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Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

Liao-Chan S, Daine-Matsuoka B, Heald N, Wong T, Lin T, Cai AG, Lai M, D'Alessio JA, Theunissen JW - PLoS ONE (2015)

Bottom Line: Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization.The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen.Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.

View Article: PubMed Central - PubMed

Affiliation: Department of Discovery Research, Igenica Biotherapeutics, Burlingame, California, United States of America.

ABSTRACT
Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs) that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.

No MeSH data available.


Related in: MedlinePlus

Surface-bound 1C1 is internalized, while 3035 shows little cellular uptake.Surface-bound 3035 or 1C1 was internalized in PC-3 cells for 0 or 1 hr at 37°C and processed for confocal immunofluorescence microscopy with the lysosomal marker LAMP1 (red color) and the actin cytoskeleton marker Phalloidin (blue color). Scale bar, 45 micron. White arrows illustrate colocalization. Representative staining from one of multiple independent experiments is shown.
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pone.0124708.g005: Surface-bound 1C1 is internalized, while 3035 shows little cellular uptake.Surface-bound 3035 or 1C1 was internalized in PC-3 cells for 0 or 1 hr at 37°C and processed for confocal immunofluorescence microscopy with the lysosomal marker LAMP1 (red color) and the actin cytoskeleton marker Phalloidin (blue color). Scale bar, 45 micron. White arrows illustrate colocalization. Representative staining from one of multiple independent experiments is shown.

Mentions: While a large portion of 1C1 internalized after 1 hr, little to no 3035 appeared to be internalized (Fig 5). Some internalized 1C1 localized to the lysosome based on the lysosomal marker LAMP1 (Fig 5), a marker commonly used to assess antibody and ADC internalization [7, 11–13]. Both at the 0-hr and 1-hr time points, 3035 was predominantly found on the cell surface (Fig 5). Thus, microscopy-based assessment of mAb internalization was consistent with the flow-based internalization assay. Importantly, the flow-based internalization assay allowed a more rapid assessment of internalization compared to immunofluorescence microscopy.


Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

Liao-Chan S, Daine-Matsuoka B, Heald N, Wong T, Lin T, Cai AG, Lai M, D'Alessio JA, Theunissen JW - PLoS ONE (2015)

Surface-bound 1C1 is internalized, while 3035 shows little cellular uptake.Surface-bound 3035 or 1C1 was internalized in PC-3 cells for 0 or 1 hr at 37°C and processed for confocal immunofluorescence microscopy with the lysosomal marker LAMP1 (red color) and the actin cytoskeleton marker Phalloidin (blue color). Scale bar, 45 micron. White arrows illustrate colocalization. Representative staining from one of multiple independent experiments is shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403856&req=5

pone.0124708.g005: Surface-bound 1C1 is internalized, while 3035 shows little cellular uptake.Surface-bound 3035 or 1C1 was internalized in PC-3 cells for 0 or 1 hr at 37°C and processed for confocal immunofluorescence microscopy with the lysosomal marker LAMP1 (red color) and the actin cytoskeleton marker Phalloidin (blue color). Scale bar, 45 micron. White arrows illustrate colocalization. Representative staining from one of multiple independent experiments is shown.
Mentions: While a large portion of 1C1 internalized after 1 hr, little to no 3035 appeared to be internalized (Fig 5). Some internalized 1C1 localized to the lysosome based on the lysosomal marker LAMP1 (Fig 5), a marker commonly used to assess antibody and ADC internalization [7, 11–13]. Both at the 0-hr and 1-hr time points, 3035 was predominantly found on the cell surface (Fig 5). Thus, microscopy-based assessment of mAb internalization was consistent with the flow-based internalization assay. Importantly, the flow-based internalization assay allowed a more rapid assessment of internalization compared to immunofluorescence microscopy.

Bottom Line: Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization.The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen.Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.

View Article: PubMed Central - PubMed

Affiliation: Department of Discovery Research, Igenica Biotherapeutics, Burlingame, California, United States of America.

ABSTRACT
Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs) that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.

No MeSH data available.


Related in: MedlinePlus