Introduction: Multi-cell targeting is a promising approach increasingly being developed for therapies addressing multiple diseases, where drug combinations often improve physiological responses compared to traditional drug combinations.¹ This project uses an innovative, low-cost approach with high potential for clinical translation: we apply bioorthogonal click reactions to generate “antibody click” pairs for simplified and affordable dual-cell targeting.^2,3 As a proof of concept, we show an “antibody click” pair that includes the tumor targeting trastuzumab antibody and the T-cell-targeting anti-PD-1 antibody pembrolizumab. We use PET imaging to monitor the distribution of the “antibody click” pair.

Methods: Trastuzumab was conjugated with trans-cyclooctyne PEG₄‑NHS ester (TCO), and the human reactive pembrolizumab or a mouse reactive anti-PD-1 antibody was conjugated with p-SCN-Bn-Deferoxamine (DFO) and tetrazine (Tz). Gel electrophoresis and FPLC were performed for all conjugates to confirm antibody integrity. MALDI was performed to calculate the ratio of TCO, DFO, or Tz per antibody. Transmission electron microscopy was performed to image the unbound and clicked antibodies. Anti-PD-1-DFO-Tz was radiolabeled with [⁸⁹Zr]-oxalate, and purity was measured via ITLC. Trastuzumab-TCO was injected via tail vein into BALB/c mice xenografted with CT26-hHER2 cells followed 24 h later by [⁸⁹Zr]-PD1-DFO-Tz. Additional no-click groups included the intravenous injection of trastuzumab, non-specific IgG-TCO, or saline prior to injection of [⁸⁹Zr]-PD1-DFO-Tz. “Antibody click” biodistribution was also studied in healthy BALB/c mice. Mice underwent PET/CT imaging at 24 h, 48, and 120 h post injection of the radiotracer. Organs were collected for biodistribution 144 h after injection. Tumors were fixed for IHC and western blot for HER2, PD1, and PD-L1 quantification in the future. ROIs were hand drawn to quantify tumor uptake from PET images; max %ID/g was calculated for each group.

Results: The dual-cell antibody click approach is shown in Fig. 1, along with immunofluorescence imaging (presented at WMIC 2023). Conjugations and antibody integrity were confirmed by gel electrophoresis and FPLC (Persuasive Data, PD A). “Antibody click” was confirmed by TEM and gel electrophoresis. The conjugates have 17 TCO per trastuzumab, and 5.0 Tz per PD1 antibody (Fig. 1). Radiolabeling and radiochemical purity were measured by ITLC; the radiolabeled antibody purity was ~99% and this was maintained following purification by PD-10 column and concentration by molecular weight cutoff spin filter (PD A). Organs were collected from healthy BALB/c mice 144 h after tracer injection for biodistribution; the uptake is as expected for an antibody tracer at that timepoint (PD B). CT26-hHER2 tumor mice were injected according to the same schedule and underwent PET/CT imaging three times before ex vivo biodistribution. The biodistribution in tumor mice was similar to healthy mice. Tumor uptake was 42.45 ± 26.77 %ID/g (mean ± SEM). No significant statistical difference was observed in the PET signal of tumors between 24 h and 120 h (PD B). In all imaging groups, the PET and biodistribution uptake was strongly correlated with tumor size (%ID vs tumor volume shown in PD B, R²=0.9419; Bq/mL vs tumor volume R²=0.9316).

Conclusion: Dual-cell antibody click is a promising approach for simultaneous cancer-immune cell targeting. Using PET imaging, we follow the biodistribution of the antibody click pair; this approach can be applied to other antibody combinations. In the future, we will perform IHC and western blots to quantify the HER2, PD1, and PD-L1 in the tumor samples and characterize and quantify the immune cells in the tumor microenvironment using flow cytometry. We will apply this approach to therapy and measure the in vitro cytotoxicity of the click pair. Future studies will use CD8-PET and CD4-PET imaging to compare immune cell accumulation in tumors in the click versus no click.

Image/Figure:
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Image/Figure Caption:

A. Scheme of the dual cell antibody click; a cancer cell and T cell may be brought together via specific antibodies targeting each cell and a click reaction between the antibodies. The chemical structure after reaction is also shown. Here, the tumor-targeting anti-human epidermal growth factor receptor 2 (HER2) is conjugated with the click moiety transcyclooctene (TCO). An anti-PD-1 targeting antibody (e.g. pembrolizumab: T cell targeting) is conjugated with tetrazine (Tz).

B. Immunofluorescence image of NCIN87 cells and PBMCs (WMIC 2023). Cancer cells and peripheral mononuclear cells (PBMCs) are co-incubated in the presence of the click antibodies trastuzumab-TCO and pembrolizumab-Tz. The cancer cells are bound to trastuzumab with a green fluorophore and CD8+ T cells are bound to a CD8 antibody with a red fluorophore. DAPI was used to stain nuclei and PBMCs (CD8+) are also shown. The scale bar represents 40 µm.

C. Scheme for our dual cell antibody click PET imaging approach using trastuzumab-TCO and pembrolizumab-DFO-Tz using the CT26 cancer cells that stably express human HER2 (CT26-hHER2).

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