Info
Background:
Human epidermal growth factor receptor (HER2) is overexpressed in up to 20% of breast and gastric cancer patients and is associated with increased aggressiveness.1 Trastuzumab-deruxtecan (T-DXd) is a HER2-targeting antibody drug conjugate that has shown significant improvements in patients with HER2-positive, HER2-low, and HER2-ultralow breast tumors.2,3 However, not all tumors show a response to T-DXd. Previous studies have shown that dual-antibody click combining EGFR-targeting antibody panitumumab with T-DXd can improve efficacy in EGFR-positive tumors with variable HER2 expression. Previously, PET imaging was performed to monitor antibody click in vivo, but only T-DXd was radiolabeled and imaged.

Hypothesis/Objective:
To monitor the in vivo biodistribution of dual antibody click targeting EGFR and HER2 (Fig. 1A), we used fluorescence to image the antibodies in an EGFR+/HER2-ultralow pancreatic tumor model.

Methods:
Antibodies were conjugated in two steps with a click moiety for bioorthogonal chemistry and a fluorophore for in vivo imaging. EGFR-targeting panitumumab was conjugated with trans-cyclooctene (TCO, 15:1 molar excess) and IRDye680RD (LICOR Bio, 3:1 molar excess), both via NHS ester reaction with lysine residues to produce pan-TCO-680RD. HER2-targeting trastuzumab was conjugated with tetrazine (Tz) and IRDye800CW (LICOR Bio) via NHS ester-lysine reaction and using the same molar ratios as above, respectively, to produce Trast-Tz-800CW. Conjugates were purified with PD-10 columns and spin filters. Fluorescence lifetime was measured for each dye and antibody conjugate (Horiba Fluorolog-3, Fig. 1B). Molecular weights were monitored by gel electrophoresis (Fig. 1C). Five million MiaPaCa2 cells (EGFR-high, HER2-ultralow) were injected subcutaneously into nude mice 12 days prior to imaging. Mice (n=4) were injected intravenously with 50 µg of pan-TCO-680RD; 24 h later, 50 µg of trast-Tz-800CW was injected. A LICOR Pearl Trilogy imaging system was used for in vivo imaging. Imaging was performed before and after each injection up to 72 h after trastuzumab-Tz-IRDye800CW (Fig. 1D). Organs were collected for ex vivo fluorescence quantification following the final time point. Mice were snap-frozen in a bath of hexane and dry ice for post-mortem cryofluorescence tomography (CFT, Emit Xerra).

Results:
The fluorescence lifetime of pan-TCO-680RD is 1.12 ns and trast-Tz-800CW is 0.45 ns. The antibody conjugates show a slight increase in lifetime compared with free dyes (Fig. 1B, 0.77 ns for Dye680RD and 0.39 ns for Dye800CW). Gel electrophoresis shows the click reaction occurred and fluorescent signal of the dye is observed on molecular weights corresponding to the heavy and light chain of the antibodies (Fig.1C). Imaging shows antibody uptake in the tumor and retention in the liver starting at 4 h until 72 h (Persuasive Data A, B). Biodistribution data align with the images. CFT shows retention in the liver and signal from the tumor and lymph nodes from trast-Tz-800CW (Persuasive Data B).

Conclusion:
In this pilot study, we have shown that in vivo NIR fluorescence imaging enables tracking the biodistribution of two fluorescently labeled antibodies in vivo. Previous work demonstrated that site-specific modifications reduce antibody click accumulation in the liver, and therefore, our ongoing studies are using site-specific conjugation approaches to generate antibody click pairs and tracking both antibodies through fluorescence imaging.

Image/Figure:
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Image/Figure Caption:
Figure 1. A. EGFR/HER2 scheme shows antibody binding through direct binding to targets and via click reaction between the two antibodies. B. Fluorescence lifetime of the dyes and antibody conjugates was measured. The fluorescence lifetime of pan-TCO-680RD is 1.12 ns and trast-Tz-800CW is 0.45 ns. There is a slight increase in the lifetime for both compared to free dyes: 0.77 ns for Dye680RD and 0.39 ns for Dye800CW. Gel electrophoresis (C) was performed to monitor the click reaction. The antibodies were also denatured to see dye signal on heavy and light chains. In vivo imaging was performed; the approach is shown in panel D and images are shown in the Persuasive Data file.

Author
Carmen Azevedo
Washington University in St. Louis