Antibody-Drug Conjugate Using Ionized Cys-Linker-MMAE as the Potent Payload Shows Optimal Therapeutic Safety
Abstract
:1. Introduction
2. Results
2.1. Design and Synthesis of the Cys-Linker-MMAE-Based ADC Payloads
2.2. Tubulin Polymerization Inhibition Study
2.3. Evaluation of the Antibody-Drug Conjugates (ADCs) Preparation
2.4. Stability Assays of the Conjugate in Plasma
2.5. Drug Release Study at the Cellular Level
2.6. Flow Cytometry Analysis
2.7. In Vitro Potency Assay of the ADC, Antibody, MMAE, and Cys-Linker-MMAE Conjugate
2.8. Trafficking Assay by Fluorescence Microscopy
2.9. In Vivo Potency in Xenografted Nude Mice
2.10. In Vivo Imaging of Fluorescein-Labeled ADC
2.11. Hematological Analysis
2.12. In Vivo Tolerability Study
3. Discussion
4. Materials and Methods
4.1. Reagents and Antibody
4.2. Synthesis of the Linker-MMAE Conjugates
4.2.1. Synthesis of 3-(2,5-Dioxo-2,5-Dihydro-1H-Pyrrol-1-yl)Propanoic Acid (4)
4.2.2. Synthesis of 6-(2,5-Dioxo-2,5-Dihydro-1H-Pyrrol-1-yl)Hexanoic Acid (5)
4.2.3. Synthesis of 3-(2-(2,5-Dioxo-2,5-Dihydro-1H-Pyrrol-1-yl)Ethoxy)Propanoic Acid (6)
4.2.4. Synthesis of 3-(2,5-Dioxo-2,5-Dihydro-1H-Pyrrol-1-yl)-N-(4-(Hydroxymethyl)Phenyl)Propanamide (7)
4.2.5. Synthesis of 6-(2,5-Dioxo-2,5-Dihydro-1H-Pyrrol-1-yl)-N-(4-(Hydroxymethyl)Phenyl)Hexanamide (8)
4.2.6. Synthesis of 4-(3-(2,5-Dioxo-2,5-Dihydro-1H-Pyrrol-1-yl)Propanamido)Benzyl (4-Nitrophenyl) Carbonate (9)
4.2.7. Synthesis of 4-(6-(2,5-Dioxo-2,5-Dihydro-1H-Pyrrol-1-yl)Hexanamido)Benzyl (4-Nitrophenyl) Carbonate (10)
4.2.8. Synthesis of (S)-2-((S)-2-(3-(2,5-Dioxo-2,5-Dihydro-1H-Pyrrol-1-yl)-N-Methylpropanamido)-3-Methylbutanamido)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-Hydroxy-1-Phenylpropan-2-yl)amino)-1-Methoxy-2-Methyl-3-Oxopropyl)Pyrrolidin-1-yl)-3-Methoxy-5-Methyl-1-Oxoheptan-4-yl)-N,3-Dimethylbutanamide (11)
4.2.9. Synthesis of 6-(2,5-Dioxo-2,5-Dihydro-1H-Pyrrol-1-yl)-N-((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-Hydroxy-1-Phenylpropan-2-yl)Amino)-1-Methoxy-2-Methyl-3-Oxopropyl)Pyrrolidin-1-yl)-3-Methoxy-5-Methyl-1-Oxoheptan-4-yl)(Methyl)Amino)-3-Methyl-1-Oxobutan-2-yl)Amino)-3-Methyl-1-Oxobutan-2-yl)-N-Methylhexanamide (12)
4.2.10. Synthesis of 4-(3-(2-(2,5-Dioxo-2,5-Dihydro-1H-Pyrrol-1-yl)Ethoxy)Propanamido)Benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-Hydroxy-1-Phenylpropan-2-yl)Amino)-1-Methoxy-2-Methyl-3-Oxopropyl)Pyrrolidin-1-yl)-3-Methoxy-5-Methyl-1-Oxoheptan-4-yl)(Methyl)Amino)-3-Methyl-1-Oxobutan-2-yl)Amino)-3-Methyl-1-Oxobutan-2-yl)(Methyl)Carbamate (13)
4.2.11. Synthesis of 4-(3-(2,5-Dioxo-2,5-Dihydro-1H-Pyrrol-1-yl)Propanamido)Benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-Hydroxy-1-Phenylpropan-2-yl)Amino)-1-Methoxy-2-Methyl-3-Oxopropyl)Pyrrolidin-1-yl)-3-Methoxy-5-Methyl-1-Oxoheptan-4-yl)(Methyl)Amino)-3-Methyl-1-Oxobutan-2-yl)Amino)-3-Methyl-1-Oxobutan-2-yl)(Methyl)Carbamate (14)
4.2.12. Synthesis of 4-(6-(2,5-Dioxo-2,5-Dihydro-1H-Pyrrol-1-yl)Hexanamido)Benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-Hydroxy-1-Phenylpropan-2-yl)Amino)-1-Methoxy-2-Methyl-3-Oxopropyl)Pyrrolidin-1-yl)-3-Methoxy-5-Methyl-1-Oxoheptan-4-yl)(Methyl)Amino)-3-Methyl-1-Oxobutan-2-yl)Amino)-3-Methyl-1-Oxobutan-2-yl)(Methyl)Carbamate (15)
4.3. Microtubule Polymerization Assay
4.4. Preparation of Reagents and Antibody-Drug Conjugates
4.5. Characterization of the ADC
4.6. Stability Assays of the ADC in Plasma
4.7. Cellular Drug Release Process
4.8. Evaluation of the ADC for Tumor Cell Killing In Vitro
4.9. Confocal Analysis for Intracellular Localization
4.10. Cell Cycle Arrest and Apoptosis Analysis
4.11. In Vivo Antitumor Activity in Human Gastric Xenograft Tumors
4.12. In Vivo Fluorescence Imaging Experiment
4.13. Hematological Analysis during Treatment
4.14. In Vivo Tolerability Experiments
4.15. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Linker-MMAE | | |
---|---|---|
X | Y | |
11 | -CH2- | - |
12 | -(CH2)4- | - |
13 | -CH2O(CH2)2- | - |
14 | -CH2- | |
15 | -(CH2)4- | |
16 | -(CH2)4- | |
Cell Lines | HER2 Status | mil40-15 | mil40 | MMAE | Cys-15 | ||||
---|---|---|---|---|---|---|---|---|---|
IC50 (nM) | Max. Inhibition | IC50 (nM) | Max. Inhibition | IC50(nM) | Max. Inhibition | IC50 (nM) | Max. Inhibition | ||
BT-474 | HER2+++ | 0.35 | 84.49% | 0.78 | 65.45% | 0.32 | 79.09% | 6.88 | 82.62% |
HCC1954 | HER2+++ | 0.09 | 96.29% | 1753.53 | 70.36% | 0.07 | 97.54% | 2.31 | 98.86% |
NCI-N87 | HER2+++ | 0.12 | 95.01% | 0.19 | 87.90% | 0.07 | 89.37% | 8.67 | 94.86% |
MCF-7 | HER2− | 110.54 | 88.06% | >3438.25 | 49.43% | 0.29 | 93.14% | 51.23 | 91.13% |
MDA-MB-468 | HER2− | 97.62 | 87.42% | 1388.43 | 59.13% | 0.17 | 87.37% | 11.11 | 85.75% |
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Wang, Y.; Liu, L.; Fan, S.; Xiao, D.; Xie, F.; Li, W.; Zhong, W.; Zhou, X. Antibody-Drug Conjugate Using Ionized Cys-Linker-MMAE as the Potent Payload Shows Optimal Therapeutic Safety. Cancers 2020, 12, 744. https://doi.org/10.3390/cancers12030744
Wang Y, Liu L, Fan S, Xiao D, Xie F, Li W, Zhong W, Zhou X. Antibody-Drug Conjugate Using Ionized Cys-Linker-MMAE as the Potent Payload Shows Optimal Therapeutic Safety. Cancers. 2020; 12(3):744. https://doi.org/10.3390/cancers12030744
Chicago/Turabian StyleWang, Yanming, Lianqi Liu, Shiyong Fan, Dian Xiao, Fei Xie, Wei Li, Wu Zhong, and Xinbo Zhou. 2020. "Antibody-Drug Conjugate Using Ionized Cys-Linker-MMAE as the Potent Payload Shows Optimal Therapeutic Safety" Cancers 12, no. 3: 744. https://doi.org/10.3390/cancers12030744