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BACKGROUND: CD1d is a monomorphic major histocompatibility complex class I-like molecule that presents lipid antigens to distinct T-cell subsets and can be expressed by various malignancies. Antibody-mediated targeting of CD1d on multiple myeloma cells was reported to induce apoptosis and could therefore constitute a novel therapeutic approach. METHODS: To determine how a CD1d-specific single-domain antibody (VHH) enhances binding of the early apoptosis marker annexin V to CD1d+ tumor cells we use in vitro cell-based assays and CRISPR-Cas9-mediated gene editing, and to determine the structure of the VHH1D17-CD1d(endogenous lipid) complex we use X-ray crystallography. RESULTS: Anti-CD1d VHH1D17 strongly enhances annexin V binding to CD1d+ tumor cells but this does not reflect induction of apoptosis. Instead, we show that VHH1D17 enhances presentation of phosphatidylserine (PS) in CD1d and that this is saposin dependent. The crystal structure of the VHH1D17-CD1d(endogenous lipid) complex demonstrates that VHH1D17 binds the A'-pocket of CD1d, leaving the lipid headgroup solvent exposed, and has an electro-negatively charged patch which could be involved in the enhanced PS presentation by CD1d. Presentation of PS in CD1d does not trigger phagocytosis but leads to greatly enhanced binding of T-cell immunoglobulin and mucin domain containing molecules (TIM)-1 to TIM-3, TIM-4 and induces TIM-3 signaling. CONCLUSION: Our findings reveal the existence of an immune modulatory CD1d(PS)-TIM axis with potentially unexpected implications for immune regulation in both physiological and pathological conditions.

Microbicides offer a promising way to prevent HIV infection in developing countries. Llama heavy chain antibody fragments (VHHs) that neutralize HIV are an ideal candidate for usage as active microbicide component. A VHH is the smallest intact antigen binding domain which allows it to reach for antigen surfaces that can not be reached by conventional antibody fragments and allows a more efficient tissue penetration. VHHs display a good performance at many different conditions. The usage of VHHs as microbicide component is dependent on many factors; high affinity and neutralization capacity, stability at different temperatures, functionality at a low pH, and efficient production in high quantities at low costs. The GRAS organism Saccharomyces cerevisiae is often used for the production of VHHs. However, the production yield in this organism often is too low for commercially viable large scale application. Understanding the causes of these low production yields and improvement of the yield is an important topic within this thesis. The characteristics of VHHs are studied in silico by typical bioinformatics and genomics approaches as well as in vitro and in vivo by mutational and cultivation studies to find correlations between protein sequence and stability or production level. We found correlations between low production yield and non-optimal codon usage, as well as framework four encoded by J-gene segment 7. To facilitate a more extensive study of all available VHH sequences a bioinformatics infrastructure was set up, resulting in the antibody variable domain database and the VHH 3DM, a molecular-class-specific information system that creates an accurate structure-based multiple sequence alignment. This sequence alignment revealed a set of eight key residues important for proper folding, which was validated in vitro and in vivo. These data were combined with known and predicted mutation effects resulting in a generic 'VHH-mutability' table, which flawlessly predicted

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