抗體藥物複合體（Antibody-Drug Conjugates，ADCs）為一種有潛力的抗癌試劑，將單株抗體與小分子毒殺藥物透過連接子（linker）把兩者接合形成複合體。單株抗體是由多種胺基酸所組成，而離胺酸（lysine）就有大約70-90個位點，因此是最常被修飾的位點，但是也因為反應位點很多，因此ADC以離胺酸為主要修飾位點的連接子，都會呈現非均一（heterogeneous）的特性，可能會影響到藥物的穩定性、藥物動力學或是與抗原的專一性結合，所以希望可以製作出修飾位點較集中的ADC產物。
本篇就是比較的2種連接子分別為N-Hydroxysuccinimide-linker（NHS-linker）以及aldehyde-linker（AD-linker）都是會修飾於蛋白上胺基的N端與離胺酸的連接子，NHS-linker是利用NHS-ester的一步驟反應接合至蛋白上，發現所得到的質量圖譜較雜亂，不易判斷藥物抗體接合比率（drug to antibody ratio，DAR）；另一種連接子是aldehyde-linker（AD-linker）是利用調控還原胺化反應的方法，而所得到的圖譜非常清晰，可以輕易計算出DAR值。
利用不同濃度相同反應時間，將兩種連接子樣品以由下而上的質譜法來處理，發現AD-linker修飾的位點比NHS-linker集中。當相同連接子濃度下，調控反應時間發現NHS-linker即使拉長反應時間，DAR值也沒有增加；而AD-linker則是隨著反應時間增加DAR值增加，甚至可以搭配注射針緩慢的加入AD-linker，使修飾位點更加集中，此現象在高濃度下更加的明顯。說明AD-linker可以利用多種不同控制方式使修飾位點較集中，而且其圖譜清晰容易計算DAR值，所以較適合被選為ADC所使用的連接子。

Antibody-Drug Conjugates (ADCs), which small molecule drug and a monoclonal antibody (mAb) is conjugated by linker, is a promising anticancer treatment reagent in cancer therapy. Monoclonal antibody is consisted by different kinds of amino acid, and there are around 70-90 lysine sites may be conjugated. Because of the high number of lysines on one mAb, ADC conjugated to these sites results in heterogeneous mixtures. It probably influence ADC pharmaceutical stability, Pharmacokinetics parameters, and reduced efficiency. Therefore, to manufacture homogenous ADC products is the major purpose.
Here we compared two kinds of lysine-based ADC linker N-Hydroxy- succinimide-linker (NHS-linker) and aldehyde-linker (AD-linker), respectively. NHS-linker is NHS-ester reaction and the mass spectrometry is too messy to distinguish drug of antibody ratio (DAR). On the other hand AD-linker is controlled by reductive amination, the mass spectrometry and DAR are easy to distinguish.
There are two different methods to compared, the first one using different stoichiometry, and the same reaction time is one hour. The result found that AD-linker reacts with the fewer modified sites than NHS-linker. The second method is changing reaction time at the same linker stoichiometry. AD-linker’s DAR will increases as the reaction time increase, but DAR of NHS-linker will not increases. Moreover AD-linker can couple with syring to control the rate of linker reagent, AD-linker focus on specific site. The phenomena is more obvious at the high concentration of AD-linker. The AD-linker not only easier to distinguish DAR but also more focus on the specific modification site. The products of AD-linker is more homogeneous than NHS-linker, AD-linker is a good candidate to be an ADC linker.

1. Schwartz, R. S., Paul Ehrlich's magic bullets. New Engl J. Med. 2004, 350 (11), 1079-1080.
2. Jhan, S. Y.; Huang, L. J.; Wang, T. F.; Chou, H. H.; Chen, S. H., Dimethyl Labeling Coupled with Mass Spectrometry for Topographical Characterization of Primary Amines on Monoclonal Antibodies. Anal. Chem. 2017, 89 (7), 4255-4263.
3. Gupta, R.; Dey, A.; Vijan, A.; Gartia, B., In Silico Structure Modeling and Characterization of Hypothetical Protein YP_004590319.1 Present in Enterobacter aerogens. J. Proteomics. Bioinform. 2017, 10 (6).
4. Konermann, L.; Ahadi, E.; Rodriguez, A. D.; Vahidi, S., Unraveling the mechanism of electrospray ionization. Anal. Chem. 2013, 85 (1), 2-9.
5. Kellie, J. F.; Tran, J. C.; Lee, J. E.; Ahlf, D. R.; Thomas, H. M.; Ntai, I.; Catherman, A. D.; Durbin, K. R.; Zamdborg, L.; Vellaichamy, A.; Thomas, P. M.; Kelleher, N. L., The emerging process of Top Down mass spectrometry for protein analysis: biomarkers, protein-therapeutics, and achieving high throughput. Mol. Biosyst. 2010, 6 (9), 1532-9.
6. Hsu, J. L.; Chen, S. H., Stable isotope dimethyl labelling for quantitative proteomics and beyond. Philos. Trans. A. Math. Phys. Eng. Sci. 2016, 374 (2079).
7. Hsu, J. L.; Huang, S. Y.; Chow, N. H.; Chen, S. H., Stable-isotope dimethyl labeling for quantitative proteomics. Anal. Chem. 2003, 75 (24), 6843-52.
8. Hsu, J. L.; Huang, S. Y.; Chen, S. H., Dimethyl multiplexed labeling combined with microcolumn separation and MS analysis for time course study in proteomics. Electrophoresis. 2006, 27 (18), 3652-60.
9. Fu, Q.; Li, L. J., De novo sequencing of neuropeptides using reductive isotopic methylation and investigation of ESI QTOF MS/MS fragmentation pattern of neuropeptides with N-terminal dimethylation. Anal. Chem. 2005, 77 (23), 7783-7795.
10. Kleifeld, O.; Doucet, A.; Keller, U. A. D.; Prudova, A.; Schilling, O.; Kainthan, R. K.; Starr, A. E.; Foster, L. J.; Kizhakkedathu, J. N.; Overall, C. M., Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products. Nat. Biotechnol. 2010, 28 (3), 281-U144.
11. Huang, S. Y.; Chen, S. F.; Chen, C. H.; Huang, H. W.; Wu, W. G.; Sung, W. C., Global Disulfide Bond Profiling for Crude Snake Venom Using Dimethyl Labeling Coupled with Mass Spectrometry and RADAR Algorithm. Anal. Chem. 2014, 86 (17), 8742-8750.
12. Huang, S. Y.; Hsieh, Y. T.; Chen, C. H.; Chen, C. C.; Sung, W. C.; Chou, M. Y.; Chen, S. F., Automatic disulfide bond assignment using a1 ion screening by mass spectrometry for structural characterization of protein pharmaceuticals. Anal. Chem. 2012, 84 (11), 4900-6.
13. Sievers, E. L.; Senter, P. D., Antibody-Drug Conjugates in Cancer Therapy. Annu. Rev. Med. 2013, 64 (1), 15-29.
14. Wagner-Rousset, E.; Janin-Bussat, M. C.; Colas, O.; Excoffier, M.; Ayoub, D.; Haeuw, J. F.; Rilatt, I.; Perez, M.; Corvaia, N.; Beck, A., Antibody-drug conjugate model fast characterization by LC-MS following IdeS proteolytic digestion. MAbs. 2014, 6 (1), 273-85.
15. Jain, N.; Smith, S. W.; Ghone, S.; Tomczuk, B., Current ADC Linker Chemistry. Pharm. Res. 2015, 32 (11), 3526-40.
16. Johansson, M. P.; Maaheimo, H.; Ekholm, F. S., New insight on the structural features of the cytotoxic auristatins MMAE and MMAF revealed by combined NMR spectroscopy and quantum chemical modelling. Sci. Rep. 2017, 7 (1), 15920.
17. LoRusso, P. M.; Weiss, D.; Guardino, E.; Girish, S.; Sliwkowski, M. X., Trastuzumab Emtansine: A Unique Antibody-Drug Conjugate in Development for Human Epidermal Growth Factor Receptor 2–Positive Cancer. Clin. Cancer. Res. 2011, 17 (20), 6437-6447.
18. Abdollahpour-Alitappeh, M.; Lotfinia, M.; Razavi-Vakhshourpour, S.; Jahandideh, S.; Najminejad, H.; Sineh Sepehr, K.; Moazami, R.; Shams, E.; Habibi-Anbouhi, M.; Abolhassani, M., Evaluation of Factors Influencing Antibody Reduction for Development of Antibody Drug Conjugates. Iranian. Biomedical. Journal. 2017, 21 (4), 270-274.
19. Zimmerman, E. S.; Heibeck, T. H.; Gill, A.; Li, X.; Murray, C. J.; Madlansacay, M. R.; Tran, C.; Uter, N. T.; Yin, G.; Rivers, P. J.; Yam, A. Y.; Wang, W. D.; Steiner, A. R.; Bajad, S. U.; Penta, K.; Yang, W.; Hallam, T. J.; Thanos, C. D.; Sato, A. K., Production of site-specific antibody-drug conjugates using optimized non-natural amino acids in a cell-free expression system. Bioconjug. Chem. 2014, 25 (2), 351-61.
20. Bondt, A.; Rombouts, Y.; Selman, M. H.; Hensbergen, P. J.; Reiding, K. R.; Hazes, J. M.; Dolhain, R. J.; Wuhrer, M., Immunoglobulin G (IgG) Fab glycosylation analysis using a new mass spectrometric high-throughput profiling method reveals pregnancy-associated changes. Mol. Cell. Proteomics. 2014, 13 (11), 3029-39.
21. Abdollahpour-Alitappeh, M.; Lotfinia, M.; Gharibi, T.; Mardaneh, J.; Farhadihosseinabadi, B.; Larki, P.; Faghfourian, B.; Sepehr, K. S.; Abbaszadeh-Goudarzi, K.; Abbaszadeh-Goudarzi, G.; Johari, B.; Zali, M. R.; Bagheri, N., Antibody-drug conjugates (ADCs) for cancer therapy: Strategies, challenges, and successes. J. Cell. Physiol. 2019, 234 (5), 5628-5642.
22. Kohler, G.; Milstein, C., Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity. Nature. 1975, 256 (5517), 495-497.
23. Bostrom, J.; Yu, S.-F.; Kan, D.; Appleton, B. A.; Lee, C. V.; Billeci, K.; Man, W.; Peale, F.; Ross, S.; Wiesmann, C.; Fuh, G., Variants of the Antibody Herceptin That Interact with HER2 and VEGF at the Antigen Binding Site. Science. 2009, 323 (5921), 1610-1614.
24. Lyon, R. P.; Bovee, T. D.; Doronina, S. O.; Burke, P. J.; Hunter, J. H.; Neff-LaFord, H. D.; Jonas, M.; Anderson, M. E.; Setter, J. R.; Senter, P. D., Reducing hydrophobicity of homogeneous antibody-drug conjugates improves pharmacokinetics and therapeutic index. Nat. Biotechnol. 2015, 33 (7), 733-5.
25. Schapira, L., Simple rules can improve prognostic accuracy. J. Clin. Oncol. 2011, 29 (4), 347-9.
26. Jaracz, S.; Chen, J.; Kuznetsova, L. V.; Ojima, I., Recent advances in tumor-targeting anticancer drug conjugates. Bioorg. Med. Chem. 2005, 13 (17), 5043-54.
27. Gebleux, R.; Casi, G., Antibody-drug conjugates: Current status and future perspectives. Pharmacol. Ther. 2016, 167, 48-59.
28. Tsuchikama, K.; An, Z., Antibody-drug conjugates: recent advances in conjugation and linker chemistries. Protein. Cell. 2018, 9 (1), 33-46.
29. Dosio, F.; Brusa, P.; Cattel, L., Immunotoxins and anticancer drug conjugate assemblies: the role of the linkage between components. Toxins. (Basel) 2011, 3 (7), 848-83.
30. Junutula, J. R.; Raab, H.; Clark, S.; Bhakta, S.; Leipold, D. D.; Weir, S.; Chen, Y.; Simpson, M.; Tsai, S. P.; Dennis, M. S.; Lu, Y.; Meng, Y. G.; Ng, C.; Yang, J.; Lee, C. C.; Duenas, E.; Gorrell, J.; Katta, V.; Kim, A.; McDorman, K.; Flagella, K.; Venook, R.; Ross, S.; Spencer, S. D.; Lee Wong, W.; Lowman, H. B.; Vandlen, R.; Sliwkowski, M. X.; Scheller, R. H.; Polakis, P.; Mallet, W., Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat. Biotechnol .2008, 26 (8), 925-32.
31. Behrens, C. R.; Ha, E. H.; Chinn, L. L.; Bowers, S.; Probst, G.; Fitch-Bruhns, M.; Monteon, J.; Valdiosera, A.; Bermudez, A.; Liao-Chan, S.; Wong, T.; Melnick, J.; Theunissen, J. W.; Flory, M. R.; Houser, D.; Venstrom, K.; Levashova, Z.; Sauer, P.; Migone, T. S.; van der Horst, E. H.; Halcomb, R. L.; Jackson, D. Y., Antibody-Drug Conjugates (ADCs) Derived from Interchain Cysteine Cross-Linking Demonstrate Improved Homogeneity and Other Pharmacological Properties over Conventional Heterogeneous ADCs. Mol. Pharm. 2015, 12 (11), 3986-98.
32. Bryden, F.; Maruani, A.; Savoie, H.; Chudasama, V.; Smith, M. E.; Caddick, S.; Boyle, R. W., Regioselective and stoichiometrically controlled conjugation of photodynamic sensitizers to a HER2 targeting antibody fragment. Bioconjug. Chem. 2014, 25 (3), 611-7.
33. Swee, L. K.; Guimaraes, C. P.; Sehrawat, S.; Spooner, E.; Barrasa, M. I.; Ploegh, H. L., Sortase-mediated modification of alphaDEC205 affords optimization of antigen presentation and immunization against a set of viral epitopes. Proc. Natl. Acad .Sci. U. S. A. 2013, 110 (4), 1428-33.
34. Beerli, R. R.; Hell, T.; Merkel, A. S.; Grawunder, U., Sortase Enzyme-Mediated Generation of Site-Specifically Conjugated Antibody Drug Conjugates with High In Vitro and In Vivo Potency. PLoS. One. 2015, 10 (7), e0131177.
35. Zhou, Q.; Stefano, J. E.; Manning, C.; Kyazike, J.; Chen, B.; Gianolio, D. A.; Park, A.; Busch, M.; Bird, J.; Zheng, X.; Simonds-Mannes, H.; Kim, J.; Gregory, R. C.; Miller, R. J.; Brondyk, W. H.; Dhal, P. K.; Pan, C. Q., Site-specific antibody-drug conjugation through glycoengineering. Bioconjug. Chem. 2014, 25 (3), 510-20.
36. van Geel, R.; Wijdeven, M. A.; Heesbeen, R.; Verkade, J. M.; Wasiel, A. A.; van Berkel, S. S.; van Delft, F. L., Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody-Drug Conjugates. Bioconjug. Chem. 2015, 26 (11), 2233-42.
37. Kovtun, Y. V.; Goldmacher, V. S., Cell killing by antibody–drug conjugates. Cancer. Letters. 2007, 255 (2), 232-240.
38. Chalouni, C.; Doll, S., Fate of Antibody-Drug Conjugates in Cancer Cells. J Exp Clin. Cancer. Res. 2018, 37 (1), 20.
39. Wakankar, A.; Chen, Y.; Gokarn, Y.; Jacobson, F. S., Analytical methods for physicochemical characterization of antibody drug conjugates. MAbs. 2011, 3 (2), 161-72.
40. Furuki, K.; Toyo'oka, T., Determination of thiol-to-protein ratio and drug-to-antibody ratio by in-line size exclusion chromatography with post-column reaction. Anal. Biochem. 2017, 527, 33-44.
41. Sang, H.; Lu, G.; Liu, Y.; Hu, Q.; Xing, W.; Cui, D.; Zhou, F.; Zhang, J.; Hao, H.; Wang, G.; Ye, H., Conjugation site analysis of antibody-drug-conjugates (ADCs) by signature ion fingerprinting and normalized area quantitation approach using nano-liquid chromatography coupled to high resolution mass spectrometry. Anal. Chim. Acta. 2017, 955, 67-78.
42. Koniev, O.; Wagner, A., Developments and recent advancements in the field of endogenous amino acid selective bond forming reactions for bioconjugation. Chem. Soc. Rev. 2015, 44 (15), 5495-551.