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The advent of biologic therapies, particularly antibody therapeutics, has revolutionized the pharmacological treatment of many rheumatic diseases. Antibody discovery began with the immunization of mice for the production of rodent immunoglobulins, but advances in protein and genetic engineering have now made it possible to generate fully human antibodies, which are better tolerated by patients. For most clinical applications in rheumatology, antibodies have been used as blocking agents capable of neutralizing the function of pro-inflammatory proteins, such as TNF. The latest strategies involve antibody products armed with effector moieties, such as anti-inflammatory drugs or cytokines, or antibody products that are specific for multiple targets for the selective inhibition of inflammation at sites of disease. Antibodies are some of the best-selling drugs in the world, and with further advances in antibody development, engineering of armed antibodies and bispecific products will have an important role in the treatment of rheumatic diseases.Antibody therapeutics are a mainstay of treating patients with rheumatic diseases but are mostly basic monoclonal structures. What can the history of and modern developments in antibody technology tell us about the next generation of antibody-based therapeutics?

Engineered cytokine products represent a growing class of therapeutic proteins which need to be tested for biological activity at various stages of pharmaceutical development. In most cases, dedicated biological assays are established for different products, in a process that can be time-consuming and cumbersome. Here we describe the development and implementation of a universal cell-based reporter system for various classes of immunomodulatory proteins. The novel system capitalizes on the fact that the signaling of various types of pro-inflammatory agents (e.g., cytokines, chemokines, Toll-like receptor agonists) may involve transcriptional activation by NF-κB. Using viral transduction, we generated stably-transformed cell lines of B or T lymphocyte origin and compared the new reporter cell lines with conventional bioassays. The experimental findings with various interleukins and with members of the TNF superfamily revealed that the newly-developed “universal” bioassay method yielded bioactivity data which were comparable to the ones obtained with dedicated conventional methods. The engineered cell lines with reporters for NF-κB were tested with several antibody-cytokine fusions and may be generally useful for the characterization of novel immunomodulatory products. The newly developed methodology also revealed a mechanism for cytokine potentiation, based on the antibody-mediated clustering of TNF superfamily members on tumor-associated extracellular matrix components.

The CRISPR-Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision. The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via error-prone end-joining pathways rather than via homology-directed repair mechanisms, however, leads to relatively low rates of precise editing from donor DNA. Here we show that spatial and temporal co-localization of the donor template and Cas9 via covalent linkage increases the correction rates up to 24-fold, and demonstrate that the effect is mainly caused by an increase of donor template concentration in the nucleus. Enhanced correction rates were observed in multiple cell types and on different genomic loci, suggesting that covalently linking the donor template to the Cas9 complex provides advantages for clinical applications where high-fidelity repair is desired. Genome editing allows scientists to change an organism’s genetic information by adding, replacing or removing sections of its DNA sequence. The CRISPR-Cas9 system is a genome-editing tool that has had a large impact on biological research in recent years, and also shows promise for the treatment of patients with genetic disorders. The tool works as follows: a small piece of RNA (a close cousin to DNA) is used to guide an enzyme called the Cas9 endonuclease to the desired region of the genome. Then, like a pair of molecular scissors, the enzyme cuts the DNA, breaking both strands of its double helix. The cell naturally starts to repair the damaged DNA, and one way to do this is to use another similar piece of intact DNA as a template. Scientists can exploit this repair mechanism (known as homology-directed repair) by giving the cell extra DNA that carries their desired sequence change, with the hope that the cell will use it as a template and edit its own genome in precisely the same way. However, it turns out that mammalian cells rarely use the template DNA to repair the damage. Instead, mammals tend to fix double-stranded breaks in DNA by simply joining the broken ends together, a method that is prone to errors. To overcome this specific issue, Savic, Ringnalda et al. tested the effect of physically linking the template DNA to the Cas9 enzyme, so that the DNA was already nearby when the enzyme made the cut. Experiments with human cells confirmed that this new approach increased the frequency of homology-directed repair up to 24-fold compared to leaving the enzyme and the template DNA separate. Improving the CRISPR-Cas9 system in this manner makes it more likely that genome editing may one day become a routine treatment for patients with genetic disorders. But first, more preclinical studies are needed to assess the safety of the CRISPR-Cas9 technology for gene editing in patients.

In nature, specific antibodies can be generated as a result of an adaptive selection and expansion of lymphocytes with suitable protein binding properties. We attempted to mimic antibody–antigen recognition by displaying multiple chemical diversity elements on a defined macrocyclic scaffold. Encoding of the displayed combinations was achieved using distinctive DNA tags, resulting in a library size of 35,393,112. Specific binders could be isolated against a variety of proteins, including carbonic anhydrase IX, horseradish peroxidase, tankyrase 1, human serum albumin, alpha-1 acid glycoprotein, calmodulin, prostate-specific antigen and tumour necrosis factor. Similar to antibodies, the encoded display of multiple chemical elements on a constant scaffold enabled practical applications, such as fluorescence microscopy procedures or the selective in vivo delivery of payloads to tumours. Furthermore, the versatile structure of the scaffold facilitated the generation of protein-specific chemical probes, as illustrated by photo-crosslinking.

We describe the development of OncoFAP, an ultra-high-affinity ligand of fibroblast activation protein (FAP) for targeting applications with pan-tumoral potential. OncoFAP binds to human FAP with affinity in the subnanomolar concentration range and cross-reacts with the murine isoform of the protein. We generated various fluorescent and radiolabeled derivatives of OncoFAP in order to study biodistribution properties and tumor-targeting performance in preclinical models. Fluorescent derivatives selectively localized in FAP-positive tumors implanted in nude mice with a rapid and homogeneous penetration within the neoplastic tissue. Quantitative in vivo biodistribution studies with a lutetium-177–labeled derivative of OncoFAP revealed a preferential localization in tumors at doses of up to 1,000 nmol/kg. More than 30% of the injected dose had already accumulated in 1 g of tumor 10 min after intravenous injection and persisted for at least 3 h with excellent tumor-to-organ ratios. OncoFAP also served as a modular component for the generation of nonradioactive therapeutic products. A fluorescein conjugate mediated a potent and FAP-dependent tumor cell killing activity in combination with chimeric antigen receptor (CAR) T cells specific to fluorescein. Similarly, a conjugate of OncoFAP with the monomethyl auristatin E-based Vedotin payload was well tolerated and cured tumor-bearing mice in combination with a clinical-stage antibody-interleukin-2 fusion. Collectively, these data support the development of OncoFAP-based products for tumor-targeting applications in patients with cancer.

In contrast to standard fragment-based drug discovery approaches, dual-display DNA-encoded chemical libraries have the potential to identify fragment pairs that bind simultaneously and benefit from the chelate effect. However, the technology has been limited by the difficulty in unambiguously decoding the ligand pairs from large combinatorial libraries. Here we report a strategy that overcomes this limitation and enables the efficient identification of ligand pairs that bind to a target protein. Small organic molecules were conjugated to the 5′ and 3′ ends of complementary DNA strands that contain a unique identifying code. DNA hybridization followed by an inter-strand code-transfer created a stable dual-display DNA-encoded chemical library of 111,100 members. Using this approach we report the discovery of a low micromolar binder to alpha-1-acid glycoprotein and the affinity maturation of a ligand to carbonic anhydrase IX, an established marker of renal cell carcinoma. The newly discovered subnanomolar carbonic anhydrase IX binder dramatically improved tumour targeting performance in vivo . A method to identify pairs of ligands that simultaneously bind to a target protein has been developed. The method uses two DNA-encoded chemical sub-libraries that self-assemble to form stable dual-display structures, and an encoding system that can be decoded by DNA sequencing and enables both ligands to be identified.

Antibody–cytokine fusion proteins (immunocytokines) are innovative biopharmaceutical agents, which are being considered for the therapy of cancer and chronic inflammatory conditions. Immunomodulatory fusion proteins capable of selective localization at the sites of rheumatoid arthritis (RA) are of particular interest, as they may increase the therapeutic index of the cytokine payload. The F8 antibody recognizes the alternatively spliced extra domain A of fibronectin, a marker of angiogenesis, which is strongly overexpressed at sites of arthritis. In this study, we investigated the targeting and therapeutic activity of the immunocytokine F8-IL4 in the mouse model of collagen-induced arthritis. Different combination regimes were tested and evaluated by the analysis of serum and tissue cytokine levels. We show that F8-IL4 selectively localizes to neovascular structures at sites of rheumatoid arthritis in the mouse, leading to high local concentrations of IL4. When used in combination with dexamethasone, F8-IL4 was able to cure mice with established collagen-induced arthritis. Response to treatment was associated with an elevation of IL13 levels and decreased IL6 plasma concentrations. A fully human version of F8-IL4 is currently being developed for clinical investigations.

A strategy for creating drugs that can be quickly neutralized is demonstrated for anticoagulants.

The encoding of chemical compounds with amplifiable DNA tags facilitates the discovery of small-molecule ligands for proteins. To investigate the impact of stereo- and regiochemistry on ligand discovery, we synthesized a DNA-encoded library of 670,752 derivatives based on 2-azido-3-iodophenylpropionic acids. The library was selected against multiple proteins and yielded specific ligands. The selection fingerprints obtained for a set of protein targets of pharmaceutical relevance clearly showed the preferential enrichment of ortho-, meta- or para-regioisomers, which was experimentally verified by affinity measurements in the absence of DNA. The discovered ligands included novel selective enzyme inhibitors and binders to tumour-associated antigens, which enabled conditional chimeric antigen receptor T-cell activation and tumour targeting.A DNA-encoded chemical library based on regio- and stereoisomers of phenylalanine has been synthesized and used for affinity-based selections against multiple target proteins. This approach led to the isolation and validation of potent ligands capable of CAR T-cell activation and tumour targeting.

A strategy for creating drugs that can be quickly neutralized is demonstrated for anticoagulants.