B.1.1.529 escapes the majority of SARS-CoV-2 neutralizing antibodies of diverse epitopes

The SARS-CoV-2 B.1.1.529 variant (Omicron) contains 15 mutations on the receptor-binding domain (RBD). How Omicron would evade RBD neutralizing antibodies (NAbs) and humoral immunity requires immediate investigation. Here, we used high-throughput yeast display screening to determine the RBD escaping mutation profiles for 247 human anti-RBD NAbs identified from SARS-CoV/SARS-CoV-2 convalescents and vaccinees. Based on the results, NAbs could be unsupervised clustered into six epitope groups (A-F), which is highly concordant with knowledge-based structural classifications. Strikingly, various single mutations of Omicron could impair NAbs of different epitope groups. Specifically, NAbs in Group A-D, whose epitope overlaps with ACE2-binding motif, are largely escaped by K417N, N440K, G446S, E484A, Q493K, and G496S. Group E (S309 site) and F (CR3022 site) NAbs, which often exhibit broad sarbecovirus neutralizing activity, are less affected by Omicron, but still, a subset of NAbs are escaped by G339D, S371L, and S375F. Furthermore, B.1.1.529 pseudovirus neutralization and RBD binding assay showed that single mutation tolerating NAbs could also be escaped due to multiple synergetic mutations on their epitopes. In total, over 85% of the tested NAbs are escaped by Omicron. Regarding NAb drugs, LY-CoV016/LY-CoV555 cocktail, REGN-CoV2 cocktail, AZD1061/AZD8895 cocktail, and BRII-196 were escaped by Omicron, while VIR7831 and DXP-604 still function at reduced efficacy. Together, data suggest Omicron could cause significant humoral immune evasion, while NAbs targeting the sarbecovirus conserved region remain most effective. Our results offer instructions for developing NAb drugs and vaccines against Omicron and future variants.

B.1.1.529 escapes the majority of SARS-CoV-2 neutralizing antibodies of diverse epitopes

The SARS-CoV-2 B.1.1.529 variant (Omicron) contains 15 mutations on the receptor-binding domain (RBD). How Omicron would evade RBD neutralizing antibodies (NAbs) and humoral immunity requires immediate investigation. Here, we used high-throughput yeast display screening1,2 to determine the RBD escaping mutation profiles for 247 human anti-RBD NAbs identified from SARS-CoV/SARS-CoV-2 convalescents and vaccinees. Based on the results, NAbs could be unsupervised clustered into six epitope groups (A-F), which is highly concordant with knowledge-based structural classifications3-5. Strikingly, various single mutations of Omicron could impair NAbs of different epitope groups. Specifically, NAbs in Group A-D, whose epitope overlaps with ACE2-binding motif, are largely escaped by K417N, N440K, G446S, E484A, Q493K, and G496S. Group E (S309 site)6 and F (CR3022 site)7 NAbs, which often exhibit broad sarbecovirus neutralizing activity, are less affected by Omicron, but still, a subset of NAbs are escaped by G339D, S371L, and S375F. Furthermore, B.1.1.529 pseudovirus neutralization and RBD binding assay showed that single mutation tolerating NAbs could also be escaped due to multiple synergetic mutations on their epitopes. In total, over 85% of the tested NAbs are escaped by Omicron. Regarding NAb drugs, LY-CoV016/LY-CoV555 cocktail, REGN-CoV2 cocktail, AZD1061/AZD8895 cocktail, and BRII-196 were escaped by Omicron, while VIR7831 and DXP-604 still function at reduced efficacy. Together, data suggest Omicron could cause significant humoral immune evasion, while NAbs targeting the sarbecovirus conserved region remain most effective. Our results offer instructions for developing NAb drugs and vaccines against Omicron and future variants.

Native llama Nanobody Library Panning Performed by Phage and Yeast Display Provides Binders Suitable for C-Reactive Protein Detection

C-reactive protein (CRP) is an inflammation biomarker that should be quantified accurately during infections and healing processes. Nanobodies are good candidates to replace conventional antibodies in immunodiagnostics due to their inexpensive production, simple engineering, and the possibility to obtain higher binder density on capture surfaces. Starting from the same pre-immune library, we compared the selection output resulting from two independent panning strategies, one exclusively exploiting the phage display and another in which a first round of phage display was followed by a second round of yeast display. There was a partial output convergence between the two methods, since two clones were identified using both panning protocols but the first provided several further different sequences, whereas the second favored the recovery of many copies of few clones. The isolated anti-CRP nanobodies had affinity in the low nanomolar range and were suitable for ELISA and immunoprecipitation. One of them was fused to SpyTag and exploited in combination with SpyCatcher as the immunocapture element to quantify CRP using electrochemical impedance spectroscopy. The sensitivity of the biosensor was calculated as low as 0.21 μg/mL.

Directed evolution of and structural insights into antibody-mediated disruption of a stable receptor-ligand complex

Antibody drugs exert therapeutic effects via a range of mechanisms, including competitive inhibition, allosteric modulation, and immune effector mechanisms. Facilitated dissociation is an additional mechanism where antibody-mediated “disruption” of stable high-affinity macromolecular complexes can potentially enhance therapeutic efficacy. However, this mechanism is not well understood or utilized therapeutically. Here, we investigate and engineer the weak disruptive activity of an existing therapeutic antibody, omalizumab, which targets IgE antibodies to block the allergic response. We develop a yeast display approach to select for and engineer antibody disruptive efficiency and generate potent omalizumab variants that dissociate receptor-bound IgE. We determine a low resolution cryo-EM structure of a transient disruption intermediate containing the IgE-Fc, its partially dissociated receptor and an antibody inhibitor. Our results provide a conceptual framework for engineering disruptive inhibitors for other targets, insights into the failure in clinical trials of the previous high affinity omalizumab HAE variant and anti-IgE antibodies that safely and rapidly disarm allergic effector cells.

Nanobody Activators of Von Willebrand Factor Via Targeting Its Autoinhibitory Module

Introduction: Von Willebrand factor (VWF) is a multimeric plasma glycoprotein responsible for platelet arrest during injury, especially at high shear. After immobilization to the vessel wall, a VWF multimer is unfurled and elongated. This leads to exposure of the A1 domain therein that in turn binds to platelet receptor GPIbα and starts the aggregation process. Recently, it was suggested that VWF activation involves force-dependent disruption of the autoinhibitory module (AIM) that flanks the A1 domain on both sides. In this scenario, the AIM could be targeted for both VWF inhibition (Caplacizumab) and activation (ristocetin), although the exact mechanism and binding site of ristocetin still remains murky. If the quasi-stable structure of the AIM is important to VWF autoinhibition, specific disruption of its confirmation may be able to activate VWF. To this end, we sought to identify AIM-targeting activators using yeast surface display of a llama nanobody library. Methods: One adult Lama glama was immunized with recombinant human VWF AIM-A1 protein produced from transfected Expi293F cells. VHH specific genes were amplified from cDNAs prepared from PBMCs of the animal and electroporated into EBY100 cells. The resulting yeast display library was screened for AIM-specific binders via selection against binding to recombinant A1 protein without an intact AIM, and then for binding to the complex of AIM-A1 with GPIbα. Positive hits were produced as His-tagged monomeric nanobodies in E. coli and purified with nickel-affinity and gel filtration chromatography. The affinity of nanobodies to AIM-A1 was determined using bio-layer interferometry. Platelet-rich plasma from healthy donors was used to assess the effect of nanobodies on platelet aggregation in a light transmission aggregometer with comparison to that of ristocetin. Results: An AIM-A1-specific nanobody yeast display library was established. Several rounds of flow cytometry-based cell sorting of yeast cells with aforementioned binding properties produced AIM-binding nanobodies. Nanobodies encoded in three single clones have been expressed from E. coli and they exhibited differential binding affinities towards AIM-A1. Clone 6C4 showed the lowest affinity (K D 120 ± 3 nM), 6D12 showed intermediate affinity (K D 31 ± 0.8 nM), and 6C11 showed the highest affinity (K D 13.5 ± 0.2 nM) as shown in Figure 1. These nanobodies showed no detectable affinity towards recombinant A1-CAIM protein (residues 1268-1493), indicating that their epitopes are located in the N-terminal portion of the AIM (residues 1238-1267). When added to human platelet-rich plasma, each nanobody dose-dependently activated platelets and rapidly induced full platelet aggregation at concentrations exceeding the affinity of the nanobody for VWF (Figure 2). The aggregation could be inhibited by the addition of antibodies that block the interaction between VWF and GPIbα. Plots of extents of aggregation as a function of nanobody concentration produced EC 50 values of ~100 nM for 6C11 and 6D12. Conclusion: By isolating nanobodies that can bind specifically to the AIM and activate plasma VWF, we add supporting evidence that the AIM protects the A1 domain from binding to platelets. Interestingly, these nanobodies bind to the NAIM, on the opposite side of the module compared to ristocetin, the only known AIM-activating agent until now. With higher VWF-binding affinities than ristocetin and a robust profile as stable monomers, these nanobodies may prove useful in VWF-related research and diagnostics. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Discovery and Preclinical Characterization of CC-95251, an Anti-SIRPα Antibody That Enhances Macrophage-Mediated Phagocytosis of Non-Hodgkin Lymphoma (NHL) Cells When Combined with Rituximab

Introduction: The transmembrane protein CD47 is ubiquitously expressed on normal cells and often overexpressed on cancer cells, including NHL. Binding of CD47 to signal regulatory protein-α (SIRPα) on macrophages induces an anti-phagocytic signal enabling tumor cells to escape the innate immune response. Macrophage antitumor activity can be restored by simultaneously blocking the CD47-SIRPα signaling axis and inducing a pro-phagocytic signal with the use of tumor-opsonizing antibodies. Targeting SIRPα on monocytes and macrophages rather than the ubiquitously expressed CD47 may overcome some toxicities associated with anti-CD47 therapies. Here, we report the discovery and characterization of a fully human anti-SIRPα antibody and its preclinical activity in combination with the opsonizing antibody rituximab in CD20+ diffuse large B-cell lymphoma (DLBCL) cell lines. Methods: A total of ~ 10 10 fully human immunoglobulin G antibodies were screened for binding to the extracellular domain of recombinant human SIRPα using a yeast display platform. Surface plasmon resonance was used to determine CC-95251 binding coverage across SIRPα haplotypes. The ability of CC-95251 to block CD47-SIRPα interaction was measured using Octet ® and Biacore™ assays. We determined the crystal structure of SIRPα in complex with the CC-95251 Fab to characterize its epitope and to define the structural basis for CD47-SIRPα interaction blockade. To identify tumor types likely susceptible to CD47-SIRPα axis disruption, expression levels of CD47-SIRPα and CD163 were assessed in bulk tumor samples using The Cancer Genome Atlas (TCGA) data. The antitumor effects of CC-95251 in combination with rituximab were examined by measuring the percentage of phagocytic macrophages in co-culture experiments of differentiated macrophages and CD20+ DLBCL cell lines (OCI-Ly3, RIVA, Pfeiffer, and Karpas 422). To confirm CC-95251 binding to monocytes, immunophenotyping of peripheral blood mononuclear cells from healthy donors and cynomolgus monkeys was performed using multiparameter flow cytometry. Lastly, pharmacokinetics and hematologic effects were analyzed in cynomolgus monkeys after treatment with 10, 30, or 100 mg/kg CC-95251. Results: Initial screening by yeast display yielded ~ 350 candidates. The top 24 clones were characterized fully and CC-95251 was selected as the lead monoclonal antibody exhibiting high binding affinity across the 6 most prevalent SIRPα human haplotypes. CC-95251 potently blocked CD47-SIRPα binding in a dose-dependent manner, with a concentration of 100 nM inhibiting CD47 binding almost completely. Co-crystallization modeling showed that CC-95251 engages SIRPα in a region overlapping the CD47 binding site, demonstrating a mechanism for CD47-SIRPα blockade. DLBCL was identified as a suitable tumor type for CC-95251 treatment based on CD47-SIRPα expression and macrophage infiltration. Co-culture experiments of donor macrophages and several DLBCL cell lines showed that CC-95251 monotherapy had weak-to-moderate antitumor activity. However, when combined with rituximab, the levels of phagocytic macrophages were markedly increased in a CC-95251 dose-dependent manner, suggesting that inhibition of the CD47-SIRPα anti-phagocytic axis with CC-95251 and activation of pro-phagocytic signaling with rituximab provide an enhanced antitumor effect in DLBCL cell lines. CC-95251 predominantly bound to cells of myeloid origin, including monocytes and, to a lesser extent, myeloid dendritic cells, whereas no binding to natural killer cells was observed. Toxicology studies in cynomolgus monkeys showed safe intravenous delivery of CC-95251 at therapeutic doses, with no evidence of white blood cell, monocyte, lymphocyte, or red blood cell depletion. Conclusions: CC-95251 is a novel, high-affinity, fully human monoclonal anti-SIRPα antibody that blocks the binding of CD47 to SIRPα. When combined with the therapeutic opsonizing antibody rituximab, CC-95251 enhances macrophage phagocytic activity against DLBCL cell lines in co-culture models. These results support the clinical evaluation of CC-95251 + rituximab for relapsed or refractory NHL. A phase 1 dose-escalation and -expansion study of CC-95251 for the treatment of advanced solid and hematologic malignancies is underway (NCT03783403). Disclosures Chan: Bristol Myers Squibb: Current Employment. Trout: Bristol Myers Squibb: Ended employment in the past 24 months. Mikolon: Bristol Myers Squibb: Current Employment. Adams: Bristol Myers Squibb: Current Employment. Guzman: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Fenalti: Bristol Myers Squibb: Current Employment. Mavrommatis: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Abbasian: Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Dearth: Bristol Myers Squibb: Current Employment. Fox: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Sivakumar: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Hariharan: Bristol Myers Squibb: Current Employment.

A Four-Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotypes C and D

Human botulism can be caused by botulinum neurotoxin (BoNT) serotypes A to G. Here, we present an antibody-based antitoxin composed of four human monoclonal antibodies (mAbs) against BoNT/C, BoNT/D, and their mosaic toxins. This work built on our success in generating protective mAbs to BoNT /A, B and E serotypes. We generated mAbs from human immune single-chain Fv (scFv) yeast-display libraries and isolated scFvs with high affinity for BoNT/C, BoNT/CD, BoNT/DC and BoNT/D serotypes. We identified four mAbs that bound non-overlapping epitopes on multiple serotypes and mosaic BoNTs. Three of the mAbs underwent molecular evolution to increase affinity. A four-mAb combination provided high-affinity binding and BoNT neutralization of both serotypes and their mosaic toxins. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing and neutralizing BoNT/C and BoNT/D serotypes and their mosaic toxins. A derivative of the four-antibody combination (NTM-1634) completed a Phase 1 clinical trial (Snow et al., Antimicrobial Agents and Chemotherapy, 2019) with no drug-related serious adverse events.