key: cord-0988209-slhrpxzj authors: Minenkova, Olga; Santapaola, Daniela; Milazzo, Ferdinando Maria; Anastasi, Anna Maria; Battistuzzi, Gianfranco; Chiapparino, Caterina; Rosi, Antonio; Gritti, Giuseppe; Borleri, Gianmaria; Rambaldi, Alessandro; Dental, Clélia; Viollet, Cécile; Pagano, Bruno; Salvini, Laura; Marra, Emanuele; Luberto, Laura; Rossi, Antonio; Riccio, Anna; Pich, Emilio Merlo; Santoro, Maria Gabriella; De Santis, Rita title: Human inhalable antibody fragments neutralizing SARS-CoV-2 variants for COVID-19 therapy date: 2022-02-12 journal: Mol Ther DOI: 10.1016/j.ymthe.2022.02.013 sha: 66cd91e578a480aca557eae83885ac14b6b4b6bb doc_id: 988209 cord_uid: slhrpxzj As of December 2021, coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains global emergency and novel therapeutics are urgently needed. Here we describe human single chain variable fragment (scFv) antibodies (76clAbs) that block an epitope of the SARS-CoV-2 spike protein essential for ACE2-mediated entry into cells. 76clAbs neutralize the delta variant and other variants being monitored (VBMs) and inhibit spike-mediated pulmonary cell-cell fusion, a critical feature of COVID-19 pathology. In two independent animal models, intranasal administration counteracted the infection. Due to high efficiency, remarkable stability, resilience to nebulization and low cost of production, 76clAbs may become a relevant tool for rapid, self-administrable early intervention in SARS-CoV-2-infected subjects independently of their immune status. select affinity-matured antibody species, we profiled the immune response to SARS-CoV-2 of 81 ten COVID-19 convalescent donors from the Papa Giovanni XXIII Hospital in Bergamo, 82 Italy. As shown in Figure 1B left panel, six out of ten COVID-19 sera (CS) were found to 83 strongly bind to the SARS-COV-2 spike (Wuhan strain). These six sera also proved to be the 84 most potent at inhibiting the spike-hACE2 interaction ( Figure 1B right panel) and the most 85 effective at neutralizing viral infectivity in Vero E6 cells (Table S1 ). Total RNA, extracted 86 from lymphocytes of the six best responders, was converted to cDNA and the 87 immunoglobulin genes were amplified and cloned in scFv antibody format for phage display, 88 as depicted in Figure S1A . After selection cycles by panning, binders were subjected to 89 affinity maturation by error-prone mutagenesis of the heavy chain and to light chain shuffling 90 followed by repeated panning on the RBD under stressing conditions (low antigen 91 J o u r n a l P r e -p r o o f 5 concentration, low pH washing, high temperature). Selected scFv5, scFv86 and scFv76 92 antibodies, as well as affinity matured derivatives from scFv76 (i.e., scFv76-46, scFv76-55, 93 scFv76-57, scFv76-58 and scFv76-77), here coded as scFv76-cluster antibodies (76clAbs), all 94 binding the SARS-CoV-2 spike in the sub and low nanomolar range, were produced in 95 soluble form and purified according to the procedure schematized in Figure S1B . The amino 96 acid sequences of CDR3 and germline gene repertoire of the selected scFv antibodies are 97 reported in Table S2 that shows 76clAbs having identical VH CDR3 originating from the 98 IGVH3-53 germline, with minor differences in the remaining VH sequence. Diversity is 99 contributed by VL sequences that are derived from the IGVK3-20 germline except for 100 scFv76-55 and scFv76-57 antibodies that originate VL from IGVK3-15 and IGVK1-9 101 germlines, respectively. Moreover, scFv5 and scFv86 antibodies share identical VH, derived 102 from the IGVH1-46 germline, and have VL derived from the IGVL2-14 and IGVK3-11 103 germlines, respectively. recognize a different antigenic epitope. In a set of independent experiments, binding affinity 118 of scFv antibodies for the SARS-CoV-2 spike was measured by Surface Plasmon Resonance 119 (SPR). Kinetic data on mutated spike and RBD proteins are reported in Table 1, Figures S3, 120 S4 and Table S4 , showing KD values ranging between sub-nanomolar and 1-digit nanomolar 121 concentrations, except for scFv76-55 that exhibits a KD of 14.9 nM for S1beta. SPR analyses 122 did not reveal additive binding of the 76clAbs, suggesting that they might recognize the same 123 or a very close antigenic epitope, while scFv5 binds to an independent epitope ( Figure S5A ). 124 To test the ability of 76clAbs to recognize a SARS-CoV-2 native spike, HEK293T cells were 125 transfected with the WT S1 spike-encoding plasmid. High content screening (HCS) 126 fluorescent imaging showed the scFv76 antibody binding to HEK293T spike+ cells ( Figure 127 S5B). Similar data were obtained with all 76clAbs and confirmed by cytofluorimetry 128 indicating the ability of such antibodies to recognize the spike expressed on the cell surface 129 ( Figure S5C ). The 76clAbs were then evaluated for their ability to inhibit the binding of the 130 viral spike to hACE2 receptor and for their capacity to neutralize viral infectivity. Results in 131 Figure 1C show that 76clAbs, but not scFv5 or scFv86, blocked spike/hACE2 interaction and 132 that, except for scFv76-55, they were highly resilient to mutations of the Delta variant, as well 133 as to other mutations of VBMs ( Figure S6 ). The IC50 values of 76clAbs, ranging from 0.36 to 134 4.30 nM, are reported in Table S5 . ScFv5 and scFv86 antibodies did not interfere with spike 135 or RBD binding to ACE2 at concentrations ≥40 nM. Interestingly, and consistently with 136 binding and SPR affinity data, these results indicate that 76clAbs, except for scFv76-55, 137 substantially maintain the capacity to inhibit the binding of the spike variants and mutated 138 RBD to hACE2. 139 To investigate the potential escape of virus variants to 76clAbs neutralization, infection of 140 cells with pseudotyped as well as real viruses was performed. The scFv5 antibody, which did 141 not inhibit the spike binding to hACE2, was selected as a negative control. Human colon 142 Caco-2 cells stably expressing the hACE2 receptor (Caco-2 hACE2) were infected with 143 J o u r n a l P r e -p r o o f luciferase-expressing SARS-CoV-2 S-pseudoviruses bearing the D614G mutation or the 144 Delta spike variant. Results in Figure 1D confirmed that 76clAbs, but not scFv5, inhibited the 145 infection of both pseudoviruses as well as the infection of pseudoviruses bearing spike 146 mutations of several VBMs ( Figure S7A ). Consistently with previous neutralization data, 147 scFv76-55 was the least effective of the cluster and the most sensitive to mutations. Similar 148 results were obtained comparing the neutralization potency of scFV76 and scFV76-55 on 149 pseudoviruses bearing the Alpha, Beta and Gamma spike variants ( Figure S7B ). 150 Neutralization of infectivity of the real SARS-CoV-2 virus was then tested by 151 microneutralization assay of cytopathic effect (CPE) in Vero E6 cells. Interestingly, all 152 76clAbs proved to be effective with MN50 (50% microneutralization titer) <15 nM against 153 the delta variant ( Figure 1E ). Neutralization potency of scFv76, scFv76-46 and scFv76-58 154 antibodies was then further evaluated by RT-qPCR in human pulmonary Calu-3 cells infected 155 with the SARS-CoV-2 Wuhan strain, D614G, or Alpha variants. Results in Table S6 show 156 that when the 76clAbs were added to the cell culture 1 h after the infection they were 157 effective at inhibiting all virus strains with IC50 below 22 nM, while when added 1 h before 158 infection, the IC50 was below 2.2 nM. A similar difference in the neutralization potency 159 before or after infection was observed with the immune sera pooled from the six COVID-19 160 convalescent donors ( Figure 1B and Table S1 ). Human scFv antibodies counteract cell-cell fusion 162 The ability of 76clAbs to prevent SARS-CoV-2 spike-induced fusion of pulmonary cells was 163 tested in vitro ( Figure S8A ). This analysis has high translational value, since syncytia 164 formation in the lung is considered a most relevant pathologic hallmark of COVID-19, For binding experiments, Nunc maxisorp plates-96 wells were coated with 100 µL/well of 325 SARS-CoV-2 spike S1 [SARS-CoV-2 spike glycoprotein S1 sheep Fc-tag, His-tagged spike 326 S1 (D614G), spike S1(HV69-70del/Y144del/ N501Y, A570D, D614G, P681H), spike 327 S1(K417N, E484K, N501Y, D614G), Spike S1 (T19R, G142D, E156G, 157-158 deletion, Similar experiments were performed for assessing the binding specificity of 76clAbs by using 339 SARS-CoV Spike/S1, MERS-CoV Spike/S1 or HCoV-HKU1 spike/S1 His-tagged proteins 340 (all from Sino Biological). 341 For competition experiments, Nunc maxisorp plates-96 wells were coated with 100 µL/well of 342 spike S1 [SARS-CoV-2 spike glycoprotein S1 sheep Fc-tag, His-tagged spike S1 (D614G), 343 spike S1(HV69-70del/Y144del/ N501Y, A570D, D614G, P681H), spike S1(K417N, E484K, J o u r n a l P r e -p r o o f SARS-CoV-2 Variants and Vaccines Effectiveness of Covid-19 Vaccines against 672 the B.1.617.2 (Delta) Variant Covid-19: Delta variant doubles risk of hospital admission compared with 674 alpha variant, study shows The SARS-CoV-2 Spike Glycoprotein as a Drug 676 and Vaccine Target: Structural Insights into Its Complexes with ACE2 and Antibodies The biogenesis of SARS-CoV-2 spike 679 glycoprotein: multiple targets for host-directed antiviral therapy Neutralizing monoclonal antibodies for treatment of COVID-19 SARS-CoV-2-neutralising 686 monoclonal antibodies for treatment of COVID-19 CoV-2 variant Delta to antibody neutralization An infectivity-enhancing site on the SARS-CoV-2 693 spike protein targeted by antibodies Robust SARS-CoV-2 infection in nasal turbinates after treatment 696 with systemic neutralizing antibodies Challenges and opportunities for antiviral monoclonal antibodies as COVID-19 therapy Nanobodies from camelid mice and llamas neutralize 702 SARS-CoV-2 variants Inhalable Nanobody (PiN-21) 705 prevents and treats SARS-CoV-2 infections in Syrian hamsters at ultra-low doses A capillary electrophoresis-based approach for the 709 identification of anti-drug antibodies against camelid VHH biologics (Nanobodies®) Protein engineering of antibody 713 binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue 714 produced in Escherichia coli Persistence of viral RNA, pneumocyte syncytia 718 and thrombosis are hallmarks of advanced COVID-19 pathology. EBioMedicine 61, 103104 Broadly neutralizing antibodies 721 overcome SARS-CoV-2 Omicron antigenic shift Considerable escape of 725 SARS-CoV-2 Omicron to antibody neutralization Antibody resistance of SARS-CoV-2 variants B.1.351 and 729 B.1.1.7 In vivo monoclonal antibody efficacy against 732 SARS-CoV-2 variant strains Accurate secondary structure prediction and fold recognition for circular dichroism 735 spectroscopy Reaction of Human Monoclonal 737 Antibodies to SARS-CoV-2 Proteins with Tissue Antigens: Implications for Autoimmune 738 Diseases Defining variant-resistant epitopes targeted 741 by SARS-CoV-2 antibodies: A global consortium study Learning from past failures: 743 Challenges with monoclonal antibody therapies for COVID-19 Highly-specific memory B cells 747 generation after the 2 nd dose of BNT162b2 vaccine compensate for the decline of serum 748 antibodies and absence of mucosal IgA Tumor-infiltrating B lymphocytes 751 as an efficient source of highly specific immunoglobulins recognizing tumor cells Vector for efficient selection and/or maturation of an 754 antibody and uses thereof New display vector 756 reduces biological bias for expression of antibodies in E. coli Determination of 50% endpoint titer using a simple formula Impairment of SARS-CoV-2 spike glycoprotein maturation and fusion activity by the broad-761 spectrum anti-infective drug nitazoxanide SARS-CoV-2 spike-protein D614G 764 mutation increases virion spike density and infectivity Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune 767 cross-reactivity with SARS-CoV Syncytia formation CoV-2-infected cells A versatile viral system for expression 773 and depletion of proteins in mammalian cells A high-throughput shotgun mutagenesis approach to 775 mapping B-cell antibody epitope Molecular Cloning: A Laboratory 777 Secondary 813 clones (blue circles) are highlighted for clones that did not meet the set thresholds but 814 whose decreased binding activity and proximity to critical residues suggested that the 815 mutated residue may be part of the antibody epitope. Critical residues (red spheres) for 816 scFv antibodies binding, and secondary residues (blue spheres) that may contribute to 817 binding, are also visualized on crystal structure of the SARS-CoV-2 spike protein 818 trimer (PDB ID # 6XCN) (center) and on SARS-CoV-2 spike protein receptor binding 819 domain 2 on TSKgel G3000 SWXL 825 30 cm x 7.8 mm column (Tosoh Bioscience). (B) Total ionic current (TIC) 826 chromatogram from SEC-HPLC analysis of scFv76 in 20 mM ammonium formate, pH 827 6.8 on Mab Pac SEC-1 column (Thermo). Inset, mass spectrum as average of spectra 828 recorded between 5 and 7 min. (C) Binding to SARS-CoV-2 RBD of thermally 829 stressed (1 mg/mL concentration for 1 h at indicated temperatures) and not treated 830 (NT) 76clAbs by ELISA. Data from one representative experiment. (D) Far-UV 831 circular dichroism spectra of antibodies. Analysis was performed in PBS buffer at 20 832 (solid lines) and 90 (dashed lines) °C. (E) UV chromatograms at 280 nm from SEC-833 HPLC analysis, as in A, of scFv76 pre-and post-nebulization at 1 mg/mL. (F) Binding 834 of scFv76, pre-and post-nebulization at 5 mg/mL or 1 mg/mL De Santis and colleagues describe engineered human antibody fragments (scFv), which are 859 extremely effective at neutralizing SARS-CoV-2 variants. Due to high stability and efficacy in 860 preclinical models, intranasal or aerosol delivery of scFv antibodies represents a promising 861 approach for halting SARS-CoV-2 infection at an early stage regardless of vaccination status