Strategic Trastuzumab Mediated Crosslinking Driving Concomitant HER2 and HER3 Endocytosis and Degradation in Breast Cancer

Wymant, Jennifer; Sayers, Edward J.; Muir, Duncan; Jones, Arwyn Tomos

doi:10.7150/jca.32470

“…Next, since the internalization of cell-surface bound antigen‒antibody complex is induced upon antigen binding, we then treated cells with chlorpromazine, an inhibitor of clathrin assembly/disassembly, to interfere with T-DM1 internalization. In line with the clathrin-mediated endocytosis of trastuzumab 22 , intracellular concentration of lys-MCC-DM1 following the chlorpromazine treatment shows a significant decrease ( Fig. 4 C).…”

Section: Resultssupporting

confidence: 68%

Target-responsive subcellular catabolism analysis for early-stage antibody–drug conjugates screening and assessment

Sang

¹

,

Liu²,

Zhou³

et al. 2021

Acta Pharmaceutica Sinica B

3

0

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Events including antibody‒antigen affinity, internalization, trafficking and lysosomal proteolysis combinatorially determine the efficiency of antibody–drug conjugate (ADC) catabolism and hence the toxicity. Nevertheless, an approach that conveniently identifies proteins requisite for payload release and the ensuing toxicity for mechanistic studies and quality assessment is lacking. Considering the plethora of ADC candidates under development, we developed a target-responsive subcellular catabolism (TARSC) approach that examines ADC catabolism and probes changes in response to targeted interferences of proteins of interest. We firstly applied TARSC to study the commercial T-DM1 and the biosimilar. We recorded unequivocal catabolic behaviors regardless of the absence and presence of the targeted interferences. Their negligible differences in TARSC profiles agreed with their undifferentiated anti-tumoral efficacy according to further in vitro viability and in vivo tumor growth assays, highlighting TARSC analysis as a useful tool for biosimilarity assessment and functional dissection of proteins requisite for ADC catabolism. Additionally, we employed TARSC to investigate the catabolic behavior of a new trastuzumab–toxin conjugate. Collectively, TARSC can not only characterize ADC catabolism at (sub)cellular level but also comprehensively determine which protein targets affect payload release and therapeutic outcomes. Future use of TARSC is thus anticipated in early-stage screening, quality assessment and mechanistic investigations of ADCs.

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“…Next, since the internalization of cell-surface bound antigen-antibody complex is induced upon antigen binding, we then treated cells with a clathrin inhibitor, chlorpromazine, to interfere with T-DM1 internalization. In line with the clathrin-mediated trastuzumab endocytosis 28 , Fig. 4C shows that intracellular concentration of lys-MCC-DM1 following the chlorpromazine treatment significantly decreased, and the concentration was reduced to comparable levels for both the CD and BS T-DM1-treated cells.…”

Section: Tarsc Analysis Of T-dm1s Empowers Mechanistic Studies and Bioequivalence Assessmentsupporting

confidence: 75%

TArget-Responsive Subcellular Catabolism Analysis for Early-stage Antibody-Drug Conjugates Screening and Assessment

Sang

¹

,

Liu

²

,

Zhou

³

et al. 2020

Preprint

0

View full text Add to dashboard Cite

Key events including antibody-antigen affinity, ADC internalization, trafficking and lysosomal proteolysis-mediated payload release combinatorially determine the therapeutic efficacy and safety for ADCs. Nevertheless, a universal technology that efficiently and conveniently evaluates the involvement of these above elements to ADC payload release and hence the final therapeutic outcomes for mechanistic studies and quality assessment is lacking. Considering the plethora of ADC candidates under development owing to the ever-evolving linker and drug chemistry, we developed a TArget-Responsive Subcellular Catabolism (TARSC) approach that measures catabolites kinetics for given ADCs and elaborates how each individual step ranging from antigen binding to lysosomal proteolysis affects ADC catabolism by targeted interferences. Using a commercial and a biosimilar ado-trastuzumab emtansine (T-DM1) as model ADCs, we recorded unequivocal catabolites kinetics for the two T-DM1s in the presence and absence of the targeted interferences. Their negligible differences in TARSC profiles fitting with their undifferentiated therapeutic outcomes suggested by in vitro viability assays and in vivo tumor growth assays, highlighting TARSC analysis as a good indicator of ADC efficacy and bioequivalency. Lastly, we demonstrated the use of TARSC in assessing payload release efficiency for a new Trastuzumab-toxin conjugate. Collectively, we demonstrated the use of TARSC in characterizing ADC catabolism at (sub)cellular level, and in systematically depicting whether given target proteins affect ADC payload release and hence therapeutic efficacy. We anticipate its future use in high-throughput screening, quality assessment and mechanistic understanding of ADCs for drug R&D before proceeding to costly in vivo experiments.

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“…Fig.4Bshows the reduction of intracellular lys-MCC-DM1 exhibited no significant difference between the two, suggesting that HER2 silencing modulated ADC delivery and catabolism to similar extents.Next, since the internalization of cell-surface bound antigen-antibody complex is induced upon antigen binding, we then treated cells with a clathrin inhibitor, chlorpromazine, to interfere with T-DM1 internalization. In line with the clathrin-mediated trastuzumab endocytosis28 , Fig.4Cshows that intracellular concentration of lys-MCC-DM1 following the chlorpromazine treatment significantly decreased, and the concentration was reduced to comparable levels for both the CD and BS T-DM1-treated cells. Once T-DM1 enters the target cells, it is transported in endosomes to lysosomes.…”

supporting

confidence: 76%

TArget-Responsive Subcellular Catabolism Analysis for Early-stage Antibody-Drug Conjugates Screening and Assessment

Shi¹,

Liu²,

Zhou³

et al. 2020

Preprint

0

View full text Add to dashboard Cite

Key events including antibody-antigen affinity, ADC internalization, trafficking and lysosomal proteolysis-mediated payload release combinatorially determine the therapeutic efficacy and safety for ADCs. Nevertheless, a universal technology that efficiently and conveniently evaluates the involvement of these above elements to ADC payload release and hence the final therapeutic outcomes for mechanistic studies and quality assessment is lacking. Considering the plethora of ADC candidates under development owing to the ever-evolving linker and drug chemistry, we developed a TArget-Responsive Subcellular Catabolism (TARSC) approach that measures catabolites kinetics for given ADCs and elaborates how each individual step ranging from antigen binding to lysosomal proteolysis affects ADC catabolism by targeted interferences. Using a commercial and a biosimilar ado-trastuzumab emtansine (T-DM1) as model ADCs, we recorded unequivocal catabolites kinetics for the two T-DM1s in the presence and absence of the targeted interferences. Their negligible differences in TARSC profiles fitting with their undifferentiated therapeutic outcomes suggested by in vitro viability assays and in vivo tumor growth assays, highlighting TARSC analysis as a good indicator of ADC efficacy and bioequivalency. Lastly, we demonstrated the use of TARSC in assessing payload release efficiency for a new Trastuzumab-toxin conjugate. Collectively, we demonstrated the use of TARSC in characterizing ADC catabolism at (sub)cellular level, and in systematically depicting whether given target proteins affect ADC payload release and hence therapeutic efficacy. We anticipate its future use in high-throughput screening, quality assessment and mechanistic understanding of ADCs for drug R&D before proceeding to costly in vivo experiments.

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Strategic Trastuzumab Mediated Crosslinking Driving Concomitant HER2 and HER3 Endocytosis and Degradation in Breast Cancer

Cited by 19 publications

References 55 publications

Target-responsive subcellular catabolism analysis for early-stage antibody–drug conjugates screening and assessment

Target-responsive subcellular catabolism analysis for early-stage antibody–drug conjugates screening and assessment

TArget-Responsive Subcellular Catabolism Analysis for Early-stage Antibody-Drug Conjugates Screening and Assessment

TArget-Responsive Subcellular Catabolism Analysis for Early-stage Antibody-Drug Conjugates Screening and Assessment

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