A biomolecular toolbox for antibodies: chop, craft, dress, furnish, graft, mill, polish, plane, veneer!
A very warm welcome to our fourth blog!
Since I started working for Immunoprecise Antibodies (IPA) and was kindly informed of the hidden treasures in their working place, I started wondering: what makes a IPA R&D scientist different from a carpenter or furniture maker? Hopefully you do not find me frivolous for making this surprising analogy. Allow me to explain the comparison in this blog and the many blogs to follow.
What we do at IPA with antibodies may seem mysterious and impossible from the outside looking at our Talem pipeline, but it also involves just handicraft. It is just on a much smaller scale than furniture. The antibody type attracting the most attention, IgG, is only 10 to 15 nanometers (10‑9 m) in size and, obviously, requires much finer tools than a carpenter uses. Although the tools are of great interest, the focus of this blog post is what is achievable with antibodies. The contemporary toolbox allows the creative protein engineer to chop, craft, dress, furnish, graft, mill, polish, plane, and veneer his antibodies as his imagination prompts. The possibilities are endless, and this versatility of the ‘starting material’ and the available advanced tools is one of the main reasons that therapeutic antibodies have become the fastest market-growing class of biopharmaceuticals1.
Antibodies consist of discrete domains located on two chains, referred to as the light (L) and heavy (H) chain. Duplicates of each chain are organized in a tetrameric IgG structure (see ‘IgG’ in Figure 1). Each chain consists of domains, referred to as CH1, CH2, CH3, CL, VH and VL, that each have a certain structural and/or biological function. Most excitingly, each domain and region therein, is discretely genetically coded the same way in all mammals, including humans. This gives the opportunity to combine the genetic codes, and thus the protein parts of the antibody, as desired, even between species, and to express these as de novo proteins. They can even be combined with non-antibody proteins to give fusion proteins with completely new xenobiotic functions.
The exploitation of the genetic toolbox by the creative bioengineer has resulted in a long list of synthetic, symmetric and asymmetric, anomalous and sometimes bizarre new proteinaceous binding entities which are coined diabody, heavy-chain-only antibodies, minibody, nanobody, triabody, tetrabody, unibody et cetera (Figure 1). Although constructing binders in this way using protein domains, regions, and fragments as if they are Lego building blocks gives the impression of scientific fun, the motivation for doing so is very serious and highly encouraging. Bispecific antibodies give Talem possibilities to bring a malignant, cancer cell in close contact with its cellular killer. Smaller antibodies provide a better penetration of degenerated and dense tissue, like that of solid tumors. Smaller molecules are also able to pass the blood brain barrier (BBB), which is usually a difficult hurdle to take for therapeutic biologics.
As every furniture maker planes and polishes his creation, created antibodies are also prudently adapted to improve their function, safety, specificity, developability and/or manufacturability. To start with the latter, a fraction of two components in our TATX‑03 multi-antibody cocktail showed undesirable decorations. These unwanted N-glycans were attached to the antibodies in the cells while produced in a process called co- and post-translational modification giving microheterogeneity of the expressed protein. As we identified the location in the variable part of the antibody, we took out the responsible amino acid anchor with a pincer and mutated this location in the gene by replacing the code with that of an inactive amino acid without affecting the binding capacity of the modified antibodies3.
This is already impressive bioengineering, but even more impressive is that, sometimes with the aid of computational modelling, other features such as affinity and specificity can be improved by rationally replacing certain amino acids. The used tools come in handy for another reason. Although antibodies are considered very safe compared to all other drug alternatives, even therapeutic antibodies that have a fully human origin can still elicit a response in the treated person. Most of these anti-drug antibodies (ADAs) do not cause adverse events in the patient, but they can reduce the efficacy of the immunotherapy in a small portion of the recipients, in particular when the treatment needs to be repeated to achieve a full therapeutic effect. To avoid such undesirable immunogenic reactions, we modify the backbone of the antibody in such a way that its specificity and functionality is conserved. This requires good eye for proportions as a carpenter commonly has, as in silico predictions of ‘bad’ sequences is still in its infancy and needs the experience and trial-and-error experimentation of the skilled R&D scientist.
One of the aimed functions of antibodies is activation of the immune cascade and recruitment of immune cells to neutralize and clear a potential threat, such as a micro-organism or tumor cell. The antibody does this through its so-called crystallizable fragment (Fc) which, unlike the variable fragment discussed above, needs N-glycosylation to be able to activate immune effector functions. Through the engineering of these glycans these effector functions can be fueled tremendously. In addition, the structure of the antibody-attached N‑glycans gives the possibility to silence inflammatory reactions. Strangely enough, such silencing ‘sugar’ structures are obtained by deletion of amino acids. In addition, glycosylation influences the biological half-life of the therapeutic binder as well, which gives another possibility for further finetuning of the designed antibody.
With respect to decorating an antibody, specific bioactive molecules can be attached chemically covalently or non-covalently to the protein backbone, or even through the forementioned N‑glycans. In this way, antibodies can be weaponized with, for example, cytotoxic drugs. This currently booming development in immunotherapies offers the possibility to combine the specificity of an antibody with the tumor-killing capacity of an anti-cancer drug thereby reducing the adverse effects of the latter in patients. It is without further explanation that Talem invests in this area as well.
Naturally occurring antibodies are the starting material for Talem like wood is for the furniture maker. But boy, we can bend and change their forms and functions to how we see fit. We do this at a scale which is almost a billion times smaller than a regular chair or cabinet. But our aims are very similar to those of a craftsperson, we also chop, craft, dress, furnish, graft, mill, polish, plane and veneer the antibodies until the crafted molecule is fit-for-its-purpose and maximally acceptable, convenient and safe for the user at the same time. Are you not interested in our next piece of furniture at molecular scale as well?
Figure 1. The ‘carpenter’ at work: only a few examples of the virtually infinite possible antibody formats. In the lower left corner, the starting material: immunoglobulin G, IgG, in which the domains are recognizable as colored ovals. The short light (L) and the long heavy (H) chain cross each other in the upper Y-shaped part of an IgG molecule. Be aware of the difference between mono-, bi-, trivalent etc. on the one hand, and mono-, bi-, trispecific etc. on the other hand. For example, others have synthesized for example bispecific trivalent minibodies. Fab, antigen-binding fragment; scFv, single chain variable fragment; VH, variable domain of the heavy chain; VL, variable domain of the light chain. Adapted from reference Holliger and Hudson, 20052.
REFERENCES
1. Global Market Insights (2021) Antibody Therapy Market Size By Type (Monoclonal Antibodies [mAbs] {Oncology, Autoimmune Diseases, Infectious Diseases}, Antibody-drug Conjugates [ADCs]), By End-use (Hospitals, Specialty Centers), Industry Analysis Report, Regional Outlook, Growth Potential, COVID-19 Impact Analysis, Price Trends, Competitive Market Share & Forecast, 2022 – 2028. See: https://www.gminsights.com/industry-analysis/antibody-therapy-market
2. Holliger P, Hudson P (2005) Engineered antibody fragments and the rise of single domains. Nat Biotechnol 23, 1126–36. DOI: 10.1038/nbt1142
3. Immunoprecise Antibodies (2021) ImmunoPrecise Update on its SARS-CoV-2 PolyTope™ Multi-Antibody Cocktail Development Program. News release of November 30, 2021. https://www.immunoprecise.com/ipa-update-on-its-sars-cov-2-polytope-multi-antibody-cocktail-development-program/