Isolation and optimization of camelid single-domain antibodies: Dirk Saerens’ work on nanobodies

Abstract

It is well established that all camelids have unique antibodies circulating in their blood. Unlike antibodies from all other species, these special antibodies are devoid of light chains, and are composed of a heavy chain homodimer. These so-called heavy-chain antibodies (HCAbs) are expressed after a V-D-J rearrangement and require dedicated constant gamma genes. An immune response is raised in these HCAbs following a classical immunization protocol. These HCAbs are easily purified from serum, and their antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies. The antigen binding site of the dromedary HCAb comprises one single domain, referred to as VHH or nanobody (Nb), therefore, a strategy was designed to clone the Nb repertoire of an immunized dromedary and to select the Nb with specificity for our target antigens. The monoclonal Nb is produced well in bacteria, is very stable and highly soluble, and it binds the antigen with high affinity and specificity. Currently, the recombinant Nb has been developed successfully for research purposes, as a probe in biosensors, to diagnose infections, or to treat diseases such as cancer or trypanosomiasis.

Key Words: Heavy-chain antibody, Single-domain antibody, Monoclonal antibody, Nanobody

INTRODUCTION AND EDUCATIONAL EXPERIENCE

Dirk Saerens started his scientific career at the Department of Cellular and Molecular Immunology at the Vrije Universiteit Brussel (VUB) - Flanders Institute for Biotechnology (VIB) as a researcher on an IWT scholarship to obtain a PhD in applied biological sciences. The Department of Cellular and Molecular Immunology is part of VUB and VIB. VUB is a dynamic and modern university with two parkland campuses in the Brussels Capital Region. VIB is a research institute with 1200 employees. The main objective of VIB is to gather innovative basic knowledge on the normal and abnormal or pathological processes that occur in cells, organs and organisms (humans, plants, or microorganisms), using gene technology.

Dirk Saerens’ PhD project fitted in the frame of an EU research project (Figure 1). The aims were to isolate camelid single-domain antibodies [i.e. nanobodies (Nbs)] against diagnostically relevant antigens[1-3]. These Nbs were subsequently engineering to improve affinity, stability[4,5] and immobilization potential[6-8]. Several publications in high-ranked journals, e.g. Journal of Molecular Biology, Journal of Biological Chemistry, Bioconjugate Chemistry & Analytical Chemistry, were produced in collaboration with national (i.e. IMEC, Leuven, Belgium and Center for Protein Engineering, ULg, Liège, Belgium) and international (Center for Nanobiotechnology, Vienna, Austria) research groups.

Figure 1 Dirk Saerens, PhD, Department of Cellular and Molecular Immunology, Vrije Universiteits Brussel-Vlaams Instituut voor Biotechnology, Pleinlaan 2, B-1050 Brussel, Belgium.

After obtaining his PhD, Saerens’ research focussed further on isolating Nbs and engineering them for diagnostic and therapeutic applications[9-12]. Several collaborations were initiated with national and international research groups. Together with researchers from the Institute for Tropical Medicine (Antwerpen, Belgium), Nbs were isolated against diagnostically relevant antigens for parasite detection[13]. Work on the development of Nbs was supervised and this collaboration resulted in a publication in a peer-reviewed journal (International Journal of Parasitology). Another project was in collaboration with the Pasteur institute in Tunis. It consisted in isolating Nbs to scorpion toxin and subsequently using these Nbs to neutralize the toxin[14-17]. Two different PhD students from Tunis were supervised during Nb isolation, characterization and optimization. This collaborative research project resulted in publications in peer-reviewed journals (e.g. Molecular Immunology & Biochemical Journal). Moreover, a research project for a PhD student was initiated, based on the engineering of the Nbs into formats for application in fusion proteins and molecular switches.

ACADEMIC STRATEGIES AND GOALS

During this postdoctoral period, several invitations to communicate Nb research updates during oral presentations were accepted, e.g. 2nd Annual Conference on Recombinant Therapeutics in Berlin (Germany) and VIB Science Clubs in several universities in Belgium. Moreover, poster presentations were given during international congresses, e.g. International Symposium - Recombinant Antibodies: new developments for future challenges, in Madrid (Spain), Fourth Annual PEGS - The essential protein engineering summit, Boston (USA), and Cambridge Healthtech Institute’s Inaugural Antibodies - Europe, Vienna (Austria).

In addition, several invitations to write reviews and book chapters on single-domain antibodies or Nbs in particular were accepted. This resulted in several reviews in peer-reviewed journals[18-21] (i.e. Current Opinions in Pharmacology, Sensors & Briefings in Functional Genomics and Proteomics) and several book chapters. More importantly, currently, Dirk Saerens is co-editor of the volume “Single Domain Antibodies” for Methods in Molecular Biology of Humana Press (Springer, 2011).

Recent research has focused on understanding of the heavy-chain antibodies (HCAbs), i.e. reconstituted full-sized antibody format, and novel scaffolds to complement the repertoire of Nbs and antibodies in general.

ACADEMIC ACHIEVEMENTS

Single-domain antibodies as building blocks for novel therapeutics

Antibodies are large and complex molecules, with two identical parts that bind independently of each other onto the antigen, and the third part of the molecule that dictates the effector function. To improve the therapeutic value of antibodies, protein-engineering endeavours have reduced the size of the antigen-binding moiety to a single-domain unit. Occasionally, it has been demonstrated that the single-domain antigen-binding derivatives of antibodies can have, on their own, an agonistic (or antagonistic) effect on their target. The small size and strict monomeric behavior, in combination with other biochemical properties such as high solubility and high specificity and affinity for the cognate antigen, make single-domain antibodies ideal to design novel artificial conjugates harnessed with innovative effector functions outside the reach of classical antibodies[18].

Parallel selection of multiple anti-infectome Nbs without access to purified antigens

A strategy was developed to isolate Nbs, camelid-derived single-domain antibody fragments, against the parasite infectome without a priori knowledge of the antigens nor having access to the purified antigens. From a dromedary, infected with Trypanosoma evansi (T. evansi), we cloned a pool of Nbs and selected after phage display 16 different Nbs specific for a single antigen, i.e. variant surface glycoprotein of T. evansi. Fourteen Nbs were isolated by panning on different total parasite lysates. Thus, this anti-infectome experiment generated Nbs, monospecific for one Trypanosoma species, whereas others were pan-reactive to various species. Several Nbs could label specifically the coat of a set of Trypanosoma species. The recognized targets are present in glycophosphatidylinositol-linked membrane fractions of bloodstream- and fly-form parasites. Due to the omnipresence of these targets on different parasite species and forms, these antibody fragments are a valuable source for validation of novel, not-yet-identified targets to design new diagnostics and therapeutics[1].

Disulfide bond introduction for general stabilization of immunoglobulin heavy-chain variable domains

Several antibody fragment engineering techniques aim at intrinsic stability enhancement, but are not applied in a truly generic way. Here, a strategy is proposed whereby consistent gain in stability is accomplished by introducing a specific disulfide bond between two opposite beta-strands in the hydrophobic core of the immunoglobulin heavy-chain variable domain of HCAbs (Nbs). Besides the rational design of a disulfide bond between residues 39 and 87, an Nb that harbors an extra naturally occurring cystine between residues 54 and 78 was compared to an equivalent Nb without that cystine. Both novel disulfide cross-links were introduced in several Nbs in various combinations. Only the extra naturally occurring cystine consistently increased the conformational and thermal stabilities of wild-type Nbs[4].

Engineering camel single-domain antibodies and immobilization chemistry for human prostate-specific antigen sensing

The specificity and affinity characteristics of antibodies make them excellent probes in biosensor applications. Unfortunately, their large size, unstable behavior, and random immobilization properties create numerous problems. The single-domain antigen-binding fragment derived from HCAbs of camelids (termed VHH) offers special advantages in terms of size, stability, and ease of generating different antibody constructs. In this paper, we show the potential of these VHHs in sensing human prostate-specific antigen (hPSA) by surface plasmon resonance technology. Different VHH constructs were immobilized onto commercial and custom-built sensor surfaces by metal chelation, biotin-streptavidin interaction, or covalent coupling. The detection of subnanogram per milliliter hPSA concentrations could be attained on a covalently coupled 3D dextran surface. Moreover, the ratio of different hPSA isoform concentrations could be assessed via a sandwich assay and resulted in the detection of clinically significant antigen concentrations within 15 min. In addition, for the first time, the intrinsic protein stability is presented as an important probe design factor, because our results reveal that higher intrinsic stability offers higher resistance to harsh regeneration conditions. In conclusion, we present VHHs as a novel class of biosensor probes that rival conventional antibodies and their derived antibody fragments[6-8].

Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies

Camel single-domain antibody fragments (VHHs) are promising tools in numerous biotechnological and medical applications. However, some conditions under which antibodies are used are so demanding that they can be met by only the most robust VHHs. A universal framework that offers the required properties for use in various applications (e.g. as intrabody, probe in biosensors, or on microarrays) is very valuable and might be further implemented when employment of VHHs in human therapy is envisaged. We identified the VHH framework of cAbBCII10 as a potential candidate, useful for the exchange of antigen specificities by complementarity determining region (CDR) grafting. Due to the large number of CDR-H loop structures present on VHHs, this grafting technique was expected to be rather unpredictable. Nonetheless, the plasticity of the cAbBCII10 framework allows successful transfer of antigen specificity from donor VHHs onto its scaffold. The cAbBCII10 was chosen essentially for its high level of stability (47 kJ/mol), good expression level (5 mg/L in Escherichia coli), and its ability to be functional in the absence of the conserved disulfide bond. All five chimeras that are generated by grafting CDR-Hs, from donor VHHs that belong to subfamily 2 that encompass 75% of all antigen-specific VHHs, on the framework of cAbBCII10 are functional and generally have increased thermodynamic stability. The grafting of CDR-H loops from VHHs belonging to other subfamilies results in chimeras of reduced antigen-binding capacity[5].