The emergence of escape variants of SARS-CoV-2 carrying mutations in the spike protein poses a challenge for therapeutic antibodies. Here, we show that through the comprehensive engineering of the variable region of the neutralizing monoclonal antibody 5A6, the engineered antibody, 5A6CCS1, is able to neutralize SARS-CoV-2 variants that escaped neutralization by the original 5A6 antibody. In addition to the improved affinity against variants, 5A6CCS1 was also optimized to achieve high solubility and low viscosity, enabling a high concentration formulation for subcutaneous injection. In cynomolgus monkeys, 5A6CCS1 showed a long plasma half-life and good subcutaneous bioavailability through engineering of the variable and constant region. These data demonstrate that 5A6CCS1 is a promising antibody for development against SARS-CoV-2 and highlight the importance of antibody engineering as a potential method to counteract escape variants.

To combat infectious diseases, vaccines are considered the best prophylactic strategy for a wide range of the population, but even when vaccines are effective, the administration of therapeutic antibodies against viruses could provide further treatment options, particularly for vulnerable groups whose immunity against the viruses is compromised. Therapeutic antibodies against dengue are ideally engineered to abrogate binding to Fcγ receptors (FcγRs), which can induce antibody-dependent enhancement (ADE). However, the Fc effector functions of neutralizing antibodies against SARS-CoV-2 have recently been reported to improve post-exposure therapy, while they are dispensable when administered as prophylaxis. Hence, in this report, we investigated the influence of Fc engineering on anti-virus efficacy using the anti-dengue/Zika human antibody SIgN-3C and found it affected the viremia clearance efficacy against dengue in a mouse model. Furthermore, we demonstrated that complement activation through antibody binding to C1q could play a role in anti-dengue efficacy. We also generated a novel Fc variant, which displayed the ability for complement activation but showed very low FcγR binding and an undetectable level of the risk of ADE in a cell-based assay. This Fc engineering approach could make effective and safe anti-virus antibodies against dengue, Zika and other viruses.

Antibody drugs blocking the complement classical pathway (CP) often require frequent intravenous administration to overcome the high abundance or rapid turnover of complement proteins. This makes treatment compliance burdensome for patients. Screening was performed to identify anti-C1s antibodies specific for the CUB domain of C1s with CP neutralising function. Antibody engineering was performed on the anti-C1s antibody to prolong its half-life in circulation. The variable region was mutated to confer pH-dependent binding to C1s, and the antibody Fc was modified to lower its isoelectric point and to have enhanced binding to the neonatal Fc receptor. A combination of pH-dependent binding to C1s and optimisation of charge characteristics was required for the final engineered antibody, RAY121, to achieve a long half-life in circulation and long-lasting neutralisation of the CP. In male cynomolgus monkeys, a single subcutaneous injection suppressed CP activity for more than a month. RAY121 neutralises the CP by using a unique mechanism of displacing C1q from the C1 complex and blocking its reassembly. Structure analysis by cryo-electron microscopy revealed that the RAY121 epitope on C1s is distinct from the C1q binding site, and that C1q displacement is mediated by the Fab arm sterically obstructing C1q binding to C1s. RAY121 is able to neutralise the CP for an extended duration and is effective at doses that can be administered subcutaneously for convenient treatment. These data present RAY121 as a promising agent for the treatment of CP-driven autoimmune disorders. Chugai Pharmaceutical Co. Ltd.