All animal experiments were complied with relevant ethical regulations regarding animal research. Pharmacokinetics study protocols in monkeys were approved by Institutional Animal Care and Use Committee(IACUC) and the Approval Number is IACUC-A2020131-K001-01. The animal experiments for in vivo efficacy studies were conducted in Academy of Military Medical Sciences and approved by the Experimental Animal Committee of Laboratory Animal Center, AMMS (approval number: IACUC-DWZX-2020-001). Protocols of mice experiments for immunization and pharmacokinetics study were approved by LUYE PHARMA Animal Experimentation.
Regents, Mice, Cells and Viruses
Reagents, cell lines and viral strains used in this study are listed in Supplementary Table 1.
Human antibody transgenic mice were generated by Boan biotechnology. Mice were bred and kept under specific-pathogen free conditions. Seven human antibody transgenic mice were immunized in 10-day intervals with recombinant Spike S1+S2 (Cat 40589-V08B1, Sino Biological), RBD protein (Cat 40592-V05H, Sino Biological), Spike S1 protein (Cat 40591-V02H, Sino Biological) and Spike S2 protein (Cat 40590-V08B, Sino Biological) from SARS-CoV-2 strain (Wuhan-1 strain, GenBank: MN_908947), respectively. After 3 rounds immunization and one boost, spleen cells were harvested after three days of the last boost for phage libraries construction.
Phage display library construction
RNA was extracted from spleen cells of immunized mice by Trizol method. cDNA synthesis was performed using Transcriptor First Strang cDNA Synthesis Kit. The construct of the phage library was carried out according to the method described in Phage Display: A laboratory manual 32 The variable regions of the heavy and light chains were obtained from the cDNA by PCR. Scfv were obtained by overlapping the variable regions of heavy and light chains by PCR, and then were digested with SfiI and ligated into plasmid pCOMB3x. The ligation products are electro transfected into E. coli TG1 competent cells. After adding helper phage and culturing overnight, the supernatant was precipitated with PEG8000 and NaCl. Libraries were got by resuspending the pellet.
Panning of phage libraries
Plates coated with immunogen (Spike S1+S2 or Spike S1 or Spike RBD) or streptavidin- magnetic beads loading biotin-protein were used to capture phages with interest ScFvs. After washing by PBST, captured phage was eluted and then used to infect E.Coli TG1. Scfvs was expressed and its binding and blocking activity was tested by ELISA. Positive hits were obtained and sequenced.
Production of human monoclonal antibody
Recombinant Vector Construction
Recombinant antibody heavy chain variable region and light chain variable region were amplified (2 × Phanta Max Master Mix, Vazyme, P515-01) using the positive clones screened from the library as the template. Overlap PCR was conducted to assemble variable region and signal peptide. Purified gene fragments were separately fused (ClonExpress II One Step Cloning Kit, Vazyme, C112-01) into the linearized pcDNA3.4 vectors with constant regions. The recombinant plasmid was prepared for production. (For recombinant human mAb production, the cDNA’s encoding the CA521 mAb variable regions of the heavy and light chains were cloned into expression.)
Antibody Expression and Purification
Candidate antibodies were expressed with Expi-CHO Expression system(gibco) for 12days and the supernatant was harvested and purified by protein A resin (GE healthcare). The antibodies were further purified by Q FF (GE healthcare) and Capto S ImpAct (GE healthcare) sequentially and then changed to buffer containing 10mM CH3COONa▪3H2O, 30mM NaCl, 5% sucrose, 0.03% tween-20, pH5.0/6.0.
ELISA -based receptor-binding inhibition assay
High binding ELISA plates were coated with 0.5 μg/mL recombinant soluble 2019-nCoV Spike RBD at 4oC overnight, and then were blocked with 3% skim milk powder in PBST (PBS containing 0.05%Tween-20) at 37oC for 1 hour, following two times washing with PBST. Serially diluted CA521 was mixed with 0.04 μg/mL (final concentration) biotinylated recombinant human ACE2 and then was incubated with coated RBD in the plates at 37oC for 1 hour. After washing, the biotinylated ACE2 binding to coated RBD was detected by HRP-conjugated Strep second mAb. Inhibition rate%=(OD450 of no antibody – OD450)/ OD450 of no antibody*100%. Irrelevant mAb with the same constant region of CA521 was used as isotype. Experiments were performed in triplicate, value=Mean±standard error.
Cross-reactivity by Elisa analysis
Recombinant SARS-CoV-2 Spike S1+S2 protein (40589-V08B1, Sino Biological), SARS Spike protein (SPN-S52H5, Acro) and MERS-CoV Spike protein (40069-V08B, Sino Biological) were coated on high binding ELISA plates with 0.5 μg/mL at 4oC overnight. Plates were blocked with 3% skim milk powder in PBST at 37oC for 1 hour and then washed two times with PBST. Serially dilutions of mAbs were added following incubation at 37oC for 1 h. Plates were washed two times and then HRP-goat anti-human IgG (H+L) mAb was used to detect antibodies binding to the Spikes. Irrelevant mAb with the same constant region of CA521 was used as isotype. Experiments were performed in triplicate, value=Mean±standard error.
Cell based binding for CA521
SARS-CoV-2 Spike protein transfected CHO cells were harvested and washed by FACS buffer (0.2% BSA in PBS) two times. 1E5 CHO-SARS-CoV-2-Spike cells were stained with isotype control IgG or CA521 at a concentration of 0.74 μg/mL at 4 oC for 1 h. After washing by FACS buffer two times, cells were incubated in dark with FITC-anti-human IgG Fc 2nd mAb at 4 oC for 30 min and then analyzed by NovoCyte 2060R flow cytometry. Irrelevant mAb with the same constant region of CA521 was used as isotype.
Experiments were performed in triplicate, value=Mean±standard error.
Affinity to SARS-CoV-2 Spike RBD mutants from different virus strain variants
The binding kinetics were assessed by Surface Plasmon Resonance (SPR) assay using the BIAcore 8K system. The measured equilibrium constant (KD) Measurements were performed at room temperature with CM5 chip, which was amino coupled by human antibody capture kit. HBS-EP+ buffer (150 mM NaCl, 10 mM HEPES, 3 mM EDTA and 0.05% (v/v) surfactant P20 pH 7.4) was used as running buffer. The blank channel of the chip served as the negative control. CA521 was captured on the chip at 400-500 response units. Serial dilutions of SARS-CoV-2-RBD mutants (from 50 nM to 3.125 nM with 2-fold dilution) were applied to flow over the chip surface which was regenerated with 3 M MgCl2 after each cycle. The affinity was calculated using a 1:1 (Langmuir) binding fit model with BIAevaluation software.
Fortebio analysis of antibody binding to CoV spike antigens RBD
Antibodies to be tested were diluted to the concentration of 4 μg/mL with PBST and then immobilized onto Octet Fab2G biosensors for real-time association and dissociation. After arriving the Signal Change Threshold 1.1 nm and washed in PBST biosensor tips were immersed into the wells containing RBD protein (40592-V05H, Sino Biological) of serial dilutions and allowed to associate for 200 seconds followed by a dissociation step of 400 seconds. KD was calculated using a 1:1 binding model in Octet Data Acquisition 22.214.171.124 (Sartorius AG).
Fortebio analysis of antibody binding to CoV spike antigens Spike-Trimer
Antibodies were diluted to the concentration of 3 μg/mL with PBST and then immobilized onto ProA biosensors. After arriving at the Signal Change Threshold 0.8 nm and washing with PBST, biosensor tips were immersed into the wells containing Spike-Trimer protein of serial dilutions and allowed to associate for 200 seconds followed by a dissociation step of 400 seconds. The KD wascalculated using a 1:1 binding model in Octet Data Acquisition 126.96.36.199.
Antibody-Dependent Cellular Phagocytosis (ADCP)
Antibody-Dependent Cellular Phagocytosis (ADCP) was conducted with SARS-CoV-2 Spike protein transfected CHO-K1 cells as target cells and macrophage derived from CD14+ monocytes as effector cells. Target cells were stained with CFSE and macrophages were stained with APC-anti-CD206. Double stained macrophages are considered to have phagocytized target cells. Phagocytosis rate (in red font) = Q2-2%/(Q2-1%+Q2-2%)*100%.
ELISA-based C1q binding assay
High binding ELISA plates were coated with 100 μL 5 μg/mL CA521 subtypes at 4 °C overnight. Plates were washed four times with PBS and blocked with PBST containing 1%BSA at 37oC for 1.5 hour. Fourfolds serial dilutions of mAbs starting at 40 µg/ml were added and plates were incubated for 2 hour at 37oC with shaking of 180 rpm. HRP labeled anti human C1q secondary antibody diluted in 1:300 with PBST was used to detect C1q binding with mAbs.
Affinity of CA521 with different subtype to FcγRs
The binding kinetics were assessed by Surface Plasmon Resonance (SPR) assay using the BIAcore 8K system. The measured equilibrium constant (KD) Measurements were performed at room temperature with CM5 chip, which was amino coupled by His Capture Kit. HBS-EP+ buffer (150 mM NaCl, 10 mM HEPES, 3 mM EDTA and 0.05% (v/v) surfactant P20 pH 7.4) was used as running buffer. The blank channel of the chip served as the negative control. FcγRs was captured on the chip. Twofolds serial dilutions of mAbs starting at different concentration (Supplementary Table 2) were applied to flow over the chip surface which was regenerated with 10 mM glycine-HCl (pH 1.5) after each cycle. The affinity was calculated using a 1:1 (Langmuir) binding fit model with BIA evaluation software.
Pharmacokinetics studies of CA521LALA
A single intravenous injection dose of CA521 was conducted in rhesus monkeys (N = 3, 2 females&1 male，age 3-5 years，body weight 3.00~3.90 kg) and C57BL/6 mice (N = 4, 4 males，age 5-7 weeks) at 50 mg/kg and 10 mg/kg. PK samples were collected at predose and 5 min, 30 min, 1 h, 3 h, 6 h, 24 h, 48 h, 72 h, 120 h, 168 h, 216 h, 264 h, 336 h, 504 h, 840 h, 1008 h postdose from moneys, and at predose and 3 min, 15min, 30min, 1h, 2h, 6h, 24h, 72h,120h,168h, 240h, 336h, 504h, 672h, 840, 1008h postdose. ELISA (enzyme-linked immunosorbent assay) was used to determine the concentration of CA521LALA in serum. In this method, SARS-CoV-2(2019-nCoV) spike protein was used as the capture reagent, and goat anti-human IgG, monkey ads-HRP was detecting agent. Results are shown as mean ± standard error (N = 3). The main PK kinetic parameters were calculated using Phoenix WinNonlin.
Pseudoviruses (80033) purchased from Beijing SanYao Science & Technology Development Company were produced and titrated as described previously 33.
SARS-CoV-2 pseudovirus were incubated with 3-fold serially diluted CA521 at 37 °C for 1 hour, and then cells suspension of Huh-7(Japanese Collection of Research Bioresources [JCRB], 0403) or HEK-293T-hACE2(CHENGDU NB BIOLAB CO., LTD) were added to the mixtures. After 24 h incubation at 37°C, neutralizations potencies of mAbs were evaluated in a luciferase assay. Luciferase activity was measured using Bio-Glo Assay reagent as a substrate (Promega). The percentage of infectivity was calculated as ratio of luciferase readout in the presence of mAbs normalized to luciferase readout in the absence of mAb. The half maximal inhibitory concentrations (IC50) were determined using 4-parameter logistic regression (GraphPad Prism). Experiments were performed in triplicate.
Infectious SARS-CoV-2 Neutralization assay
Neutralizing activity of mAbs was measured using a standard plaque reduction neutralization with Vero cells. Briefly, 5-fold serial dilutions of mAbs were added to approximately 100 PFU of SARS-CoV-2 and incubated for 1 h at 37 °C. Then, the mixture was added to Vero cell monolayers in a 24-well plate in duplicate and incubated for 1 h at 37 °C. The mixture was removed, and 1 ml of 1.0% (w/v) LMP agarose (Promega) in DMEM plus 4% (v/v) FBS was layered onto the infected cells. After further incubation at 37 °C for 2 days, the wells were stained with 1% (w/v) crystal violet dissolved in 4% (v/v) formaldehyde to visualize the plaques. PRNT50 values were determined using non-linear regression analysis. All experiments were performed followed the standard operating procedures of the approved Biosafety Level-3 facility.
Infection and antibody treatment of mice
Experiments involving live SARS-CoV-2 viruses were performed in the enhanced biosafety level 3 (P3+) facilities in the Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences.
Specific pathogen-free 6-8-week female Balb/C mice were lightly anesthetized with isoflurane and transduced intranasally with 2×103 PFU of SARS-CoV-2 mouse adaped strain (MASCp6) in 30 µl DMEM. Groups of Balb/C mice that received SARS-CoV-2 challenge were treated intraperitoneally with a single dose of 20 mg/kg of CA521 (n=4) or PBS (n=6) at 2 h post infection. The lung and trachea tissues of mice were collected at 3 dpi for virus titer and autopsy test.
Viral RNA quantitation
Viral burden in lung and trachea from mice were measured as described previously. Briefly, lung and trachea tissue homogenates were clarified by centrifugation at 6,000 rpm for 6 min and viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer’s protocol. Viral burden in each tissue sample was performed by quantitative reverse transcription PCR (RT-qPCR) targeting the S gene of SARS-CoV-2. RT-qPCR was performed using One Step PrimeScript RT-PCR Kit (Takara). The determination of the detection limit was based on the lowest level at which viral RNA was detected and remained within the range of linearity of a standard curve (Ct value of 38). RT-qPCR was performed using One Step PrimeScript RT PCR Kit (Takara, Japan) with the following primers and probes: CoV-F3 (5‘-TCCTGGTGATTCTTCTTCAGGT-3’); CoV-R3 (5‘-TCTGAGAGAGGGTCAAGTGC-3’); and CoV-P3 (5‘-AGCTGCAGCACCAG CTGTCCA-3’).The 20μl reaction mixtures were set up with 2μl of RNA. Cycling conditions were as follows: 42 °C for 5 min, 95 °C for 10 s, followed by 40 cycles of 95 °C for 5s and 60 °C for 20s.
Histology and Immunostaining
Lung tissues were excised and fixed with 10% neutral buffered formaline, dehydrated and embedded in paraffin. Sections at a thickness of 4 μm were stained with hematoxylin and eosin (H & E) according to standard histological procedures. Images were captured using Olympus BX51 microscope equipped with a DP72 camera.