將抗體連接於奈米粒子、晶片等固相載體上，稱為抗體固化；而連接螢光、
毒素、蛋白質等功能分子，則稱為抗體修飾。由於不同抗體能專一地辨認其對應
的抗原，因此兩項技術能應用於生化檢測、萃取目標蛋白及開發抗體藥物複合體
等多種重要用途上。在固化及修飾的過程中，如何維持抗體的抗原辨識活性，為
重要的研究課題。
本論文藉由抗體天然的金屬結合位與光親和性標記基團，開發了新的抗體固
化及修飾方法。金屬結合位位於抗體Fc 區域底部，遠離抗原結合區域。在此位
點進行固化或修飾能最大幅度保留抗原辨識活性。而藉由激發光親和性標記基團，
能使抗體與載體或探針生成共價鍵結，增加連結的穩定性。
在抗體固化方法中，我們成功將Trastuzumab、anti-SAA mAb、Cetuximab 三
種不同抗體固化於NTA/Dia-2(1/1)@MNP 上。在Fab曝露量及萃取SAA 蛋白實
驗中，相比於隨機固化方法，分別可得9.2 倍及22 倍訊號提升。此外針對具有
Fab glycan 的抗體如Cetuximab，此方法仍能維持抗體位向性，EGFR 蛋白萃取量
相較於過去硼酸誘導固化方法，有6 倍（SDS-PAGE分析）及3 倍（LC-MS/MS）
差距，並能應用於分析蛋白質訊號傳遞上。
在抗體修飾方法中，我們合成兩代氮[基]三醋酸誘導光親和探針（NTA
probe）。並成功利用第二代NTA probe 將炔基修飾在Trastuzumab 上。藉由活
性測試分析，此修飾方法並不會影響抗原辨識活性。此外利用點擊反應，可連接
螢光基團，並分析得到螢光對抗體比例約1.8。利用SDS-PAGE分析亦證明大部
分炔基皆修飾在抗體Fc區域。
總結來說，本論文成功開發出抗體固化及修飾方法，能應用於不同抗體上。
無需複雜的基因工程技術，便可快速製備不同的生化工具。

The immobilization of antibodies on solid supports, such as nanoparticles, glass
slides, etc., is called antibody immobilization The incorporation of functional groups
on antibodies, such as fluorescence dyes, toxins, other protein, etc., is called antibody
modification. By harnessing the specific antigen recognition ability of antibodies, these
two technologies can be applied to many biochemical tools such as diagnostic assays,
protein purification, and the development of antibody-drug conjugates. However, to
maintain the original bioactivity of antibody is one of the most significant challenges
in antibody immobilization and modification.
In this thesis, we developed a novel antibody immobilization and modification
method using the native metal binding site of antibody coupled with a photoaffinity
labeling (PAL) group. The metal binding site is located at the Fc domain of antibodies,
which is far from its antigen binding domain, thus performing immobilization or
modification at this site can retain the bioactivity of antibody. Furthermore, the PAL
group can allow antibodies to form stable covalent linkages with a solid support or
functional probes via irradiation with UV light.
For antibody immobilization, three antibodies (Trastuzumab, anti-SAA mAb, and
Cetuximab) were successfully immobilized on fabricated nitrilotriacetic acid/diazirinefunctionalized
magnetic nanoparticles (NTA/Dia-2(1/1)@MNP). Compared to random
antibody immobilization methods, our method achieved a 9.2- and 22-fold signal
improvement for Fab domain exposure and the extraction of SAA protein, respectively.
More importantly, for antibodies which contain a Fab glycan like Cetuximab, this
method retains the antibody orientation. Furthermore, compared to previous boronic
acid-directed immobilization methods, our immobilization method improved the
extraction yield for EGFR 6-fold in SDS-PAGE analysis and 3-fold in LC-MS/MS
analysis, and can be applied to the analysis of protein signal transduction.
For antibody modification, we synthesized two NTA probes and successfully
created an alkyne-modified antibody (Trastuzumab) using our 2nd generation NTA
probe. The binding affinity test revealed that the bioactivity of the modified antibody
is the same as intact antibody, indicating that the developed method does not affect
antigen binding affinity. Furthermore, the modified antibody could be attached with a
fluorescence dye via click reaction, and results show that much of the modification
occurred at the Fc domain of the antibody, with a dye to antibody ratio of around 1.8.
In this thesis research, we have developed a new antibody immobilization and
modification method which can be applied to various native antibodies. The method
does not require complex genetic engineering of the antibody, which enables the
efficient creation of various biochemical tools.

摘要 ............................................................................................................ I
Abstract ..................................................................................................... II
謝誌 ......................................................................................................... III
目錄 ........................................................................................................... V
圖目錄 ....................................................................................................... X
流程圖目錄 .......................................................................................... XVI
縮寫對照表（Abbreviation） ............................................................ XVII
第一章 緒論 ............................................................................................. 1
1.1 抗體（ANTIBODY） ............................................................................ 1
1.1.1IgG 抗體的結構 ................................................................................ 1
1.1.2 抗原結合區段（Fab）及結晶區段（Fc） .................................... 2
1.2 抗體固化的重要性與應用 ................................................................. 3
1.3 抗體固化方法 ..................................................................................... 3
1.3.1 物理吸附方法 .................................................................................. 5
1.3.2 胺基酸化學耦聯方法 ...................................................................... 6
1.3.2.1 離胺酸（Lysine, Lys） ................................................................ 6
1.3.2.2 半胱胺酸（Cysteine, Cys） ......................................................... 8
1.3.3 藉由抗體上醣體之固化方法 ........................................................ 10
1.3.3.1 化學氧化法 ................................................................................ 11
1.3.3.2 硼酯固化法 ................................................................................. 13
1.3.4 藉由抗體結合蛋白或胜肽之固化方法 ........................................ 17
1.3.5 其他抗體固化方法 ....................................................................... 20
1.4 抗體修飾的重要性與應用 ............................................................... 21
1.5 抗體修飾方法 ................................................................................... 22
1.5.1 胺基酸化學耦聯方法 .................................................................... 24
1.5.1.1 離胺酸 ......................................................................................... 24
1.5.1.2 半胱胺酸 ..................................................................................... 26
1.5.1.3 其他胺基酸 ................................................................................. 28
1.5.2 藉由抗體上醣體之修飾方法 ........................................................ 30
1.5.2.1 化學氧化法 ................................................................................. 30
1.5.2.2 酵素修飾法 ................................................................................. 31
1.5.2.3 硼酸輔助法 ................................................................................. 32
1.5.3 藉由抗體結合胜肽（antibody binding peptide）之修飾方法 .... 33
1.6 抗體的金屬結合位（metal binding site） ...................................... 34
1.6.1 抗體金屬結合位之應用 ................................................................ 35
1.7 光親和性標記基團（photoaffinity labeling group, PAL group） . 41
1.7.1 氮烯 ................................................................................................ 42
1.7.2 羰自由基 ....................................................................................... 43
1.7.3 碳烯 ................................................................................................ 44
第二章 氮[基]三醋酸及光親和性磁性奈米粒子應用於天然抗體位向
性固化 ..................................................................................................... 47
2.1 研究動機 ........................................................................................... 47
2.2 研究結果與討論 ............................................................................... 48
2.2.1 氮[基]三醋酸及光親和性固化的概念與設計 ............................. 48
2.2.2 NTA, Dia-1, Dia-2 之合成 ............................................................. 49
2.2.3 NTA@MNP、Dia-1@MNP 和Dia-2@MNP 之製備與鑑定 ..... 52
2.2.4 抗體與NTA@MNP 吸附測試 ..................................................... 55
2.2.5 Trastuzumab-Ni2+-NTA@MNP 之競爭實驗測試 ........................ 58
2.2.6 抗體與Dia-1@MNP、Dia-2@MNP 吸附測試 ........................... 60
2.2.7 探討不同NTA/Dia-2 比例對抗體固化效率之影響 .................... 63
2.2.8 探討照光反應時間及抗體濃度對抗體固化效率之影響 ............ 66
2.2.9 比較不同抗體固化方法之Fab 曝露量 ........................................ 69
2.2.10 以anti-SAA mAb-NTA/Dia-2(1/1)@MNP 萃取血清中SAA 蛋
白並與其他固化方法比較 ..................................................................... 72
2.2.11 以Cetuximab-NTA/Dia-2@MNP 萃取細胞中EGFR 蛋白並與
其他固化方法比較 ................................................................................. 74
2.3 總結 ................................................................................................... 80
2.4 實驗部分 ........................................................................................... 82
第三章 氮[基]三醋酸誘導光親和探針應用於天然抗體修飾 ............ 93
3.1 研究動機 ........................................................................................... 93
3.2 研究結果與討論 ............................................................................... 94
3.2.1 第一代氮[基]三醋酸誘導光親和探針結構設計與合成 ............ 94
3.2.2 利用第一代探針進行抗體修飾 ................................................... 98
3.2.2.1 NTA 誘導及光親和標記之概念驗證 ........................................ 99
3.2.2.2 抗體光親和標記效率之最佳化測試 ....................................... 101
3.2.2.3 修飾抗體的純化 ....................................................................... 102
3.2.2.4 純化抗體的釋放 ...................................................................... 104
3.2.2.5 小結 ........................................................................................... 105
3.2.3 第二代探針結構設計與合成 ...................................................... 106
3.2.4 利用第二代探針進行抗體修飾 .................................................. 108
3.2.4.1 抗體光親和性標記效率之最佳化測試 ................................... 111
3.2.4.2 修飾抗體的純化 ....................................................................... 112
3.2.4.3 抗體的回收與再標記 ............................................................... 114
3.2.4.4 純化抗體的釋放 ....................................................................... 117
3.2.5 以螢光染劑驗證炔基修飾抗體 .................................................. 119
3.2.6 探討修飾抗體上螢光染劑對抗體比例（Dye to antibody ratio,
DAR） .................................................................................................. 122
3.2.7 抗體修飾前後抗原辨識能力之比較 ......................................... 124
3.3 總結 ................................................................................................. 126
3.4 實驗步驟 ........................................................................................ 128
第四章 結論 ......................................................................................... 143
參考文獻 ............................................................................................... 145

1. Hoffman, W.; Lakkis, F. G.; Chalasani, G. B Cells, Antibodies, and More. Clin. J. Am. Soc. Nephrol. 2016, 11, 137-154.
2. Reth, M. Matching cellular dimensions with molecular sizes. Nat. Immunol. 2013, 14, 765-767.
3. Kurien, B. T.; Scofield, R. H. Western blotting. Methods 2006, 38, 283-293.
4. Weiner, L. M.; Surana, R.; Wang, S. Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat. Rev. Immunol. 2010, 10, 317-327.
5. Sung, H.-J.; Ahn, J.-M.; Yoon, Y.-H.; Rhim, T.-Y.; Park, C.-S.; Park, J.-Y.; Lee, S.-Y.; Kim, J.-W.; Cho, J.-Y. Identification and Validation of SAA as a Potential Lung Cancer Biomarker and its Involvement in Metastatic Pathogenesis of Lung Cancer. J. Proteome Res. 2011, 10, 1383-1395.
6. Arora, S.; Saxena, V.; Ayyar, B. V. Affinity chromatography: A versatile technique for antibody purification. Methods 2017, 116, 84-94.
7. Janeway, C. A. T., P.; Walport, M.; Shlomchik, M.J. Immunobiology: Tge Immune System in Health and Disease. 5th edition. (Grarland Science, New York, 2001).
8. Al-Lazikani, B.; Lesk, A. M.; Chothia, C. Standard conformations for the canonical structures of immunoglobulins. J. Mol. Biol. 1997, 273, 927-948.
9. Nimmerjahn, F.; Ravetch, J. V. Fcγ receptors as regulators of immune responses. Nat. Rev. Immunol. 2008, 8, 34-47.
10. Dunkelberger, J. R.; Song, W.-C. Complement and its role in innate and adaptive immune responses. Cell Res. 2010, 20, 34-50.
11. Lin, P.-C.; Chou, P.-H.; Chen, S.-H.; Liao, H.-K.; Wang, K.-Y.; Chen, Y.-J.; Lin, C.-C. Ethylene Glycol-Protected Magnetic Nanoparticles for a Multiplexed Immunoassay in Human Plasma. Small 2006, 2, 485-489.
12. Lee, H.; Sun, E.; Ham, D.; Weissleder, R. Chip-NMR biosensor for detection and molecular analysis of cells. Nat. Med. 2008, 14, 869-874.
13. Kagawa, T.; Matsumi, Y.; Aono, H.; Ohara, T.; Tazawa, H.; Shigeyasu, K.; Yano, S.; Takeda, S.; Komatsu, Y.; Hoffman, R. M.; Fujiwara, T.; Kishimoto, H. Immuno-hyperthermia effected by antibody-conjugated nanoparticles selectively targets and eradicates individual cancer cells. Cell Cycle 2021, 20, 1221-1230.
14. Cho, M. H.; Lee, E. J.; Son, M.; Lee, J.-H.; Yoo, D.; Kim, J.-w.; Park, S. W.; Shin, J.-S.; Cheon, J. A magnetic switch for the control of cell death signalling in in vitro and in vivo systems. Nat. Mater. 2012, 11, 1038-1043.
15. Liu, S.; Chen, X.; Bao, L.; Liu, T.; Yuan, P.; Yang, X.; Qiu, X.; Gooding, J. J.; Bai, Y.; Xiao, J.; Pu, F.; Jin, Y. Treatment of infarcted heart tissue via the capture and local delivery of circulating exosomes through antibody-conjugated magnetic nanoparticles. Nat. Biomed. Eng. 2020, 4, 1063-1075.
16. Yang, L.; Guo, S.; Li, Y.; Zhou, S.; Tao, S. Protein microarrays for systems biology. Acta Biochim. Biophys. 2011, 43, 161-171.
17. He, H.-J.; Zong, Y.; Bernier, M.; Wang, L. Sensing the insulin signaling pathway with an antibody array. Proteomics Clin. Appl. 2009, 3, 1440-1450.
18. Rucker, V. C.; Havenstrite, K. L.; Herr, A. E. Antibody microarrays for native toxin detection. Anal. Biochem. 2005, 339, 262-270.
19. Chen, Z.; Dodig-Crnković, T.; Schwenk, J. M.; Tao, S.-C. Current applications of antibody microarrays. Clin. Proteomics 2018, 15, 7.
20. Trilling, A. K.; Beekwilder, J.; Zuilhof, H. Antibody orientation on biosensor surfaces: a minireview. Analyst 2013, 138, 1619-1627.
21. Vashist, S. K.; Marion Schneider, E.; Lam, E.; Hrapovic, S.; Luong, J. H. T. One-step antibody immobilization-based rapid and highly-sensitive sandwich ELISA procedure for potential in vitro diagnostics. Sci. Rep. 2014, 4, 4407.
22. Li, Z.; Yi, Y.; Luo, X.; Xiong, N.; Liu, Y.; Li, S.; Sun, R.; Wang, Y.; Hu, B.; Chen, W.; Zhang, Y.; Wang, J.; Huang, B.; Lin, Y.; Yang, J.; Cai, W.; Wang, X.; Cheng, J.; Chen, Z.; Sun, K.; Pan, W.; Zhan, Z.; Chen, L.; Ye, F. Development and application of a rapid IgM-IgG combined antibody test for SARS-CoV-2 infection diagnosis. J. Med. Virol. 2020, 92, 1518-1524.
23. Choi, J.-S.; Jang, W. S.; Park, J.-S. Comparison of adsorption and conjugation of Herceptin on poly(lactic-co-glycolic acid) nanoparticles-Effect on cell internalization in breast cancer cells. Mater. Sci. Eng. C 2018, 92, 496-507.
24. Yoon, T.-J.; Yu, K. N.; Kim, E.; Kim, J. S.; Kim, B. G.; Yun, S.-H.; Sohn, B.-H.; Cho, M.-H.; Lee, J.-K.; Park, S. B. Specific Targeting, Cell Sorting, and Bioimaging with Smart Magnetic Silica Core–Shell Nanomaterials. Small 2006, 2, 209-215.
25. Szijj, P. A.; Bahou, C.; Chudasama, V. Minireview: Addressing the retro-Michael instability of maleimide bioconjugates. Drug Discov. Today. Technol. 2018, 30, 27-34.
26. Alonso, R.; Jiménez-Meneses, P.; García-Rupérez, J.; Bañuls, M.-J.; Maquieira, Á. Thiol–ene click chemistry towards easy microarraying of half-antibodies. ChemComm. 2018, 54, 6144-6147.
27. Barrientos, G.; Habazin, S.; Novokmet, M.; Almousa, Y.; Lauc, G.; Conrad, M. L. Changes in subclass-specific IgG Fc glycosylation associated with the postnatal maturation of the murine immune system. Sci. Rep. 2020, 10, 15243.
28. Hoffman, W. L.; O'Shannessy, D. J. Site-specific immobilization of antibodies by their oligosaccharide moieties to new hydrazide derivatized solid supports. J. Immunol. Methods 1988, 112, 113-120.
29. Prieto-Simón, B.; Saint, C.; Voelcker, N. H. Electrochemical Biosensors Featuring Oriented Antibody Immobilization via Electrografted and Self-Assembled Hydrazide Chemistry. Anal. Chem. 2014, 86, 1422-1429.
30. Yuan, Y.; Yin, M.; Qian, J.; Liu, C. Site-directed immobilization of antibodies onto blood contacting grafts for enhanced endothelial cell adhesion and proliferation. Soft Matter 2011, 7, 7207-7216.
31. Peluso, P.; Wilson, D. S.; Do, D.; Tran, H.; Venkatasubbaiah, M.; Quincy, D.; Heidecker, B.; Poindexter, K.; Tolani, N.; Phelan, M.; Witte, K.; Jung, L. S.; Wagner, P.; Nock, S. Optimizing antibody immobilization strategies for the construction of protein microarrays. Anal. Biochem. 2003, 312, 113-124.
32. Kim, G.; Weiss, S. J.; Levine, R. L. Methionine oxidation and reduction in proteins. Biochim. Biophys. Acta 2014, 1840, 901-905.
33. Duval, F.; van Beek, T. A.; Zuilhof, H. Key steps towards the oriented immobilization of antibodies using boronic acids. Analyst 2015, 140, 6467-6472.
34. Lin, P.-C.; Chen, S.-H.; Wang, K.-Y.; Chen, M.-L.; Adak, A. K.; Hwu, J.-R.; Chen, Y.-J.; Lin, C.-C. Fabrication of Oriented Antibody-Conjugated Magnetic Nanoprobes and Their Immunoaffinity Application. Anal. Chem. 2009, 81, 8774-8782.
35. Adak, A. K.; Li, B.-Y.; Huang, L.-D.; Lin, T.-W.; Chang, T.-C.; Hwang, K. C.; Lin, C.-C. Fabrication of Antibody Microarrays by Light-Induced Covalent and Oriented Immobilization. ACS Appl. Mater. Interfaces 2014, 6, 10452-10460.
36. Fan, C.-Y.; Huo, Y.-R.; Adak, A. K.; Waniwan, J. T.; dela Rosa, M. A. C.; Low, P. Y.; Angata, T.; Hwang, K.-C.; Chen, Y.-J.; Lin, C.-C. Boronate affinity-based photoactivatable magnetic nanoparticles for the oriented and irreversible conjugation of Fc-fused lectins and antibodies. Chem. Sci. 2019, 10, 8600-8609.
37. Adak, A. K.; Huang, K.-T.; Li, P.-J.; Fan, C.-Y.; Lin, P.-C.; Hwang, K.-C.; Lin, C.-C. Regioselective SN2-Type Reaction for the Oriented and Irreversible Immobilization of Antibodies to a Glass Surface Assisted by Boronate Formation. ACS Appl. Bio Mater. 2020, 3, 6756-6767.
38. Fishman, J. B.; Berg, E. A. Protein A and Protein G Purification of Antibodies. Cold Spring Harb. Protoc. 2019, 2019, pdb.prot099143.
39. Moser, J. J.; Chan, E. K. L.; Fritzler, M. J. Optimization of immunoprecipitation-western blot analysis in detecting GW182-associated components of GW/P bodies. Nat. Protoc. 2009, 4, 674-685.
40. Gao, S.; Guisán, J. M.; Rocha-Martin, J. Oriented immobilization of antibodies onto sensing platforms - A critical review. Anal. Chim. Acta 2022, 1189, 338907.
41. Jung, Y.; Lee, J. M.; Kim, J.-w.; Yoon, J.; Cho, H.; Chung, B. H. Photoactivable Antibody Binding Protein: Site-Selective and Covalent Coupling of Antibody. Anal. Chem. 2009, 81, 936-942.
42. Jung, Y.; Kang, H. J.; Lee, J. M.; Jung, S. O.; Yun, W. S.; Chung, S. J.; Chung, B. H. Controlled antibody immobilization onto immunoanalytical platforms by synthetic peptide. Anal. Biochem. 2008, 374, 99-105.
43. Choe, W.; Durgannavar, T. A.; Chung, S. J. Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides. Materials (Basel) 2016, 9, 994.
44. Ericsson, E. M.; Enander, K.; Bui, L.; Lundström, I.; Konradsson, P.; Liedberg, B. Site-Specific and Covalent Attachment of His-Tagged Proteins by Chelation Assisted Photoimmobilization: A Strategy for Microarraying of Protein Ligands. Langmuir 2013, 29, 11687-11694.
45. Chari, R. V. J.; Miller, M. L.; Widdison, W. C. Antibody-Drug Conjugates: An Emerging Concept in Cancer Therapy. Angew. Chem. Int. Ed. 2014, 53, 3796-3827.
46. Walsh, S. J.; Bargh, J. D.; Dannheim, F. M.; Hanby, A. R.; Seki, H.; Counsell, A. J.; Ou, X.; Fowler, E.; Ashman, N.; Takada, Y.; Isidro-Llobet, A.; Parker, J. S.; Carroll, J. S.; Spring, D. R. Site-selective modification strategies in antibody–drug conjugates. Chem. Soc. Rev. 2021, 50, 1305-1353.
47. Lucas, A. T.; Price, L. S. L.; Schorzman, A. N.; Storrie, M.; Piscitelli, J. A.; Razo, J.; Zamboni, W. C. Factors Affecting the Pharmacology of Antibody-Drug Conjugates. Antibodies (Basel) 2018, 7, 10.
48. Beck, A.; Goetsch, L.; Dumontet, C.; Corvaïa, N. Strategies and challenges for the next generation of antibody–drug conjugates. Nat. Rev. Drug Discov. 2017, 16, 315-337.
49. Qiu, D.; Huang, Y.; Chennamsetty, N.; Miller, S. A.; Hay, M. Characterizing and understanding the formation of cysteine conjugates and other by-products in a random, lysine-linked antibody drug conjugate. mAbs 2021, 13, 1974150.
50. 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, 925-932.
51. Matos, M. J.; Oliveira, B. L.; Martínez-Sáez, N.; Guerreiro, A.; Cal, P. M. S. D.; Bertoldo, J.; Maneiro, M.; Perkins, E.; Howard, J.; Deery, M. J.;Chalker, J. M.; Corzana, F.; Jiménez-Osés, G.; Bernardes, G. J. L., Chemo- and Regioselective Lysine Modification on Native Proteins. J. Am. Chem. Soc. 2018, 140, 4004-4017.
52. Adusumalli, S. R.;Rawale, D. G.; Thakur, K.; Purushottam, L.; Reddy, N. C.; Kalra, N.; Shukla, S.; Rai, V. Chemoselective and Site-Selective Lysine-Directed Lysine Modification Enables Single-Site Labeling of Native Proteins. Angew. Chem. Int. Ed. 2020, 59, 10332-10336.
53. Vinogradova, E. V.; Zhang, C.; Spokoyny, A. M.; Pentelute, B. L.; Buchwald, S. L. Organometallic palladium reagents for cysteine bioconjugation. Nature 2015, 526, 687-691.
54. Kasper, M.-A.; Glanz, M.; Stengl, A.; Penkert, M.; Klenk, S.; Sauer, T.; Schumacher, D.; Helma, J.; Krause, E.; Cardoso, M. C.; Leonhardt, H.; Hackenberger, C. P. R. Cysteine-Selective Phosphonamidate Electrophiles for Modular Protein Bioconjugations. Angew. Chem. Int. Ed. 2019, 58, 11625-11630.
55. Kasper, M.-A.; Glanz, M.; Oder, A.; Schmieder, P.; von Kries, J. P.; Hackenberger, C. P. R. Vinylphosphonites for Staudinger-induced chemoselective peptide cyclization and functionalization. Chem. Sci. 2019, 10, 6322-6329.
56. Tessier, R.; Nandi, R. K.; Dwyer, B. G.; Abegg, D.; Sornay, C.; Ceballos, J.; Erb, S.; Cianférani, S.; Wagner, A.; Chaubet, G.; Adibekian, A.; Waser, J. Ethynylation of Cysteine Residues: From Peptides to Proteins in Vitro and in Living Cells. Angew. Chem. Int. Ed. 2020, 59, 10961-10970.
57. Badescu, G.; Bryant, P.; Bird, M.; Henseleit, K.; Swierkosz, J.; Parekh, V.; Tommasi, R.; Pawlisz, E.; Jurlewicz, K.; Farys, M.; Camper, N.; Sheng, X.; Fisher, M.; Grygorash, R.; Kyle, A.; Abhilash, A.; Frigerio, M.; Edwards, J.; Godwin, A. Bridging Disulfides for Stable and Defined Antibody Drug Conjugates. Bioconjug. Chem. 2014, 25, 1124-1136.
58. 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, 3986-3998.
59. Walsh, S. J.; Omarjee, S.; Galloway, W. R. J. D.; Kwan, T. T. L.; Sore, H. F.; Parker, J. S.; Hyvönen, M.; Carroll, J. S.; Spring, D. R. A general approach for the site-selective modification of native proteins, enabling the generation of stable and functional antibody–drug conjugates. Chem. Sci. 2019, 10, 694-700.
60. Ban, H.; Nagano, M.; Gavrilyuk, J.; Hakamata, W.; Inokuma, T.; Barbas, C. F. Facile and Stabile Linkages through Tyrosine: Bioconjugation Strategies with the Tyrosine-Click Reaction. Bioconjug. Chem. 2013, 24, 520-532.
61. Dovgan, I.; Erb, S.; Hessmann, S.; Ursuegui, S.; Michel, C.; Muller, C.; Chaubet, G.; Cianférani, S.; Wagner, A. Arginine-selective bioconjugation with 4-azidophenyl glyoxal: application to the single and dual functionalisation of native antibodies. Org. Biomol. Chem. 2018, 16, 1305-1311.
62. Laguzza, B. C.; Nichols, C. L.; Briggs, S. L.; Cullinan, G. J.; Johnson, D. A.; Starling, J. J.; Baker, A. L.; Bumol, T. F.; Corvalan, J. R. F. New antitumor monoclonal antibody-vinca conjugates LY203725 and related compounds: design, preparation, and representative in vivo activity. J. Med. Chem. 1989, 32, 548-555.
63. Zuberbühler, K.; Casi, G.; Bernardes, G. J. L.; Neri, D. Fucose-specific conjugation of hydrazide derivatives to a vascular-targeting monoclonal antibody in IgG format. ChemComm. 2012, 48, 7100-7102.
64. Solomon, B.; Koppel, R.; Schwartz, F.; Fleminger, G. Enzymic oxidation of monoclonal antibodies by soluble and immobilized bifunctional enzyme complexes. J. Chromatogr. A. 1990, 510, 321-329.
65. Li, X.; Fang, T.; Boons, G.-J. Preparation of Well-Defined Antibody–Drug Conjugates through Glycan Remodeling and Strain-Promoted Azide–Alkyne Cycloadditions. Angew. Chem. Int. Ed. 2014, 53, 7179-7182.
66. Lin, C.-W.; Tsai, M.-H.; Li, S.-T.; Tsai, T.-I.; Chu, K.-C.; Liu, Y.-C.; Lai, M.-Y.; Wu, C.-Y.; Tseng, Y.-C.; Shivatare, S. S.; Wang, C.-H.; Chao, P.; Wang, S.-Y.; Shih, H.-W.; Zeng, Y.-F.; You, T.-H.; Liao, J.-Y.; Tu, Y.-C.; Lin, Y.-S.; Chuang, H.-Y.; Chen, C.-L.; Tsai, C.-S.; Huang, C.-C.; Lin, N.-H.; Ma, C.; Wu, C.-Y.; Wong, C.-H. A common glycan structure on immunoglobulin G for enhancement of effector functions. Proc. Natl. Acad. Sci. U.S.A. 2015, 112, 10611-10616.
67. Adak, A. K.; Huang, K.-T.; Liao, C.-Y.; Lee, Y.-J.; Kuo, W.-H.; Huo, Y.-R.; Li, P.-J.; Chen, Y.-J.; Chen, B.-S.; Chen, Y.-J.; Chu Hwang, K.; Wayne Chang, W.-S.; Lin, C.-C. Investigating a Boronate-Affinity-Guided Acylation Reaction for Labelling Native Antibodies. Chem. Eur. J. 2022, 28, e202104178.
68. Park, J.; Lee, Y.; Ko, B. J.; Yoo, T. H. Peptide-Directed Photo-Cross-Linking for Site-Specific Conjugation of IgG. Bioconjug. Chem. 2018, 29, 3240-3244.
69. Muguruma, K.; Osawa, R.; Fukuda, A.; Ishikawa, N.; Fujita, K.; Taguchi, A.; Takayama, K.; Taniguchi, A.; Ito, Y.; Hayashi, Y., Development of a High-Affinity Antibody-Binding Peptide for Site-Specific Modification. ChemMedChem 2021, 16, 1814-1821.
70. Hale, J. E.; Beidler, D. E. Purification of Humanized Murine and Murine Monoclonal Antibodies Using Immobilized Metal-Affinity Chromatography. Anal. Biochem. 1994, 222, 29-33.
71. Muir, B. W.; Barden, M. C.; Collett, S. P.; Gorse, A.-D.; Monteiro, R.; Yang, L.; McDougall, N. A.; Gould, S.; Maeji, N. J. High-throughput optimization of surfaces for antibody immobilization using metal complexes. Anal. biochem. 2007, 363, 97-107.
72. Ojeda, I.; Barrejón, M.; Arellano, L. M.; González-Cortés, A.; Yáñez-Sedeño, P.; Langa, F.; Pingarrón, J. M. Grafted-double walled carbon nanotubes as electrochemical platforms for immobilization of antibodies using a metallic-complex chelating polymer: Application to the determination of adiponectin cytokine in serum. Biosens. Bioelectron. 2015, 74, 24-29.
73. Rosen, C. B.; Kodal, A. L. B.; Nielsen, J. S.; Schaffert, D. H.; Scavenius, C.; Okholm, A. H.;Voigt, N. V.; Enghild, J. J.; Kjems, J.; Tørring, T.; Gothelf, K. V. Template-directed covalent conjugation of DNA to native antibodies, transferrin and other metal-binding proteins. Nat. Chem. 2014, 6, 804.
74. Mortensen, M. R.; Skovsgaard, M. B.; Okholm, A. H.; Scavenius, C.; Dupont, D. M.; Rosen, C. B.; Enghild, J. J.; Kjems, J.; Gothelf, K. V. Small-Molecule Probes for Affinity-Guided Introduction of Biocompatible Handles on Metal-Binding Proteins. Bioconjug. Chem. 2018, 29, 3016-3025.
75. Skovsgaard, M. B.; Jeppesen, T. E.; Mortensen, M. R.; Nielsen, C. H.; Madsen, J.; Kjaer, A.; Gothelf, K. V. Affinity-Guided Conjugation to Antibodies for Use in Positron Emission Tomography. Bioconjug. Chem. 2019, 30, 881-887.
76. Hemdan, E. S.; Zhao, Y. J.; Sulkowski, E.; Porath, J. Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 1811-1815.
77. Singh, A.; Thornton, E. R.; Westheimer, F. H. The Photolysis of Diazoacetylchymotrypsin. J. Bio. Chem. 1962, 237, 3006-3008.
78. Vodovozova, E. L. Photoaffinity labeling and its application in structural biology. Biochem. (Mosc.) 2007, 72, 1-20.
79. Fleet, G. W. J.; Porter, R. R.; Knowles, J. R. Affinity Labelling of Antibodies with Aryl Nitrene as Reactive Group. Nature 1969, 224, 511-512.
80. Borden, W. T.; Gritsan, N. P.; Hadad, C. M.; Karney, W. L.; Kemnitz, C. R.; Platz, M. S. The Interplay of Theory and Experiment in the Study of Phenylnitrene. Acc. Chem. Res. 2000, 33, 765-771.
81. Dorman, G.; Prestwich, G. D. Benzophenone Photophores in Biochemistry. Biochem. 1994, 33, 5661-5673.
82. Smith, R. A. G.; Knowles, J. R. The preparation and photolysis of 3-aryl-3H-diazirines. J. Chem. Soc. Perkin Trans 2 1975, 7, 686-694.
83. Brunner, J.; Senn, H.; Richards, F. M. 3-Trifluoromethyl-3-phenyldiazirine. A new carbene generating group for photolabeling reagents. J. Bio. Chem. 1980, 255, 3313-3318.
84. Church, R. F. R.; Weiss, M. J. Diazirines. II. Synthesis and properties of small functionalized diazirine molecules. Observations on the reaction of a diaziridine with the iodine-iodide ion system. J. Org. Chem. 1970, 35, 2465-2471.
85. Hirabayashi, K.; Hanaoka, K.; Shimonishi, M.; Terai, T.; Komatsu, T.; Ueno, T.; Nagano, T. Selective Two-Step Labeling of Proteins with an Off/On Fluorescent Probe. Chem. Eur. J. 2011, 17, 14763-14771.
86. Bond, M. R.; Zhang, H.; Vu, P. D.; Kohler, J. J. Photocrosslinking of glycoconjugates using metabolically incorporated diazirine-containing sugars. Nat. Protoc. 2009, 4, 1044-1063.
87. Gunturu, K. S.; Woo, Y.; Beaubier, N.; Remotti, H. E.; Saif, M. W. Gastric cancer and trastuzumab: first biologic therapy in gastric cancer. Ther. Adv. Med. Oncol. 2013, 5, 143-151.
88. Procacci, B.; Roy, S. S.; Norcott, P.; Turner, N.; Duckett, S. B. Unlocking a Diazirine Long-Lived Nuclear Singlet State via Photochemistry: NMR Detection and Lifetime of an Unstabilized Diazo-Compound. J. Am. Chem. Soc. 2018, 140, 16855-16864.
89. Albini, A.; Kisch, H. Transition metal complexes of azo compounds V. Complexation and cleavage of the N=N bond of diazirines by iron carbonyls. J. Organomet. Chem. 1975, 94, 75-85.
90. Malle, E.; Sodin-Semrl, S.; Kovacevic, A. Serum amyloid A: an acute-phase protein involved in tumour pathogenesis. Cell Mol. Life Sci. 2009, 66, 9-26.
91. Wang, J.-Y.; Zheng, Y.-Z.; Yang, J.; Lin, Y.-H.; Dai, S.-Q.; Zhang, G.; Liu, W.-L. Elevated levels of serum amyloid A indicate poor prognosis in patients with esophageal squamous cell carcinoma. BMC Cancer 2012, 12, 365.
92. Li, Z.; Hou, Y.; Zhao, M.; Li, T.; Liu, Y.; Chang, J.; Ren, L. Serum amyloid a, a potential biomarker both in serum and tissue, correlates with ovarian cancer progression. J. Ovarian Res. 2020, 13, 67.
93. Wu, D.-C.; Wang, K.-Y.; Wang, S. S. W.; Huang, C.-M.; Lee, Y.-W.; Chen, M. I.; Chuang, S.-A.; Chen, S.-H.; Lu, Y.-W.; Lin, C.-C.; Lee, K.-W.; Hsu, W.-H.; Wu, K.-P.; Chen, Y.-J. Exploring the expression bar code of SAA variants for gastric cancer detection. PROTEOMICS 2017, 17, 1600356.
94. Chan, D. C.; Chen, C. J.; Chu, H. C.; Chang, W. K.; Yu, J. C.; Chen, Y. J.; Wen, L. L.; Huang, S. C.; Ku, C. H.; Liu, Y. C.; Chen, J. H. Evaluation of serum amyloid A as a biomarker for gastric cancer. Ann. Surg. Oncol. 2007, 14, 84-93.
95. van de Bovenkamp, F. S.; Hafkenscheid, L.; Rispens, T.; Rombouts, Y. The Emerging Importance of IgG Fab Glycosylation in Immunity. J. Immunol. 2016, 196, 1435-1441.
96. Giddens, J. P.; Lomino, J. V.; DiLillo, D. J.; Ravetch, J. V.; Wang, L.-X. Site-selective chemoenzymatic glycoengineering of Fab and Fc glycans of a therapeutic antibody. Proc. Natl. Acad. Sci. U.S.A. 2018, 115, 12023-12027.
97. You, B.; Chen, E. X. Anti-EGFR Monoclonal Antibodies for Treatment of Colorectal Cancers: Development of Cetuximab and Panitumumab. J. Clin. Pharmacol. 2012, 52, 128-155.
98. Fasano, M.; Della Corte, C. M.; Viscardi, G.; Di Liello, R.; Paragliola, F.; Sparano, F.; Iacovino, M. L.; Castrichino, A.; Doria, F.; Sica, A.; Morgillo, F.; Colella, G.; Tartaro, G.; Cappabianca, S.; Testa, D.; Motta, G.; Ciardiello, F. Head and neck cancer: the role of anti-EGFR agents in the era of immunotherapy. Ther. Adv. Med. Oncol. 2021, 13, 1758835920949418.
99. Jo, S.-M.; Noh, S.-h.; Jin, Z.; Lim, Y.; Cheon, J.; Kim, H.-S. Simple and efficient capture of EGFR-expressing tumor cells using magnetic nanoparticles. Sens. Actuators B Chem. 2014, 201, 144-152.
100. Chen, H.-L.; Hsu, F.-T.; Kao, Y.-C. J.; Liu, H.-S.; Huang, W.-Z.; Lu, C.-F.; Tsai, P.-H.; Ali, A. A. A.; Lee, G. A.; Chen, R.-J.; Chen, C.-Y. Identification of epidermal growth factor receptor-positive glioblastoma using lipid-encapsulated targeted superparamagnetic iron oxide nanoparticles in vitro. J. Nanobiotechnology 2017, 15, 86.
101. Han, C.-L.; Chien, C.-W.; Chen, W.-C.; Chen, Y.-R.; Wu, C.-P.; Li, H.; Chen, Y.-J. A Multiplexed Quantitative Strategy for Membrane Proteomics: Opportunities for Mining Therapeutic Targets for Autosomal Dominant Polycystic Kidney Disease. Mol. Cell. Proteomics 2008, 7, 1983-1997.
102. Tomohiro, T.; Inoguchi, H.; Masuda, S.; Hatanaka, Y. Affinity-based fluorogenic labeling of ATP-binding proteins with sequential photoactivatable cross-linkers. Bioorg. Med. Chem. Lett. 2013, 23, 5605-5608.
103. Li, P.-J.; Anwar, M. T.; Fan, C.-Y.; Juang, D. S.; Lin, H.-Y.; Chang, T.-C.; Kawade, S. K.; Chen, H.-J.; Chen, Y.-J.; Tan, K.-T.; Lin, C.-C. Fluorescence “Turn-on” Lectin Sensors Fabricated by Ligand-Assisted Labeling Probes for Detecting Protein–Glycoprotein Interactions. Biomacromolecules 2020, 21, 815-824.
104. Song, H.-N.; Kim, D.-H.; Park, S.-G.; Lee, M. K.; Paek, S.-H.; Woo, E.-J. Purification and characterization of Fab fragments with rapid reaction kinetics against myoglobin. Biosci., Biotechnol. Biochem. 2015, 79, 718-724.
105. Head, S. A.; Shi, W.; Zhao, L.; Gorshkov, K.; Pasunooti, K.; Chen, Y.; Deng, Z.; Li, R.-j.; Shim, J. S.; Tan, W.; Hartung, T.; Zhang, J.; Zhao, Y.; Colombini, M.; Liu, J. O. Antifungal drug itraconazole targets VDAC1 to modulate the AMPK/mTOR signaling axis in endothelial cells. Proc. Natl. Acad. Sci. U.S.A. 2015, 112, 7276-7285.