Uncontrolled self-association is a major challenge in the exploitation of proteins as therapeutics. Here we describe the development of a structural proteomics approach to identify the amino acids responsible for aberrant self-association of monoclonal antibodies and the design of a variant with reduced aggregation and increased serum persistence in vivo. We show that the human monoclonal antibody, MEDI1912, selected against nerve growth factor binds with picomolar affinity, but undergoes reversible self-association and has a poor pharmacokinetic profile in both rat and cynomolgus monkeys. Using hydrogen/deuterium exchange and cross-linking-mass spectrometry we map the residues responsible for self-association of MEDI1912 and show that disruption of the self-interaction interface by three mutations enhances its biophysical properties and serum persistence, whilst maintaining high affinity and potency. Immunohistochemistry suggests that this is achieved via reduction of non-specific tissue binding. The strategy developed represents a powerful and generic approach to improve the properties of therapeutic proteins.

One hallmark of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) is infiltration of leukocytes into the CNS, where chemokines and their receptors play a major mediatory role. CX3CR1 is a chemokine receptor involved in leukocyte adhesion and migration and hence a mediator of immune defense reactions. The role of CX3CR1 in MS and EAE pathogenesis however remains to be fully assessed. Here, we demonstrate CX3CR1 mRNA expression on inflammatory cells within active plaque areas in MS brain autopsies. To test whether blocking CNS infiltration of peripheral leukocytes expressing CX3CR1 would be a suitable treatment strategy for MS, we developed a selective, high-affinity inhibitor of CX3CR1 (AZD8797). The compound is active outside the CNS and AZD8797 treatment in Dark Agouti rats with myelin oligodendrocyte glycoprotein-induced EAE resulted in reduced paralysis, CNS pathology, and incidence of relapses. The compound is effective when starting treatment before onset, as well as after the acute phase. This treatment strategy is mechanistically similar to, but more restricted than, current very late antigen-4-directed approaches that have significant side effects. We suggest that blocking CX3CR1 on leukocytes outside the CNS could be an alternative approach to treat MS.fractalkine receptor | inflammation M ultiple sclerosis (MS) is a chronic inflammatory, demyelinating and degenerative disease of the central nervous system (CNS). It was already discovered in the early 1900s that a similar disease could be induced in different animal species by injection of spinal cord extracts or myelin-derived proteins (1-3). This group of animal models for MS, called experimental autoimmune encephalomyelitis (EAE), has provided an experimental platform for building an extensive understanding of the pathology of MS, as well as discovering strategies for intervention of the disease. A typical feature of the pathogenesis in both MS and EAE is the infiltration of leukocytes from the blood stream into the CNS (3). Leukocyte adhesion and extravasation includes several well defined steps and various adhesion molecules, chemokines and their receptors are important mediators for this process. In line with this, the recently developed therapeutic drugs natalizumab and fingolimod, which broadly target leukocyte migration to the brain, exhibit efficacy in EAE models (4, 5), and they are now established therapies in MS (6).Natalizumab, blocking the interaction between very late antigen-4 (VLA-4) and CD106 (VCAM-1), is an effective treatment both on clinical endpoints and MRI biomarkers (7). Fingolimod, the first oral drug for relapsing remitting MS (RRMS), acts on S1P receptors preventing lymphocytes from moving out of lymphoid tissue (8). Natalizumab is only approved as a second-line monotherapy in RRMS or in patients with very active disease, because it carries increased risk of developing the often fatal progressive multifocal leukoencephalopathy (7, 9). Treatment with fingolimod is associated with side effects such as...

The TrkA-PathHunter cell-based assay was used in high-throughput screening (HTS) to identify compounds that inhibit nerve growth factor (NGF)/TrkA signaling. The assay was conducted in a 384-well format, and typical Z' values during HTS ranged from 0.3 to 0.8. The reproducibility of IC50 values was good, and the use of cryopreserved cells was well tolerated, as judged by assay parameters such as Z' and S/B and by comparison of IC50 values obtained with cells in culture. During hit deconvolution, TrkA-kinase inhibitors were identified with ATP-competitive as well as non-ATP-competitive mechanisms of action. Furthermore, other mechanisms of action such as NGF and TrkA antagonists were also identified. Because of the different molecular mechanisms identified, it is possible that subsequent optimization work to increase affinity and selectivity might lead to compounds that could have a better chance to evoke clinical efficacy without the adverse effects observed for nonselective TrkA inhibitors.

More knowledge regarding persistence of antibody response to SARS-CoV-2 infections in the general population with mild symptoms is needed. We measured and compared levels of SARS-CoV-2 spike- and nucleocapsid-specific IgG-antibodies in serum samples from 145 laboratory-confirmed COVID-19 cases and 324 non-cases. The IgG-antibody levels against the spike protein in cases were stable over the time-period studied (14 to 256 days), while antibody levels against the nucleocapsid protein decreased over time.