An antibody-dependent enhancement (ADE) activity eliminated neutralizing antibody with potent prophylactic and therapeutic efficacy against SARS-CoV-2 in rhesus monkeys

Shuang Wang
Yun Peng
Rongjuan Wang
Shasha Jiao
Min Wang
Weijin Huang
Chao Shan
Wen Jiang
Zepeng Li
Chunying Gu
Ben Chen
Xue Hu
Yanfeng Yao
Juan Min
Huajun Zhang
Ying Chen
Ge Gao
Peipei Tang
Gang Li
An Wang
Lan Wang
Shuo Chen
Xun Gui
Jinchao Zhang
Zhiming Yuan
Datao Liu

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Abstract

Efficacious interventions are urgently needed for the treatment of COVID-19. Here, we report a monoclonal antibody (mAb), MW05, showing high SARS-CoV-2 neutralizing activity by disrupting the interaction of receptor binding domain (RBD) with angiotensin-converting enzyme 2 (ACE2) receptor. Crosslinking of Fc with FcγRIIB mediates antibody-dependent enhancement (ADE) activity by MW05. This activity was eliminated by introducing the LALA mutation to the Fc region (MW05/LALA). Most importantly, potent prophylactic and therapeutic effects against SARS-CoV-2 were observed in rhesus monkeys. A single dose of MW05/LALA completely blocked the infection of SARS-CoV-2 in a study of its prophylactic effect and totally cleared SARS-CoV-2 in three days in a treatment setting. These results pave the way for the development of MW05/LALA as an effective strategy for combating COVID-19.

SciScore for 10.1101/2020.07.26.222257: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	IACUC: Monkey studies were carried out in an animal biosafety level 4 (ABSL-4) facility with protocols approved by the Laboratory Animal Welfare and Ethics Committee of the Chinese Academy of Sciences. Consent: The blood was taken from a convalescent COVID-19 patient after got his signature for the informed consent form.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	Nine 6 or 7 year-old rhesus monkeys (3 females and 6 males) were divided into 3 groups: a control group (one female and two males), a pre-exposure group (one female and two males) and a post-exposure group (one female and two males).
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
For the generation of human ACE2-hFc and SARS-CoV-2 RBD-mFc recombinant proteins, RBD or ACE2 sequence (1-615aa, accession number: NP_068576.1) was cloned into mouse IgG1 or human IgG1 Fc backbone in pKN293E expression vectors and transiently transfected into HEK293 cells followed by media collection and purification using MabSelect SuRe antibody purification resin (Cat: 29-0491-04, GE Healthcare).	ACE2 suggested: None IgG1 suggested: None human IgG1 suggested: None
After washing twice with 1 × PBS, cells were stained with 1/200 diluted Goat Anti human IgG Fc-FITC antibody (Cat: F9512, Sigma) for 45 min and analyzed using flow cytometry (CytoFLEX, Beckman Coulter).	Anti human IgG suggested: (Sigma-Aldrich Cat# F9512, RRID:AB_259808)
Then 10 μL anti-FcγRI antibody-FITC (Cat: 10256-R401-F, Sino Biological), anti-FcγRIIa antibody-FITC (Cat: 10374-MM02-F, Sino Biological), anti-FcγRIIIa antibody-FITC (Cat: 10389-MM41-F, Sino Biological) and FITC-labeled anti-FcγRIIb antibody (Cat: NBP2-14905, Biotechne; Cat: MX488AS100-1KT, Sigma-Aldrich) were added into cells (1×106 cells/sample in 100 μL) and incubated for 60 min at 2-6°C and analyzed using flow cytometry (CytoFLEX, Beckman Coulter)	anti-FcγRIIa suggested: None antibody-FITC suggested: (Alomone Labs Cat# AAR-007-F, RRID:AB_2756537) anti-FcγRIIIa suggested: None anti-FcγRIIb suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Cells and viruses: HEK293 (ATCC, CRL-3216) cells, Huh7 (Institute of Basic Medical Sciences CAMS, 3111C0001CCC000679) cells and Vero E6 (ATCC, CRL-1586) cells were cultured at 37 °C in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS).	HEK293 suggested: None Vero E6 suggested: None
Raji (ATCC, CCL-86) cells, THP-1 (ATCC, TIB-202) cells and K562 (ATCC, CCL-243) cells were cultured at 37 °C in RPMI 1640 Medium with 10% FBS.	THP-1 suggested: None
antibodies, heavy chain and light chain plasmids were transiently co-transfected into HEK293 cells or stably expressed in CHO cells followed by purification with Protein A resin.	CHO suggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)
The TCID50 was determined by the transduction of pseudovirus into Huh7 cells.	Huh7 suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)
Antibody-dependent enhancement (ADE) assay: The ADE assays were performed using Raji, THP-1 and K562 cell lines.	K562 suggested: NCI-DTP Cat# K-562, RRID:CVCL_0004)
Software and Algorithms
Sentences	Resources
Maokang Biological) for 30 min at RT.	Maokang Biological suggested: None
The 50% neutralization titer (NT50) was calculated using GraphPad Prism 7.0.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
No protocol registration statement was detected.

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SciScore for 10.1101/2020.07.26.222257: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement

Monkey studies were carried out in an animal biosafety level 4 (ABSL-4) facility with protocols approved by the Laboratory Animal Welfare and Ethics Committee of the Chinese Academy of Sciences.

Randomization

not detected.

Blinding

not detected.

Power Analysis

not detected.

Sex as a biological variable

Nine 6 or 7 year-old rhesus monkeys (3 females and 6 males) were divided into 3 groups: a control group (one female and two males), a pre-exposure group (one female and two males) and a post-exposure group (one female and two males).

Cell Line Authentication

not detected.

Table 2: Resources

Antibodies
Sentences	Resources
For the generation of human ACE2-hFc and SARS-CoV-2 RBD-mFc recombinant proteins , RBD or ACE2 sequence (1-615aa, accession number: NP_068576.1) was cloned into mouse IgG1 or human IgG1 Fc backbone in pKN293E expression vectors and transiently transfected into HEK293 cells followed by media collection and purification using MabSelect SuRe antibody purification resin (Cat: 29-0491-04, GE Healthcare).	ACE2 suggested: None IgG1 suggested: None human IgG1 suggested: None
After washing twice with 1 × PBS, cells were stained with 1/200 diluted Goat Anti human IgG Fc-FITC antibody (Cat: F9512, Sigma) for 45 min and analyzed using flow cytometry (CytoFLEX, Beckman Coulter).	Anti human IgG suggested: (Sigma-Aldrich Cat# F9512, RRID:AB_259808)
Then 10 μL anti-FcγRI antibody-FITC (Cat: 10256-R401-F, Sino Biological), anti-FcγRIIa antibodyFITC (Cat: 10374-MM02-F, Sino Biological), anti-FcγRIIIa antibody-FITC (Cat: 10389-MM41-F, Sino Biological) and FITC-labeled anti-FcγRIIb antibody (Cat: NBP2-14905, Biotechne; Cat: MX488AS100-1KT, Sigma-Aldrich) were added into cells (1×106 cells/sample in 100 μL) and incubated for 60 min at 2-6℃ and analyzed using flow cytometry (CytoFLEX, Beckman Coulter)	antibody-FITC suggested: (Alomone Labs Cat# AAR-007-F, RRID:AB_2756537) anti-FcγRIIa suggested: None anti-FcγRIIIa suggested: None anti-FcγRIIb suggested: None
Experimental Models: Cell Lines
Sentences	Resources
HEK293 (ATCC, CRL-3216) cells, Huh7 (Institute of Basic Medical Sciences CAMS, 3111C0001CCC000679) cells and Vero E6 (ATCC, CRL-1586) cells were cultured at 37 °C in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS).	Huh7 suggested: None
Raji (ATCC, CCL-86) cells, THP-1 (ATCC, TIB-202) cells and K562 (ATCC, CCL-243) cells were cultured at 37 °C in RPMI 1640 Medium with 10% FBS.	THP-1 suggested: None K562 suggested: ATCC Cat# CCL-243, RRID:CVCL_0004
For the generation of human ACE2-hFc and SARS-CoV-2 RBD-mFc recombinant proteins , RBD or ACE2 sequence (1-615aa, accession number: NP_068576.1) was cloned into mouse IgG1 or human IgG1 Fc backbone in pKN293E expression vectors and transiently transfected into HEK293 cells followed by media collection and purification using MabSelect SuRe antibody purification resin (Cat: 29-0491-04, GE Healthcare).	HEK293 suggested: None
For preparation of MW05 and MW07 recombinant antibodies, heavy chain and light chain plasmids were transiently cotransfected into HEK293 cells or stably expressed in CHO cells followed by purification with Protein A resin.	CHO suggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213
For SARS-CoV-2 authentic virus neutralization assay, Vero E6 cells were diluted and seeded into a 96well plate with 1×104 cells/well in 100 µL volume at 37 °C. 16 h later, cells were washed by 1× PBS for 3 times and added diluted antibodies in equal volume with the concentration ranging from 0.1 μg/mL to 100 μg/mL. 100 TCID50 SARS-CoV-2 authentic virus was used for each well.	Vero E6 suggested: None
Software and Algorithms
Sentences	Resources
Maokang Biological) for 30 min at RT.	Maokang Biological suggested: None
The 50% neutralization titer (NT50) was calculated using GraphPad Prism 7.0.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from JetFighter: We did not find any issues relating to colormaps.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

Read the original source

SciScore for 10.1101/2020.07.26.222257: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement

Monkey studies were carried out in an animal biosafety level 4 (ABSL-4) facility with protocols approved by the Laboratory Animal Welfare and Ethics Committee of the Chinese Academy of Sciences.

Randomization

not detected.

Blinding

not detected.

Power Analysis

not detected.

Sex as a biological variable

Cell Line Authentication

not detected.

Table 2: Resources

Antibodies

Sentences Resources

For the generation of human ACE2-hFc and SARS-CoV-2 RBD-mFc recombinant proteins , RBD or ACE2 sequence (1-615aa, accession number: NP_068576.1) was cloned into mouse IgG1 or human IgG1 Fc backbone in pKN293E expression vectors and transiently transfected into HEK293 cells followed by media collection and purification using MabSelect SuRe antibody purification resin (Cat: 29-0491-04, GE Healthcare).

ACE2

suggested: None

      <div style="margin-bottom:8px">
        <div><b>IgG1</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>human IgG1</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washing twice with 1 × PBS, cells were stained with 1/200 diluted Goat Anti human IgG Fc-FITC antibody (Cat: F9512, Sigma) for 45 min and analyzed using flow cytometry (CytoFLEX, Beckman Coulter).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Anti human IgG</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Then 10 μL anti-FcγRI antibody-FITC (Cat: 10256-R401-F, Sino Biological), anti-FcγRIIa antibodyFITC (Cat: 10374-MM02-F, Sino Biological), anti-FcγRIIIa antibody-FITC (Cat: 10389-MM41-F, Sino Biological) and FITC-labeled anti-FcγRIIb antibody (Cat: NBP2-14905, Biotechne; Cat: MX488AS100-1KT, Sigma-Aldrich) were added into cells (1×106 cells/sample in 100 μL) and incubated for 60 min at 2-6℃ and analyzed using flow cytometry (CytoFLEX, Beckman Coulter)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>antibody-FITC</b></div>
        <div>suggested: (Alomone Labs Cat# AAR-007-F, <a href="https://scicrunch.org/resources/Any/search?q=AB_2756537">AB_2756537</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-FcγRIIa</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-FcγRIIIa</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-FcγRIIb</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">As expected, MW05 and MW07 blocked authentic SARS-CoV-2 entry into Vero E6 cells, with 100% neutralization titer (NT100) around 1 μg/ml for MW05 and 5 μg/ml for MW07 (Fig. 2, C and D).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Vero E6</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To evaluate ADE activities of MW05 and MW07, we assessed the infection of SARS-CoV-2 pseudovirus and mAbs complex in THP-1, K562 and Raji cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>K562</b></div>
        <div>suggested: NCI-DTP Cat# K-562, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0004">CVCL_0004</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">FACS data revealed that Raji cells, which showed ADE activity for MW05, only express a relatively high level of FcγRIIB; THP-1 cells express high levels of FcγRIA and FcγRIIA; and K562 cells only express high level of FcγRIIA (Fig. 3C).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Raji</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For preparation of MW05 and MW07 recombinant antibodies, heavy chain and light chain plasmids were transiently cotransfected into HEK293 cells or stably expressed in CHO cells followed by purification with Protein A resin.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HEK293</b></div>
        <div>suggested: CLS Cat# 300192/p777_HEK293, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0045">CVCL_0045</a></div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>CHO</b></div>
        <div>suggested: CLS Cat# 603479/p746_CHO, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0213">CVCL_0213</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The FcγR expression profiles of Raji, THP-1 and K562 were determined by FACS.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>THP-1</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All mAbs were tested in concentrations ranging from 0.55 ng/mL to 28 µg/mL in the context of Huh7 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Huh7</b></div>
        <div>suggested: CLS Cat# 300156/p7178_HuH7, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0336">CVCL_0336</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Maokang Biological) for 30 min at RT.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Maokang Biological</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The 50% neutralization titer (NT50) was calculated using GraphPad Prism 7.0.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>GraphPad Prism</b></div>
        <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
      </div>
    </td></tr></table>

Data from additional tools added to each annotation on a weekly basis.

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Version published to 10.1101/2020.07.26.222257v1 on bioRxiv
Jul 27, 2020

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