Bioluminescent Antibodies for Point‐of‐Care Diagnostics

We introduce a general method to transform antibodies into ratiometric, bioluminescent sensor proteins for the no‐wash quantification of analytes. Our approach is based on the genetic fusion of antibody fragments to NanoLuc luciferase and SNAP‐tag, the latter being labeled with a synthetic fluorescent competitor of the antigen. Binding of the antigen, here synthetic drugs, by the sensor displaces the tethered fluorescent competitor from the antibody and disrupts bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore. The semisynthetic sensors display a tunable response range (submicromolar to submillimolar) and large dynamic range (ΔR _max>500 %), and they permit the quantification of analytes through spotting of the samples onto paper followed by analysis with a digital camera.     

Efficient Innate Immune Killing of Cancer Cells Triggered by Cell‐Surface Anchoring of Multivalent Antibody‐Recruiting Polymers

Binding of monoclonal antibodies (mAbs) onto a cell surface triggers antibody‐mediated effector killing by innate immune cells through complement activation. As an alternative to mAbs, synthetic systems that can recruit endogenous antibodies from the blood stream to a cancer cell surface could be of great relevance. Herein, we explore antibody‐recruiting polymers (ARPs) as a novel class of immunotherapy. ARPs consist of a cell‐binding motif linked to a polymer that contains multiple small molecule antibody‐binding motifs along its backbone. As a proof of concept, we employ a lipid anchor that inserts into the phospholipid cell membrane and make use of a polymeric activated ester scaffold onto which we substitute dinitrophenol as an antibody‐binding motif. We demonstrate that ARPs allow for high avidity antibody binding and drive antibody recruitment to treated cells for several days. Furthermore, we show that ARP‐treated cancer cells are prone to antibody‐mediated killing through phagocytosis by macrophages.     

Three‐Dimensional Ordered Antibody Arrays Through Self‐Assembly of Antibody–Polymer Conjugates

Three‐dimensional (3D) ordered arrays of human immunoglobulin G (IgG) were fabricated using well‐defined full‐length antibody–polymer conjugates (APCs). The conjugates were prepared through a two‐step sequential click approach with a combination of oxime ligation and strain promoted alkyne–azide cycloaddition. They were able to self‐assemble into lamellar nanostructures with alternating IgG and poly(N ‐isopropylacrylamide) (PNIPAM) nanodomains. As a proof‐of‐concept, these materials were fabricated into thin films and their specific binding ability was tested. The nanostructure not only improves the packing density and the proper orientation of the IgG, but also provides nanochannels to facilitate substrate transport.     

Peptide‐Directed DNA‐Templated Protein Labelling for The Assembly of a Pseudo‐IgM

The development of methods for conjugation of DNA to proteins is of high relevance for the integration of protein function and DNA structures. Here, we demonstrate that protein‐binding peptides can direct a DNA‐templated reaction, selectively furnishing DNA–protein conjugates with one DNA label. Quantitative conversion of oligonucleotides is achieved at low stoichiometries and the reaction can be performed in complex biological matrixes, such as cell lysates. Further, we have used a star‐like pentameric DNA nanostructure to assemble five DNA–Rituximab conjugates, made by our reported method, into a pseudo‐IgM antibody structure that was subsequently characterized by negative‐stain transmission electron microscopy (nsTEM) analysis.     

Multifunctional Antibody Agonists Targeting Glucagon‐like Peptide‐1, Glucagon,and Glucose‐Dependent Insulinotropic Polypeptide Receptors

Glucagon‐like peptide‐1 (GLP‐1) receptor (GLP‐1R), glucagon (GCG) receptor (GCGR), and glucose‐dependent insulinotropic polypeptide (GIP, also known as gastric inhibitory polypeptide) receptor (GIPR), are three metabolically related peptide hormone receptors. A novel approach to the generation of multifunctional antibody agonists that activate these receptors has been developed. Native or engineered peptide agonists for GLP‐1R, GCGR, and GIPR were fused to the N‐terminus of the heavy chain or light chain of an antibody, either alone or in pairwise combinations. The fusion proteins have similar in vitro biological activities on the cognate receptors as the corresponding peptides, but circa 100‐fold longer plasma half‐lives. The GLP‐1R mono agonist and GLP‐1R/GCGR dual agonist antibodies both exhibit potent effects on glucose control and body weight reduction in mice, with the dual agonist antibody showing enhanced activity in the latter.