MOG - CCR4-ND ELISA
3.3.3 Reliability test
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3.3.3 Reliability test
To check whether the kit functions adequately in stressed condition, we tested the kit by adding MOG from human host ranging from 0.1 nM to 3.35 nM and anti-CCR antibodies from goat (CCR1,2,5,6,7) and mouse (CCR3) hosts (each with 3.35 nM, total value of 23,35 nM). Each well in this kit was loaded with 100 µL of 128 nM. As illustrated in Fig 12 and 13, MOG absorbance showed linear trend and was higher than others absorbance.
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Figure 12. Schematic of reliability test
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0 0.05 0.1 0.15 0.2 0.25 0.3
0.1 1 10
Absorbance (450nm)
Ab concentration (nM)
CCR Antibodies - CCR4-ND ELISA
Human (MOG) Goat (CCR2,3,5,6,7) Mouse (CCR1)
Figure 13. Absorbance reading of MOG + anti-CCR antibodies against CCR4-ND
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Chapter 4
Conclusion
GPCR’s inherent structural complexity was hard to mimic in vivo and so industry has been using peptides of extracellular domain to screen anti-GPCR antibody candidates. We offer a new way of screening anti-GPCR antibody via whole GPCR reconstituted into nanodisc platform. By using the entirety of GPCR, antibody derivatives screened from this system would have more specificity to complex structure of GPCR and affinity to the target than those from conventional way.
This research demonstrates the function of the kit in three criteria. The efficiency of the kit was verified by screening antibody of interest from two closely related antibodies. The usefulness of the kit was verified by rising HRP signals as the amount of antibody of interest increases. The reliability of the kit was finally verified by successfully screening antibody of interest from pool of antibodies where the amount of non-binding antibodies was 6-fold that of antibody of interest. Therefore, this kit can be useful in discovering antibody candidates for all unexplored GPCR.
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Reference
[1] Hauser, A. S., Attwood, M. M., Rask-Andersen, M., Schiöth, H. B., & Gloriam, D. E. (2017). Trends in GPCR drug discovery:
new agents, targets and indications. Nature Reviews Drug Discovery, 16(12), 829–842.
[2] Hutchings, C. J., Koglin, M., Olson, W. C., & Marshall, F. H.
(2017). Opportunities for therapeutic antibodies directed at G- protein-coupled receptors. Nature Reviews Drug Discovery, 16(11), 787–810.
[3] Niwa, R., Shoji-Hosaka, E., Sakurada, M., Shinkawa, T., Uchida, K., Nakamura, K., Matsushima, K., Ueda, R., Hanai, N., &
Shitara, K. (2004). Defucosylated Chimeric Anti-CC Chemokine Receptor 4 IgG1 with Enhanced Antibody-Dependent Cellular Cytotoxicity Shows Potent Therapeutic Activity to T-Cell Leukemia and Lymphoma. Cancer Research, 64(6), 2127–2133.
[4] Shi, L., Lehto, S. G., Zhu, D. X. D., Sun, H., Zhang, J., Smith, B. P., Immke, D. C., Wild, K. D., & Xu, C. (2015). Pharmacologic Characterization of AMG 334, a Potent and Selective Human Monoclonal Antibody against the Calcitonin Gene-Related Peptide Receptor. Journal of Pharmacology and Experimental Therapeutics, 356(1), 223–231.
32
5] Okamoto, H., Kim, J., Aglione, J., Lee, J., Cavino, K., Na, E., Rafique, A., Kim, J. H., Harp, J., Valenzuela, D. M., Yancopoulos, G. D., Murphy, A. J., & Gromada, J. (2015). Glucagon Receptor Blockade With a Human Antibody Normalizes Blood Glucose in Diabetic Mice and Monkeys. Endocrinology, 156(8), 2781–2794.
[6] Kuhne, M. R., Mulvey, T., Belanger, B., Chen, S., Pan, C., Chong, C., Cao, F., Niekro, W., Kempe, T., Henning, K. A., Cohen, L. J., Korman, A. J., & Cardarelli, P. M. (2012). BMS- 936564/MDX-1338: A Fully Human Anti-CXCR4 Antibody Induces Apoptosis In Vitro and Shows Antitumor Activity In Vivo in Hematologic Malignancies. Clinical Cancer Research, 19(2), 357–366.
[7] Denisov, I. G., & Sligar, S. G. (2016). Nanodiscs for structural and functional studies of membrane proteins. Nature Structural & Molecular Biology, 23(6), 481–486.
[8] Schuler, M. A., Denisov, I. G., & Sligar, S. G. (2012).
Nanodiscs as a New Tool to Examine Lipid–Protein Interactions.
Methods in Molecular Biology, 415–433.
[9] Frauenfeld, J., Löving, R., Armache, J. P., Sonnen, A. F. P., Guettou, F., Moberg, P., Zhu, L., Jegerschöld, C., Flayhan, A., Briggs, J. A. G., Garoff, H., Löw, C., Cheng, Y., & Nordlund, P.
(2016). A saposin-lipoprotein nanoparticle system for
33
(2016). A saposin-lipoprotein nanoparticle system for membrane proteins. Nature Methods, 13(4), 345–351.
[10] Yang, H., Kim, D., Kim, J., Moon, D., Song, H. S., Lee, M., Hong, S., & Park, T. H. (2017). Nanodisc-Based Bioelectronic Nose Using Olfactory Receptor Produced in Escherichia coli for the Assessment of the Death-Associated Odor Cadaverine. ACS Nano, 11(12), 11847–11855.
[11] Bhattacharya, P., Grimme, S., Ganesh, B., Gopisetty, A., Sheng, J. R., Martinez, O., Jayarama, S., Artinger, M., Meriggioli, M., & Prabhakar, B. S. (2010). Nanodisc-Incorporated Hemagglutinin Provides Protective Immunity against Influenza Virus Infection. Journal of Virology, 84(1), 361–371.
[12] Lee, S. H., Jin, H. J., Song, H. S., Hong, S., & Park, T. H.
(2012). Bioelectronic nose with high sensitivity and selectivity using chemically functionalized carbon nanotube combined with human olfactory receptor. Journal of Biotechnology, 157(4), 467–472.
[13] Park, S. J., Kwon, O. S., Lee, S. H., Song, H. S., Park, T. H.,
& Jang, J. (2012). Ultrasensitive Flexible Graphene Based Field-Effect Transistor (FET)-Type Bioelectronic Nose. Nano Letters, 12(10), 5082–5090.
[14] Duvic, M., Evans, M., & Wang, C. (2016). Mogamulizumab
34
[14] Duvic, M., Evans, M., & Wang, C. (2016). Mogamulizumab for the treatment of cutaneous T-cell lymphoma: recent advances and clinical potential. Therapeutic Advances in Hematology, 7(3), 171–174.
[15] Viney, J. M., Andrew, D. P., Phillips, R. M., Meiser, A., Patel, P., Lennartz-Walker, M., Cousins, D. J., Barton, N. P., Hall, D. A.,
& Pease, J. E. (2014). Distinct Conformations of the Chemokine Receptor CCR4 with Implications for Its Targeting in Allergy.
The Journal of Immunology, 192(7), 3419–3427.
[16] Mariani, M., Lang, R., Binda, E., Panina-Bordignon, P., &
D’Ambrosio, D. (2004). Dominance of CCL22 over CCL17 in induction of chemokine receptor CCR4 desensitization and internalization on human Th2 cells. European Journal of Immunology, 34(1), 231–240.
[17] Mainland, J. D., Keller, A., Li, Y. R., Zhou, T., Trimmer, C., Snyder, L. L., … Matsunami, H. (2013). The missense of smell:
functional variability in the human odorant receptor repertoire.
Nature Neuroscience, 17(1), 114–120.
[18] Li, Y.R., & Matsunami, H. (2011). Activation state of the M3 muscarinic acetylcholine receptor modulates mammalian odorant receptor signaling, Science signaling, 4(155).
[19] Kwon, O.S., Lee, S.H., Park, S.J., An, J.H., Song, H.S., Kim,
35
RraA-Mediated Enhancement of Recombinant Membrane Protein Production in the Engineered Escherichia coli Strains SuptoxD and SuptoxR.
[25] Gialama, D., Kostelidou, K., Michou, M., Delivoria, D. C., Kolisis, F. N., & Skretas, G. (2016). Development of Escherichia coli Strains That Withstand Membrane Protein-Induced Toxicity and Achieve High-Level Recombinant Membrane Protein Production. ACS Synthetic Biology, 6(2), 284–300.
국 문 초 록
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국 문 초 록
GPCR이 포함된 나노디스크를 통 한 약물항체 스크리닝
김 수 서울대학교 대학원 협동과정 바이오엔지니어링 전공
G 단백질 연결 수용체 (G protein-coupled receptors, GPCR)를 타겟으로 하는 약물의 개발은 제약 산업에서 중요한 주제이며, 현재까지 대부분의 경우 소분자 합성 의약품들이 개발되어 사용되어지고 있다. 그러나 바이오의약품, 특히 항체의 개발은 큰 진전이 없이 2021년 기준 단 2종만이 미국 FDA에 승인받은 상태이다. 이는 항체 약물개발에 항원인 GPCR을 자연적인 형태로 만들어 개발에 사용하기 어려움이 있고 설사 어렵게 만들었다 하여도 그 방식들이 대량생산을 하는데 어려움을 가졌기 때문이다. 본 연구는,
E.coli-발현 시스템을 사용하여 나노디스크 플랫폼으로 재조합한
GPCR을 기반으로 한 키트를 제작하고 항체를 스크리닝 하였다. 이 키트는 간접적 효소면역측정법 (Indirect ELISA) 방식을 기반으로 설계되어, 항원인 GPCR이 포함된 나노디스크가 표면에 붙어있는 상태에서 확인하고자 하는 항체들을 분별한 뒤 특정 파장을 유도하는 표시 항체를 후처리 하는 방식으로 진행하였다. 제작된 키트는 미국 FDA에서 승인받은 항체인 Mogamulizumab (MOG)과 6 종류의 대- 케모카인 수용체 항체들, 그리고 타겟이 되는 GPCR인 cc-
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그리고 타겟이 되는 GPCR인 cc-chemokine receptor type 4를 항원으로 사용하여 그 기능을 확인하였다. 대량생산이 가능한 나노디스크 방식의 재조합 GPCR은 약물개발 방식에 새로운 패러다임을 여는데 기여할 것이다.
주요어: 약물, 대량생산, 항체 스크리닝, G 단백질 연결 수용체, 효소면역측정법, 나노디스크
학번: 2020-21101