Platform for Orthogonal N-Cysteine-Specific Protein Modification Enabled by Cyclopropenone Reagents.

Protein conjugates are valuable tools for studying biological processes or producing therapeutics, such as antibody-drug conjugates. Despite the development of several protein conjugation strategies in recent years, the ability to modify one specific amino acid residue on a protein in the presence of other reactive side chains remains a challenge. We show that monosubstituted cyclopropenone (CPO) reagents react selectively with the 1,2-aminothiol groups of N-terminal cysteine residues to give a stable 1,4-thiazepan-5-one linkage under mild, biocompatible conditions. The CPO-based reagents, all accessible from a common activated ester CPO-pentafluorophenol (CPO-PFP), allow selective modification of N-terminal cysteine-containing peptides and proteins even in the presence of internal, solvent-exposed cysteine residues. This approach enabled the preparation of a dual protein conjugate of 2×cys-GFP, containing both internal and N-terminal cysteine residues, by first modifying the N-terminal residue with a CPO-based reagent followed by modification of the internal cysteine with a traditional cysteine-modifying reagent. CPO-based reagents enabled a copper-free click reaction between two proteins, producing a dimer of a de novo protein mimic of IL2 that binds to the ß-IL2 receptor with low nanomolar affinity. Importantly, the reagents are compatible with the common reducing agent dithiothreitol (DTT), a useful property for working with proteins prone to dimerization. Finally, quantum mechanical calculations uncover the origin of selectivity for CPO-based reagents for N-terminal cysteine residues. The ability to distinguish and specifically target N-terminal cysteine residues on proteins facilitates the construction of elaborate multilabeled bioconjugates with minimal protein engineering.

Affinity Selections of DNA-Encoded Chemical Libraries on Carbonic Anhydrase IX-Expressing Tumor Cells Reveal a Dependence on Ligand Valence.

DNA-encoded chemical libraries are typically screened against purified protein targets. Recently, cell-based selections with encoded chemical libraries have been described, commonly revealing suboptimal performance due to insufficient recovery of binding molecules. We used carbonic anhydrase IX (CAIX)-expressing tumor cells as a model system to optimize selection procedures with code-specific quantitative polymerase chain reaction (qPCR) as selection readout. Salt concentration and performing PCR on cell suspension had the biggest impact on selection performance, leading to 15-fold enrichment factors for high-affinity monovalent CAIX binders (acetazolamide; KD =8.7 nM). Surprisingly, the homobivalent display of acetazolamide at the extremities of both complementary DNA strands led to a substantial improvement of both ligand recovery and enrichment factors (above 100-fold). The optimized procedures were used for selections with a DNA-encoded chemical library comprising 1 million members against tumor cell lines expressing CAIX, leading to a preferential recovery of known and new ligands against this validated tumor-associated target. This work may facilitate future affinity selections on cells against target proteins which might be difficult to express otherwise.

Targeting an engineered cytokine with interleukin-2 and interleukin-15 activity to the neovasculature of solid tumors.

There is a growing interest in the antibody-based delivery of cytokines to the tumor environment as a means to boost the anti-cancer activity of tumor-resident T cells and NK cells. Here, we describe the expression and characterization of fusion proteins, featuring the L19 antibody (specific to the alternatively-spliced EDB domain of fibronectin) and an engineered cytokine with interleukin-2 and interleukin-15 properties. The cytokine moiety was fused either at the N-terminal or at the C-terminal extremity and both fusion proteins showed a selective tumor accumulation in a quantitative biodistribution experiment. The N-terminal fusion inhibited tumor growth in immunocompetent mice bearing F9 carcinomas or WEHI-164 sarcomas when used as single agent. The anticancer activity was compared to the one of the same cytokine payload used as recombinant protein or fused to an anti-hen egg lysozyme antibody, serving as negative control of irrelevant specificity in the mouse. These results indicate that the antibody-based delivery of engineered cytokines to the tumor neovasculature may mediate a potent anticancer activity.

Introduction of an Aldehyde Handle on Nanobodies by Affinity-Guided Labeling.

Chemically modified antigen-binding proteins are widely applied for their targeting abilities in the fields of biotechnology, medicine, and diagnostics. However, the production of site-selectively modified proteins remains a challenge. Here, we have designed a chemical probe for the introduction of a reactive aldehyde on nanobodies by metal-complex-guided conjugation. The probe design allows for purification of the conjugates, and the aldehyde constitutes an efficient handle for further modification of the nanobodies. In vitro experiments confirmed the binding activity and selectivity of fluorescent conjugates toward the native antigen. Furthermore, the modification strategy allowed for production of a nanobody-drug conjugate that was active in vitro.

Aptamer-Directed Conjugation of DNA to Therapeutic Antibodies.

A method for aptamer directed conjugation of DNA to therapeutic antibodies has been developed. In the method, an antibody selective aptamer binds to a specific site in the constant domain of human IgG1 antibodies and is used for both templated and direct conjugation to the antibodies. Through optimization of the design and reaction conditions, the antibody-DNA conjugates could be produced efficiently using equal stoichiometry of protein and DNA. Three different antibodies were evaluated, and the conjugates were characterized by anion exchange chromatography and SDS-PAGE. The conjugation sites for one of the antibodies were determined by MS/MS analysis of the digested conjugate. The antibody-DNA conjugate was also tested for receptor binding on cell surfaces showing retained binding.

Considerations on Probe Design for Affinity-Guided Protein Conjugation.

The plethora of methods developed for the creation of protein conjugates often differs significantly with regard to the heterogeneity of the resulting products, in the degree of genetic manipulation of the protein required, and in the technical skills required to perform the conjugation procedure. Affinity-guided protein conjugation is a protein labeling methodology based on noncovalent binding interactions between a labeling probe and the protein of interest. These interactions increase the local concentration of a reactive group in the probe on the protein surface thus facilitating the conjugation in proximity of the complexation site. The ability to produce high-quality conjugates from nongenetically modified proteins both in vitro, but also in cells, demonstrates the power of affinity-guided protein conjugation. Here, we present the progress of affinity-guided protein conjugation in relation to selective protein labeling in living systems and the formation of high-quality protein conjugates. Furthermore, the probe design will be discussed in relation to the utility of the probe for labeling in vitro or in living systems.

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.

Affinity-Guided Conjugation to Antibodies for Use in Positron Emission Tomography.

The radionuclide copper-64 is widely used in combination with biomolecules, such as antibodies, for positron emission tomography (PET). Copper-64 is ideal for the imaging of biomolecules with long circulation times due to its relatively long half-life, and when conjugated to an antibody, specific cells can be targeted in vivo. Here, we have prepared a trastuzumab-chelator conjugate by using affinity-guided conjugation, in which an azide was attached to the antibody prior to a strain promoted azide-alkyne cycloaddition reaction with DBCO-PEG4-NOTA. The conjugate was benchmarked against a standard nonspecific labeled trastuzumab-NOTA conjugate. The conjugates were tested for incorporation of copper-64, stability in buffer and plasma, and tumor targeting in vivo using PET imaging of mice with xenograft tumors expressing HER2. Both conjugates showed good incorporation of copper-64 and a high stability with less than 10% degradation after 36 h. Furthermore, both conjugates showed accumulation at the tumor site with mean uptake of 7.2 ± 2.4%ID/g and 5.2 ± 1.3%ID/g after 40 h for the affinity-guided labeled trastuzumab and the nonspecific labeled trastuzumab, respectively.

Small-Molecule Probes for Affinity-Guided Introduction of Biocompatible Handles on Metal-Binding Proteins.

Protein conjugates of high heterogeneity may contain species with significantly different biological properties, and as a consequence, the focus on methods for production of conjugates of higher quality has increased. Here, we demonstrate an efficient and generic approach for the modification of metal-binding proteins with biocompatible chemical handles without the need for genetic modifications. Affinity-guided small-molecule probes are developed for direct conjugation to off-the-shelf proteins and for installing different chemical handles on the protein surface. While purification of protein conjugates obtained by small molecule conjugation is troublesome, the affinity motifs of the probes presented here allow for purification of the conjugates. The versatility of the probes is demonstrated by conjugation to several His-tagged and natural metal-binding proteins, including the efficient and area-selective conjugation to three therapeutically relevant antibodies.

DNA-Templated Introduction of an Aldehyde Handle in Proteins.

Many medical and biotechnological applications rely on protein labeling, but a key challenge is the production of homogeneous and site-specific conjugates. This can rarely be achieved by simple residue-specific random labeling, but generally requires genetic engineering. Using site-selective DNA-templated reductive amination, we created DNA-protein conjugates with control over labeling stoichiometry and without genetic engineering. A guiding DNA strand with a metal-binding functionality facilitates site-selectivity by directing the coupling of a second reactive DNA strand in the vicinity of a protein metal-binding site. We demonstrate DNA-templated reductive amination for His6 -tagged proteins and metal-binding proteins, including IgG1 antibodies. We also used a cleavable linker between the DNA and the protein to remove the DNA and introduce a single aldehyde on the protein. This functions as a handle for further modifications with desired labels. In addition to directing the aldehyde positioning, the DNA provides a straightforward route for purification between reaction steps.

Intracellular Delivery of a Planar DNA Origami Structure by the Transferrin-Receptor Internalization Pathway.

DNA origami provides rapid access to easily functionalized, nanometer-sized structures making it an intriguing platform for the development of defined drug delivery and sensor systems. Low cellular uptake of DNA nanostructures is a major obstacle in the development of DNA-based delivery platforms. Herein, significant strong increase in cellular uptake in an established cancer cell line by modifying a planar DNA origami structure with the iron transport protein transferrin (Tf) is demonstrated. A variable number of Tf molecules are coupled to the origami structure using a DNA-directed, site-selective labeling technique to retain ligand functionality. A combination of confocal fluorescence microscopy and quantitative (qPCR) techniques shows up to 22-fold increased cytoplasmic uptake compared to unmodified structures and with an efficiency that correlates to the number of transferrin molecules on the origami surface.