Over the past two decades, antibodies (Abs) have firmly established their clinical value and now account for a large share of the biopharmaceutical market. More recently, alternative formats such as single-domain antibodies derived from camelids, also known as nanobodies or VHHs, have attracted growing interest because of their small size and their presumed low immunogenic potential. However, recent clinical studies have revealed unwanted immune responses directed against several of these nanobodies administered to patients.
In this study, the authors sought to determine whether VHH76, a nanobody designed to target the SARS-CoV-2 receptor-binding domain and block viral entry, presented a risk of immunogenicity. To address this question, they monitored the response of CD4 T cells, which activate antibody-producing B lymphocytes, toward VHH76. They examined the cellular response to three versions of this antibody: the original version, a humanized version, and a germline version (see Box). The humanized variant contains six human substitutions, whereas the germline variant, obtained through screening of a combinatorial VHH76 sequence library, contains sixteen. The VHH76 variants were tested for their ability to trigger T-lymphocyte responses using cells collected from healthy donors. All three variants elicited T-cell responses; however, the response was weaker in the case of the germline variant. Two important regions of VHH76 were identified: a hypervariable loop involved in virus recognition, and a VHH-specific region intrinsic to their structural framework.
Using a sensitive and specific detection assay, the researchers demonstrated that a single-domain antibody carries a risk of immunogenicity, and that humanization of the sequences reduces this risk. By identifying two key regions of VHH76, these findings could contribute to the development of new de-immunization strategies.
Research on the immunogenic potential of VHHs is still in its early stages, and further investigations are needed to fully characterize and mitigate the risks of unwanted immune responses associated with the therapeutic use of these new antibody formats.
Joliot contacts : bernard.maillere@cea.fr / herve.nozach@cea.fr
- Immunogenicity is the ability of a compound to trigger an immune response. This property is undesirable for therapeutic antibodies.
- Single-domain antibodies, also called nanobodies, are antibody fragments composed of a single monomeric variable domain. Like a full antibody, they can selectively bind an antigen, but they are much smaller. The first single-domain antibodies were derived from antibodies found in camelids and are referred to as VHH fragments.
- Humanized antibodies. When a monoclonal antibody is produced in a mouse, it can be humanized by replacing certain mouse DNA sequences with human DNA sequences, without losing the antibody's affinity or specificity.
- Germline antibodies. To make new therapeutic antibody formats as close as possible to original human germline sequences, amino acids in the hypervariable region (which interacts with the antigen) are replaced by residues from the germline lineage.
In all cases, the goal is to reduce the ability of therapeutic antibodies to trigger an immune response.