Reconby: Nanobody de novo discovery by artificial CDR recombination

Nanobodies are proteins that selectively bind to an antigen. Their smaller size and lower complexity compared to conventional antibodies result in beneficial economic and biochemical properties. Therefore, they have a great potential for therapy or diagnostics, among many other applications.
Complementarity determining regions or CDRs (CDR1, CDR2, CDR3) are unique nanobody regions involved in antigen binding. Nevertheless, even nanobodies that bind to the same antigen have different binding affinities, as they present different CDRs.
Our goal is to create a library of artificially developed nanobodies that bind to a specific antigen. Inspired by nature, this is achieved by random in vitro recombination of the different CDR genetic sequences of already characterized nanobodies that bind to said antigen.
These newly formed nanobody gene fragments are expressed in E. coli, generating a library for the screening of nanobodies with higher binding affinity than the starting ones. This way, our system improves the traditional method for nanobody production, being faster, cheaper, and without using animals.

Figure 1. Project abstract.

Background

Antibodies: structure, production, and applications

Antibodies are very special proteins capable of binding to different molecules called antigens (Ag). They are widely used for many applications including research, diagnosis, and therapy, since they can be elicited against all possible targets, associating to them with high specificity and affinity. Their characteristics and their enormous number of applications make them very valuable molecules, although the traditional way of obtaining them has certain limitations. Producing monoclonal antibodies against a single epitope requires developing hybridomas (Figure 2). This process is very tedious, expensive, time-consuming (from months to years), and requires using animals (1).

Figure 2. Hybridoma development based on polyethyleneglycol (PEG). The fusion of B lymphocytes in spleen cells with cancerous myeloma cells gives rise to hybridoma cells, which continuously produce antibodies in vitro. After cell fusions, there is a multi-step screening process to identify antigen-specific hybridomas, which are then propagated.

An alternative to the use of hybridomas is the recombinant expression of antibodies in cell cultures. Unfortunately, the large molecular weight, heterotetrameric composition, and multiple disulfide bonds that conventional immunoglobulins-γ (IgG) have, prevent a facile production in bacteria and even in the cytoplasm of eukaryotic cells (2). However, bacteria can successfully express antibody fragments thanks to their simpler structures and components (3).
The overall structure of conventional IgG (the most abundant antibodies in mammalian blood) consists of two heavy (H)-chain and two light (L)-chain polypeptides (Figure 3). The domain at the N-terminal end of each chain is variable, designated as VH or VL depending on whether it is in the H or L chain, respectively. A few years ago an exception to this conventional IgG structure was discovered in the sera of Camelidae (Figure 4) (4). These sera contain IgG antibodies that lack the L-chains and the first constant domain of the heavy ones (CH1). These IgG are called heavy-chain antibodies (HCAbs), and they contain at the N-terminal end of each chain a variable domain called VHH, also known as nanobody (Figure 5). Nanobodies are the functional and structural equivalent of the antigen-binding fragment of conventional antibodies (5).

Figure 3. Structure of conventional IgG (IgG1) in mammals. They contain two heavy (H) and two light (L) chains, each composed of a variable (V) and constant (C) domains. The heavy chains are made up of the VH, CH1, hinge, CH2, and CH3 domains and the light chains are made up of the VL and CL domains. The VL, CL, VH, and CH1 domains together constitute the antigen-binding fragment (Fab). A VH-VL pair linked by an oligopeptide constitutes the smallest intact functional antigen-binding fragment that can be generated from conventional antibodies, and it is known as scFv (single-chain variable fragment).

Figure 5. Structure of the two types of HCAbs that are present in sera of Camelidae. Each H chain is composed of the VHH, hinge, CH2, and CH3 domains. The hinge of the IgG2 fraction is longer than that of the IgG3 type.

Figure 4. Members of the Camelidae family.

Since nanobodies are small and less complex antibody-like molecules, it becomes feasible to produce them in an active and functional recombinant form via Escherichia coli expression systems, which are simpler and cheaper than other expression platforms, such as yeasts, insects, or mammalian cells (Figure 6).

Figure 6. Some of the most commonly used systems for producing recombinant proteins (6).

Nanobody libraries

In order to store nanobodies against different antigens and have them readily available, libraries were created to save their sequences, for example, cloned into plasmids inside bacteria. Three different types of libraries (Figure 7) are used to find antigen-specific nanobodies (2):

Naïve libraries are constructed after isolating B cells from unimmunized healthy donors. Their mRNA is retrotranscribed to cDNA and used to amplify the nanobody genetic region. The library is formed when these amplicons are cloned into a vector and transformed into suitable bacteria cells.
Immune libraries require injecting adult animals several times with the desired target mixed with an adjuvant. It is recommended to immunize more than one animal in order to obtain a larger panel of nanobodies, from which the best performing one will be chosen. After the immunization, a small aliquot of blood is taken and the following steps are similar to those of naïve libraries.
Synthetic libraries are constructed by selecting stable and well-expressed nanobody scaffolds whose complementarity determining regions (CDR) are randomized. CDR1, CDR2, and CDR3 are unique nanobody regions involved in antigen binding. The sequences of randomized scaffolds are amplified by PCR and cloned into vectors that are used to transform bacteria. This is kind of library constitutes an animal-free strategy. However, a fraction of the CDR three-dimensional structure is already determined by the scaffold nanobody structure, reducing the variety of nanobodies obtained.

Figure 7. Types of nanobodies libraries.

Each of these libraries has some positive and negative points. However, none of them allows generating nanobodies with a high stability and/or affinity without having to use animals.

Our approach

Our project goal is the creation of a library of newly formed nanobodies that bind to a specific antigen: GFP, as 'proof-of-concept'. This is achieved by random in vitro recombination of the different CDR genetic sequences of already known nanobodies that bind to that same antigen - even nanobodies that bind to the same antigen do not have the same affinity, as they present differences in their CDR aminoacidic composition. These newly formed nanobody gene fragments are cloned and nanobodies are expressed in E. coli membrane, allowing the final selection of those with higher binding affinity to said antigen.
With our new nanobody generation method, not only the CDRs are variable but also the whole nanobody structure, allowing us to obtain a huge variety of nanobodies. Moreover, we are able to design nanobodies using sequences from different species, increasing like that the options without using experimentation animals, solving one of the immune and naïve libraries problems.
Our library is special because it addresses one of the main problems of synthetic libraries: the absence of the antibody affinity maturation process. With our strategy, we try to optimize existing nanobodies to improve their affinity for their target antigen.

Figure 8. Project approach.

Inspiration

Choosing which project to undertake was not an easy task. We had several options in mind, but given the context, there was one thing always present in our thoughts: the pandemic. For us, the world would have been a very different place these past two years if a highly effective antibody against SARS-CoV-2 had been available when the pandemic began. This made us think big and we decided that we wanted to develop a method that would allow us to obtain antibodies quickly, cheaply, and without having to use animals.

References

Parray HA, Shukla S, Samal S, Shrivastava T, Ahmed S, Sharma C, et al. Hybridoma technology a versatile method for isolation of monoclonal antibodies, its applicability across species, limitations, advancement and future perspectives. Int Immunopharmacol. 2020 Aug 1;85:106639.
Muyldermans S. A guide to: generation and design of nanobodies. FEBS J. 2021;288(7):2084–102.
Sandomenico A, Sivaccumar JP, Ruvo M. Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments. Int J Mol Sci. 2020 Sep 1;21(17):1–39.
Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hammers C, Songa EB, et al. Naturally occurring antibodies devoid of light chains. Nature. 1993;363(6428):446–8.
Muyldermans S. Nanobodies: Natural single-domain antibodies. Annu Rev Biochem. 2013;82:775–97.
Recombinant Protein and Its Expression Systems - Creative BioMart [Internet]. [cited 2021 Oct 14]. Available from: https://www.creativebiomart.net/resource/articles-recombinant-protein-and-its-expression-systems-365.htm

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