862.2024.2443538