An ACE2-IgG4 Fc Fusion Protein Demonstrates Strong Binding to All Tested SARS-CoV-2 Variants and Reduced Lung Inflammation in Animal Models of SARS-CoV-2 and Influenza

Main Article Content

Emmanuel Y. Merigeon
Dong Yang
Elizabeth A. Ihms
Leda C. Bassit
Elizabeth A. Fitzpatrick
Colleen B. Jonsson
Raymond F. Schinazi
David S. Block
Henrik S. Olsen

Abstract

Background: The continued emergence of SARS-CoV-2 variants has caused concern that a constantly evolving virus will escape vaccines and antibody therapies. New approaches are needed.


Methods: We created and manufactured an ACE2 extracellular domain (ECD) fragment Fc fusion drug candidate, G921, and engineered the compound for enhanced delivery of drug to peripheral tissues by minimizing the size of the ACE2 ECD and by incorporating an Fc domain to enhance transcytosis. G921 was assessed for binding, neutralization, in vivo anti-inflammatory effect, and pharmacokinetic profile.


Results: G921 was expressed as an IgG4 Fc fusion protein presenting two ACE2 domains to ACE2 ligands while avoiding risk of infection via antibody-dependent enhancement. G921 strongly binds to the SARS-CoV-2 Wuhan-Hu-1 spike protein and demonstrates further diminished off rate to the spike protein from each of the currently identified variants of concern. G921 demonstrates ACE2 enzymatic activity comparable to positive control and binding to the neonatal Fc receptor (FcRn) without binding to low affinity Fc-gamma receptors (FcγRs). G921 is effective in a concentration-dependent manner in a focus reduction neutralization assay with EC50=16.3±4.2 μg/mL without cytotoxicity in Vero E6 cells when tested at 200 μg/mL in an MTS cell proliferation assay. G921 demonstrates statistically significant reduction of lung inflammation in relevant models of both SARS-CoV-2 and influenza. The pharmacokinetic profile demonstrated dose-dependent exposure with a multi-day half-life in monkeys and rats.


Conclusion: G921 data are consistent with both antiviral and anti-inflammatory modes of action. G921 is a novel approach for the prevention and treatment of COVID-19 and possible other diseases characterized by deficiency of ACE2.

Downloads

Download data is not yet available.

Article Details

Section
Articles

References

1. Shang J, Wan Y, Luo C, Ye G, Geng Q, Auerbach A, Li F. Cell entry mechanisms of SARS-CoV-2. Proc Natl Acad Sci U S A. 2020;117(21):11727-34. doi: 10.1073/pnas.2003138117. PubMed PMID: 32376634; PMCID: PMC7260975.

2. Barros EP, Casalino L, Gaieb Z, Dommer AC, Wang Y, Fallon L, Raguette L, Belfon K, Simmerling C, Amaro RE. The flexibility of ACE2 in the context of SARS-CoV-2 infection. Biophys J. 2021;120(6):1072-84. doi: 10.1016/j.bpj.2020.10.036. PubMed PMID: 33189680; PMCID: PMC7661960.

3. Yan R, Zhang Y, Li Y, Xia L, Zhou Q. Structure of dimeric full-length human ACE2 in complex with B0AT1. bioRxiv. 2020:2020.02.17.951848. doi: 10.1101/2020.02.17.951848.

4. Guney C, Akar F. Epithelial and Endothelial Expressions of ACE2: SARS-CoV-2 Entry Routes. J Pharm Pharm Sci. 2021;24:84-93. doi: 10.18433/jpps31455. PubMed PMID: 33626315.

5. Asselah T, Durantel D, Pasmant E, Lau G, Schinazi RF. COVID-19: Discovery, diagnostics and drug development. J Hepatol. 2021;74(1):168-84. doi: 10.1016/j.jhep.2020.09.031. PubMed PMID: 33038433; PMCID: PMC7543767.

6. Vieira C, Nery L, Martins L, Jabour L, Dias R, Simoes ESAC. Downregulation of Membrane-bound Angiotensin Converting Enzyme 2 (ACE2) Receptor has a Pivotal Role in COVID-19 Immunopathology. Curr Drug Targets. 2021;22(3):254-81. doi: 10.2174/1389450121666201020154033. PubMed PMID: 33081670.

7. Verdecchia P, Cavallini C, Spanevello A, Angeli F. The pivotal link between ACE2 deficiency and SARS-CoV-2 infection. Eur J Intern Med. 2020;76:14-20. doi: 10.1016/j.ejim.2020.04.037. PubMed PMID: 32336612; PMCID: PMC7167588.

8. Vickers C, Hales P, Kaushik V, Dick L, Gavin J, Tang J, Godbout K, Parsons T, Baronas E, Hsieh F, Acton S, Patane M, Nichols A, Tummino P. Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase. J Biol Chem. 2002;277(17):14838-43. doi: 10.1074/jbc.M200581200. PubMed PMID: 11815627.

9. Chung MK, Karnik S, Saef J, Bergmann C, Barnard J, Lederman MM, Tilton J, Cheng F, Harding CV, Young JB, Mehta N, Cameron SJ, McCrae KR, Schmaier AH, Smith JD, Kalra A, Gebreselassie SK, Thomas G, Hawkins ES, Svensson LG. SARS-CoV-2 and ACE2: The biology and clinical data settling the ARB and ACEI controversy. eBioMedicine. 2020;58:102907. doi: 10.1016/j.ebiom.2020.102907. PubMed PMID: 32771682; PMCID: PMC7415847.

10. Domingo P, Mur I, Pomar V, Corominas H, Casademont J, de Benito N. The four horsemen of a viral Apocalypse: The pathogenesis of SARS-CoV-2 infection (COVID-19). eBioMedicine. 2020;58:102887. doi: 10.1016/j.ebiom.2020.102887. PubMed PMID: 32736307; PMCID: PMC7387269.

11. Gan R, Rosoman NP, Henshaw DJE, Noble EP, Georgius P, Sommerfeld N. COVID-19 as a viral functional ACE2 deficiency disorder with ACE2 related multi-organ disease. Med Hypotheses. 2020;144:110024. doi: 10.1016/j.mehy.2020.110024. PubMed PMID: 32758871; PMCID: PMC7308773.

12. Tracking SARS-CoV-2 Variants. World Health Organization. Updated October 7, 2021. Accessed October 11, 2021. Available from: https://www.who.int/en/activities/tracking-SARS-CoV-2-variants.

13. Ramanathan M, Ferguson ID, Miao W, Khavari PA. SARS-CoV-2 B.1.1.7 and B.1.351 Spike variants bind human ACE2 with increased affinity. bioRxiv. 2021:2021.02.22.432359. doi: 10.1101/2021.02.22.432359. PubMed PMID: 33655251; PMCID: PMC7924271.

14. Salleh MZ, Derrick JP, Deris ZZ. Structural Evaluation of the Spike Glycoprotein Variants on SARS-CoV-2 Transmission and Immune Evasion. Int J Mol Sci. 2021;22(14):7425. doi: 10.3390/ijms22147425. PubMed PMID: 34299045; PMCID: PMC8306177.

15. Liu L, Wei Q, Lin Q, Fang J, Wang H, Kwok H, Tang H, Nishiura K, Peng J, Tan Z, Wu T, Cheung KW, Chan KH, Alvarez X, Qin C, Lackner A, Perlman S, Yuen KY, Chen Z. Anti-spike IgG causes severe acute lung injury by skewing macrophage responses during acute SARS-CoV infection. JCI Insight. 2019;4(4). doi: 10.1172/jci.insight.123158. PubMed PMID: 30830861; PMCID: PMC6478436.

16. Lee WS, Wheatley AK, Kent SJ, DeKosky BJ. Antibody-dependent enhancement and SARS-CoV-2 vaccines and therapies. Nat Microbiol. 2020;5(10):1185-91. doi: 10.1038/s41564-020-00789-5. PubMed PMID: 32908214.

17. Kruse RL. Therapeutic strategies in an outbreak scenario to treat the novel coronavirus originating in Wuhan, China. F1000Res. 2020;9:72. doi: 10.12688/f1000research.22211.2. PubMed PMID: 32117569; PMCID: PMC7029759.

18. Baum A, Fulton BO, Wloga E, Copin R, Pascal KE, Russo V, Giordano S, Lanza K, Negron N, Ni M, Wei Y, Atwal GS, Murphy AJ, Stahl N, Yancopoulos GD, Kyratsous CA. Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with individual antibodies. Science. 2020;369(6506):1014-8. doi: 10.1126/science.abd0831. PubMed PMID: 32540904; PMCID: PMC7299283.

19. Baum A, Ajithdoss D, Copin R, Zhou A, Lanza K, Negron N, Ni M, Wei Y, Mohammadi K, Musser B, Atwal GS, Oyejide A, Goez-Gazi Y, Dutton J, Clemmons E, Staples HM, Bartley C, Klaffke B, Alfson K, Gazi M, Gonzalez O, Dick E, Jr., Carrion R, Jr., Pessaint L, Porto M, Cook A, Brown R, Ali V, Greenhouse J, Taylor T, Andersen H, Lewis MG, Stahl N, Murphy AJ, Yancopoulos GD, Kyratsous CA. REGN-COV2 antibodies prevent and treat SARS-CoV-2 infection in rhesus macaques and hamsters. Science. 2020;370(6520):1110-5. doi: 10.1126/science.abe2402. PubMed PMID: 33037066; PMCID: PMC7857396.

20. Jones BE, Brown-Augsburger PL, Corbett KS, Westendorf K, Davies J, Cujec TP, Wiethoff CM, Blackbourne JL, Heinz BA, Foster D, Higgs RE, Balasubramaniam D, Wang L, Zhang Y, Yang ES, Bidshahri R, Kraft L, Hwang Y, Zentelis S, Jepson KR, Goya R, Smith MA, Collins DW, Hinshaw SJ, Tycho SA, Pellacani D, Xiang P, Muthuraman K, Sobhanifar S, Piper MH, Triana FJ, Hendle J, Pustilnik A, Adams AC, Berens SJ, Baric RS, Martinez DR, Cross RW, Geisbert TW, Borisevich V, Abiona O, Belli HM, de Vries M, Mohamed A, Dittmann M, Samanovic MI, Mulligan MJ, Goldsmith JA, Hsieh CL, Johnson NV, Wrapp D, McLellan JS, Barnhart BC, Graham BS, Mascola JR, Hansen CL, Falconer E. The neutralizing antibody, LY-CoV555, protects against SARS-CoV-2 infection in nonhuman primates. Sci Transl Med. 2021;13(593):eabf1906. doi: 10.1126/scitranslmed.abf1906. PubMed PMID: 33820835; PMCID: PMC8284311.

21. Sugawara A, Shimada H, Otsubo Y, Kouketsu T, Suzuki S, Yokoyama A. The usefulness of angiotensin-(1-7) and des-Arg(9)-bradykinin as novel biomarkers for metabolic syndrome. Hypertens Res. 2021;44(8):1034-6. doi: 10.1038/s41440-021-00671-9. PubMed PMID: 34045691.

22. Liu X, Yang N, Tang J, Liu S, Luo D, Duan Q, Wang X. Downregulation of angiotensin-converting enzyme 2 by the neuraminidase protein of influenza A (H1N1) virus. Virus Res. 2014;185:64-71. doi: 10.1016/j.virusres.2014.03.010. PubMed PMID: 24662240; PMCID: PMC7114376.

23. Zou Z, Yan Y, Shu Y, Gao R, Sun Y, Li X, Ju X, Liang Z, Liu Q, Zhao Y, Guo F, Bai T, Han Z, Zhu J, Zhou H, Huang F, Li C, Lu H, Li N, Li D, Jin N, Penninger JM, Jiang C. Angiotensin-converting enzyme 2 protects from lethal avian influenza A H5N1 infections. Nat Commun. 2014;5:3594. doi: 10.1038/ncomms4594. PubMed PMID: 24800825; PMCID: PMC7091848.

24. Ricke DO. Two Different Antibody-Dependent Enhancement (ADE) Risks for SARS-CoV-2 Antibodies. Front Immunol. 2021;12:640093. doi: 10.3389/fimmu.2021.640093. PubMed PMID: 33717193; PMCID: PMC7943455.

25. Halstead SB, Katzelnick L. COVID-19 Vaccines: Should We Fear ADE? J Infect Dis. 2020;222(12):1946-50. doi: 10.1093/infdis/jiaa518. PubMed PMID: 32785649; PMCID: PMC7454712.

26. Pyzik M, Sand KMK, Hubbard JJ, Andersen JT, Sandlie I, Blumberg RS. The Neonatal Fc Receptor (FcRn): A Misnomer? Front Immunol. 2019;10:1540. doi: 10.3389/fimmu.2019.01540. PubMed PMID: 31354709; PMCID: PMC6636548.

27. Wysocki J, Schulze A, Batlle D. Novel Variants of Angiotensin Converting Enzyme-2 of Shorter Molecular Size to Target the Kidney Renin Angiotensin System. Biomolecules. 2019;9(12). doi: 10.3390/biom9120886. PubMed PMID: 31861139; PMCID: PMC6995632.

28. A Dose-escalation Study in Subjects With Pulmonary Arterial Hypertension (PAH). ClinicalTrials.gov identifier: NCT03177603. Updated April 21, 2020. Accessed July 2022. https://ClinicalTrials.gov/show/NCT03177603.

29. Recombinant Human Angiotensin-converting Enzyme 2 (rhACE2) as a Treatment for Patients With COVID-19. ClinicalTrials.gov identifier: NCT04335136. Updated August 2, 2021. Accessed July 2022. https://ClinicalTrials.gov/show/NCT04335136.

30. Khan A, Benthin C, Zeno B, Albertson TE, Boyd J, Christie JD, Hall R, Poirier G, Ronco JJ, Tidswell M, Hardes K, Powley WM, Wright TJ, Siederer SK, Fairman DA, Lipson DA, Bayliffe AI, Lazaar AL. A pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome. Crit Care. 2017;21(1):234. doi: 10.1186/s13054-017-1823-x. PubMed PMID: 28877748; PMCID: PMC5588692.

31. Hemnes AR, Rathinasabapathy A, Austin EA, Brittain EL, Carrier EJ, Chen X, Fessel JP, Fike CD, Fong P, Fortune N, Gerszten RE, Johnson JA, Kaplowitz M, Newman JH, Piana R, Pugh ME, Rice TW, Robbins IM, Wheeler L, Yu C, Loyd JE, West J. A potential therapeutic role for angiotensin-converting enzyme 2 in human pulmonary arterial hypertension. Eur Respir J. 2018;51(6). doi: 10.1183/13993003.02638-2017. PubMed PMID: 29903860; PMCID: PMC6613216.