Modular, defined assembly of antibody-based constructs using click chemistry
Chris Langsdorf¹, Robert Aggeler¹, Brian Agnew¹, Aimei Chen¹, Nicholas Dolman¹, Yi-Zhen Hu¹, Lynn Kamen², Shanhua Lin¹, Bhaskar Mandavilli¹, Benjamin Ordonia², Marcy Wickett¹, Terry Zhang¹ and Wenjun Zhou¹
¹Thermo Fisher Scientific, ²Genentech, Inc.
Abstract
Immunoglobulin antibodies contribute to a broad range of research assays and provide the foundation for diverse biopharmaceutical agents. This document describes the use of bio-orthogonal click chemistry reactions to synthesize common antibody-based structures and demonstrates their application in cell-based assays. Site-specific antibody-fluorophore conjugates were synthesized using SiteClick™ conjugation chemistry to attach Alexa Fluor™ and iFL pHrodo™ dyes to therapeutic antibodies. Analytical characterization confirmed efficient, reproducible payload attachment without affecting antibody functionality. Flow cytometry and high content analysis experiments demonstrate performance in receptor occupancy, antibody internalization, and effector function assays. A double conjugation strategy was employed to synthesize antibody drug conjugates (ADCs) that become brightly fluorescent upon endocytosis. ADC uptake and targeted cell killing were demonstrated using cell line and spheroid models. Applications are shown using flow cytometry for suspension cells, and high content analysis or live cell microscopy for adherent cells and spheroid models.
SiteClick Antibody Labeling System
SiteClick antibody-labeling technology enables simple and site-specific attachment of compounds, including fluorescent dyes or toxins, to the carbohydrate domains present only on the heavy chains of IgG antibodies, regardless of isotype and host species.
Three-step Labeling Process
- The SiteClick antibody-labeling process begins with the removal of terminal galactose residues from heavy-chain N-linked glycans using β-galactosidase, exposing modifiable GlcNAc residues.
- Free terminal GlcNAc residues are activated with azide tags by enzymatic attachment of GalNAz using the GalT (Y289L) enzyme.
- Azide residues are reacted via click chemistry with a dibenzocyclooctyne (sDIBO)-functionalized probe of choice (e.g., Alexa Fluor 488 sDIBO alkyne).
Key Applications and Data
SiteClick Conjugation Does Not Modify F(ab')2 Domains
Mass spectra comparing F(ab')2 fragments of DIBO-modified trastuzumab species to unmodified trastuzumab F(ab')2 fragments, demonstrating that SiteClick conjugation does not alter the F(ab')2 domains.
SiteClick Labeling: Consistent Conjugate-Antibody Ratio
Data showing consistent Drug-to-Antibody Ratios (DAR1, DAR2, DAR3, DAR4) for Trastuzumab-MMAE conjugates, with DAR4 exceeding 70%. Mass spectra analysis of Trastuzumab-MMAE SiteClick conjugate is also presented, conducted by LC-MS.
New pHrodo™ iFL Sensor Dyes Report pH Change
Two graphs illustrate the pH response of trastuzumab labeled with amine-reactive pHrodo Red iFL STP or pHrodo Green iFL STP. These conjugates are minimally fluorescent at neutral pH outside cells but become brightly fluorescent upon specific endosomal internalization, reporting on pH changes. A diagram shows the recycling pathway from early endosomes (pH ~6.3) to late endosomes.
Binding to FcγRIIIA – V158 by SiteClick-modified IgG1 Antibody
An ELISA binding curve shows the affinity of SiteClick variants to FcγRIIIA – V158. The data indicates that SiteClick antibody labeling has minimal impact on IgG affinity for Fc receptors.
Bright, Specific Signal Indicates Antibody Internalization
An internalization assay compares trastuzumab labeled with iFL pHrodo Red via amine or SiteClick conjugation. Results show that iFL pHrodo Red labeled trastuzumab specifically internalizes into HER2+ cells, regardless of conjugation chemistry. A bar graph displays the mean average membrane intensity (red spots per cell) for HER2+ and HER2- cells treated with SiteClick conjugate versus amine conjugate.
SiteClick Labeled Antibodies Specifically Bind to Cells
A cell binding assay compares Trastuzumab (Herceptin) and Obinutuzumab (Gazyva) conjugated with Alexa Fluor 488 sDIBO using SiteClick labeling. Analysis on SKBR3 (HER2+) and MDA-MB-231 (HER2-) cells shows specific binding to HER2+ cells, represented by mean average membrane intensity.
Antibody Internalization in HER2+ SKBR3 Cells
A live confocal image of SKBR3 cells treated with a trastuzumab-iFL pHrodo Red conjugate. The image displays internalized red fluorescence from the antibody, along with green fluorescence for lysosomes (CellLight™ Lysosomes-GFP) and blue fluorescence for the nucleus (NucBlue™ Live ReadyProbes® Reagent), with the plasma membrane stained in deep red (CellMask™ Deep Red Plasma Membrane Stain).
Visualize Antibody Penetration of Spheroids
An image of a HER2+ SKBR3 cell spheroid treated with iFL pHrodo Red labeled Trastuzumab. Red fluorescence indicates antibody penetration into the spheroid.
Direct Indication of ADC Cell Penetration and Killing
An image of a SKBR3 spheroid treated with a Trastuzumab-MMAE antibody-drug conjugate (ADC) labeled with iFL pHrodo Red. Red fluorescence indicates antibody penetration, and green fluorescence (CellEvent Caspase 3/7 Green) indicates apoptosis, demonstrating targeted cell killing.
Conclusions
- SiteClick IgG antibody-labeling technology enables reproducible site-specific attachment of compounds without altering F(ab')2 structure or compromising affinity for Fc receptors.
- When conjugated to antibodies, iFL pHrodo Red sDIBO alkyne provides a direct fluorescent indication of antibody penetration into cells.
- Dual-labeled antibodies, such as fluorescent antibody-drug conjugates, can be created using a mixture of two different sDIBO alkyne species.
Related Products
| Product | Size | Cat. No. |
|---|---|---|
| pHrodo™ iFL Red STP ester, amine reactive | 1mg | P36010 |
| pHrodo™ iFL Red STP ester, amine reactive | 3 X 100 ug | P36011 |
| pHrodo™ IFL Green STP ester, amine reactive | 1mg | P36012 |
| pHrodo™ IFL Green STP ester, amine reactive | 3 x 100 ug | P36013 |
| pHrodo™ iFL Red Microscale Protein Labeling Kit | 1 Kit | P36014 |
| pHrodo™™ iFL Green Microscale Protein Labeling Kit | 1 Kit | P36015 |
| SiteClick™ Antibody Azido Modification Kit | 1 Kit | S20026 |
| Click-iT™ Alexa Fluor™ 488 sDIBO Alkyne for Antibody Labeling | 1 Kit | C20027 |
| Click-iT™ Alexa Fluor™ 555 sDIBO Alkyne for Antibody Labeling | 1 Kit | C20028 |
| Click-iT™ Alexa Fluor™ 647 sDIBO Alkyne for Antibody Labeling | 1 Kit | C20029 |
| Click-iT™ Biotin sDIBO Alkyne for Antibody Labeling | 1 Kit | C20030 |
| Click-iT™ Amine sDIBO Alkyne for Antibody Labeling | 1 Kit | C20031 |
| Click-iT™ SDP Ester SDIBO Alkyne for Antibody Labeling | 1 Kit | C20032 |
| Click-iT™ pHrodo™ iFL Red sDIBO Alkyne for Antibody Labeling | 1 Kit | C20034 |
| SiteClick™ Biotin Antibody Labeling Kit | 1 Kit | S20033 |
| Zenon™ pHrodo™ iFL green mouse IgG labeling kit | 1 Kit | Z25609 |
| Zenon™ pHrodo™ iFL red mouse IgG labeling kit | 1 Kit | Z25610 |
| Zenon™™ pHrodo™ iFL green human IgG labeling kit | 1 Kit | Z25611 |
| Zenon™™ pHrodo™ iFL red human IgG labeling kit | 1 Kit | Z25612 |
