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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

Internalization interplay between two anti-EphA2 mAbs measured with anti-Alexa Fluor mAbs.(A) Cellular uptake of 1C1-A488 in the presence of an IgG1-A594 control, unlabeled 3035 or 3035-A594 over a 4-hr time course. Surface A488 fluorescence was quenched with anti-A488-19A in the presence of the chimeric IgG1 isotype control or anti-A594-1A. (B) Cellular uptake of 3035-A594 in the presence of an IgG1-A488 control, unlabeled 1C1 or 1C1-A488 over a 4-hr time course. Surface A594 fluorescence was quenched with anti-A594-1A in the presence of the chimeric IgG1 isotype control or anti-A488-19A. The sample incubated with 1C1-A488 and 3035-A594 and quenched with anti-A488-19A and anti-A594-1A is analyzed for A488 fluorescence in A and A594 fluorescence in B. One representative experiment of multiple is shown. (C, D) Independent experiment in which the layout is reciprocal to the layout shown in A & B: 1C1 is labeled with A594 (C) instead of A488 (A), and the anti-Alexa Fluor mAbs and controls are changed accordingly. 3035 is labeled with A488 (D) instead of A594 (A), and the anti-Alexa Fluor mAbs and controls are changed accordingly. The sample incubated with 1C1-A594 and 3035-A488 and quenched with anti-A594-1A and anti-A488-19A is analyzed for A594 fluorescence in C and A488 fluorescence in D. One representative experiment of multiple is shown.
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pone.0124708.g006: Internalization interplay between two anti-EphA2 mAbs measured with anti-Alexa Fluor mAbs.(A) Cellular uptake of 1C1-A488 in the presence of an IgG1-A594 control, unlabeled 3035 or 3035-A594 over a 4-hr time course. Surface A488 fluorescence was quenched with anti-A488-19A in the presence of the chimeric IgG1 isotype control or anti-A594-1A. (B) Cellular uptake of 3035-A594 in the presence of an IgG1-A488 control, unlabeled 1C1 or 1C1-A488 over a 4-hr time course. Surface A594 fluorescence was quenched with anti-A594-1A in the presence of the chimeric IgG1 isotype control or anti-A488-19A. The sample incubated with 1C1-A488 and 3035-A594 and quenched with anti-A488-19A and anti-A594-1A is analyzed for A488 fluorescence in A and A594 fluorescence in B. One representative experiment of multiple is shown. (C, D) Independent experiment in which the layout is reciprocal to the layout shown in A & B: 1C1 is labeled with A594 (C) instead of A488 (A), and the anti-Alexa Fluor mAbs and controls are changed accordingly. 3035 is labeled with A488 (D) instead of A594 (A), and the anti-Alexa Fluor mAbs and controls are changed accordingly. The sample incubated with 1C1-A594 and 3035-A488 and quenched with anti-A594-1A and anti-A488-19A is analyzed for A594 fluorescence in C and A488 fluorescence in D. One representative experiment of multiple is shown.

Mentions: When targeting a surface antigen with two mAbs, it is important to understand interplay between the two mAbs and their target [21–23]. With the two different anti-Alexa Fluor mAbs in place, we assessed interplay between the slowly internalizing mAb 3035 and the rapidly internalizing mAb 1C1. First, PC-3 cells were labeled with 1C1-A488 and 3035-A594. After incubating cells with mAbs for up to 4 hr, surface fluorescence was quenched with anti-A488-19A and anti-A594-1A. Relative to the control in which PC-3 cells were stained with 1C1-A488 and the chimeric IgG1 isotype control labeled with A594, 1C1-A488’s half-time increased from 23 to 116 min by co-incubation with 3035-A594 (Fig 6A, circle versus triangle, respectively). A second control in which 3035-A594 was replaced with unlabeled 3035 showed a similar reduction in 1C1-A488’s internalization with a half-time of 124 min, demonstrating that dye conjugation to 3035 did not affect its activity (Fig 6A, square). The half-time of the third control sample in which cells were co-incubated with 1C1-A488 and 3035-A594 and only A488 fluorescence was quenched was 110 min, equivalent to the 121 min half-time of the sample in which cells were co-incubated with 1C1-A488 and 3035-A594 and both A488 and A594 fluorescence were quenched (Fig 6A, inverted triangle versus triangle, respectively), confirming that anti-A594-1A does not affect A488 fluorescence (S4 Fig). On the other hand, cellular uptake of 3035-A594 was increased by co-incubation with conjugated or unconjugated 1C1 (Fig 6B). 3035’s half-time decreased from 392 min to a half-time between 154 and 194 min by addition of naked or dye-conjugated 1C1.


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)

Internalization interplay between two anti-EphA2 mAbs measured with anti-Alexa Fluor mAbs.(A) Cellular uptake of 1C1-A488 in the presence of an IgG1-A594 control, unlabeled 3035 or 3035-A594 over a 4-hr time course. Surface A488 fluorescence was quenched with anti-A488-19A in the presence of the chimeric IgG1 isotype control or anti-A594-1A. (B) Cellular uptake of 3035-A594 in the presence of an IgG1-A488 control, unlabeled 1C1 or 1C1-A488 over a 4-hr time course. Surface A594 fluorescence was quenched with anti-A594-1A in the presence of the chimeric IgG1 isotype control or anti-A488-19A. The sample incubated with 1C1-A488 and 3035-A594 and quenched with anti-A488-19A and anti-A594-1A is analyzed for A488 fluorescence in A and A594 fluorescence in B. One representative experiment of multiple is shown. (C, D) Independent experiment in which the layout is reciprocal to the layout shown in A & B: 1C1 is labeled with A594 (C) instead of A488 (A), and the anti-Alexa Fluor mAbs and controls are changed accordingly. 3035 is labeled with A488 (D) instead of A594 (A), and the anti-Alexa Fluor mAbs and controls are changed accordingly. The sample incubated with 1C1-A594 and 3035-A488 and quenched with anti-A594-1A and anti-A488-19A is analyzed for A594 fluorescence in C and A488 fluorescence in D. One representative experiment of multiple is shown.
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Related In: Results  -  Collection

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pone.0124708.g006: Internalization interplay between two anti-EphA2 mAbs measured with anti-Alexa Fluor mAbs.(A) Cellular uptake of 1C1-A488 in the presence of an IgG1-A594 control, unlabeled 3035 or 3035-A594 over a 4-hr time course. Surface A488 fluorescence was quenched with anti-A488-19A in the presence of the chimeric IgG1 isotype control or anti-A594-1A. (B) Cellular uptake of 3035-A594 in the presence of an IgG1-A488 control, unlabeled 1C1 or 1C1-A488 over a 4-hr time course. Surface A594 fluorescence was quenched with anti-A594-1A in the presence of the chimeric IgG1 isotype control or anti-A488-19A. The sample incubated with 1C1-A488 and 3035-A594 and quenched with anti-A488-19A and anti-A594-1A is analyzed for A488 fluorescence in A and A594 fluorescence in B. One representative experiment of multiple is shown. (C, D) Independent experiment in which the layout is reciprocal to the layout shown in A & B: 1C1 is labeled with A594 (C) instead of A488 (A), and the anti-Alexa Fluor mAbs and controls are changed accordingly. 3035 is labeled with A488 (D) instead of A594 (A), and the anti-Alexa Fluor mAbs and controls are changed accordingly. The sample incubated with 1C1-A594 and 3035-A488 and quenched with anti-A594-1A and anti-A488-19A is analyzed for A594 fluorescence in C and A488 fluorescence in D. One representative experiment of multiple is shown.
Mentions: When targeting a surface antigen with two mAbs, it is important to understand interplay between the two mAbs and their target [21–23]. With the two different anti-Alexa Fluor mAbs in place, we assessed interplay between the slowly internalizing mAb 3035 and the rapidly internalizing mAb 1C1. First, PC-3 cells were labeled with 1C1-A488 and 3035-A594. After incubating cells with mAbs for up to 4 hr, surface fluorescence was quenched with anti-A488-19A and anti-A594-1A. Relative to the control in which PC-3 cells were stained with 1C1-A488 and the chimeric IgG1 isotype control labeled with A594, 1C1-A488’s half-time increased from 23 to 116 min by co-incubation with 3035-A594 (Fig 6A, circle versus triangle, respectively). A second control in which 3035-A594 was replaced with unlabeled 3035 showed a similar reduction in 1C1-A488’s internalization with a half-time of 124 min, demonstrating that dye conjugation to 3035 did not affect its activity (Fig 6A, square). The half-time of the third control sample in which cells were co-incubated with 1C1-A488 and 3035-A594 and only A488 fluorescence was quenched was 110 min, equivalent to the 121 min half-time of the sample in which cells were co-incubated with 1C1-A488 and 3035-A594 and both A488 and A594 fluorescence were quenched (Fig 6A, inverted triangle versus triangle, respectively), confirming that anti-A594-1A does not affect A488 fluorescence (S4 Fig). On the other hand, cellular uptake of 3035-A594 was increased by co-incubation with conjugated or unconjugated 1C1 (Fig 6B). 3035’s half-time decreased from 392 min to a half-time between 154 and 194 min by addition of naked or dye-conjugated 1C1.

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