Recombinant antibody formats, expressions, and applications
Recombinant antibodies (rAbs), also known as genetically engineered antibodies, are generated by in vitro cloning of the antibody heavy and light chain DNA sequences. Compared to monoclonal antibodies produced using traditional hybridoma techniques, rAbs offer advantages, such as high lot-to-lot consistency, animal-free manufacturing, engineering advancement possibilities, and continuous supply. Given the importance, recombinant antibodies are becoming indispensable tools for basic research, diagnostics, and clinical applications.
Chimeric antibodies
Antibody research and clinical development were revolutionised by the discovery of hybridoma technology in 1975. However, for therapeutic purposes, the efficacy of murine-derived antibodies is limited by human anti-mouse antibody responses, in which the murine antibodies are identified as foreign molecules by the human immune system. In 1984, the first chimeric antibody, also recognised as the first version of recombinant antibodies, was constructed by genetic engineering to reduce the immunogenicity of murine antibodies in humans. A total of 30%-35% of the molecules are derived from mouse antibody sequences and 65%-70% are from human antibody sequences. The resulting chimeric antibodies retain the antigen-binding ability of the parental mouse antibodies. Antibody chimerisation is the first step in developing therapeutic humanised antibodies. Using complementarity-determining region grafting technology and computer-aided molecular modelling, Sino Biological provides high-quality monoclonal antibody humanization services that enable a high degree of successful humanisation (>90%).
Antibody fragments
Each full-length immunoglobulin (IgG) molecule contains two heavy and two light chains linked by disulphide bonds (Figure 1). Antibody fragments, such as Fab, scFv, and VHH, have a small size, providing better penetration of tissues or tumours than their full-length counter- parts. This gives them a promising future in immunotherapy, especially in solid tumours. Furthermore, they also have a short half-life, which is useful as radioactive imaging agents. However, due to the lack of Fc regions, they cannot elicit Fc-mediated antibody effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).
Enzymatic digestion was initially used to fragment IgG antibodies. Pepsin cleaves the IgG heavy chains in the hinge regions after the disulphide bonds, creating a bivalent Fab fragment known as F(ab’)2. Then, the fragment can be cleaved into two identical Fab fragments by papain. However, this enzymatic cleavage method is limited by the types of antibody fragments generated. Besides, it is unsuitable for industrial antibody production and purification. Thanks to advances in antibody engineering techniques, these problems can be solved by producing antibody fragments recombinantly. After successfully cloning and sequencing the antibody genes, antibody fragments can be expressed in microbial expression systems, such as E. coli and mammalian systems (i.e., HEK293 cells), through transient transfection.
With rich experience and expertise in recombinant production, Sino Biological has built a high-throughput (HTP) VHH expression platform (Figure 2) that delivers numerous VHH antibody production projects, with an overall success rate of more than 90%. In addition to common VHH formats, we can express dual- and multi-targeting VHHs (Figure 3). Furthermore, Sino Biological can express various other fragments with high specificity and affinity, such as scFv and Fab.
Bispecific antibodies
Unlike conventional monoclonal antibodies, bispecific antibodies (bsAbs) are those with two binding sites that can recognize two different antigens or epitopes on the same antigen. Due to this unique feature, bispecific antibodies have attracted much attention from researchers and the drug industry. To this date, four bsAb drugs have been approved by the Food and Drug Administration (FDA), and over 160 bsAbs currently undergoing clinical trials for cancer, diabetes, Alzheimer's disease, and other diseases.
Initially, bispecific antibodies were generated by quadroma technology, but it poses a significant challenge to downstream antibody manufacturing and purification. Following the development of recombinant DNA technology in the last 20 years, several bispecific antibody formats have emerged to suit the desired target–product profile. To solve the heavy chain mismatching problem, Genentech first proposed the “knob-into-hole” (KiH) technology, which involves engineering CH3 domains to create either a “knob” or a “hole” in each heavy chain to induce heterodimerisation. Similarly, other technologies, such as common light chain and CrossMab are employed to tackle the light chain mispairing problem. Expressing bispecific antibodies is predominantly generated in mammalian cells. Due to various structural similarities between monoclonal and bispecific antibodies, many established purification processes for conventional mAbs are compatible with bispecifics.
Fc-fusion proteins
Fc-fusion proteins (also known as Fc chimeric fusion proteins, Fc-Ig’s, and Fc-tag proteins) are homodimers consisting of an IgG-Fc domain fused to a protein of interest, such as a ligand, peptide, and enzyme. Although monoclonal antibodies are at the focal point of therapeutic biologics development, Fc-fusion proteins are also a successful class of biopharmaceutical products, with at least thirteen drugs approved by the European Medicines Agency (EMA) and FDA. In addition to therapeutic applications, Fc-fusion proteins serve as detection reagents in basic research, including flow cytometry, immunohistochemistry, and protein binding assays. In fact, linkage to the Fc domain can improve the solubility and stability of some binding partners. Given the size and need for glycosylation (most are glyco-proteins), Fc-fusion proteins are mainly produced in mammalian expression systems.
Concluding remarks
Recently, advancements in antibody engineering technologies have greatly enhanced the generation of recombinant antibodies in various formats as therapeutic agents. There are already more than 100 antibody-based drugs approved by the FDA, and numerous antibodies are currently in their late-stage clinical studies. Moreover, engineered recombinant antibodies can be used in many research applications: western blotting (WB), immunohistochemistry (IHC), immunofluorescence (IF), flow cytometry (FC), and surface plasmon resonance (SPR).
As molecular biological and immunological technologies are continuously developing, recombinant antibodies will be widely used in basic scientific research and disease prevention, diagnosis, and treatment, thereby providing strong support for scientific research and pursuing a healthier future for humans.
References
1. Frenzel, A., et al . (2013). Expression of recombinant antibodies. Frontiers in immunology, 4, 217.
2. Basu, K., et al . (2019). Why recombinant antibodies—benefits and applications. Current opinion in biotechnology, 60, 153-158.
3. Butler, M. (2004). Animal cell culture and technology. Taylor & Francis.
4. Ma, H., et al . (2017). Recombinant antibody fragment production. Methods, 116, 23-33.
5. Zhao, L., et al . (2019). Genetic Engineering Antibody: Principles and Application. In IOP Conference Series: Materials Science and Engineer- ing (Vol. 612, No. 2, p. 022045). IOP Publishing.
6. Little, M. (2009). Recombinant antibodies for immunotherapy. Cambridge University Press.
7. Ma, J., et al . (2021). Bispecific antibodies: from research to clinical application. Frontiers in Immunology, 12, 1555.
8. Czajkowsky, D. M., et al. (2012). Fc-fusion proteins: new developments and future perspectives. EMBO molecular medicine, 4(10), 1015-1028.
