Antibody engineering is the use of molecular biology techniques to develop antibodies with enhanced or modified properties compared to their naturally occurring counterparts. By engineering antibodies, scientists can improve their affinity, specificity, stability, and other characteristics to better suit a particular application. In the past few years, various engineered antibody drugs have been approved or are in phase II and III clinical trials.1
The field of antibody engineering has made significant strides in creating molecules that are increasingly effective and safe for therapeutic applications. The engineering of therapeutic antibodies involves several sophisticated techniques aimed at producing antibodies that are not only highly specific to their target antigens but also compatible with the human immune system to minimize the risk of adverse reactions.
There are three primary types of engineered antibodies used in therapeutics:
These are produced by fusing the variable region (Fab) of a mouse antibody (binding to the target antigen) with the constant region (Fc) of a human antibody. This design helps reduce the immune response against the antibody compared to fully mouse antibodies, but some individuals might still show reactions. Examples of therapeutics include Erbitux (cetuximab) for colorectal cancer and Rituxan (rituximab) for certain autoimmune diseases and types of cancer.
To further decrease immunogenicity, humanized antibodies are created by grafting only the antigen-binding sites (complementarity-determining regions, or CDRs) of a mouse antibody onto a human antibody framework. This approach retains the specificity of the mouse antibody while greatly increasing its compatibility with the human immune system. The extent of humanization influences their immunogenicity level, with more humanized versions offering lower risk. Therapeutics include Herceptin (trastuzumab) for breast cancer, and Humira (adalimumab) for rheumatoid arthritis. These therapies are part of a wide range of approved monoclonal antibodies.
Developed using advanced genetic engineering techniques, fully human antibodies offer the lowest risk of immunogenicity as they are entirely derived from human sequences. Examples are Darzalex (daratumumab) for multiple myeloma and Hemlibra (emicizumab) for hemophilia A.2
Genetic engineering plays a pivotal role in discovering and optimizing antibody candidates. Techniques such as phage display technology, transgenic mice, and single B-cell cloning are instrumental in this process. Additionally, newer methods like directed evolution and computational antibody design are emerging, further enhancing the capabilities of antibody engineering.3
This technique displays diverse antibody sequences on the surface of bacteriophages. Researchers can then "pan" these libraries against specific antigens, selecting antibody candidates with desired binding properties. This is also a key approach in generating antibody fragments and screening for effective light chains.
Genetically modified mice with human antibody genes create diverse human-like antibodies. B-cells from these mice can be screened for antigen-specific antibodies of interest.
Identifying and isolating individual B-cells producing desirable antibodies allows researchers to clone and express the exact antibody sequences for further development. This is especially useful for isolating rare, high-affinity antibodies, as single B cell screening directly isolates and characterizes antibodies from individual B cells of immunized or infected donors without the need to create artificial libraries.
Mimicking natural selection in the lab, this technique introduces random mutations into antibody genes and selects beneficial variants with improved properties like higher affinity or lower immunogenicity. Adjustments often focus on the amino acids within the light chains to optimize binding and stability.
Powerful algorithms predict and design antibody sequences with desired characteristics, accelerating the optimization process.
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As of now, there are many different antibody formats. This began when a methodology was developed to express smaller antibody molecules: Fab and Fv fragments. Various vectors were used with competent Escherichia coli for recombinant antibody construction.4 The process involves isolating the antibody genes, inserting them into a bacterial expression vector, and transforming the vector into E. coli cells. Under suitable conditions, the bacteria will produce the desired antibody fragments.
The success of Fab and Fv fragments opened the door to further innovation in antibody design, leading to the development of additional formats like single-chain variable fragments (scFv) and bispecific antibodies. Each of these formats offers distinct benefits. For example, scFvs consist of variable regions from the heavy and light chains of an antibody connected by a short linker, which allows for smaller size and greater tissue penetration.
Bispecific antibodies represent one of the most exciting advances in antibody engineering. These molecules can bind two different antigens simultaneously, which has significant implications for cancer immunotherapy, as they can bring immune cells directly to the tumor cells for more effective destruction.
Antibody engineering combines biology and laboratory techniques to design safer and more effective therapeutic agents. By using methods like phage display, genetic engineering, and humanization, researchers can develop highly specific and low-immunogenicity drugs tailored for patient needs.
These innovations not only improve therapeutic performance but also reduce development risks and support better outcomes in clinical applications.
H. Saeed, F. U., Wang, R., Ling, S., & Wang, S. (2017). Antibody Engineering for Pursuing a Healthier Future. Frontiers in Microbiology, 8. https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2017.00495/full
Harding, F. A., Stickler, M. M., Razo, J., & DuBridge, R. B. (2010). The immunogenicity of humanized and fully human antibodies: Residual immunogenicity resides in the CDR regions. MAbs, 2(3), 256-265. https://www.tandfonline.com/doi/full/10.4161/mabs.2.3.11641
Frenzel, A., Schirrmann, T., & Hust, M. (2016). Phage display-derived human antibodies in clinical development and therapy. MAbs, 8(7), 1177-1194. https://www.tandfonline.com/doi/full/10.1080/19420862.2016.1212149
Okamoto, T., Mukai, Y., Yoshioka, Y., Shibata, H., Kawamura, M., Yamamoto, Y., Nakagawa, S., Kamada, H., Hayakawa, T., Mayumi, T., & Tsutsumi, Y. (2004). Optimal construction of non-immune scFv phage display libraries from mouse bone marrow and spleen established to select specific scFvs efficiently binding to antigen. Biochemical and Biophysical Research Communications, 323(2), 583-591. https://www.sciencedirect.com/science/article/abs/pii/S0006291X04018741
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