Microbial Expression in Antibody Discovery
Antibody drug discovery and development is the process of identifying new therapeutic antibodies to combat different diseases such as cancers, HIV, autoimmune, hereditary, and more. The technologies used to discovery these antibody-based drug candidates has revolutionized the science conducted by industries and academia labs alike. When discussing the discovery process, it can broadly be divided into five stages, as listed in Figure 1. After the discovery and development process, the new therapeutic antibody drug enters the clinical phase, where it is manufactured for clinical testing and tested through clinical trials. There are three phases in clinical trials, Phase 1: where they assess the safety of the drug, Phase II: where they test the efficacy of the drug, and Phase III: where they test the efficacy of the drug and how it compares to the current treatment options. After a drug candidate successfully passes the three phases of clinical trials, it is approved for distribution to patients. These revolutionary antibody therapeutics can be come in a number of different formats, such as full length antibodies, bispecific antibodies, antibody fragments, and more. Each of these formats have certain advantages and disadvantages, but for the purpose of this article, we will just focus on antibody fragments used in drug discovery1.
Figure 1: Overview of the discovery process for therapeutic antibodies.
What are antibody fragments?
One of the biggest disadvantages to using full sized monoclonal antibodies as therapeutic antibodies is the lack of delivery, especially against cancerous tumors. Full length antibodies are oftentimes prevented from reaching the center of a solid tumor due to their substantial physical barriers. This causes a reduction of the therapeutic effects2. This is one of the reasons that lead to researchers looking into antibody fragments. Smaller fragments are able to penetrate deeper within the tumor and allow for radiolabeled imaging or successful therapeutic antibody delivery3.
Antibody fragments, in particular the scFvs, Fabs and VHH/VH retain full antigen-binding capacity and superior properties for research, diagnostic and therapeutic applications. Fragments are particularly useful in applications where epitope binding is sufficient for the desired effect including therapeutic applications such as virus neutralization or receptor blocking. The smallest antigen binding fragment that retains its complete antigen binding site is the Fv fragment, which consists entirely of variable (V) regions. A soluble and flexible amino acid peptide linker is used to connect the V regions to a scFv (single chain fragment variable) fragment for stabilization of the molecule, or the constant (C) domains are added to the V regions to generate a Fab fragment [fragment, antigenbinding]. scFv and Fab are widely used fragments that can be easily produced in prokaryotic hosts. Other Ab formats include disulfide-bond stabilized scFv (ds-scFv), single chain Fab (scFab), as well as di- and multimeric antibody formats like dia-, tria- and tetra-bodies, or minibodies (miniAbs) that comprise different formats consisting of scFvs linked to oligomerization domains. The smallest fragments are VHH/VH of camelid heavy chain Abs and single domain Abs (sdAb). Antibody fragments are important for the development of therapeutic antibodies and the most effective type of fragment for depends on the disease form.
Figure 2: Schematic depicting different antibody formats. The figure uses the standard IgG molecule and Camelid heavy chain IgG (hcIgG) as the bases of which the fragments are derived from.
How to produce antibody fragments?
Due to the rapidly growing therapeutic antibody market, there have been a number of biotechnological advancements aimed to improve the antibody production process. Improving yields, structural stability, functional reliability, increasing specificity are just a few of the ways these production processes are becoming more advanced. For the production of antibody fragments researchers have the ability to choose between chemical synthesis or biological synthesis in microorganisms. There are several advantages to using microorganisms over other methods such as they can produce high molecular weight proteins, the use of native enzymatic machinery to synthesis proteins, and they do not produce organic or inorganic pollutants4. Antibody fragments are primary used in the initial discovery phase of antibody drug discovery because the full length antibody have several disadvantages. Full length mAbs compared to antibody fragments such as long synthesis time, complicated glycosylation, and longer retention in non-tissue specific areas. Antibody fragments such as Fab and Fv regions exhibit antigen binding, usually have shorter synthesis time, less complicated production, increased tissue penetration, and faster engineering in different host organisms such as microbial5,6.
Prokaryotic expression systems are widely used for recombinant protein production due to their well understood genetics, high cell densities available, low costs, and easy genetic manipulation. E.coli was actually the first microbial organism used for the production of recombinant proteins and is still one of the most commonly used microbial systems7. A wide variety of proteins can be successfully produced in E.coli. These include full length prokaryotic and eukaryotic proteins including enzymes, antibodies and their fragments, protein complexes, and even some human integral membrane proteins. It turns out that up to 50% of proteins from eubacteria and 10% of proteins from eukarya can be expressed in E.coli in a soluble form8. Proteins that cannot be successfully produced in soluble form may precipitate in the E.coli cytoplasm as inclusion bodies (Figure 3). When that happens you could try a cumbersome process called in vitro protein refolding and still get the necessary outcome or we could attempt optimization at the process or molecular levels. At the process level you can adopt methods that do not require target engineering, for example we could play around with the expression conditions like trying out different strains or adjusting the growth and induction conditions, changing media, buffers, and chaperone co-expression and in some cases even improving the cell lysis conditions.
Figure 3: Bacterial transient protein expression workflow.
How to use antibody fragments in drug discovery?
One application of antibody fragments created in E.coli for the antibody drug discovery process, is using cell screening for therapeutic antibody engineering. Currently for antibody discovery, there are two approaches that are consistently used: animal immunization, where an animal is infected with an antigen to produce an immune response to generate antibodies, and surface display methodologies, that use phage display or microbial cell surfaces for in vitro selection of naïve or immune-libraries. Antibody fragments or engineered antibodies are usually selected from combinatorial antibody-libraries using various display libraries. These antibodies are usually optimized for enhance tissue penetration, pharmacokinetics, or an effector. In E.coli, antibody fragments are secreted into the bacterial periplasm and chaperones and other machinery are needed for the fragments to display on a phage9. Another application of that uses E.coli for antibody discovery is using bacterial cells and peptides for antibody biomarker identification. A study published by Ballew et al, described the use of bacterial cell-displayed peptide libraries to bind to serum antibodies from patients with celiac disease. These bacterial cells were then screened using cell –sorting to identify two distinct consensus epitopes for celiac disease10. Antibody fragments can also be used as biosensor to detect cancer-specific antigens. These fragments can also be used as immunotherapy options because of their ability to bind to certain antigens and some are able to resist degradation for a longer period of time11. There is constant need of new techniques for antibody engineering for a number of innovative applications. Antibody therapeutics is a revolutionizing field and it only continues to grow over time.