Sequence-defined antibody-recruiting macromolecules

Abstract

Antibody-recruiting molecules represent a novel class of therapeutic agents that mediate the recruitment of endogenous antibodies to target cells, leading to their elimination by the immune system. Compared to single-ligand copies, macromolecular scaffolds presenting multiple copies of an antibody-binding ligand offer advantages in terms of increased complex avidity. In this study, we describe the synthesis of sequence-defined macromolecules designed for antibody recruitment, utilising dinitrophenol (DNP) as a model antibody-recruiting motif. The use of discrete macromolecules gives access to varying the spacing between DNP motifs while maintaining the same chain length. This characteristic enables the investigation of structure-dependent binding interactions with anti-DNP antibodies. Through solid-phase thiolactone chemistry, we synthesised a series of oligomers with precisely localised DNP motifs along the backbone and a terminal biotin motif for surface immobilisation. Utilising biolayer interferometry analysis, we observed that oligomers with adjacent DNP motifs exhibited enhanced avidity for anti-DNP antibodies. Molecular modelling provided insights into the structures and dynamics of the various macromolecules, shedding light on the accessibility of the ligands to the antibodies. Overall, our findings highlight that the use of sequence-defined macromolecules can contribute to our understanding of structure-activity relationships and provide insights for the design of novel antibody-recruiting therapeutic agents.

Scheme 1. Iterative solid-phase synthesis of sequence-defined antibody recruiting macromolecules bearing DNP and non-functional monomers at different position along the backbone, as well as a biotin unit at one extremity (blue, pink and yellow balls, respectively): (i) aminolysis of the thiolactone ring with ethanolamine and nucleophilic aromatic substitution with DNP-Cl; (ii) aminolysis of the thiolactone ring with ethanolamine and thiol-Michael reaction using either methyl- or 1-ethoxyethyl- acrylate; (iii) chain extension with an isocyanate-functionalised thiolactone (TLa-NCO); (iv) addition of biotin as a terminal unit; (v) cleavage from the Rink amide support and simultaneous deprotection of the acetal unit when using the acetal-containing 1-ethoxyethyl acrylate (M2–M5). The dashed box schematizes the proposed concept of using sequence-defined macromolecules in the area of antibody recruiting.

Fig. 1. The obtained BLI results for oligomer M1 with a methyl ester (left), displaying a significantly lower binding affinity compared to M2 with a carboxylic acid (right) side group (see ESI for the synthesis procedure).

Fig. 2. The final structure of three unique heptamers (same exact mass = 2528 Da) synthesised using an optimised procedure with DNP-Cl and 1-EA. Note the increase in spacing between the DNP units from M3 to M5 (see Fig. S3–S5 and ESI for their synthesis).

Fig. 3. BLI sensorgrams of anti-DNP antibody binding to oligomers M3–M5, displaying the highest binding affinity for M3.

Fig. 4. (a) Snapshot of the final MD frame (water molecules not shown for clarity). The DNP ligands are coloured in blue, the carboxylate residues in dark pink and the biotin moieties in gold. The grey spheres are located at the geometric centre of the aromatic cycle of the DNPs. The blue dashed lines measure the distances between the DNP with the labelled value in Å. The blue value inside the triangle represents the area of the triangle in Ų. (b) Box-and-whisker plot of the area between DNPs for the 3 oligomers. The first line of the box represents the first quartile (Q1), the second represents the median (Q2) and the third represents the third quartile (Q3). The small diamonds indicate the outliers, i.e. data values below Q1 − 1.5 × IQR or above Q3 + 1.5 × IQR (c) box-and-whisker plot of the SASA of each DNP for the 3 oligomers.