Biology subjects

Dekkers, G.

Publications and source records attributed to Dekkers, G..

4 recordsLinked to original sources

bioRxiv2026

Affinity maturation of a single-domain antibody via rational mutagenesis informed by artificial intelligence

Antibodies can originate from various sources such as animal immunizations, patient material, DNA/RNA libraries and (AI-based) de novo design. Pre-made (e.g. synthetic) DNA libraries have substantial advantages over traditional immunization campaigns in terms of speed, cost, animal welfare, amount of required antigen and the possibility to select against otherwise problematic (e.g. toxic, unstable or expensive) antigens. The main downside is that selections from these non-immune libraries often result in binders with relatively low affinity due to the absence of in vivo affinity maturation. Now that in silico structure prediction tools are coming of age, the resulting models could conceivably be used to rationally design mutations, e.g. to enhance binding affinity. Here we used a synthetic single-domain antibody (sdAb) library to select new binders against interleukin-12 (IL-12). One of the selected candidates, 2F3, was predicted to bind to the p40 subunit of IL-12 in a manner that would compete with binding of the receptor protein IL-12R{beta}1. Intriguingly, this epitope is also targeted by the clinically approved antibodies Briakinumab and Ustekinumab, which are used to treat a variety of inflammation disorders. The predicted structure of the IL-12/sdAb complex was used to rationally design eleven point mutations, of which three showed to enhance the binding affinity. By combining these mutations, we could enhance the affinity by eightfold compared to the original lead clone. This shows that correctly (AI-)predicted protein structural models can be used for rational mutagenesis to enhance binding affinity to relevant drug targets.

biochemistry↗

bioRxiv2026

Identification of a C3b-specific nanobody that does not bind C3 and blocks alternative pathway convertases

The human complement system is a protein network in blood and other body fluids that fights invading pathogens and is involved in maintaining homeostasis. A central step in the complement cascade is the conversion of complement protein C3 to C3b by convertase enzymes. Upon cleavage of C3, the nascent C3b molecule undergoes a large conformational change which exposes new epitopes. Molecules that discriminate between C3b and C3 could provide a powerful tool to selectively bind surface-bound complement activation products while leaving the circulating precursors untouched. In this study, we developed C3b-specific nanobodies by generating phage libraries from llamas immunized with purified and surface-bound C3b. We describe UNbC3b-1 as a high-affinity binder that recognizes soluble and surface-bound C3b molecules but does not bind to C3 in its native state. A 4.2 [A] structure of the UNbC3b-1:C3b complex generated by cryogenic electron microscopy shows that UNbC3b-1 binds C3b at the interface of MG3, MG4, and MG6, overlapping with the complement receptor immunoglobulin (CRIg) binding site. Functionally, we show that UNbC3b-1 inhibits complement activity in the alternative pathway (AP), likely by preventing association between substrate C3 and the AP C3 convertase (C3bBb). Altogether, nanobody UNbC3b-1 is a valuable tool to specifically bind C3b molecules and to selectively inhibit the AP convertases of the complement system.

immunology↗

bioRxiv2025

Identification of allo- or orthosteric VHH/single-domain antibodies that enhance or block pathogen binding to Siglec-1 on dendritic cells

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/695420v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@e85819org.highwire.dtl.DTLVardef@1eff6b4org.highwire.dtl.DTLVardef@12daa92org.highwire.dtl.DTLVardef@11959e5_HPS_FORMAT_FIGEXP M_FIG C_FIG BackgroundSiglec-1 (Sialoadhesin/CD169) is expressed on myeloid cells and plays a key role in host defences by capturing incoming sialylated-pathogens such as Campylobacter jejuni. However, binding to Siglec-1 has also been exploited by pathogens such as SARS-CoV-2 for further dissemination. ResultsHere we identified high-affinity VHHs also known as single-domain antibodies or Nanobodies that bind to Siglec-1 and allo- or orthosterically modulate ligand binding. VHH 2C2 was shown to bind directly to the ligand binding site of Siglec-1 and blocked binding of ganglioside liposomes and Campylobacter jejuni to monocyte-derived dendritic cells (moDCs) and ex vivo Siglec-1+ DCs. VHH 2C2 also blocked SARS-CoV-2 binding of moDCs. In contrast, the VHHs 1B5 and 1C1 interacted with Siglec-1 outside the ligand binding site and acted as positive allosteric modulators of Siglec-1 ligand interactions, as was illustrated by increased ganglioside liposome and Campylobacter jejuni binding by moDCs. Our data suggests that mechanistically, the VHH 1B5 and 1C1 interfere with the cis-binding sialic acids present on the Siglec-1-expressing cell and thereby enhance trans-interactions with ligands. ConclusionIn conclusion, we have isolated VHH that enhance or block Siglec-1 ligand binding to a variety of sialylated-pathogens enabling further interrogation of Siglec-1 function. Moreover, unlike conventional blocking antibodies targeting specific pathogens, Siglec-1 binding VHH could potentially serve as broad-spectrum pathogen blocking agents.

immunology↗

bioRxiv2020

Unravelling the effect of a potentiating anti-Factor H antibody on atypical hemolytic uremic syndrome associated factor H variants

The complement system plays an important role in our innate immune system. Complement activation results in clearance of pathogens, immune complex and apoptotic cells. The host is protected from complement-mediated damage by several complement regulators. Factor H (FH) is the most important fluid-phase regulator of the alternative pathway of the complement system. Heterozygous mutations in FH are associated with complement-related diseases such as atypical hemolytic uremic syndrome (aHUS) and age-related macular degeneration. We recently described an agonistic anti-FH monoclonal antibody that can potentiate the regulatory function of FH. This antibody could serve as a potential new drug for aHUS patients and alternative to C5 blockade by Eculizumab. However, it is unclear whether this antibody can potentiate FH mutant variants in addition to wild type FH. Here, the functionality and potential of the agonistic antibody in the context of pathogenic aHUS-related FH mutant proteins was investigated. The binding affinity of recombinant WT FH, and the FH variants, W1183L, V1197A, R1210C, and G1194D to C3b was increased upon addition of the potentiating antibody and similarly, the decay accelerating activity of all mutants is increased. The potentiating anti-FH antibody is able to restore the surface regulatory function of most of the tested FH mutants to WT FH levels. In conclusion, our potentiating anti-FH is broadly active and able to enhance both WT FH function as well as most aHUS-associated FH variants tested in this study.

immunology↗