Generation of a Nebulizable CDR-Modified MERS-CoV Neutralizing Human Antibody
Abstract
:1. Introduction
2. Results
2.1. Generation of Antibodies Reactive to Recombinant MERS-CoV RBD Protein From Patients
2.2. Selection of MERS-CoV Neutralizing Antibodies
2.3. Modification of CDR Residues to Enhance Antibody Stability
2.4. Neutralizing Potency After Nebulization
3. Discussion
4. Materials and Methods
4.1. Ethics Statement
4.2. Construction of A Human scFv Phage-Display Library and Three Randomization Libraries
4.3. Biopanning
4.4. High-Throughput Retrieval of scFv Clones and Phage ELISA
4.5. Expression of scFv-hFc and IgG1
4.6. ELISA
4.7. Nebulization
4.8. Microneutralization Assay
4.9. Flow Cytometry
4.10. SE-HPLC
4.11. DLS Assay
4.12. PRNT Assay
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
MERS-CoV | Middle East respiratory syndrome coronavirus |
DPP4 | dipeptidyl peptidase 4 |
RBD | receptor-binding domain |
i.v. | intravenous |
mAb | monoclonal antibody |
CDR | complementarity-determining region |
scFv | single-chain variable fragment |
PBMC | peripheral blood mononuclear cell |
SE-HPLC | size-exclusion high-performance liquid chromatography |
DLS | dynamic light scattering |
PRNT | plaque reduction neutralization test |
References
- Zaki, A.M.; van Boheemen, S.; Bestebroer, T.M.; Osterhaus, A.D.; Fouchier, R.A. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N. Engl. J. Med. 2012, 367, 1814–1820. [Google Scholar] [CrossRef] [PubMed]
- Van Boheemen, S.; de Graaf, M.; Lauber, C.; Bestebroer, T.M.; Raj, V.S.; Zaki, A.M.; Osterhaus, A.D.; Haagmans, B.L.; Gorbalenya, A.E.; Snijder, E.J.; et al. Genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans. mBio 2012, 3. [Google Scholar] [CrossRef] [PubMed]
- Raj, V.S.; Mou, H.; Smits, S.L.; Dekkers, D.H.; Muller, M.A.; Dijkman, R.; Muth, D.; Demmers, J.A.; Zaki, A.; Fouchier, R.A.; et al. Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature 2013, 495, 251–254. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- The FANTOM Consortium and the RIKEN PMI and CLST (DGT). A promoter-level mammalian expression atlas. Nature 2014, 507, 462–470. [Google Scholar] [Green Version]
- Chan, J.F.; Choi, G.K.; Tsang, A.K.; Tee, K.M.; Lam, H.Y.; Yip, C.C.; To, K.K.; Cheng, V.C.; Yeung, M.L.; Lau, S.K.; et al. Development and Evaluation of Novel Real-Time Reverse Transcription-PCR Assays with Locked Nucleic Acid Probes Targeting Leader Sequences of Human-Pathogenic Coronaviruses. J. Clin. Microbiol. 2015, 53, 2722–2726. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Chan, J.F.; Lau, S.K.; Woo, P.C. The emerging novel Middle East respiratory syndrome coronavirus: The “knowns” and “unknowns”. J. Med. Assoc. 2013, 112, 372–381. [Google Scholar] [CrossRef]
- Oh, M.D.; Park, W.B.; Choe, P.G.; Choi, S.J.; Kim, J.I.; Chae, J.; Park, S.S.; Kim, E.C.; Oh, H.S.; Kim, E.J.; et al. Viral Load Kinetics of MERS Coronavirus Infection. N. Engl. J. Med. 2016, 375, 1303–1305. [Google Scholar] [CrossRef]
- Guery, B.; Poissy, J.; el Mansouf, L.; Sejourne, C.; Ettahar, N.; Lemaire, X.; Vuotto, F.; Goffard, A.; Behillil, S.; Enouf, V.; et al. Clinical features and viral diagnosis of two cases of infection with Middle East Respiratory Syndrome coronavirus: A report of nosocomial transmission. Lancet 2013, 381, 2265–2272. [Google Scholar] [CrossRef]
- Oh, M.D.; Park, W.B.; Park, S.W.; Choe, P.G.; Bang, J.H.; Song, K.H.; Kim, E.S.; Bin Kim, H.; Kim, N.J. Middle East respiratory syndrome: What we learned from the 2015 outbreak in the Republic of Korea. Korean J. Intern. Med. 2018, 33, 233–246. [Google Scholar] [CrossRef]
- Kindler, E.; Jonsdottir, H.R.; Muth, D.; Hamming, O.J.; Hartmann, R.; Rodriguez, R.; Geffers, R.; Fouchier, R.A.; Drosten, C.; Muller, M.A.; et al. Efficient replication of the novel human betacoronavirus EMC on primary human epithelium highlights its zoonotic potential. MBio 2013, 4, 11–12. [Google Scholar] [CrossRef]
- Zielecki, F.; Weber, M.; Eickmann, M.; Spiegelberg, L.; Zaki, A.M.; Matrosovich, M.; Becker, S.; Weber, F. Human cell tropism and innate immune system interactions of human respiratory coronavirus EMC compared to those of severe acute respiratory syndrome coronavirus. J. Virol. 2013, 87, 5300–5304. [Google Scholar] [CrossRef] [PubMed]
- Corti, D.; Misasi, J.; Mulangu, S.; Stanley, D.A.; Kanekiyo, M.; Wollen, S.; Ploquin, A.; Doria-Rose, N.A.; Staupe, R.P.; Bailey, M.; et al. Protective monotherapy against lethal Ebola virus infection by a potently neutralizing antibody. Science 2016, 351, 1339–1342. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Gunn, B.M.; Yu, W.H.; Karim, M.M.; Brannan, J.M.; Herbert, A.S.; Wec, A.Z.; Halfmann, P.J.; Fusco, M.L.; Schendel, S.L.; Gangavarapu, K.; et al. A Role for Fc Function in Therapeutic Monoclonal Antibody-Mediated Protection against Ebola Virus. Cell Host Microbe. 2018, 24, 221–233. [Google Scholar] [CrossRef] [PubMed]
- Walker, L.M.; Burton, D.R. Passive immunotherapy of viral infections: ‘super-antibodies’ enter the fray. Nat. Rev. Immunol. 2018, 18, 297–308. [Google Scholar] [CrossRef] [PubMed]
- Du, L.; Yang, Y.; Zhou, Y.; Lu, L.; Li, F.; Jiang, S. MERS-CoV spike protein: A key target for antivirals. Expert. Opin. Targets 2017, 21, 131–143. [Google Scholar] [CrossRef] [PubMed]
- Wang, N.; Shi, X.; Jiang, L.; Zhang, S.; Wang, D.; Tong, P.; Guo, D.; Fu, L.; Cui, Y.; Liu, X.; et al. Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4. Cell Res. 2013, 23, 986–993. [Google Scholar] [CrossRef] [Green Version]
- Corti, D.; Zhao, J.; Pedotti, M.; Simonelli, L.; Agnihothram, S.; Fett, C.; Fernandez-Rodriguez, B.; Foglierini, M.; Agatic, G.; Vanzetta, F.; et al. Prophylactic and postexposure efficacy of a potent human monoclonal antibody against MERS coronavirus. Proc. Natl. Acad. Sci. USA 2015, 112, 10473–10478. [Google Scholar] [CrossRef] [Green Version]
- Du, L.; Zhao, G.; Yang, Y.; Qiu, H.; Wang, L.; Kou, Z.; Tao, X.; Yu, H.; Sun, S.; Tseng, C.T.; et al. A conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in Middle East respiratory syndrome coronavirus spike protein. J. Virol. 2014, 88, 7045–7053. [Google Scholar] [CrossRef]
- Jiang, L.; Wang, N.; Zuo, T.; Shi, X.; Poon, K.M.; Wu, Y.; Gao, F.; Li, D.; Wang, R.; Guo, J.; et al. Potent neutralization of MERS-CoV by human neutralizing monoclonal antibodies to the viral spike glycoprotein. Sci Transl Med. 2014, 6, 59. [Google Scholar] [CrossRef]
- Luke, T.; Wu, H.; Zhao, J.; Channappanavar, R.; Coleman, C.M.; Jiao, J.A.; Matsushita, H.; Liu, Y.; Postnikova, E.N.; Ork, B.L.; et al. Human polyclonal immunoglobulin G from transchromosomic bovines inhibits MERS-CoV in vivo. Sci. Transl. Med. 2016, 8. [Google Scholar] [CrossRef]
- Pascal, K.E.; Coleman, C.M.; Mujica, A.O.; Kamat, V.; Badithe, A.; Fairhurst, J.; Hunt, C.; Strein, J.; Berrebi, A.; Sisk, J.M.; et al. Pre- and postexposure efficacy of fully human antibodies against Spike protein in a novel humanized mouse model of MERS-CoV infection. Proc. Natl. Acad. Sci. USA 2015, 112, 8738–8743. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Tang, X.C.; Agnihothram, S.S.; Jiao, Y.; Stanhope, J.; Graham, R.L.; Peterson, E.C.; Avnir, Y.; Tallarico, A.S.; Sheehan, J.; Zhu, Q.; et al. Identification of human neutralizing antibodies against MERS-CoV and their role in virus adaptive evolution. Proc. Natl. Acad. Sci. USA 2014, 111, 2018–2026. [Google Scholar] [CrossRef] [PubMed]
- Ying, T.; Du, L.; Ju, T.W.; Prabakaran, P.; Lau, C.C.; Lu, L.; Liu, Q.; Wang, L.; Feng, Y.; Wang, Y.; et al. Exceptionally potent neutralization of Middle East respiratory syndrome coronavirus by human monoclonal antibodies. J. Virol. 2014, 88, 7796–7805. [Google Scholar] [CrossRef] [PubMed]
- Agrawal, A.S.; Ying, T.; Tao, X.; Garron, T.; Algaissi, A.; Wang, Y.; Wang, L.; Peng, B.H.; Jiang, S.; Dimitrov, D.S.; et al. Passive Transfer of A Germline-like Neutralizing Human Monoclonal Antibody Protects Transgenic Mice Against Lethal Middle East Respiratory Syndrome Coronavirus Infection. Sci. Rep. 2016, 6, 31629. [Google Scholar] [CrossRef]
- Cockrell, A.S.; Yount, B.L.; Scobey, T.; Jensen, K.; Douglas, M.; Beall, A.; Tang, X.C.; Marasco, W.A.; Heise, M.T.; Baric, R.S. A mouse model for MERS coronavirus-induced acute respiratory distress syndrome. Nat. Microbiol. 2016, 2, 16226. [Google Scholar] [CrossRef]
- Fan, C.; Wu, X.; Liu, Q.; Li, Q.; Liu, S.; Lu, J.; Yang, Y.; Cao, Y.; Huang, W.; Liang, C.; et al. A Human DPP4-Knockin Mouse’s Susceptibility to Infection by Authentic and Pseudotyped MERS-CoV. Viruses 2018, 10. [Google Scholar] [CrossRef]
- Houser, K.V.; Gretebeck, L.; Ying, T.; Wang, Y.; Vogel, L.; Lamirande, E.W.; Bock, K.W.; Moore, I.N.; Dimitrov, D.S.; Subbarao, K. Prophylaxis With a Middle East Respiratory Syndrome Coronavirus (MERS-CoV)-Specific Human Monoclonal Antibody Protects Rabbits From MERS-CoV Infection. J. Infect. Dis. 2016, 213, 1557–1561. [Google Scholar] [CrossRef]
- Johnson, R.F.; Bagci, U.; Keith, L.; Tang, X.; Mollura, D.J.; Zeitlin, L.; Qin, J.; Huzella, L.; Bartos, C.J.; Bohorova, N.; et al. 3B11-N, a monoclonal antibody against MERS-CoV, reduces lung pathology in rhesus monkeys following intratracheal inoculation of MERS-CoV Jordan-n3/2012. Virology 2016, 490, 49–58. [Google Scholar] [CrossRef]
- Li, Y.; Wan, Y.; Liu, P.; Zhao, J.; Lu, G.; Qi, J.; Wang, Q.; Lu, X.; Wu, Y.; Liu, W.; et al. A humanized neutralizing antibody against MERS-CoV targeting the receptor-binding domain of the spike protein. Cell Res. 2015, 25, 1237–1249. [Google Scholar] [CrossRef] [Green Version]
- Qiu, H.; Sun, S.; Xiao, H.; Feng, J.; Guo, Y.; Tai, W.; Wang, Y.; Du, L.; Zhao, G.; Zhou, Y. Single-dose treatment with a humanized neutralizing antibody affords full protection of a human transgenic mouse model from lethal Middle East respiratory syndrome (MERS)-coronavirus infection. Antivir. Res. 2016, 132, 141–148. [Google Scholar] [CrossRef] [Green Version]
- Hart, T.K.; Cook, R.M.; Zia-Amirhosseini, P.; Minthorn, E.; Sellers, T.S.; Maleeff, B.E.; Eustis, S.; Schwartz, L.W.; Tsui, P.; Appelbaum, E.R.; et al. Preclinical efficacy and safety of mepolizumab (SB-240563), a humanized monoclonal antibody to IL-5, in cynomolgus monkeys. J. Allergy Clin. Immunol. 2001, 108, 250–257. [Google Scholar] [CrossRef] [PubMed]
- Koleba, T.; Ensom, M.H. Pharmacokinetics of intravenous immunoglobulin: A systematic review. Pharmacotherapy 2006, 26, 813–827. [Google Scholar] [CrossRef] [PubMed]
- Guilleminault, L.; Azzopardi, N.; Arnoult, C.; Sobilo, J.; Herve, V.; Montharu, J.; Guillon, A.; Andres, C.; Herault, O.; Le Pape, A.; et al. Fate of inhaled monoclonal antibodies after the deposition of aerosolized particles in the respiratory system. J. Control. Release 2014, 196, 344–354. [Google Scholar] [CrossRef] [PubMed]
- Bitonti, A.J.; Dumont, J.A. Pulmonary administration of therapeutic proteins using an immunoglobulin transport pathway. Adv. Drug Deliv. Rev. 2006, 58, 1106–1118. [Google Scholar] [CrossRef] [PubMed]
- Bitonti, A.J.; Dumont, J.A.; Low, S.C.; Peters, R.T.; Kropp, K.E.; Palombella, V.J.; Stattel, J.M.; Lu, Y.; Tan, C.A.; Song, J.J.; et al. Pulmonary delivery of an erythropoietin Fc fusion protein in non-human primates through an immunoglobulin transport pathway. Proc. Natl. Acad. Sci. USA 2004, 101, 9763–9768. [Google Scholar] [CrossRef] [Green Version]
- Low, S.C.; Nunes, S.L.; Bitonti, A.J.; Dumont, J.A. Oral and pulmonary delivery of FSH-Fc fusion proteins via neonatal Fc receptor-mediated transcytosis. Hum. Reprod. 2005, 20, 1805–1813. [Google Scholar] [CrossRef]
- Van Heeke, G.; Allosery, K.; De Brabandere, V.; De Smedt, T.; Detalle, L.; de Fougerolles, A. Nanobodies(R) as inhaled biotherapeutics for lung diseases. Pharm. Ther. 2017, 169, 47–56. [Google Scholar] [CrossRef]
- Kim, Y.S.; Aigerim, A.; Park, U.; Kim, Y.; Rhee, J.Y.; Choi, J.P.; Park, W.B.; Park, S.W.; Kim, Y.; Lim, D.G.; et al. Sequential Emergence and Wide Spread of Neutralization Escape Middle East Respiratory Syndrome Coronavirus Mutants, South Korea, 2015. Emerg. Infect. Dis. 2019, 25, 1161–1168. [Google Scholar] [CrossRef]
- Noh, J.; Kim, O.; Jung, Y.; Han, H.; Kim, J.E.; Kim, S.; Lee, S.; Park, J.; Jung, R.H.; Kim, S.I.; et al. High-throughput retrieval of physical DNA for NGS-identifiable clones in phage display library. MAbs 2019, 11, 532–545. [Google Scholar] [CrossRef]
- Ying, T.; Prabakaran, P.; Du, L.; Shi, W.; Feng, Y.; Wang, Y.; Wang, L.; Li, W.; Jiang, S.; Dimitrov, D.S.; et al. Junctional and allele-specific residues are critical for MERS-CoV neutralization by an exceptionally potent germline-like antibody. Nat. Commun. 2015, 6, 8223. [Google Scholar] [CrossRef] [Green Version]
- Pommie, C.; Levadoux, S.; Sabatier, R.; Lefranc, G.; Lefranc, M.P. IMGT standardized criteria for statistical analysis of immunoglobulin V-REGION amino acid properties. J. Mol. Recognit. 2004, 17, 17–32. [Google Scholar] [CrossRef] [PubMed]
- Tartaglia, G.G.; Cavalli, A.; Pellarin, R.; Caflisch, A. Prediction of aggregation rate and aggregation-prone segments in polypeptide sequences. Protein Sci. 2005, 14, 2723–2734. [Google Scholar] [CrossRef] [PubMed]
- Igawa, T.; Tsunoda, H.; Tachibana, T.; Maeda, A.; Mimoto, F.; Moriyama, C.; Nanami, M.; Sekimori, Y.; Nabuchi, Y.; Aso, Y.; et al. Reduced elimination of IgG antibodies by engineering the variable region. Protein Eng. Des. Sel. 2010, 23, 385–392. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Feige, M.J.; Hendershot, L.M.; Buchner, J. How antibodies fold. Trends Biochem. Sci. 2010, 35, 189–198. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Sormanni, P.; Amery, L.; Ekizoglou, S.; Vendruscolo, M.; Popovic, B. Rapid and accurate in silico solubility screening of a monoclonal antibody library. Sci. Rep. 2017, 7, 8200. [Google Scholar] [CrossRef] [PubMed]
- Larios Mora, A.; Detalle, L.; Gallup, J.M.; Van Geelen, A.; Stohr, T.; Duprez, L.; Ackermann, M.R. Delivery of ALX-0171 by inhalation greatly reduces respiratory syncytial virus disease in newborn lambs. MAbs 2018, 10, 778–795. [Google Scholar] [CrossRef] [Green Version]
- Respaud, R.; Marchand, D.; Parent, C.; Pelat, T.; Thullier, P.; Tournamille, J.F.; Viaud-Massuard, M.C.; Diot, P.; Si-Tahar, M.; Vecellio, L.; et al. Effect of formulation on the stability and aerosol performance of a nebulized antibody. MAbs 2014, 6, 1347–1355. [Google Scholar] [CrossRef] [Green Version]
- Moussa, E.M.; Panchal, J.P.; Moorthy, B.S.; Blum, J.S.; Joubert, M.K.; Narhi, L.O.; Topp, E.M. Immunogenicity of Therapeutic Protein Aggregates. J. Pharm. Sci. 2016, 105, 417–430. [Google Scholar] [CrossRef] [Green Version]
- Beck-Broichsitter, M.; Kleimann, P.; Schmehl, T.; Betz, T.; Bakowsky, U.; Kissel, T.; Seeger, W. Impact of lyoprotectants for the stabilization of biodegradable nanoparticles on the performance of air-jet, ultrasonic, and vibrating-mesh nebulizers. Eur. J. Pharm. Biopharm. 2012, 82, 272–280. [Google Scholar] [CrossRef]
- Rosenberg, A.S. Effects of protein aggregates: An immunologic perspective. AAPS J. 2006, 8, 501–507. [Google Scholar] [CrossRef]
- Ewert, S.; Huber, T.; Honegger, A.; Pluckthun, A. Biophysical properties of human antibody variable domains. J. Mol. Biol. 2003, 325, 531–553. [Google Scholar] [CrossRef]
- Honegger, A.; Malebranche, A.D.; Rothlisberger, D.; Pluckthun, A. The influence of the framework core residues on the biophysical properties of immunoglobulin heavy chain variable domains. Protein Eng. Des. Sel. 2009, 22, 121–134. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Chennamsetty, N.; Voynov, V.; Kayser, V.; Helk, B.; Trout, B.L. Design of therapeutic proteins with enhanced stability. Proc. Natl. Acad. Sci. USA 2009, 106, 11937–11942. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Wang, X.; Das, T.K.; Singh, S.K.; Kumar, S. Potential aggregation prone regions in biotherapeutics: A survey of commercial monoclonal antibodies. MAbs 2009, 1, 254–267. [Google Scholar] [CrossRef]
- Wu, S.J.; Luo, J.; O’Neil, K.T.; Kang, J.; Lacy, E.R.; Canziani, G.; Baker, A.; Huang, M.; Tang, Q.M.; Raju, T.S.; et al. Structure-based engineering of a monoclonal antibody for improved solubility. Protein Eng. Des. Sel. 2010, 23, 643–651. [Google Scholar] [CrossRef] [Green Version]
- Jespers, L.; Schon, O.; Famm, K.; Winter, G. Aggregation-resistant domain antibodies selected on phage by heat denaturation. Nat. Biotechnol. 2004, 22, 1161–1165. [Google Scholar] [CrossRef]
- Perchiacca, J.M.; Bhattacharya, M.; Tessier, P.M. Mutational analysis of domain antibodies reveals aggregation hotspots within and near the complementarity determining regions. Proteins 2011, 79, 2637–2647. [Google Scholar] [CrossRef]
- Dudgeon, K.; Rouet, R.; Kokmeijer, I.; Schofield, P.; Stolp, J.; Langley, D.; Stock, D.; Christ, D. General strategy for the generation of human antibody variable domains with increased aggregation resistance. Proc. Natl. Acad. Sci. USA 2012, 109, 10879–10884. [Google Scholar] [CrossRef] [Green Version]
- Agrawal, A.S.; Garron, T.; Tao, X.; Peng, B.H.; Wakamiya, M.; Chan, T.S.; Couch, R.B.; Tseng, C.T. Generation of a transgenic mouse model of Middle East respiratory syndrome coronavirus infection and disease. J. Virol. 2015, 89, 3659–3670. [Google Scholar] [CrossRef]
- Li, K.; Wohlford-Lenane, C.; Perlman, S.; Zhao, J.; Jewell, A.K.; Reznikov, L.R.; Gibson-Corley, K.N.; Meyerholz, D.K.; McCray, P.B., Jr. Middle East Respiratory Syndrome Coronavirus Causes Multiple Organ Damage and Lethal Disease in Mice Transgenic for Human Dipeptidyl Peptidase 4. J. Infect. Dis. 2016, 213, 712–722. [Google Scholar] [CrossRef]
- Zhao, G.; Jiang, Y.; Qiu, H.; Gao, T.; Zeng, Y.; Guo, Y.; Yu, H.; Li, J.; Kou, Z.; Du, L.; et al. Multi-Organ Damage in Human Dipeptidyl Peptidase 4 Transgenic Mice Infected with Middle East Respiratory Syndrome-Coronavirus. PLoS ONE 2015, 10, 145561. [Google Scholar] [CrossRef] [PubMed]
- Li, K.; Wohlford-Lenane, C.L.; Channappanavar, R.; Park, J.E.; Earnest, J.T.; Bair, T.B.; Bates, A.M.; Brogden, K.A.; Flaherty, H.A.; Gallagher, T.; et al. Mouse-adapted MERS coronavirus causes lethal lung disease in human DPP4 knockin mice. Proc. Natl. Acad. Sci. USA 2017, 114, E3119–E3128. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kanof, M.E.; Smith, P.D.; Zola, H. Isolation of whole mononuclear cells from peripheral blood and cord blood. Curr. Protoc. Immunol. 2001. [Google Scholar] [CrossRef]
- Barbas, C.F.; Burton, D.R.; Scott, J.K.; Silverman, G.J. Phage Display: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York, NY, USA, 2001. [Google Scholar] [CrossRef]
- Andris-Widhopf, J.; Steinberger, P.; Fuller, R.; Rader, C.; Barbas, C.F., 3rd. Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences. Cold Spring Harb. Protoc. 2011, 2011. [Google Scholar] [CrossRef]
- Lee, Y.; Kim, H.; Chung, J. An antibody reactive to the Gly63-Lys68 epitope of NT-proBNP exhibits O-glycosylation-independent binding. Exp. Mol. Med. 2014, 46, 114. [Google Scholar] [CrossRef]
- Lee, S.; Yoon, I.H.; Yoon, A.; Cook-Mills, J.M.; Park, C.G.; Chung, J. An antibody to the sixth Ig-like domain of VCAM-1 inhibits leukocyte transendothelial migration without affecting adhesion. J. Immunol. 2012, 189, 4592–4601. [Google Scholar] [CrossRef]
- Jin, J.; Park, G.; Park, J.B.; Kim, S.; Kim, H.; Chung, J. An anti-EGFR x cotinine bispecific antibody complexed with cotinine-conjugated duocarmycin inhibits growth of EGFR-positive cancer cells with KRAS mutations. Exp. Mol. Med. 2018, 50, 67. [Google Scholar] [CrossRef]
- Reed, L.J.; Muench, H. A Simple Method for Estimating Fifty Per Cent. Endpoints. Am. J. Epidemiol. 1938, 27, 493–497. [Google Scholar] [CrossRef]
Antibody | SE-HPLC (% Monomer/% Aggregates) | DLS (% Monomer ± SD/% Aggregates ± SD) | ||
---|---|---|---|---|
Pre-Nebulization | Post-Nebulization | Pre-Nebulization | Post-Nebulization | |
C-8 | 100.0/0 | 97.9/2.1 | 100.0 ± 0/0 | 78.4 ± 3.5/21.6 ± 3.5 |
C-8-2-4B-10D | 100.0/0 | 100.0/0 | 99.2 ± 0.7/0.8 ± 0.7 | 98.6 ± 0.4/1.4 ± 0.4 |
m336 | 100.0/0 | 99.4/0.6 | 96.6 ± 0.6/3.4 ± 0.6 | 77.5 ± 2.3/22.5 ± 2.3 |
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Kim, S.I.; Kim, S.; Kim, J.; Chang, S.Y.; Shim, J.M.; Jin, J.; Lim, C.; Baek, S.; Min, J.-Y.; Park, W.B.; et al. Generation of a Nebulizable CDR-Modified MERS-CoV Neutralizing Human Antibody. Int. J. Mol. Sci. 2019, 20, 5073. https://doi.org/10.3390/ijms20205073
Kim SI, Kim S, Kim J, Chang SY, Shim JM, Jin J, Lim C, Baek S, Min J-Y, Park WB, et al. Generation of a Nebulizable CDR-Modified MERS-CoV Neutralizing Human Antibody. International Journal of Molecular Sciences. 2019; 20(20):5073. https://doi.org/10.3390/ijms20205073
Chicago/Turabian StyleKim, Sang Il, Sujeong Kim, Jinhee Kim, So Young Chang, Jung Min Shim, Jongwha Jin, Chungsu Lim, Songyi Baek, Ji-Young Min, Wan Beom Park, and et al. 2019. "Generation of a Nebulizable CDR-Modified MERS-CoV Neutralizing Human Antibody" International Journal of Molecular Sciences 20, no. 20: 5073. https://doi.org/10.3390/ijms20205073