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Human Immunodeficiency Virus type 1 (HIV-1) is one of the major health problems worldwide and has been for over thirty years. Most (67%) of the people infected with HIV-1 are living in sub-Saharan Africa. Here, the access to treatments is limited and most women are not in a position to protect themselves from getting infected. The aim of this study was to identify VHH that are both broad and potent HIV-1 neutralizers, which can be used in the fight against HIV-1. A VHH is an antibody fragment containing the antigen binding domain derived from a heavy chain only antibody. This special type of antibody can be found in members of the camel family. The advantage of VHH is that they are relatively small, stable, cheap to produce and easy to work with. In order to raise VHH against HIV-1, two llamas were immunized with a mix of its envelope proteins. After cloning of the llama’s VHH repertoire into a phagemid expression system, multiple different strategies were followed to identify neutralizing VHH. In total, twelve families of neutralizing VHH were found and characterized in more detail. These VHH target four independent areas on the HIV-1 envelope protein. The best VHH of them being J3, which neutralized 96 out of a 100 tested viruses with a median IC50 of 0.9 µg/ml and is targeting the CD4 binding site. The CD4 binding site on the envelope protein is considered an important area as the first interaction between virus and its target cell occurs via CD4. Previous studies have shown that linking two VHH together into so-called biheads may increase their breadth and potency. For this reason we have linked two different VHH, targeting independent epitopes, together to form bi-specific biheads. Several combinations of VHH have been tried, the best (J3 linked to 2E7) yielded upto 1400 times increase in potency compared to a mix of the individual VHH. Another bihead (J3 linked to 1F10) was capable of neutralizing a viral strain J3 alone could not neutralize, possibly improving the breadth of this molecule above 96%.

Alzheimer's Disease (AD) is the most common form of dementia among elderly in the Western world. AD is a devastating neurodegenerative disease where patients starting with episodic memory problems end up completely bedridden and care dependent. At present there is no real therapy stopping or reversing AD progression. Roughly 100 years ago Alois Alzheimer described the deposition of amyloid-beta (Aβ) in post-mortem brains, one of the hallmarks of AD. Since this description AD has had great attention. However, the exact mechanism of AD remains unclear, a general consensus is reached over Aβ as a peptide involved in AD progression. Aβ is derived from amyloid-beta precursor protein (APP) by the proteolytic cleavage activity of beta-secretase BACE1. Interesting, next to AD patients, healthy individuals also produce Aβ in their brain. So next to the production and presence of Aβ, an extra mechanism is in place to exert toxicity and therefore AD progression. The hydrophobic nature of Aβ makes this peptide prone to aggregate. Overwhelming evidence point to an aggregated form of Aβ (oligomer) as the culprit of toxicity. This makes aggregation of Aβ the mechanism responsible for the generation of toxic Aβ species. Members from the Camelidae family possess, next to conventional IgGs, a heavy chain only antibody. The antigen-binding domain of this antibody (VHH) is fully function while being about ten times smaller compared to a conventional antibody. Next to size, VHH have several other benefits like stability, production costs and genetic modulation. Next to that, VHH are found that inhibit protein aggregation and enzyme activity. We exploited the capability of VHH to inhibit protein aggregation by selecting and generation VHH against Aβ. Llamas were immunized with patient material containing severe Aβ deposition. The subsequently generated VHH library yielded one VHH that inhibits aggregation, and reduced Aβ toxicity. Mutation studies and an additional crystal structure led to the hypothesis that this VHH binds to a small aggregated form of Aβ. A secondary strategy to prevent Aβ toxicity is to inhibit Aβ production by targeting BACE1. We selected a VHH that inhibits BACE1 activity in vitro and in vivo. The in vivo pilot experiment showed a reduced concentration of Aβ and therefore might be of therapeutical value. Problematic is the delivery of compounds to the brain due to the blood brain barrier (BBB). We provided a proof of principle to deliver compounds over cell layers that were BBB models. Transferrin Receptor (TfR) was used to piggy bag an anti-TfR covalently coupled to an anti-Aβ over the cell layer. The aim of this thesis was to find VHH against Ab targets and characterize these VHH. We made great progress in characterizing VHH G7 in vitro and explain characteristics needed for aggregation and toxicity inhibition. Besides this we found a VHH inhibiting BACE1 activity in vitro and in vivo providing a secondary therapeutic strategy for AD. Furthermore, proof of principle is provided to deliver compounds over cellular monolayers modeling the blood brain barrier.

Delivery of drugs to sites of disease can be hampered by the presence of cellular barriers in the human body. The presence of such barriers reduces the therapeutic activity of drugs on the disease target. The barrier present between the bloodstream and the brain for instance, stringently controls what goes into the brain and what not. Diseases that take place in such enclosed systems may be difficult to reach for pharmaceutical compounds because of these barriers and this can reduce their therapeutic effectivity. In this thesis we have described the development of molecules that interact with specific barriers in the human body and that mediate translocation across these barriers. As platform we used antibodies from the llama. Llama antibodies have several favourable properties as compared with other mammalian antibodies. The llama antibody platform was used in the described studies to make entities capable of translocating across cellular barriers. Part of the described studies has looked at the removal of compounds from the body by efflux via the intestinal wall (a cellular barrier). We developed molecules which can interact specifically with that barrier and mediate the passage of unwanted molecules. These molecules can be used for example for the removal of viruses that have infected the body. Fusion of the translocation molecules with compounds that bind viruses would enable the resulting compounds to both bind the virus and mediate its translocation across the intestinal wall. Such therapeutic strategies could tackle the infections of persistent viruses such as HIV. The other part of the described studies has looked at the delivery of blood borne compounds across the blood brain barrier to the central nervous system. Efficient platforms that mediate brain delivery from the bloodstream are scarce/ non-existent. We found molecules which have favourable properties in artificial model systems mimicking the blood-brain barrier. Application of these molecules to mice showed that there was a significant proportion of the injected dose able to accumulate in the brain, which indicates that our molecules indeed can target the brain. Further genetic adaptation of the antibodies might improve the translocation process to the brain environment and deliver a bigger pool of therapeutics to the site of disease. Development of these molecules might yield entities capable of delivering therapeutically relevant compounds to the brain. At the moment, brain delivery of pharmaceuticals is still a feature that is hardly possible without invasive procedures. Due to this aberrant delivery, there is no proper treatment possible of diseases like Alzheimer’s, Parkinson’s and brain cancer. We hope that with further development, our molecules will be able to give a solution to the brain delivery problem and thereby aid in the treatment of brain afflictions. This thesis has given examples of how antibodies from the llama can be used as retargeting agents. We have shown that they can be designed to translocate across cellular barriers and to take along cargo in this process. Development of the antibodies may yield molecules which can help in the diagnosis and treatment of several diseases.

Diagnosis of the hepatitis B Virus (HBV) is important for treatment and prevention of further spread of the virus. Today, detection of hepatitis B virus surface antigen (HBsAg) is the method of choice for the screening and initial diagnosis of HBV. Genetic diversity and discovery of HBV variants with mutations in the immunological dominant region of HBsAg caused none reactivity in some diagnostic assays. Characterization of anti-HBs monoclonal antibodies lead to discovery of a unique monoclonal antibody revealing the true topology of the small s-protein. The knowledge of the antibody recognition spectra, lead to the development of a diagnostics assay showing for the first time detection of all HBsAg mutants based on three different epitope regions. The three epitope regions should in theory enable detection of all known HBsAg mutants, which was proven after testing HBsAg mutants in direct comparison to other diagnostic assay lacking complete mutant detection. The improved HBsAg screening assay permitted better and earlier diagnosis of HBV infections, leaving the diagnosed HBV patient for treatment. Today the main motivation for intervening in an on-going HBV infection is in case the liver is compromised. Depending on the status of the patient, antiviral therapy is started or in worse case when the liver stops to function, a liver transplantation is the final choice. Transplantation of the liver is preceded by antiviral therapy in combination with Hepatitis B immunoglobulin (HBIg) additions. In a quest for an alternative source for HBig we isolated and selected single-domain antibodies (VHHs) that recognize the major small s-protein of HBV. Testing of five VHH in an in-vitro neutralization experiment identified one VHH that could neutralize HBV comparable with good neutralizing anti-HBs reference antibodies. Pepscan analysis and amino acid substitution experiments suggested mutual recognition of the VHH of both the “a”-determinant and c-terminus of the s-protein, fixating the structure and preventing conformational changes needed during viral entry. Alternatively binding to a yet to be discovered HBV (co-) receptor is blocked by the VHH. Ab initio modeling of the dimer “a”-determinant sequence (aa100-155) with VHH S5 revealed a tempting structure that could explain our and published observations. Mainly the location of the cysteine residues in the newly found structure was striking, showing possible formation of inter and intra chain disulfide bridges. Separate analysed c-terminal hydrophobic region (155-226) showed structural homology with Arfaptin 2, a BAR domain protein. The homologous structure predicted importance of the region giving curvature to the aggregated s-proteins in the HBsAg particles. We conclude from the modelling data that the protruding spikes are dimers of the “a”-determinant with peripheral stabilized c-terminal s-protein fragments on the 3- and 5-fold axis.

Heavy-chain antibodies are present in llamas next to conventional immunoglobulins. Their variable domain, also known as VHH is a small, single-chain globular protein with full antigen-binding capacity. Its single-domain nature and some structural differences from conventional immunoglobulins make it a tool with high potential for a number of biotechnological and medical applications. The HIV pandemic represents since three decades a dramatic health problem throughout the world and especially in resource-limited countries. As an alternative to vaccination, prevention is regarded as the best way to control the pandemic, both from a healthcare and economic point of view. The development of microbicides that help preventing sexually transmitted infections of HIV (the most common way the virus is transmitted) is therefore highly desirable. In this thesis, several aspects of the development of llama heavy-chain antibody fragments as active compound of topical microbicides are investigated. In chapter one the 3D structure of the HIV neutralizing VHH D7 was determined. The extension, flexibility and amino acid-composition of its CDRs were compared to other known (human) neutralizing antibodies in order to define the way D7 neutralizes the virus by binding gp120. Structural comparison suggested diverse modes of interaction. Mutational analysis identified CDR3 as key area of the interaction with gp120, whose conformational flexibility is likely to accommodate multiple modes of antibody binding inside the CD4 binding site. Comparison of the related VHH A12 and D7 revealed broad and narrow cross-clade neutralization phenotypes respectively. In chapter two a family-specific phage library was generated and a number of homologous VHH was selected with varying neutralizing profiles. Analysis of their sequences and their recognition of a panel of different HIV-1 subtypes, allowed the understanding of the molecular basis of the different neutralization behaviors. In particular the important role played by the last two C-terminal amino acids of CDR3 was demonstrated. Secretion efficiency of VHH is a particularly important factor when large amounts are needed at low production cost. In chapter three we analyzed databases of structures and sequences to find sequence-related factors that influence the yield of VHH when produced in the host S. cerevisiae. We showed how the combined presence of five key residues and specific J segments is fundamental for proper folding. We also suggested that interactions with ER-resident chaperones are necessary to achieve high secretion efficiency. In the last chapter, VHH A12 and D7 were used as models to study their availability, activity and stability in a VHH-based topical microbicide. We systematically analyzed likely challenges that VHH will have to face in case of use in sub-tropical settings of resource-limited countries, as HIV preventative drug. VHH proved to be readily available when formulated as tablets or aqueous gel and very stable after extended incubation at physiological temperature and at low pH. We also investigated the tissue-penetration kinetics of VHH in order to assess whether the drug would be present in the sub-mucosal layers of the vagina and once there, carry out its protective action.

This first part of this thesis, ’Removing substances’, describes a novel immunoadsorption technique that uses variable domains of heavy chain llama antibodies (VHH) to remove a toxic agent, Toxic Shock Syndrome Toxin-1 (TSST-1) from the circulation. This study was designed as a proof-of-principle that immunoadsorption of substances by VHH is a feasible, effective technique. We chose the removal of TSST-1 from a porcine sepsis model because in this system, there was a direct relation between the (one) substance (TSST-1) and the disease (sepsis). The second part of this thesis, ’Removing excess volume’, describes a novel method to measure absolute plasma refill rates during hemodialysis, using a feedback-regulated ultrafiltration system coupled to a dialysis machine. We designed this system because we wanted to be able to non-invasively measure absolute plasma refill rates continuously during dialysis. This system is the first to do so, and, using the method, we investigated the influence of blood-pressure stabilizing interventions on refill. Finally, the application of the method as a novel determinant of the patient’s dry weight was investigated

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 the best producing variants of a set HIV neutralizing VHHs. The alignment, combined with structural data, also made a clear definition of the VHH CDR regions possible. A more detailed in silico and in vitro and in vivo studies of two similar VHHs demonstrated that the following optimisation possibilities; improved codon usage, framework four replacement, and yeast fermentation on ethanol, resulted in a higher production yield and secretion efficiency in yeast. Combined with electron microscopy and mammalian cell expression results some light was shed on the possible mechanisms causing the low production levels in yeast. With the availability of Lama glama and Lama pacos germline sequences the maturation pathway of VHHs was studied. For L. glama 23 V-genes were identified, as well as typical llama hotspot motifs. Evidence was found for gene conversion of D-gene segments. Knowledge of the maturation pathway will eventually assist the selection and design of even better VHHs against HIV. Combined with the in silico screening methods and demonstrated optimisation possibilities, exquisite opportunities are offered to select or design the optimal VHHs for the use in microbicides and many other applications.