JP2008529504A - Neutralizing monoclonal antibody against severe acute respiratory syndrome-related coronavirus - Google Patents

Abstract

The present invention provides isolated antibodies that bind to the receptor binding region of the spike protein of severe acute respiratory syndrome associated coronavirus (SARS-CoV) and competitively inhibit SARS-CoV binding to host cells. It is characterized by that. The mAb or substrate is 1) as a passive immunizing agent for preventing SARS-CoV infection, 2) as a biological agent for diagnosing SARS-CoV infection, 3) immunotherapy for SARS-CoV infection initial treatment And 4) It can be used as a probe to study the immunity, antigenicity, structure and function of SARS-CoVS protein.
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Description

  Severe acute respiratory syndrome (SARS) is a recently recognized severe respiratory disease with fever and is caused by infection with a new type of coronavirus (SARS-CoV) (Non-Patent Documents 1-5). Although the SARS pandemic has subsided, there is concern over the possibility of future recurrence, particularly the recently reported laboratory infection (Non-Patent Document 6). However, there is currently no treatment or prevention method that can counter this deadly virus (Non-Patent Documents 7 and 8).

  Like other coronaviruses, SARS-CoV is an enveloped virus and contains a large plus-strand RNA genome. The plus-strand RNA genome consists of a viral replicase protein and a structural protein including spike (S), membrane (M), envelope (E), nucleocapsid (N) and some proteins of unknown nature (Non-Patent Documents 4, 5, 9) ). Systematic analysis has shown that SARS-CoV differs in nature from the three known coronavirus antigenic groups. Therefore, post-genomic characterization of SARS-CoV is important for creating anti-SARS therapeutics and vaccines (Non-Patent Documents 10 and 11).

  Coronavirus infection occurs when the S protein attaches to a special host receptor and induces structural changes within the S protein. SARS-CoV S protein consists of a leader (residues 1-14), an extracellular domain (residues 15-1190), a transmembrane domain (residues 1191-1227) and a short cell tail (residues 1227-1255) It is a type 1 transmembrane glycoprotein of the expected length of 1255 amino acids including (Non-patent Document 5). Unlike many other coronaviruses such as mouse hepatitis virus (MHV) (Non-Patent Documents 12 and 13), SRS of SARS-CoV (Non-Patent Documents) No typical cleavage motif is identified in 5). Nevertheless, its S1 and S2 domains were predicted by sequence alignment of other coronavirus S proteins (Non-patent Documents 5 and 14). The SARS-CoV S protein, which contains a putative fusion peptide and a region of two seven amino acid repeats (HR1 and HR2), is involved in the fusion between the virus and the target cell membrane. The HR1 and HR2 regions combine to form a 6-helix bundle structure (Non-Patent Documents 15-18). This 6-helix bundle structure resembles the nucleus of fusion activity of HIV gp41 (Non-patent Document 19) and MHV S protein (Non-patent Documents 20, 21). The S1 region of the SARS-CoV S protein mediates virus binding to angiotensin converting enzyme 2 (ACE2), which is a functional receptor for SARS-CoV, in sensitive cells (Non-Patent Document 22-25). Recently, a small fragment of 193 amino acids in the S1 region (residues 318-510) has been recognized as the receptor binding region (RBD), and this region binds to ACE2 (Non-patent Documents 26-28). .

  This application claims the benefit of US Patent Application No. 60 / 651,046, filed February 8, 2005, and is a continuation of US Patent Application No. 11 / 141,925, filed May 31, 2005. This application claims the benefit of US Patent Application No. 60 / 576,118, filed June 2, 2004. This application is cited for various references. Accordingly, in order to describe in more detail the art to which the present invention pertains, such references and their accompanying disclosures are hereby incorporated by reference in their entirety.

Drosten, C, S. Gunther, W. Preiser, WS Van Der, HR Brodt, S. Becker, H. Rabenau, M. Panning, L. Kolesnikova, RA Fouchier, A. Berger, AM Burguiere, J. Cinatl, M Eickmann, N. Escriou, K. Grywna, S. Kramme, JC Manuguerra, S. Muller, V. Rickerts, M. Sturmer, S. Vieth, HD Klenk, AD Osterhaus, H. Schmitz, and HW Doerr. 2003. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N. Engl J. Med. 348: 1967-1976. Ksiazek, TG, D. Erdman, CS. Goldsmith, SR Zaki, T. Peret, S. Emery, S. Tong, C. Urbani, JA Comer, W. Lim, PE Rollin, SF Dowell, AE Ling, CD. Humphrey , WJ. Shieh, J. Guarner, CD. Paddock, P. Rota, B. Fields, J. DeRisi, JY Yang, N. Cox, JM Hughes, JW LeDuc, WJ. Bellini, and LJ. Anderson. 2003. A novel coronavirus associated with severe acute respiratory syndrome. N. Engl. J. Med. 348: 1953-1966. Peiris, JS, ST Lai, LL Poon, Y. Guan, LY Yam, W. Lim, J. Nicholls, WK Yee, WW Yan, MT Cheung, VC Cheng, KH Chan, DN Tsang, RW Yung, TK Ng, and KY Yuen. 2003. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 361: 1319-1325. Marra, MA, S JM Jones, CR. Astell, RA Holt, A. Brooks- Wilson, YSN Butterfield, J. Khattra, JK Asano, SA Barber, SY Chan, A. Cloutier, SM Coughlin, D. Freeman, N. Girn, OL Griffith, SR Leach, M. Mayo, H. McDonald, SB Montgomery, PK Pandoh, AS Petrescu, AG Robertson, JE Schein, A. Siddiqui, DE Smailus, JM Stott, GS Yang, F. Plummer, A. Andonov, H. Artsob, N. Bastien, K. Bernard, TF Booth, D. Bowness, M. Czub, M. Drebot, L. Fernando, R. Flick, M. Garbutt, M. Gray, A. Grolla, S Jones, H. Feldmann, A. Meyers, A. Kabani, Y, Li, S. Normand, U. Stroher, GA Tipples, S. Tyler, R. Vogrig, D. Ward, B. Watson, RC Brunham, M Krajden, M. Petric, DM Skowronski, C Upton, and RL Roper. 2003. The genome sequence of the SARS-associated coronavirus. Science 300: 1399-1404. Rota, PA, MS Oberste, SS Monroe, WA Nix, R. Campagnoli, JP Icenogle, S. Penaranda, B. Bankamp, K. Maher, MH Chen, S. Tong, A. Tamin, L. Lowe, M. Frace , JL DeRisi, Q. Chen, D. Wang, DD Erdman, TCT Peret, C. Bums, TG Ksiazek, PE Rollin, A. Sanchez, S. Liffick, B. Holloway, J. Limor, K. McCaustland, M. Olsen-Rasmussen, R. Fouchier, S. Gunther, ADME Osterhaus, C. Drosten, MA Pallansch, LJ. Anderson, and WJ Bellini. 2003. Characterization of a Novel Coronavirus Associated with Severe Acute Respiratory Syndrome. Science 300: 1394-1399 . Fleck, F. 2004. SARS virus returns to China as scientists race to find effective vaccine. Bull, World Health Organ. 82: 152-153. Marshall, E. and M. Enserink. 2004. Medicine. Caution urged on SARS vaccines. Science 303: 944-946. Peiris, J. S., Y. Guan, and K.Y. Yuen. 2004. Severe acute respiratory syndrome. Nat. Med. 10: S88-S97. Qin, E., Q. Zhu, M. Yu, B. Fan, G. Chang, B. Si, B. Yang, W. Peng, f. Jiang, B. Liu, Y. Deng, H. Liu, Y Zhang, C. Wang, Y. Li, Y. Gan, X. Li, F. Lu, G. Tan, W. Cao, R. Yang, J. Wang, W. Li, Z. Xu, Y. Li , Q. Wu, W. Lin, Y. Han, G. Li, W. Li, H. Lu, J. Shi, Z. Tong, F. Zhang, S. Li, B. Liu, S. Liu, W Dong, J. Wang, GKS Wong, J. Yu, and H. Yang. 2003. A complete sequence and comparative analysis of a SARS- associated virus (isolate BJOl). Chin. Sci. Bull 48: 941-948. Holmes, K. V. and L. Enjuanes. 2003. Virology: The SARS coronavirus: a postgenomic era. Science 300: 1377-1378. Holmes, K.V. 2003.SARS coronavirus: a new challenge for prevention and therapy.J.CHn..Invest! 11: 1605-1609. Frana, M.F., J.N. Behnke, L.S. Sturman, and K.V.Holmes. 1985.Proteolytic cleavage of the E2 glycoprotein of murine coronavirus: host-dependent differences in proteolytic cleavage and cell fusion.J. Virol. 56: 912-920. Luytjes, W., LS Sturman, PJ. Bredenbeek, J. Charite, BA van der Zeijst, MC Horzinek, and WJ. Spaan. 1987. Primary structure of the glycoprotein E2 of coronavirus MHV-A59 and identification of the trypsin cleavage site. Virology 161: 479-487. Spiga, O., A. Bernini, A. Ciutti, S. Chiellini, N. Menciassi, F. Finetti, V. Causarono, F. Anselmi, F. Prischi, and N. Niccolai, 2003. Molecular modeling of Sl and S2 subunits of SARS coronavirus spike glycoprotein. Biochem. Biophys. Res. Commun. 310: 84. Bosch, BJ, BE Martina, ZR Van Der, J. Lepault, BJ. Haijema, C. Versluis, AJ. Heck, R. De Groot, AD Osterhaus, and PJ. Rottier. 2004. Severe acute respiratory syndrome coronavirus (SARS- CoV) infection inhibition using spike protein heptad repeat-derived peptides. Proc. Natl. Acad. ScI USA 101: 8455-8460. Ingallinella, P., E. Bianchi, M. Finotto, G. Cantoni, DM Eckert, VM Supekar, C. Bruckmann, A. Carfi, and A. Pessi. 2004. Structural characterization of the fusion- active complex of severe acute respiratory syndrome (SARS) coronavirus. Proc. Natl. Acad ScL USA 101: 8709-8714. Liu, S., G. Xiao, Y. Chen, Y. He, J. Niu, C. Escalante, H. Xiong, J. Farmar, AK Debnath, P. Tien, and S. Jiang. 2004. Interaction between the heptad repeat 1 and 2 regions in spike protein of SARS-associated coronavirus: implication for virus fusogenic mechanism and identification of fusion inhibitors. Lancet 363: 938-947. Tripet, B., M. W. Howard, M. Jobling, R.K.Holmes, K. V. Holmes, and R.S.Hodges. 2004.Structural characterization of the SARS -coronavirus spike S fusion protein core.J. Biol Chem. 279: 20836-20849. Chan, D.C., D. Fass, J.M.Berger, and P.S.Kim.1997.Core structure of gp41 from the HIV envelope glycoprotein.Cell 89: 263-273. Bosch, BJ., ZR van der, CA. de Haan, and PJ. Rottier. 2003. The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex. J. Virol. 77: 8801 -8811. Xu, Y., Z. Lou, Y. Liu, H. Pang, P. Tien, GF Gao, and Z. Rao. 2004.Crystal structure of SARS-CoV spike protein fusion core.J. Biol. Chem. 279: 49414-49419. Li, WH, MJ. Moore, NY Vasilieva, JH Sui, SK Wong, AM Berne, M. Somasundaran, JL Sullivan, K. Luzuriaga, TC Greenough, HY Choe, and M. Farzan. 2003. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus.Nature 426: 450-454. Dimitrov, D.S. 2003.The secret life of ACE2 as a receptor for the SARS Virus.Cell 115: 652-653. Prabakaran, P., X. Xiao, and D.S. Dimitrov. 2004. A model of the ACE2 structure and function as a SARS-CoV receptor. Biochem. Biophys. Res. Commun. 314: 235-241. Wang, P., J. Chen, A. Zheng, Y. Nie, X. Shi, W. Wang, G. Wang, M. Luo, H. Liu, L. Tan, X. Song, Z. Wang, X Yin, X. Qu, X. Wang, T. Qing, M. Ding, and H. Deng. 2004. Expression cloning of functional receptor used by SARS coronavirus. Biochem. Biophys. Res. Commun. 315: 439-444. Xiao, X., S. Chakraborti, A.S. Dimitrov, K. Gramatikoff, and D.S.Dimitrov. 2003. The SARS-CoV S glycoprotein: expression and functional characterization. Biochem. Biophys. Res. Commun. 312: 1159-1164. Wong, SK, W. Li, MJ. Moore, H. Choe, and M. Farzan. 2004. A 193-amino-acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2. J. Biol Chem. 279 : 3197-3201. Babcock, GJ, DJ.Esshaki, WD Thomas, Jr., and DM Ambrosino. 2004. Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor.J. Virol. 78: 4552-4560 . Cavanagh, D. 1995. The coronavirus surface glycoprotein. In The Coronaviridae. S. G. Siddell, editor. Plenum Press, New York and London. 73-114. Saif, LJ. 1993. Coronavirus immunogens. Vet. Microbiol. 37: 285-297. Buchholz, UJ., A. Bukreyev, L. Yang, EW Lamirande, BR Murphy, K. Subbarao, and PL Collins. 2004. Contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity.Proc. Natl. Acad. Sci. USA 101: 9804-9809. Yang, Z. Y., W.P. Kong, Y. Huang, A. Roberts, B.R. Murphy, K. Subbarao, and GJ. Nabel. 2004. A DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice.Nature 428: 561-564. Bisht, H., A. Roberts, L. Vogel, A. Bukreyev, PL Collins, BR Murphy, K. Subbarao, and B. Moss. 2004. Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice. Proc. Natl. Acad. Sci. USA 101: 6641-6646. Bukreyev, A., EW Lamirande, UJ Buchholz, LN Vogel, WR Elkins, M. St Claire, BR Murphy, K. Subbarao, and PL Collins. 2004. Mucosal immunization of African green monkeys (Cercopithecus aethiops) with an attenuated parainfluenza virus expressing the SARS coronavirus spike protein for the prevention of SARS. Lancet 363: 2122-2127. He, Y., Y. Zhou, S. Liu, Z. Kou, W. Li, M. Farzan, and S. Jiang. 2004. Receptor- binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine. Biochem. Biophys. Res. Commun. 324: 773-781. He, Y., Y. Zhou, P. Siddiqui, and S. Jiang. 2004. Inactivated SARS-CoV vaccine elicits high titers of spike protein-specific antibodies that block receptor binding and virus entry.Biochem. Biophys. Res. Commun. 325: 445-452 Morrison, S.L., M.J.Johnson, L.A. Herzenberg, and V.T.Oi. 1984. Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains.Proc. Natl. Acad. Sci. USA 81: 6851-6855. Boulianne, G.L., N. Hozumi, and M.J.Shulman. 1984. Production of functional chimaeric mouse / human antibody.Nature 312: 643-646. Nisihara, T., Y. Ushio, H. Higuchi, N. Kayagaki, N. Yamaguchi, K. Soejima, S. Matsuo, H. Maeda, Y. Eda, K. Okumura, and H. Yagita. 2001. Humanization and epitope mapping of neutralizing anti-human Fas ligand monoclonal antibodies: structural insights into Fas / Fas ligand interaction. J. Immunol. 167: 3266-3275. Winter, G. and C. Milstein. 1991. Man-made antibodies. Nature 349: 293-299. Foote, J. and G. Winter. 1992. Antibody framework residues affecting the conformation of the hypervariable loops. J. MoI. Biol. 224: 487-499. Zhang, H., G. Wang, J. Li, Y. Me, X. Shi, G. Lian, W. Wang, X. Yin, Y. Zhao, X. Qu, M. Ding, and H. Deng. 2004. Identification of an antigenic determinant on the S2 domain of the severe acute respiratory syndrome coronavirus spike glycoprotein capable of inducing neutralizing antibodies. J Virol 78: 6938-6945. Nie, Y., G. Wang, X. Shi, H. Zhang, Y. Qiu, Z. He, W. Wang, G. Lian, X. Yin, L. Du, L. Ren, J. Wang, X He, T. Li, H. Deng, and M. Ding. 2004. Neutralizing antibodies in patients with severe acute respiratory syndrome-associated coronavirus infection. J Infect. Dis. 190: 1119-1126. Hofmann, H., K. Hattermann, A. Marzi, T. Gramberg, M. Geier, M. Krumbiegel, S. Kuate, K. Uberla, M. Niedrig, and S. Pohlmann. 2004. S protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients.J Virol 78: 6134-6142. Lu, L., I. Manopo, BP Leung, HH Chng, AE Ling, LX. Chee, EE Ooi, SW Chan, and J. Kwang. 2004. Immunological characterization of the spike protein of the severe acute respiratory syndrome coronavirus. Microbiol. 42: 1570-1576. He, Y., Y. Zhou, H. Wu, B. Luo, J. Chen, W. Li, and S. Jiang. 2004. Identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (SARS) coronavirus: implication for developing SARS diagnostics and vaccines. J. Immunol. 173: 4050-4057. Wilson, J.A., M. Hevey, R. Bakken, S. Guest, M. Bray, A.L.Schmaljohn, and M.K.Hart. 2000. Epitopes involved in antibody-mediated protection from Ebola virus.Science 287: 1664-1666. Zwick, MB, LL Bonnycastle, A. Menendez, MB Irving, CF. Barbas, III, PW Parren, DR Burton, and JK Scott. 2001. Identification and characterization of a peptide that specifically binds the human, broadly neutralizing anti-human immunodeficiency virus type 1 antibody bl2. J Virol 75: 6692-6699. Sui, J., W. Li5 A. Murakami, A. Tamin, LJ. Matthews, SK Wong, MJ. Moore, AS Tallarico, M. Olurinde, H. Choe, LJ. Anderson, WJ. Bellini, M. Farzan, and WA Marasco. 2004. Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to Sl protein that blocks receptor association.Proc. Natl. Acad. Sci. USA lOl: 2536-2541. Subbarao, K., J. McAuliffe, L. Vogel, G. Fahle, S. Fischer, K. Tatti, M. Packard, WJ. Shieh, S. Zaki, and B. Murphy. 2004. Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice.J Virol 78: 3572-3577. ter Meulen, J., ABH Bakker, EN van den Brink, GJ. Weverling, BEE Martina, BL Haagmans, T. Kuiken, J. de Kruif, W. Preiser, W. Spaan, HR Gelderblom, J. Goudsmit, and ADME Osterhaus. 2004. Human monoclonal antibody as prophylaxis for SARS coronavirus infection in ferrets. Lancet 363: 2139-2141. Traggiai, E., S. Becker, K. Subbarao, L. Kolesnikova, Y. Uematsu, MR Gismondo, BR Murphy, R. Rappuoli, and A. Lanzavecchia. 2004. An efficient method to make human monoclonal antibodies from memory B cells : potent neutralization of SARS coronavirus. Nat Med. 10: 871-875. WeIt5 S., CR. Divgi, FX Real, SD Yeh, P. Garin-Chesa, CL. Finstad, J. Sakamoto, A. Cohen, ER Sigurdson, N. Kemeny, and. 1990. Quantitative analysis of antibody localization in human metastatic colon cancer: a phase I study of monoclonal antibody A33. J Clin Oncol 8: 1894-1906. Welt, S. and G. Ritter. 1999.Antibodies in the therapy of colon cancer.Semin.Oncol. 26: 683-690. Breedveld, F.C. 2000.Therapeutic monoclonal antibodies. Lancet 355: 735-740. Xu, ZK, LX Wei, LY Wang, HT Wang, and S. Jiang. 2002. The In vitro and in vivo protective activity of monoclonal antibodies directed against Hantaan virus: potential application for immunotherapy and passive immunization. Biochem. Biophys. Res. Commun. 298: 552-558.

  The S protein of coronavirus is a major antigenic determinant that induces the production of neutralizing antibodies (Non-patent Documents 29, 30). Therefore, S protein is inevitably used as an antigen for vaccine development (Non-patent Document 30). Recently, it has been found that SARS-CoV S protein is a major inducer of protective immunity among structural proteins (Non-patent Document 31). Yang, et al. (Non-Patent Document 32), DNA vaccine candidates encoding S protein are SARS-CoV neutralization (neutralizing antibody titers range from 1:25 to 1: 150) and protection in mice It was reported that immunity was induced and the protective effect was mediated by neutralizing antibodies rather than T cell dependent mechanisms. Bishto et al. (33) reported that SARS-CoV S protein expressed by attenuated vaccinia virus (MVA) is S-specific antibody and SARS-CoV infection using SARS-CoV neutralizing antibody titer 1: 284. It was demonstrated to induce protective immunity of mice against. This is indicated by the reduced titer of SARS-CoV in the respiratory tract of mice after challenge infection. Bukleyev et al. (34) show that mucosal immunity of African green monkeys using attenuated parainfluenza virus (BHPIV3) expressing SARS-CoV S protein is moderate in the range of 1: 8 to 1:16. He reported neutralizing antibodies with a summed titer and protecting animals from attack. Although these data indicate that the SARS-CoV S protein is a protective antigen capable of inducing neutralizing antibodies, its antigenic determinants remain unclear.

  Recently, we have found that the receptor binding region of SARS-CoV S protein is neutralized in SARS-CoV infected patients and animals immunized with inactivated virus and S protein (35,36). It was proved to be the main target of the antibody. Therefore, recombinant RBD of SARS-CoV S protein was used as an immunogen to induce monoclonal antibody neutralization (mAb).

  The present invention can bind to the receptor binding region (RBD) in the spike (S) protein of severe acute respiratory syndrome-related coronavirus (SARS-CoV), and the binding of SARS-CoV to the host cell is competitive. An isolated antibody is provided that is suppressible. Furthermore, the present invention provides a substance provided with the complementarity determining region of the monoclonal antibody, and the substance can recognize the same epitope as the monoclonal antibody.

  In one embodiment, the aforementioned substance is an antibody. In one preferred embodiment, the antibody is a neutralizing antibody. The present invention also provides single chain antibodies or antibody fusion constructs (humanized antibodies and chimeric antibodies as described above).

  The intent of this application is to encompass different chimeric constructs using the antibodies of the present invention. The invention also encompasses all humanized constructs of the antibody. In one example, the isolated antibody described above is linked directly or indirectly to a cytotoxic agent.

  The present invention also provides a cell containing the antibody. The present invention further provides a nucleic acid molecule encoding the antibody. The present invention further provides a nucleic acid molecule capable of hybridizing specifically with the molecule. Such nucleic acid molecules include, but are not limited to, synthetic DNA or RNA, genomic DNA and RNA.

  The present invention further provides a vector comprising the nucleic acid molecule or a portion of the nucleic acid molecule. In one embodiment, the vector is an expression vector and is capable of expressing a protein encoded by the nucleic acid molecule. The present invention further comprises a cell containing the nucleic acid molecule. The cells can be used for expression.

  The present invention is a method of producing antibodies. The antibody can bind to the receptor binding region (RBD) in the spike (S) protein of SARS-CoV, and can competitively suppress the binding between SARS-CoV and the host cell. The method comprises the step of allowing the regulatory element to express the antibody by appropriately binding the nucleic acid molecule described above to an appropriate regulatory element, and allowing the bound nucleic acid molecule to express the antibody. Subjecting to appropriate conditions and recovering the expressed antibody, thereby producing the antibody. The present invention also provides an antibody produced by the method.

  The present invention provides a composition comprising an effective amount of the monoclonal antibody and a suitable carrier. The present invention further provides a pharmaceutical composition comprising an effective amount of the monoclonal antibody and a pharmaceutically acceptable carrier.

  The present invention also provides a method of treating SARS-CoV infection using the pharmaceutical composition. The present invention further provides a method for preventing SARS-CoV infection using the pharmaceutical ingredient.

  The present invention also provides a method for detecting SARS-CoV (or cells infected with SARS-CoV). The method comprises the step of contacting with the antibody or derivative thereof, wherein the antibody or derivative thereof is capable of forming a complex with a receptor binding region (RBD) in the SARS-CoV spike (S) protein. Under certain circumstances, it can bind to the RBD of the viral S protein, allowing detection of the complex formed.

  Finally, the present invention provides a method for screening compounds. The compound can inhibit infection of the virus by blocking the binding of acute respiratory syndrome-related coronavirus (SARS-CoV) to a host cell receptor. The method comprises the steps of: (a) constructing a system in which the antibody binds to the receptor binding region (RBD) in the spike (S) protein of SARS-CoV, and (b) the compound comprises the system (a) Contacting. In this step, the compound reduces the binding of RBD to the RBD in the SARS-CoV S protein, thereby allowing the compound to prevent the binding, and the compound is caused by the RBD in the SARS-CoV S protein. It becomes possible to control infection. The present invention further provides a compound obtained as a result of screening. The compound can be used as a treatment or prevention of severe acute respiratory syndrome (SARS).

  The present invention provides a kit comprising a compartment containing an antibody capable of recognizing SARS virus.

  In the present invention, the receptor binding region (RBD) contains multiple conformation-dependent neutralizing epitopes that induce a group of potent neutralizing monoclonal antibodies (mAbs), which RBD treats SARS. Clarify that it can be used for diagnosis and prevention.

  The present invention provides isolated monoclonal antibodies. The antibody binds to the receptor binding domain (RBD) of the severe acute respiratory syndrome-related coronavirus (SARS-CoV) spike (S) protein to competitively suppress the binding of SARS-CoV to the host cell. To do.

  The present invention also provides a substance comprising the complementarity determining region of the above monoclonal antibody. The substance can bind to the same epitope as the monoclonal antibody described above. Such substances include, but are not limited to, polypeptides, small molecules, antibodies or antibody fragments. In one preferred embodiment, the antibody is a neutralizing antibody. In other examples, the antibody is a single chain antibody or an antibody fusion construct, a humanized antibody or a chimeric antibody as described above. The intent of this application is to encompass different chimeric constructs using the invented constructs. The invention also extends to all humanized antibody constructs. Techniques for making chimeric or humanized antibodies are well known. For example, refer to references (Non-Patent Documents 37 and 38) for chimeric antibodies, and references (Non-Patent Documents 39 to 41) for humanized antibodies. One of ordinary skill in the art will be able to modify the sequence of the above materials based on the present disclosure. The modification includes addition, deletion, or mutation of a specific amino acid sequence in the fragment. General methods for generating antibodies are well known to those skilled in the art. See (eg, Using Antibodies: A Laboratory Manual: Portable Protocol No. 1 by Ed. Harlow (1998)).

  In one embodiment, the isolated antibody described above is linked directly or indirectly to one or more cytotoxic agents. Such cytotoxic substances include, but are not limited to, radionucleotides or other toxins. The present invention also provides a cell comprising an antibody. The present invention further provides nucleic acid molecules encoding the above-described antibodies. Once the antibody is isolated, the gene encoding the antibody can be isolated and the nucleic acid sequence identified. Accordingly, the present invention further provides nucleic acid molecules that can specifically hybridize with the above-described molecules. Such nucleic acid molecules include, but are not limited to, synthetic DNA or RNA, genomic DNA, cDNA and RNA.

  The present invention also provides a vector comprising the above-described nucleic acid molecule or a portion thereof. The portion may be a functional portion that performs a specific function. The fragment or partial sequence can also encode a functional region of a functional protein. In one embodiment, the vector is an expression vector and is capable of expressing a protein encoded by a nucleic acid molecule. The present invention further provides a cell comprising the above-described nucleic acid molecule. The cells can also be used for expression. Vectors are known to those skilled in the art. See (eg, Graupner, U.S. Patent. No. 6,337,208, “Cloning Vector,” issued January 8, 2002). See (Schumacher et. Al., U.S. Patent. No. 6,190,906, "Expression Vector for the Regulatable Expression of Foreign Genes in Prokaryotes," issued February. 20, 2001). In one example, the vector is a plasmid.

  The present invention provides a method for generating antibodies capable of binding to the spike (S) protein receptor binding region (RBD) of SARS-CoV. The antibody competitively inhibits the binding of SARS-CoV to the host cell. The method includes the step of appropriately binding the nucleic acid molecule described above to a regulatory element so that the regulatory element expresses the antibody, and the appropriate conditions for allowing the bound nucleic acid molecule to express the antibody. Producing an antibody comprising the step of placing underneath and recovering the expressed antibody. The present invention also provides an antibody produced by the method.

  Hybridoma cell lines, 32H5 (Conf I), 31H12 (Conf II), 18D9 (Conf III), 30F9 (Conf IV), 33G4 (Conf V) and 19B2 (Conf VI), are international Deposited with the American Strains Conservation Agency (ATCC), (10801 University Blvd., Manassas, VA. 20110, USA) on January 13, 2005, based on the Butabest Convention on Approval. Cell lines 32H5 (Conf I), 31H12 (Conf II), 18D9 (Conf III), 30F9 (Conf IV), 33G4 (Conf V) and 19B2 (Conf VI) are ATCC accession numbers PTA-6525, PTA-6524, PTA-6521, PTA-6523, PTA-6526, and PTA-6522 were given, respectively.

  The present invention also provides epitopes recognized by the monoclonal antibodies described above. The sequence or conformation of the epitope is important for diagnostic or therapeutic use.

  The present invention provides a composition comprising an effective amount of the monoclonal antibody described above and a suitable carrier. The effective amount is determined by a series of experiments. The present invention further provides a pharmaceutical composition comprising an effective amount of the above monoclonal antibody and a pharmaceutically acceptable carrier. As used herein, a pharmaceutically acceptable carrier means a general pharmaceutical carrier. Examples of suitable carriers include carriers known to those skilled in the art, but are not limited and may be any general pharmaceutical carrier, including phosphate buffered saline and phosphate buffered saline containing Polysorb 80. Examples include water, water, emulsifiers such as oil / water emulsifiers and various wetting agents. Other carriers include sterile aqueous solutions, tablets, coated tablets and capsules. Generally, the carrier is starch, milk, sugar, certain clays, gelatin, stearic acid or stearate, magnesium or calcium stearate, talc, vegetable fat or vegetable oil, resin, and glycol or other Contains excipients such as known excipients. The carrier can also include flavor and color additives or other ingredients. The composition comprising the carrier is formulated using known conventional techniques.

  The present invention provides a method for treating SARS-CoV infection using the pharmaceutical composition described above. The present invention further provides a method for preventing SARS-CoV infection using the above-described pharmaceutical composition. The present invention further provides a method for detecting SARS-CoV (or SARS-CoV infected cells), which comprises the steps of contacting with an antibody or derivative thereof and detecting the complex formed. The antibody or derivative thereof can bind to the virus spike (S) protein receptor binding region (RBD) under conditions that allow the antibody or derivative thereof and SARS-CoV S protein RBD to form a complex. .

Finally, the present invention provides a method of screening for compounds that can suppress SARS-CoV infection. The suppression is accomplished by inhibiting the binding of the virus to the host cell receptor. The screening method includes the following steps.
(A) Construction of a system in which an antibody binds to a SARS-CoV spike (S) protein receptor binding region (RBD) (b) A compound is contacted with the system of (a), whereby the above antibody and SARS-CoVS protein are received Decreased binding to the body binding region (RBD) indicates that the compound can inhibit the binding, thereby suppressing SARS-CoV infection by the S protein RBD.
The present invention further provides a compound obtained as a result of screening. The compounds can be used for the treatment or prevention of severe acute respiratory syndrome (SARS).

  The present invention provides a kit comprising a compartment containing an antibody capable of recognizing SARS virus and / or a substance that competitively suppresses binding with the antibody.

  The present invention provides that RBD has multiple conformation-dependent neutralizing epitopes that induce a group of potent neutralizing monoclonal antibodies (mAbs) that are used for the treatment, diagnosis and prevention of SARS Clarify that it is possible.

  The present invention will be better understood with reference to the following experimental details, but for those skilled in the art, the details are merely illustrative and the following claims as set forth herein It should be noted that the invention defined in the section is not limited.

[Experiment details]
SP2 / 0 bone marrow cells and spleen cells derived from Balb / c mice were fused to prepare 27 hybridoma clones. Balb / c mice were immunized with a fusion protein containing an Fc fragment of human IgG1 (shown as RBD-Fc) bound to the spike (S) protein receptor binding region (RBD) of SARS-CoV. Of the 27 monoclonal antibodies (mAbs) generated from the hybridoma clones, all mAbs recognized higher-order structure-dependent epitopes except 2 mAb bound to the adjacent linear epitope. Based on the results obtained from the binding competition study, these 25 conformation specific mAbs are classified into 6 groups and designated ConfI-VI. ConfIV and ConfV mAb significantly inhibited the binding of RBD-Fc and ACE2 (SARS-CoV receptor), suggesting that these epitopes overlap with the S protein receptor binding site. Most mAbs (23/25) recognize conformational epitopes, which have strong neutralizing activity against SARS pseudovirus and have a 50% neutralization (ND ₅₀ ) of 0.005 to 6. It reaches 569 μg / ml.

The mAb that neutralizes the SARS-CoV can be used as in the following 1) to 3).
1) Immunotherapy for initial treatment of SARS-CoV infection 2) Biological agent for diagnosis of SARS-CoV infection 3) Probes for studying immunogenicity, antigenicity, structure and function of SARS-CoVS protein Of mouse mAbs can be humanized for the treatment and prevention of SARS-CoV infection.

[Materials and methods]
<Immunization of mice and generation of mAb>
5 Balb / c mice (4 weeks old) were immunized by subcutaneous injection of protein A sepharose purified RBD-Fc (20 μg) in combination with MLP + TDM adjuvant system (Sigma, Saint Louis, MI). Adjustment of protein A sepharose of RBD-Fc was performed as described above (Non-patent Document 35). Thereafter, booster immunization was performed at 10-week intervals with 10 μg of the same antigen and MLP + TDM adjuvant. Mouse antiserum was collected and anti-RBD antibody and SARS-CoV neutralizing antibody were detected.

  Hybridomas producing anti-RBD mAbs were made using conventional methods. Briefly, splenocytes were collected from immunized mice and fused with SP2 / 0 myeloma cells. Cell culture supernatants collected from wells containing hybridoma colonies were screened by enzyme-linked immunoassay (ELISA) using S1-C9 as a coated antigen as described above (Non-patent Document 35). The amount of cells in the well that showed a positive reaction was increased and remeasured. Cultures that showed a positive reaction were subcultured to create a stable hybridoma cell line. The mAb was purified from the culture supernatant using Protein A Sepharose 4 Fast Flow (Amersham Biosciences).

<ELISA and binding competition>
The reactivity of mouse serum or mAb with various antigens was determined by ELISA. Briefly, a 96-well microtiter plate (Corning Costar, Acton, MA) using 1 μg / ml recombinant protein (RBD-Fc or S1-C9) or purified human IgG (Zymed, South San Francisco, Calif.), Respectively. Was coated. The coating was carried out by standing overnight at 4 ° C. in 0.1 M carbonate buffer (pH 9.6). After blocking with 2% non-fat milk, serially diluted mouse serum or mAb was added, incubated at 37 ° C. for 1 hour, and washed 4 times with PBS containing 0.1% Tween 20. The bound antibody was HRP-conjugated goat anti-mouse IgG (Zymed), incubated at 37 ° C. for 1 hour, and detected after washing. The reaction was visualized by adding the substrate 3,3 ′, 5,5 ′, tetramethylbenzidine (TMB), and the absorbance was measured at 450 nm using an ELISA plate reader (Tecan US, Research Triangle Park, NC). did.

  To determine the effect of disulfide bond reduction on the binding of RBD-specific mAbs, ELISA plates were coated with 1 μg / ml concentration of recombinant RBD-Fc or S1-C9 and 10 mM dithiothreitol (DDT). Treated for 1 hour at 37 ° C. and washed. The wells were then treated with 50 mM iodoacetamide for 1 hour at 37 ° C. After washing, the standard ELISA described above was performed.

  A competitive ELISA was performed to determine the inhibitory activity of RBD-specific mAbs on the binding of biotinylated mAbs and RBD-Fc. Briefly, ELISA plate wells were coated with 1 μg / ml RBD-Fc as described above. A mixture containing 50 μg / ml unlabeled mAb and 1 μg / ml biotinylated mAb was added and incubated at 37 ° C. for 1 hour. Biotinylated mAb binding was detected by sequentially adding HRP-conjugated streptavidin (Zymed) and TMB. The biotinylation of mAb was carried out using an EZ-linked NHS-PEO solid phase biotinylation kit {Pierce, Rockford, IL) according to the attached procedure.

<Neutralization of SARS pseudovirus infection>
Traditional neutralization assays using live SARS-CoV are cumbersome and need to be performed in a BSL-3 facility. Therefore, the SARS-CoV pseudovirus system (Non-patent Documents 27, 32, 42, 43) was adapted in the laboratory. The assay is sensitive, quantitative and can be performed in a BSL-2 facility. SARS pseudovirus producing SARS-CoVS protein and a defective HIV-1 genome expressing luciferase as a reporter were prepared as described above (Non-patent Documents 27, 42, 43). Briefly, 293T cells were transformed into a plasmid encoding the codon-optimized SARS-CoVS protein and a plasmid encoding the HIV-1 genome lacking Env and expressing luciferase (pNL4-3.luc.RE ) And FuGENE 6 transfection reagent {Fugene 6 reagents} (Boehringer Mannheim). The supernatant containing SARS pseudovirus was cultured for 48 hours after infection and used for single cycle infection of 293T (293T / ACE2) cells transformed with ACE2. Briefly, 293T / ACE2 cells were seeded at 10 ⁴ cells / well in 96-well tissue culture plates and cultured overnight. Prior to addition to the cells, the pseudovirus-containing supernatant was pre-cultured at 37 ° C. for 1 hour using mouse serum or mAb serially diluted 2-fold. After 24 hours, a fresh medium was added and further cultured for 48 hours. The cells were washed with PBS and lysed with a lysing agent attached to the luciferase kit (Promega Madison, Wis.). An aliquot of the lysed cells was transferred to a 96-well coaster flat-bottom luminometer plate (Corning Costar, Corning, NY) and luciferase substrate (Promega) was added. Relative luminescence (RLU) was immediately determined with an Ultra 384 luminometer (Tecan US).

<Inhibition of RBD-Fc binding to receptor by mAb>
Inhibition of mAb on binding of RBD-Fc and ACE2-expressing cells was measured using flow cytometry. Briefly, 10 ⁶ 293 / ACE2 cells were detached, harvested and washed with Hank's balanced salt solution (HBSS) (Sigma, St. Louis, MO). RBD-Fc was added to the cells at a final concentration of 1 μg / ml in the presence or absence of 50 μg / ml mAb and incubated at room temperature for 30 minutes. Cells were washed with HBSS and incubated with an anti-human IgG-FITC conjugate (Zymed) diluted 1:50 for an additional 30 minutes at room temperature. After washing, the sample was fixed with PBS containing 1% formaldehyde, and analyzed with a Becton FACSC Calibur flow cytometer {Becton FACSC Calibur flow cytometer} (Mountain View, CA) using Cell Quest software {CellQuest software}.

  Inhibition of RBD-Fc binding to water-soluble ACE2 by mAb was measured using ELISA. Briefly, 96-well ELISA plates (Corning Costar) were used at 2 ° C. in 0.1 M carbonate buffer (pH 9.6) using 2 μg / ml recombinant water-soluble ACE2 (R & D systems, Inc., Minneapolis, Minn.). Left overnight to coat. After blocking with 2% non-fat milk, 1 μg / ml RBD-Fc was added to the wells in the presence or absence of 50 μg / ml mouse mAb and incubated at 37 ° C. for 1 hour. After washing, HRP-conjugated goat anti-human IgG (Zymed) was added and incubated for an additional hour. After washing, the substrate was detected using TMB.

[result]
<Isolation and initial evaluation of RBD-specific mAb>
RBD-Fc fusion protein was transiently expressed in 293T cells and purified to homogeneity using protein A. Five mice (A to E) were immunized 4 times with RBD-Fc in the presence of Ribi adjuvant. All animals showed a significant antibody response to RBD-Fc after the first boost and antibody titers increased with immunization. Antisera were collected on day 4 after the third boost and showed very strong neutralizing activity against SARS pseudovirus producing SARS-CoV and S protein.

  A group of 27 RBD-specific mAbs were prepared by fusing spleen cells and Sp2 / 0 myeloma cells from RBD-Fc immunized mice and detecting hybridomas using S1-C9 as an antigen. The epitope specificity of the mAb was initially determined by ELISA using RBD-Fc, DTT reduced RBD-Fc, S1-C9, DTT reduced S1-C9 and purified human IgG as a coated antibody (Table 1). Most mAbs (25/27) reacted with native RBD-Fc and S1-C9, whereas DTT reduced RBD-Fc and DTT reduced S1-C9 showed no response. This suggested that the mAb recognizes a disulfide bond-dependent conformational epitope expressed on the S protein RBD. The other 2 mAbs (4D5 and 17H9) recognized both natural and reduced RBD-Fc and S1-C9, suggesting that the mAb recognizes a linear epitope on RBD. None of the mAbs detected by S1-C9 reacted with human IgG, whereas control antisera from mice immunized with RBD-Fc reacted with human IgG (Table 1).

  Since mAbs 4D5 and 17H9 reacted with reduced RBD-Fc and S1-C9, it was possible that the epitope could be mapped by a synthetic peptide. Using a group of 27 overlapping peptides covering the S protein RBD, the 4D5 and 17H9 epitopes were located by ELISA. As shown in FIG. 1, 4D5 reacted with peptide 435-451 (NYNYKYRYLRHGKLRPF), and 17H9 reacted with two overlapping peptides, 442-458 (YLRHGKLRPFERDISNV) and 449-465 (RPFERDISNVPFSPDGK). The 17H9 epitope was clearly mapped to the overlapping sequence of peptides 442-458 and 449-465 (RPFERDISNV), whereas the 4D5 epitope required most of the peptide 435-451 sequence. Peptide 435-451 overlaps with partial sequences of peptides 442-458 and 449-465. Thus, the 2 mAb recognizes an adjacent linear epitope in RBD. None of the conformation-dependent mAbs reacted with the tested peptides (data not shown).

<Epitope specificity of RBD-specific mAb identified by binding competition assay>
To evaluate conformation dependent epitopes, RBD-specific mAbs were classified by binding competition assay (Table 2). First, 1 mAb (10E7) in 27 mAb was biotinylated, and the inhibitory activity of 27 mAb on the binding between 10E7 and RBD-Fc was measured. mAbs 4D5 and 17H9 recognized a linear epitope. The epitope was mapped by the peptide described above and used as a control in the competition assay. About half of the conformation-dependent mAbs (13/25) competed with biotinylated 10E7, while the remaining mAbs did not inhibit the binding of 10E7 to RBD-Fc. Four non-competing mAbs (11E12, 33G4, 45B5, and 17H9) were then biotinylated and tested as in the binding competition assay. Of the 13 mAbs, 5 competed with biotinylated 10E7 and inhibited the binding of biotinylated 45B5 and RBD-Fc, so they were named as a group. Therefore, the 25 conformation dependent mAbs were classified into 6 different competitive groups (designated ConfI-VI). The two linear epitope dependent mAbs (4D5 and 17H9) did not compete with any conformation specific mAb. These results suggest that S protein RBD has multiple antigen structures. The structure elicits an antibody specific response in mice. However, the immunodominant epitope of RBD is conformation dependent.

<Evaluation analysis of mAb inhibiting receptor binding>
RBD-Fc can bind effectively to ACE2 and water-soluble ACE2 expressed on 293T / ACE2 cells and was measured by flow cytometry and ELISA, respectively (data not shown). It was tested whether RBD-specific mAb suppresses the binding of RBD-Fc to intracellular or water-soluble ACE2. As shown in FIG. 2, all mAbs in the ConfIV group (28D6, 30F9 and 35B5) and the ConfV group (24F4, 33G4 and 38D4) are highly consistent in binding RBD-Fc to both intracellular and water-soluble ACE2. Completely inhibited. All ConfIII group 2 mAbs (11E12 and 18D9) and 2 mAb (19B2 and 45F6) in ConfV group 4 mAb partially inhibited the binding of RBD-Fc to ACE2 expressed on 293T / AEC2 cells and water-soluble ACE2. Other mAbs included 2 mAb recognizing the linear sequence, and none showed a significant inhibitory effect on receptor binding. These results suggest that ConfIV and ConfV group mAbs recognize epitopes that overlap the conformational receptor binding site of the S protein, but the mAbs themselves compete with each other in binding competition assays. There wasn't. ConfIII group mAb and ConfVI group 2 mAb (19B2 and 45F6) may bind to conformational epitopes involved in receptor binding. Neither ConfI nor ConfII group mAbs inhibit receptor binding, suggesting that the mAb recognizes a conformational epitope that does not overlap with the RBD receptor binding site. These results reveal the isomerism of the RBD-specific mAb epitope and further suggest that the S protein RBD has multiple antigenic conformations.

<RBD-specific mAb has strong neutralizing activity>
Each RBD-specific mAb was tested for neutralizing activity against SARS pseudovirus. Remarkably, most conformation dependent mAbs (23/25) have strong neutralizing activity, with 50% neutralization (ND ₅₀ ) ranging from 0.005 to 6.569 μg / ml ( Table 3), on the other hand, 2 mAbs recognizing linear epitopes (4D5 and 17H9) and ConfVI group 1 mAb (44B5) were tested at high concentrations up to 100 μg / ml but did not neutralize SARS pseudovirus infection. ConfV group 33G4 mAb and ConfIV group 30F9 mAb inhibited receptor binding and showed high neutralizing activity against pseudovirus. Interestingly, ConfVI group 45F6 had relatively low pseudovirus neutralizing activity and partially inhibited the binding of RBD-Fc to ACE2. FIG. 3 shows the dose-dependent neutralizing activity of mAb representative of each group. These results suggest that S protein RBD predominantly induces neutralizing antibodies that recognize conformational epitopes.

[Experimental considerations]
Recent studies have shown that SARS-CoV S protein is one of the major antigens that elicit an immune response during infection (Non-Patent Documents 44-46). This suggests that S protein may serve as an immunogen that induces neutralizing mAbs. In the present study, RBD-Fc, a recombinant fusion protein, was used as an immunogen to immunize mice, and a hybridoma clone was created to produce 27 mAb. Most mAbs (25/27) recognized conformational epitopes, of which 23 mAb had strong neutralizing activity. Only 2 mAb was mapped near the linear epitope by overlapping peptides and could not neutralize infection with SARS pseudovirus. Interestingly, because conformation-dependent mAbs can be classified into six different groups based on binding competition experiments (eg, ConfI-VI), there are several different structural epitopes in RBD, It is suggested that neutralizing antibodies can be induced.

  All neutralizing mAbs against RBD are expected to be able to inhibit the interaction between RBD and ACE2. ACE is a functional receptor for SARS-CoV. On the other hand, we found that only mAbs that recognize ConfF and V can effectively inhibit the binding of RBD and ACE. Some mAbs that react with ConfIII and IV partially inhibit the interaction between RBD and ACE2. This suggests that the epitope may overlap with the receptor binding site on RBD, or that the binding of the mAb to RBD may cause conformational changes in the receptor binding site. It is conceivable to suppress the binding to ACE2. A mAb that recognizes ConfI and II does not show a significant effect on the binding of RBD to ACE2, but has strong neutralizing activity, so the mAb does not affect the interaction between RBD and ACE2. Without any SARS-CoV infection. The functional mechanism of these mAbs needs to be further elucidated. These data suggest that RBD induces neutralizing antibodies not only at the receptor binding site but also at other characteristic higher-order structures, suggesting antigenic isomerism. SARS-CoVS protein RBD It is suggested that it has a secondary structural epitope and induces a strong antibody neutralization reaction.

  The conformational sensitivity of the SARS-CoV neutralizing mAbs described herein is consistent with the properties of neutralizing mAbs against other enveloped viruses, and generally results in a more natural conformation for binding. Necessary (Non-Patent Documents 47 and 48). SARS-CoVS protein RBD is a small fragment consisting of 193 amino acids but has 7 cysteines, 5 of which are essential for ACE2 binding. Disulfide bonds between cysteines form complex tertiary structures and can constitute multiple antigenic higher order structures. On the other hand, human neutralizing mAb selected from a non-immune human antibody library can react with DDT-reduced S protein and inhibit receptor binding (Non-patent Document 49). Therefore, the identification of neutralization determinants for SARS-CoVS protein RBD requires further evaluation, which provides important information for anti-SARS treatment and vaccine development.

  Reports that passive transplantation of mouse immune serum reduced lung virus replication in mice exposed to SARS-CoV (Non-patent Document 33, 50), and infection in mice and ferrets with neutralizing mAb vaccination The report that it was prevented (Non-Patent Documents 51 and 52) suggests that passive immunization with an anti-SARS antibody is useful as a strategy for controlling SARS. Therefore, mAbs with high amounts of SARS-CoV neutralizing activity can be used as an initial treatment for SARS-CoV infection. On the other hand, application of mouse mAb to humans is limited by human anti-mouse antibody (HAMA) reaction (Non-patent Documents 53-55). If a small amount of mouse mAb is used for a short period (1 to 2 weeks) in the early stages of SARS-CoV infection, severe HAMA may not occur and the emergency treatment can save the lives of SARS patients There is sex. We used a similar strategy to provide initial treatment for human hunt turn virus (HTNV) infection with mouse anti-HTNV mAb (56). Furthermore, by humanizing the mouse neutralizing mAb, it can be used as a treatment or immunological prophylaxis as a preventive effect against SARS-CoV infection in a population with a high possibility of infection.

  There are three advantages of this research. First, a plurality of very powerful RBD-specific neutralizing mAbs are created and can be developed as immunotherapy for SARS emergency treatment. Second, the mAb can be developed as a diagnostic agent for SARS-CoV infection. Third, the mAb can be used as a probe to study SARS-CoVS protein immunity, antigenicity, structure and function. The mAb can be further humanized for SARS treatment and prevention.

FIG. 1 shows the epitope mapping of mAbs 4D5 and 17H9. This mapping was performed by overlapping peptides containing the S protein RBD. Each peptide was coated with 5 μg / ml and tested with 10 μg / ml mAb. FIG. 2 shows inhibition of binding between RBD-Fc and ACE2 by mAb. The upper panel shows inhibition of binding between RBD-Fc and intracellular ACE2 expressed in 293T / ACE2 cells, and was measured by flow cytometry. The lower panel shows inhibition of binding between RBD-Fc and water-soluble ACE2, and was measured by ELISA. RBD-Fc was 1 μg / ml, mAb was 50 μg / ml, and the inhibition rate (%) was calculated for each mAb. FIG. 3 shows neutralization of SARS pseudovirus by mAb. In the figure, inhibition of SARS pseudovirus infection in 293T / ACE2 cells is shown. Inhibition was performed with a representative mAb from each group. Each mAb was tested in a 2-fold dilution series and the degree of neutralization (%) was calculated.

Claims (36)

Can bind to the receptor binding region in the spike protein of severe acute respiratory syndrome associated coronavirus (SARS-CoV), or
An isolated antibody, wherein binding between a severe acute respiratory syndrome-related coronavirus (SARS-CoV) and a receptor in a host cell or a cell-free receptor can be competitively suppressed. A substance comprising a complementarity determining region of the antibody according to claim 1,
The substance is
A substance characterized in that it can bind to the same epitope as the antibody of claim 1, or can competitively suppress the binding of the epitope like the antibody of claim 1.   The substance according to claim 2, wherein the substance is an antibody.   The antibody according to claim 1, wherein the antibody is a neutralizing antibody.   The antibody is produced by a hybridoma 18D9 having an accession number PTA-6521 of ATCC (American Strain Conservation Agency).   An epitope recognized by the antibody of claim 5.   An antibody produced by the hybridoma 19B2 having an accession number PTA-6522 of ATCC (American Strain Conservation Agency).   An epitope recognized by the antibody of claim 7.   An antibody produced by a hybridoma 30F9 having an accession number PTA-6523 of ATCC (American Strain Conservation Agency).   An epitope recognized by the antibody of claim 9.   An antibody produced by the hybridoma 31H12 having an ATCC (American Strain Conservation Agency) acquisition number PTA-6524.   An epitope recognized by the antibody of claim 11.   An antibody produced by a hybridoma 32H5 having an accession number PTA-6525 of ATCC (American Strain Conservation Agency).   An epitope characterized by being recognized by the antibody of claim 13.   An antibody produced by the hybridoma 33G4 having an accession number PTA-6526 of ATCC (American Strain Conservation Agency).   An epitope recognized by the antibody of claim 15.   The antibody according to claim 1, which is a single-chain antibody or an antibody fusion construct.   2. The antibody according to claim 1, wherein the antibody is a humanized antibody.   The antibody according to claim 1, wherein the antibody is a chimeric antibody.   2. The isolated antibody according to claim 1, wherein the antibody is directly or indirectly linked to a cytotoxic substance.   A cell comprising the antibody according to claim 1.   A nucleic acid molecule encoding the antibody of claim 1.   23. A nucleic acid molecule characterized in that it can hybridize in the molecule-specific manner according to claim 22.   The nucleic acid molecule according to claim 22, wherein the nucleic acid molecule is synthetic DNA, genomic DNA, complementary DNA, or RNA.   25. A vector comprising the nucleic acid molecule of claim 24 or a portion of the nucleic acid molecule.   A cell comprising the nucleic acid molecule according to claim 24. A method of generating an antibody comprising:
The antibody can bind to a receptor binding region in a spike protein of severe acute respiratory syndrome associated coronavirus (SARS-CoV), or
The antibody is an antibody capable of competitively suppressing the binding between a severe acute respiratory syndrome-related coronavirus (SARS-CoV) and a host cell,
The method
The nucleic acid molecule of claim 22 appropriately binds to a suitable regulatory element, whereby the regulatory element expresses the antibody;
Placing the bound nucleic acid molecule under suitable conditions to allow expression of the antibody; and
A method of producing an antibody comprising the step of recovering the expressed antibody.   28. An antibody produced by the method of claim 27.   2. A composition comprising an effective amount of the antibody of claim 1 and a suitable carrier.   A pharmaceutical composition comprising an effective amount of the antibody of claim 1 and a pharmaceutically acceptable carrier.   31. A method of treating an infection with severe acute respiratory syndrome associated coronavirus (SARS-CoV), comprising using the pharmaceutical composition of claim 30.   31. A method for preventing infection of severe acute respiratory syndrome associated coronavirus (SARS-CoV), comprising using the pharmaceutical composition of claim 30. A method for detecting cells infected with severe acute respiratory syndrome-related coronavirus (SARS-CoV) or SARS-CoV, comprising:
The method
Contacting with the antibody or derivative thereof,
In the step, the antibody or a derivative thereof is capable of forming a complex between the antibody or the derivative thereof and a receptor binding region in a spike protein of severe acute respiratory syndrome-related coronavirus (SARS-CoV). Under circumstances, is capable of binding to a receptor binding region in the viral spike protein;
The method further comprises
Detecting the formed complex. A method for screening compounds comprising:
The compound can inhibit the infection of severe acute respiratory syndrome-related coronavirus by blocking the binding of the virus to a host cell receptor,
The method
(A) constructing a system in which the antibody according to claim 1 binds to a receptor binding region in a spike protein of severe acute respiratory syndrome-related coronavirus (SARS-CoV),
(B) said compound comprising contacting said system (a), wherein said antibody according to claim 1 is receptor binding in a spike protein of severe acute respiratory syndrome associated coronavirus (SARS-CoV) By reducing binding to the region, the compound can interfere with the binding and the compound can infect the receptor binding region in the spike protein of severe acute respiratory syndrome associated coronavirus (SARS-CoV). A method of screening for a compound characterized in that it is possible to suppress the above.   35. A compound obtained by the method of claim 34.   A kit comprising a compartment containing the antibody according to claim 1.

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