Bioconjugation strategies for multivalent peptide ligands

In nature multiple weak interactions are often combined to enhance the overall affinity and specificity of binding. This effect is known as multivalency and plays a pivotal role in e.g. adhesion of viruses or bacteria to cells, immune responses and protein-protein interactions. Inspired by nature’s success, researchers nowadays often combine several low affinity ligands e.g. carbohydrates, peptides or proteins in order to create high affinity multivalent structures for specific delivery of therapeutics or imaging probes. Current challenges in this field include systematic studies concerning the number of functional groups and the influence of length and flexibility of the linker. Especially for multivalent targets of which no structural information about the position of binding sites is available, generic strategies that allow fast and easy screening of constructs with different linkers or functional groups are essential. This dissertation introduces novel concepts for the synthesis of well-defined multivalent ligands that can be applied in the field of targeted drug delivery and molecular imaging. In the first part of this thesis a modular approach is presented to arrive at well-defined multimodal dendrons. Two, three, four or five peptides were site-specifically introduced to the periphery of a polyamide dendritic wedge via native chemical ligation, while a reporter group was conjugated at the focal point. In between the ligands and the core scaffold, poly(ethylene glycol) units were used of which the length can be tuned to match inter-receptor distances. Peptides with different sequences and a variety in molecular weights were efficiently conjugated and combined with several reporter groups and spacers in order to generate a toolbox for the development of smart biomaterials. The arithmetic control over the degree of branching allowed a systematic study on the strength of multivalent interactions. In addition, our dendrons were shown to be applicable as core scaffolds for the synthesis of well-defined structures with higher valency numbers. In collaboration with the Leiden University Medical Center nonavalent peptides based on the AB3 polyamide core were prepared and evaluated for use in tumor vaccination. Although native chemical ligation proved to be very efficient for the introduction of linear peptides, the presence of thiol catalysts excludes the use of disulphide containing peptides or folded proteins. Thereto, oxime chemistry was explored in chapter 3 as an alternative and complementary bioconjugation method. Besides the fact that no redox chemistry is involved, oxime ligations use readily available starting materials and provide fast reaction kinetics. We developed variants of the original dendritic wedges reported in chapter 2 by replacing the cysteines at the periphery with aminooxy groups. Linear peptides functionalized with a glyoxyl moiety were efficiently conjugated to the dendrons thereby forming stable multivalent constructs. The introduction of folded proteins proved to be possible as well, although the conversion of the ligation seemed to be limited by steric hindrance. In a final example, native chemical and oxime ligation were combined for the development of peptide-protein hybrids. One copy of a recombinantly expressed protein with a C-terminal thioester was successfully conjugated to a cysteine present at the focal point of an AB3 wedge, while three peptides were subsequently introduced to the aminooxy-functionalized periphery. In collaboration with the University of California in Santa Barbara the tumor homing behavior of our aminooxy-functionalized dendrons was analyzed after modification with peptides developed via in vivo phage display. In the first part of chapter 4 the modification of the AB5 dendritic wedge with a short linear peptide that homes to clotted plasma proteins is described. Although in vivo experiments in mice showed that a specific receptor in tumor tissue was recognized, the extravasation was limited by the size of the construct. In contrast, a highly positively charged cyclic peptide with cell penetrating properties was capable of directing the entire dendritic architecture towards a specific receptor in tumor lymphatics. These observations are in agreement with results previously reported for micelles and nanoparticles and emphasize the influence of peptide properties and overall size on the biodistribution of multivalent macromolecules. Whether there is a beneficial effect originating from the multivalent display of the peptides still remains to be determined as the in vivo results proved to be highly dependent on factors such as tissue penetration, blood clearance rate and cellular uptake. The use of oxime chemistry as an efficient bioconjugation method was further demonstrated by the site-selective immobilization of biomolecules onto surfaces. The modification of carboxylate-functionalized surface plasmon resonance chips with an aminooxy-functionalized linker is discussed in chapter 5. Several peptides and proteins were oxidized under mild conditions using either NaIO4 or pyridoxal 5’-phosphate and introduced via their N-terminus to the surfaces. The immobilized ligands were fully active towards binding partners and the observed affinities agreed with previous measurements using independent techniques. An additional attractive property of our method is that the surface coverage is highly controllable and can be increased step-wise by performing sequential injections. This allowed us to consider the influence of protein density on the binding affinity and kinetics for multivalent ligands. Bivalent ligands connected via a long and flexible linker bound to the immobilized receptors via multivalent interactions, regardless of surface coverage. In contrast, for bivalent compounds coupled via a short and rigid linker a dramatic decrease in dissociation rate was found with increasing density. In chapter 6 the natural bivalent structure of immunoglobins was applied in a novel targeting concept. The use of antibodies directed against surface markers up-regulated in disease cells is a promising strategy for the treatment of cancer. However, this approach does not always give satisfactory results as the presence of receptors in healthy cells can cause severe side effects. We designed a generic targeting concept based on proteolytic activation of blocked antibodies in order to increase their tissue specificity. The synthesis of these constructs by covalent conjugation of epitopes at a position close to the antigen binding site was not efficient enough to produce fully blocked antibodies. In contrast, a non-covalent approach in which the antibody was blocked by effective bivalent epitopes turned out to be successful. The use of double stranded DNA as a rigid linker was investigated because of its long persistence length and the possibility to easily tune the distance by the number of base pairs. Peptide-DNA conjugates were successfully synthesized using the selective reaction between a thiol and a maleimide group and the formation of stable cyclic monomers after association with antibodies could be demonstrated using size-exclusion chromatography.