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Title Page 1
Contents 4
List of Abbreviations 14
Abstract 18
Chapter 1. Introduction 20
1.1. Historical Overview of Antibody-drug conjugates (ADCs) 20
1.2. Current Limitations of ADCs 22
1.3. Current Strategies to Expanding the Therapeutic Index of ADCs 25
1.3.1. Antibody 25
1.3.2. Linker 36
1.3.3. Payload 50
1.4. Development of Affinity-Based Linkers for Site-Selective Antibody Labeling 59
1.5. Development of a Phosphate-Based Soluble Exatecan Pro-Prodrug 61
Chapter 2. Results and Discussion 62
2.1. Fc-binding O-Acyl Hydroxamate Reagents for Site-Selective Antibody Labeling 62
2.1.1. Design and synthesis of Fc-binding O-acyl hydroxamates (OAHs) 62
2.1.2. Reactivity and stability of Fc-binding OAHs affinity labels (7a-g) 66
2.1.3. Cross-linking test of Fc-binding OAHs with trastuzumab 69
2.1.4. Reaction kinetics analysis of Fc-binding OAH 7g 71
2.1.5. Binding site analysis of trastuzumab-Fc-binding OAH 7g 73
2.1.6. Application of Fc-binding OAH for site-selective ADC 77
2.1.7. Biological efficacy test of trastuzumab-Fc binding OAH 81
2.2. Affinity-Based Catalysts for Site-Selective Antibody Labeling 84
2.2.1. Design and synthesis of affinity-based catalysts and acyl donors 84
2.2.2. Cross-linking test of affinity-based catalysts with trastuzumab 89
2.2.3. Biological efficacy test of trastuzumab-MMAE ADC 103
2.3. Phosphate-Based Soluble Exatecan Pro-Prodrug 107
2.3.1. Design and synthesis of exatecan prodrug and pro-prodrug 107
2.3.2. Conjugation of prodrug and pro-prodrug to CLDN18.2 mAb 111
2.3.3. Hydrophobicity comparison between exatecan prodrug and exatecan pro-prodrug 112
Chapter 3. Conclusions 114
Chapter 4. Materials and Methods 115
4.1. General Information 115
4.1.1. Chemicals and antibody 115
4.1.2. Purification of small molecules and peptides 116
4.1.3. Intact mass analysis of ADCs 116
4.1.4. Binding site analysis using in-solution digestion 116
4.1.5. Instruments 116
4.1.6. Instrumental parameters 117
4.1.7. Mass determination 118
4.2. Synthetic Methods 120
4.2.1. Synthesis of 5-(((tert-butoxycarbonyl)amino)oxy)-5-oxopentanoic acid. (2a–d) 120
4.2.2. Synthesis of 5-(aminooxy)-5-oxopentanoic acid. (3a-g) 121
4.2.3. Synthesis of affinity labels. (7a-g) 122
4.2.4. Synthesis of FITC-7g 123
4.2.5. Synthesis of FITC-8 123
4.2.6. Synthesis of protected beta glucuronide-MMAE. (10) 124
4.2.7. Synthesis of beta glucuronide-MMAE. (11) 124
4.2.8. Synthesis of DBCO-beta glucuronide-MMAE. (12) 124
4.2.9. Synthesis of tert-butyl 2-((2-(2-azidoethoxy)acetyl)thio)acetate. (16) 125
4.2.10. Synthesis of 2-((2-(2-azidoethoxy)acetyl)thio)acetic acid. (17) 125
4.2.11. Synthesis of 5-((benzyloxy)(methyl)amino)-5-oxopentanoic acid. (18) 126
4.2.12. Synthesis of 5-(hydroxy(methyl)amino)-5-oxopentanoic acid. (19) 126
4.2.13. Synthesis of 3-(5-methoxy-5-oxopentanamido)-2-oxopyrrolidin-1-yl methyl glutarate. (20) 127
4.2.14. Synthesis of 5-((1-hydroxy-2-oxopyrrolidin-3-yl)amino)-5-oxopentanoic acid. (21) 128
4.2.15. Synthesis of 1-azido-N-((4-nitrophenyl)sulfonyl)-3,6,9,12- tetraoxa pentadecan-15-amide. (22) 128
4.2.16. Synthesis of 1-azido-N-(4-nitrobenzyl)-N-((4-nitrophenyl)sulfonyl)-3,6,9,12- tetraoxapentadecan-15-amide. (23) 129
4.2.19. Synthesis of affinity-based catalysts 24~28. (SH series) 129
4.2.20. Synthesis of affinity-based catalysts hydroxamic acid. (29) 130
4.2.21. Synthesis of affinity-based catalysts cyclic hydroxamic acid. (30) 130
4.2.21. Synthesis of (2S,3R,4S,5S,6R)-2-(4-formyl-2-nitrophenoxy)-6-(methoxy carbonyl) tetrahydro-2H-pyran-3,4,5-triyl triace. (31) 131
4.2.22. Synthesis of (2S,3R,4S,5S,6R)-2-(4-(hydroxymethyl)-2-nitrophenoxy)- 6-(methoxy carbonyl) tetrahydro-2H-pyran-3,4,5-triyl triacetate. (32) 131
4.2.23. Synthesis of (2S,3R,4S,5S,6R)-2-(2-amino-4-(hydroxymethyl)phenoxy) -6-(methoxy carbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate. (33) 132
4.2.24. Synthesis of (2S,3R,4S,5S,6R)-2-(2-(3-((((9H-fluoren-9-yl)methoxy) carbonyl) amino)propanamido)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate. (34) 133
4.2.25. Synthesis of (2S,3R,4S,5S,6R)-2-(2-(3-((((9H-fluoren-9-yl)methoxy) carbonyl)amino)propanamido)-4-(((((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano [3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate (36) 134
4.2.26. Synthesis of (2S,3R,4S,5S,6R)-2-(2-(3-((((9H-fluoren-9-yl)methoxy) carbonyl) amino)propanamido)-4-(((((1R,9R)-9-((bis(2-cyanoethoxy)phosphanyl)oxy)-9 ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo [de]pyrano [3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenoxy)-6-(metho xycarbonyl) tetrahydro-2H-pyran-3,4,5-triyl triacetate. (37) 135
4.2.27. Synthesis of (2S,3R,4S,5S,6R)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-(((((1R,9R)-9-((bis(2-cyanoethoxy)phosphoryl)oxy)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H ,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl tria cetate. (38) 136
4.2.28. Synthesis of (2R,3S,4S,5R,6S)-6-(2-(3-aminopropanamido)-4-(((((1R,9R)-9-ethyl-5-fluoro-4-methyl-10,13-dioxo-9-(phosphonooxy)-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2bquinolin-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (39) 136
4.2.29. Synthesis of DBCO-BG-exatecan-phosphate pro-prodrug. (40) 137
4.2.30. Synthesis of (2R,3S,4S,5R,6S)-6-(2-(3-aminopropanamido)-4-(((((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexa hydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid. (41) 137
4.2.32. Synthesis of DBCO-BG-exatecan prodrug (42) 138
4.3. Products Identification 139
4.3.1. ¹H NMR and ¹³C NMR spectra of 2a-d 139
4.3.2. ¹H NMRspectra of 3a-g 143
4.3.3. Structures, HPLC chromatogram, and HRMS of Fc-binding OAHs 154
References 174
논문요약 184
Figure 1. Comparison of response between conventional chemotherapy and ADCs. Waterfall... 22
Figure 2. Mechanisms of ADC-induced toxicity. Schematic showing major toxicity pathways of... 24
Figure 3. Mechanistic classification of bispecific antibody (bsAb) therapeutic strategies. (a)... 28
Figure 4. Tumor-selective activation mechanism of probody therapeutics. Probody therapeutics ... 30
Figure 5. Comparative advantages of nanobodies over conventional mAb. While mAbs suffer... 32
Figure 6. Anti-payload antibody strategy to reduce off-target toxicity in ADC therapy. This... 35
Figure 7. Engineered cysteine for site-specific conjugation. Schematic illustration of engineered... 37
Figure 8. Site-specific antibody-drug conjugation via unnatural amino acid (UAA) incorporation... 39
Figure 9. Site-specific antibody-drug conjugation via disulfide rebridging. Schematic... 41
Figure 10. FGE-mediated introduction of reactive aldehyde groups into antibody. A schematic... 43
Figure 11. Site specific antibody conjugation via transglutaminase-mediated labeling. A... 45
Figure 12. Affinity ligands for site-selective acylation to human IgG1 Lys248. AbClick Standard... 47
Figure 13. Effect of side chain PEGylation on hydrophobicity shielding of ADC. Schematic... 49
Figure 14. Mechanism of action of PBD dimer. (a) Structure of SG2057 (PBD dimer); (b)... 53
Figure 15. Concept of prodrug and pro-prodrug. This figure illustrates the structural modification... 54
Figure 16. Platform of OPHAS linker for rapid and efficient payload release chemistry. The... 56
Figure 17. Sequence analysis and modeling of Fc-III cyclic peptides. Green: Can't modify amino... 62
Figure 18. Reactivity (N-Ac-Lys) assessment of Fc-binding OAHs 7 and 8. (a) Reactivity... 67
Figure 19. Stability assessment of Fc-binding OAH 7a-g and 8. (a) Stability of Fc-binding OAH... 68
Figure 20. Reactivity assessment of Fc-binding OAHs 7a-g and 8. Schematic illustration of... 69
Figure 21. Cross-linking optimization of Fc-binding OAH 7a-g and 8. (a) Reactivity with... 70
Figure 22. Kinetic analysis of Fc-binding OAH 7g. Reaction rates were measured at varying... 71
Figure 23. Analysis of the pseudo-first-order reaction kinetics of 7g. (a) Initial reaction velocity... 72
Figure 24. Binding site analysis of trastuzumab-FITC conjugates. (a) Schematic illustration of... 74
Figure 25. Identification of the binding site on trastuzumab by 7g. MS/MS analysis confirming... 74
Figure 26. Expected and calculated molecular weight of trastuzumab-7g. Peptide mapping... 76
Figure 27. Application of affinity label 7g for site-selective ADC construction. Schematic... 77
Figure 28. MALDI-TOF analysis of trastuzumab 13. Measured mass matches expected 79
Figure 29. MALDI-TOF analysis of trastuzumab-7g conjugate 14. Measured mass matches... 79
Figure 30. MALDI-TOF analysis of trastuzumab-MMAE ADC 15. Deconvoluted intact mass... 80
Figure 31. Antigen binding assay of trastuzumab MMAE ADC 15. Black: Trastuzumab (T); Blue:... 81
Figure 32. Cell viability assay of trastuzumab MMAE ADC 15. Black: Trastuzumab (T); Red: T-... 83
Figure 33. Reaction mechanism of native chemical ligation. (NCL) A C-terminal thioester reacts ... 84
Figure 34. Concept of affinity-based catalysts for site-selective conjugations. In this strategy, a... 86
Figure 35. Non-specific binding analysis of acyl donor. HIC-HPLC analysis showing no non-... 89
Figure 36. Cross-linking test between trastuzumab and designed linkers. HIC-HPLC analysis ... 90
Figure 37. Optimization of cross-linking efficiency by modulate reaction conditions. HIC-HPLC... 92
Figure 38. Stability assessment of Affinity-based catalyst and acyl donor. Acyl donor remained... 94
Figure 39. Cross-linking assessment of thioether type affinity-based catalyst 97
Figure 40. Cross-linking between trastuzumab and hydroxamic acid type affinity-based catalysts.... 101
Figure 41. Cross-linking optimization of affinity-based catalysts (-SH) and acyl donor. After site-... 104
Figure 42. Cell viability assay of trastuzumab-pro-BG-MMAE DAR2. Cell viability was ... 106
Figure 43. Rational design of exatecan pro-prodrug 107
Figure 44. Comparative physicochemical characterization of exatecan prodrug and exatecan pro-... 113
Figure 45. ¹H (400 MHz, CDCl₃) and ¹³C NMR (100 MHz, CDCl₃) of 2a 139
Figure 46. ¹H (400 MHz, CDCl₃) and ¹³C NMR (100 MHz, CDCl₃) of 2b 140
Figure 47. ¹H (400 MHz, CDCl₃) and ¹³C NMR (100 MHz, CDCl₃) of 2c 141
Figure 48. ¹H (400 MHz, CDCl₃) and ¹³C NMR (100 MHz, CDCl₃) of 2d 142
Figure 49. ¹H NMR (400 MHz, CDCl₃) of 3a 143
Figure 50. ¹H NMR (400 MHz, CDCl₃) of 3b 143
Figure 51. ¹H NMR (400 MHz, CDCl₃) of 3c 144
Figure 52. ¹H NMR (400 MHz, CDCl₃) of 3d 144
Figure 53. ¹H NMR (400 MHz, CDCl₃) of 3e 145
Figure 54. ¹H NMR (400 MHz, CDCl₃) of 3f 145
Figure 55. ¹H NMR (400 MHz, CDCl₃) of 3g 146
Figure 56. ¹H NMR (500 MHz, DMSO D6) and ¹³C NMR (100 MHz, DMSO D6) of thioester... 147
Figure 57. ¹H NMR (500 MHz, DMSO D6) and ¹³C NMR (100 MHz, DMSO D6) of... 148
Figure 58. ¹H NMR (500 MHz, DMSO D6) of hydroxamic acid INT 2 149
Figure 59. ¹H NMR (500 MHz, DMSO D6) of hydroxamic acid INT 3 149
Figure 60. ¹H NMR (500 MHz, DMSO D6) and ¹³C NMR (100 MHz, DMSO D6) of... 150
Figure 61. ¹H NMR (500 MHz, DMSO D6) and ¹³C NMR (100 MHz, DMSO D6) of cyclic... 151
Figure 62. ¹H NMR (500 MHz, DMSO D6) and ¹³C NMR (100 MHz, DMSO D6) of NASA... 152
Figure 63. ¹H NMR (500 MHz, DMSO D6) and ¹³C NMR (100 MHz, DMSO D6) of NASA acyl... 153
Figure 64. Structures, HPLC chromatograms, and HRMS chromatogram of 7a. HRMS (ESI-... 154
Figure 65. Structures, HPLC chromatograms, and HRMS chromatogram of 7b. HRMS... 155
Figure 66. Structures, HPLC chromatograms, and HRMS chromatogram of 7c. HRMS... 156
Figure 67. Structures, HPLC chromatograms, and HRMS chromatogram of 7d. HRMS... 157
Figure 68. Structures, HPLC chromatograms, and HRMS chromatogram of 7e. HRMS... 158
Figure 69. Structures, HPLC chromatograms, and HRMS chromatogram of 7f. HRMS... 159
Figure 70. Structures, HPLC chromatograms, and HRMS chromatogram of 7g. HRMS... 160
Figure 71. Structures, HPLC chromatograms, and HRMS chromatogram of 8. HRMS... 161
Figure 72. Structures, HPLC chromatograms, and HRMS chromatogram of FITC-7g.... 162
Figure 73. Structures, HPLC chromatograms, and HRMS chromatogram of FITC 8.... 163
Figure 74. Structures, HPLC chromatograms, and HRMS chromatogram of 12. HRMS... 164
Figure 75. Structures, HPLC chromatograms, and LC/MS of affinity-based catalyst 24 165
Figure 76. Structures, HPLC chromatograms, and LC/MS of affinity-based catalyst 25 166
Figure 77. Structures, HPLC chromatograms, and LC/MS of affinity-based catalyst 26 167
Figure 78. Structures, HPLC chromatograms, and LM/MS of affinity-based catalyst 27 168
Figure 79. Structures, HPLC chromatograms, and LC/MS of affinity-based catalyst 28 169
Figure 80. Structures, HPLC chromatograms, and LC/MS of affinity-based catalyst 29 170
Figure 81. Structures, HPLC chromatograms, and LC/MS of affinity-based catalyst 30 171
Figure 82. Structures, HPLC chromatograms, and LC/MS of exatecan prodrug 172
Figure 83. Structures, HPLC chromatograms, and LC/MS of exatecan pro-... 173
Scheme 1. Scheme of O-acyl hydroxamate (2a-d, 3a-g). (i) Glutaric anhydride, (DCM), 25 °C... 64
Scheme 2. Scheme of Fc-binding OAHs (7a-g, 8). (i) TFA:TIS:EDT:DW (distilled water) ＝... 65
Scheme 3. Scheme of FITC-affinity labels (FITC 7g, FITC 8). (i) DBCO-FITC, DMF, rt, 1 h;... 73
Scheme 4. Scheme of DBCO-beta glucuronide monomethyl auristatin E (MMAE). (10-12) (i)... 78
Scheme 5. Scheme of affinity-based catalysts. Reagents and condition: (i) TFA:TIS:EDT:EW ＝... 87
Scheme 6. Scheme of thioester-based acyl donor. To minimize non-specific interactions with the... 88
Scheme 7. Scheme of affinity-based catalyst with thioether bridge formation 95
Scheme 8. Scheme of new affinity-based catalysts and acyl donor. Structure of a newly developed... 99
Scheme 9. Scheme of reactivity modulated new acyl donors 102
Scheme 10. Scheme of DBCO-BG-exatecan-phosphate pro-prodrug 109
Scheme 11. Scheme of DBCO-BG-exatecan prodrug 111
초록보기
항체-약물 복합체(ADC)는 항체의 표적 특이성과 세포독성 약물을 결합하여, 암세포를 선택적으로 제거할 수 있는 차세대 정밀 항암제로 주목받고 있다. ADC 개발의 궁극적인 목표는 종양 선택적 약물 전달을 통해 최소 유효 용량 (MED)을 감소시키고, 비표적 독성을 최소화함으로써 최대 허용 용량 (MTD)을 증가시켜 치료 지수(TI)를 확장하는 데 있다. 그러나 실제 임상에서 많은 ADC들은 이론적으로 기대되는 TI의 확장을 달성하지 못하였으며, MTD는 기존 화학항암제와 유사한 수준에 머무는 경우가 많다. 이는 크게 두 가지 요인, 표적 항원이 정상 조직에도 발현되는 데에서 기인하는 on-target toxicity와, 링커 불안정성과 약물의 소수성으로 인해 발생하는 off-target toxicity에 기인한다. 본 연구는 이러한 제한점을 극복하고 차세대 ADC로써 안전성과 약물의 효능을 향상시키기 위해, 위치선택적 접합 기술과 약물의 수용성을 개선한 prodrug 전략을 개발하였다. 먼저, 접합 위치의 정밀한 제어를 위해 인간 IgG1 항체의 CH2-CH3 경계에 위치한 Lys248에 강하게 결합하는 Fc-Ⅲ cyclic peptide 기반의 친화성 리간드를 설계하고, O-acyl hydroxamate 유도체를 도입하여 아실화를 유도하였다. 또한, 항체에 비공유적으로 결합한 후 근접성에 기반한 아실 전이를 유도하는 affinity-based catalyst를 개발하여, 동일한 리간드가 반복적으로 반응에 참여할 수 있는 촉매적 시스템을 구축하였다. 이러한 전략은 기존 Cys 또는 Lys 기반의 무작위 접합이 가진 비균일성과 불안정성 문제를 해결하고, 안정적인 isopeptide 결합 형성을 통해 ADC 균질성과 링커 안정성을 크게 향상시켰다.
또한, off-target toxicity의 주된 원인 중 하나인 약물의 소수성 문제를 해결하기 위해, 수용성을 향상시킨 prodrug 전략을 개발하였다. 특히 Exatecan 구조에 phosphate와 β-glucuronide 기능단을 도입하여 약물의 소수성을 효과적으로 마스킹하였으며, 이로 인해 약물은 순환계 내에서 비활성화된 형태로 존재하면서도 종양 미세환경 내의 특이적 효소(예: phosphatase 또는 β-glucuronidase)에 의해 선택적으로 활성화되는 특징을 지닌다. 이 전략은 기존의 hydrophobic payload가 유도하던 비표적 세포의 macropinocytosis 기반 흡수를 현저히 줄이며, 전신 독성을 낮추고 종양 내 선택성을 향상시키는 데 기여하였다. Pro-prodrug 형태의 Exatecan은 기존 약물 대비 월등한 수용성을 보였으며, HIC-HPLC 분석에서 retention time이 의미 있게 감소하였고, 높은 DAR에서도 aggregation이 억제되었다. 이 두 가지 전략은 항체의 엔지니어링 없이도 적용 가능하고, 다양한 payload 및 적응증에 확장될 수 있어 차세대 ADC 설계에 있어 실용성과 범용성을 갖춘 전략으로 생각된다.MARC 보기
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