Globally accessible preventive and therapeutic molecules against SARS-CoV-2 are urgently needed. DARPin molecules are an emerging class of novel therapeutics based on naturally occurring repeat proteins (∼15 kDa in size) and can be rapidly produced in bacteria in large quantities. Here, we report the identification of 380 DARPin molecules specifically targeting the SARS-CoV-2 spike protein selected from a naïve library of 1012 DARPin molecules. Using extensive biophysical and biochemical characterization, (pseudo)virus neutralization assays and cryo-EM analysis, 11 mono-DARPin molecules targeting either the receptor binding domain (RBD), the S1 N-terminal-domain (NTD) or the S2 domain of the SARS-CoV-2 spike protein were chosen. Based on these 11 mono-DARPin molecules, 31 anti-SARS-CoV-2 multi-DARPin molecules were constructed which can broadly be grouped into 2 types; multi-paratopic RBD-neutralizing DARPin molecules and multi-mode DARPin molecules targeting simultaneously RBD, NTD and the S2 domain. Each of these multi-DARPin molecules acts by binding with 3 DARPin modules to the SARS-CoV-2 spike protein, leading to potent inhibition of SARS-CoV-2 infection down to 1 ng/ml (12 pM) and potentially providing protection against viral escape mutations. Additionally, 2 DARPin modules binding serum albumin, conferring an expected half-life of about 3 weeks in humans, were included in the multi-DARPin molecules. The protective efficacy of one multi-DARPin molecule was studied in a Golden Syrian hamster SARS-CoV-2 infection model, resulting in a significant reduction in viral load and pathogenesis. In conclusion, the multi-DARPin molecules reported here display very high antiviral potency, high-production yield, and a long systemic half-life, and thereby have the potential for single-dose use for prevention and treatment of COVID-19.
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with potential resistance to existing drugs emphasizes the need for new therapeutic modalities with broad variant activity. Here we show that ensovibep, a trispecific DARPin (designed ankyrin repeat protein) clinical candidate, can engage the three units of the spike protein trimer of SARS-CoV-2 and inhibit ACE2 binding with high potency, as revealed by cryo-electron microscopy analysis. The cooperative binding together with the complementarity of the three DARPin modules enable ensovibep to inhibit frequent SARS-CoV-2 variants, including Omicron sublineages BA.1 and BA.2. In Roborovski dwarf hamsters infected with SARS-CoV-2, ensovibep reduced fatality similarly to a standard-of-care monoclonal antibody (mAb) cocktail. When used as a single agent in viral passaging experiments in vitro, ensovibep reduced the emergence of escape mutations in a similar fashion to the same mAb cocktail. These results support further clinical evaluation of ensovibep as a broad variant alternative to existing targeted therapies for Coronavirus Disease 2019 (COVID-19).
Proof of Lemma 1. Note that (16) can be written asBy the Moreau decomposition (Parikh et al., 2014), we havewhere h ⇤ denotes the convex conjugate of h. We want to derive a similar identity for ⌧ h, ⌧ > 0. The convex conjugate of ⌧ h is
e14626 Background: Urelumab (BMS-663513) is a humanized monoclonal antibody binding to CD137 which, upon Fc-clustering, leads to activation of T-cells. Urelumab is currently in Phase 2 clinical development and has been reported to cause significant hepatotoxicities (around 15% Grade ≥2 ALT and AST elevation) when given as infusion every 3 weeks at doses ≥0.3 mg/kg. Currently ongoing clinical trials report decreased systemic toxicity but limited efficacy at lower doses of urelumab. We hypothesized that more effective triggering of CD137 without associated systemic toxicity may be achieved by targeting a CD137 agonistic engager without Fc to fibroblast activation protein (FAP) which is abundantly expressed in the stroma of many solid tumors. To achieve this, a targeted molecule belonging to the DARPin family of binding proteins was composed of one FAP- and two CD137-binding domains in a “beads on a string” format and tested in a mouse model with human PBMCs. Methods: Human PBMCs were used to reconstitute the immune system in NOG mice implanted subcutaneously with HT-29 human colon cancer cells. Mice were monitored for survival, body weight, and tumor size during the treatment phase of two weeks. Results: None of the mice in the control group died and no significant body weight loss was observed. Six of ten (60%) mice in the CD137 antibody group showed strong signs of graft vs. host disease and either died or reached the termination criterion of ≥20% body weight loss and were sacrificed. One of 30 (3%) mice died in the DARPin drug candidate groups but none of the animals showed body weight loss of ≥20% (p < 0.001, Log-rank test). Tumor growth inhibition was comparable for all treatment groups (around 20-30% at Day 18, p < 0.05 vs. control, Mann Whitney Test). Conclusions: This study confirms the hypothesis that systemic toxicities caused by the urelumab mode of action can be circumvented by FAP-targeting of a CD137 agonistic DARPin drug candidate while achieving comparable tumor growth inhibition. Consequently, higher clinical doses of tumor stroma-targeted agonistic DARPin drug candidates might be possible, and may result in stronger tumor growth inhibition.
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