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 discover these antibody-based drug candidates have 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 tests the safety of the drug, Phase II tests the efficacy of the drug, and Phase III tests 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 designed in a number of different formats, such as full length antibodies, bispecific antibodies, antibody fragments, and more. Each of these formats has certain advantages and disadvantages, but for the purpose of this article, we will just focus on antibody fragments used in drug discovery¹.

Therapeutic Antibody Discovery

Antibody drug development; from target identification, lead generation and optimization to preclinical testing.

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 their reduced delivery, especially against cancerous tumors. Full length antibodies are oftentimes prevented from reaching the center of a solid tumor due to substantial physical barriers, causing a reduction in therapeutic effects². This is one of the reasons leading researchers to consider 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 depends on the disease form.

Therapeutic Antibody Formats

Therapeutic antibodies may be designed in several formats to improve their delivery and to enable expanded specificities.

Figure 2: Schematic depicting different antibody formats. The figure uses the standard IgG molecule and Camelid heavy chain IgG (hcIgG) as the basis from 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 their ability to produce high molecular weight proteins, availability of native enzymatic machineries for protein synthesis, and a clean production process devoid of organic or inorganic pollutants⁴. Antibody fragments are primarily used in the initial phase of antibody discovery to avoid several disadvantages associated with full-length antibodies, such as long synthesis time, higher production cost, and lower yields. 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 microbial^5,6.

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 including full length prokaryotic and eukaryotic proteins, such as 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 precipitation occurs, a cumbersome process called in vitro protein refolding may still support production yields. Alternatively, optimization of conditions during production such as growth conditions, buffers used, strain selection, chaperone co-expression and even improving lysis conditions may be adopted to enhance yields. Additionally, other methods focused on improving the target’s expression or molecular properties may also be leveraged.

Bacterial Recombinant Protein Production Workflow

Bacterial recombinant protein production is optimized through engineering and non-engineering methods.

Figure 3: Bacterial transient protein expression workflow.

How to Use Antibody Fragments in Drug Discovery

Antibody fragments can be used as therapeutic agents for their ability to specifically bind and potentially block the function of biologically relevant targets. Functionally, antibody fragments present various advantages over full-length antibodies due to their format, which allows them to reach challenging epitopes, and have reduced immunogenicity. As cancer immunotherapeutics, their small size supports superior tumor penetration. In addition to their use as therapeutics, antibody fragments may also support diagnostic applications, such as in the detection of cancer biomarkers (e.g., HER2, CEA). However, antibody fragments can also be leveraged in the drug discovery process. For example, Luptak et al. 2019 described how they utilized both scFv and Fab fragments to facilitate the crystallization of Mcl-1, a member of the Bcl-2 family of pro-survival proteins involved in the regulation of apoptosis. By facilitating the crystallization of Mcl-1, these antibody fragments supported structure-guided drug design and the discovery of the inhibitor AZD5991, an anticancer agent.

There is a constant need for new techniques for antibody engineering to support a number of innovative applications. Antibody therapeutics is a revolutionizing field and it only continues to grow over time.