Preclinical evaluation of EpCAM-binding designed ankyrin repeat proteins (DARPins) as targeting moieties for bimodal near-infrared fluorescence and photoacoustic imaging of cancer

Abstract

Purpose: Fluorescence-guided surgery (FGS) can play a key role in improving radical resection rates by assisting surgeons to gain adequate visualization of malignant tissue intraoperatively. Designed ankyrin repeat proteins (DARPins) possess optimal pharmacokinetic and other properties for in vivo imaging. This study aims to evaluate the preclinical potential of epithelial cell adhesion molecule (EpCAM)-binding DARPins as targeting moieties for near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging of cancer.

Methods: EpCAM-binding DARPins Ac2, Ec4.1, and non-binding control DARPin Off7 were conjugated to IRDye 800CW and their binding efficacy was evaluated on EpCAM-positive HT-29 and EpCAM-negative COLO-320 human colon cancer cell lines. Thereafter, NIRF and PA imaging of all three conjugates were performed in HT-29_luc2 tumor-bearing mice. At 24 h post-injection, tumors and organs were resected and tracer biodistributions were analyzed.

Results: Ac2-800CW and Ec4.1-800CW specifically bound to HT-29 cells, but not to COLO-320 cells. Next, 6 nmol and 24 h were established as the optimal in vivo dose and imaging time point for both DARPin tracers. At 24 h post-injection, mean tumor-to-background ratios of 2.60 ± 0.3 and 3.1 ± 0.3 were observed for Ac2-800CW and Ec4.1-800CW, respectively, allowing clear tumor delineation using the clinical Artemis NIRF imager. Biodistribution analyses in non-neoplastic tissue solely showed high fluorescence signal in the liver and kidney, which reflects the clearance of the DARPin tracers.

Conclusion: Our encouraging results show that EpCAM-binding DARPins are a promising class of targeting moieties for pan-carcinoma targeting, providing clear tumor delineation at 24 h post-injection. The work described provides the preclinical foundation for DARPin-based bimodal NIRF/PA imaging of cancer.

Keywords: Colon cancer; DARPins; EpCAM; Fluorescence-guided surgery; Molecular imaging; Photoacoustic imaging.

Fig. 1

In vitro binding of EpCAM DARPin-800CW conjugates. A Binding of Ac2-800CW, Ec4.1-800CW, and Off7-800CW to HT-29 (black), and COLO-320 (grey) colon cancer cell lines at various concentrations using cell-based plate assays. Experiments were performed in triplicate. B Binding of Ac2-800CW, Ec4.1-800CW, and Off7-800CW (each at 100 nM) to HT-29 and COLO-320 cells using flow cytometry on the 800 nm channel. Dark grey curves display DARPin-800CW binding, whereas light grey curves represent unstained cells. C Immunofluorescence analysis of Ac2-800CW, Ec4.1-800CW, and Off7-800CW binding to HT-29 and COLO-320 cells. The 800CW signal representing DARPin-800CW localization is displayed in red. DAPI stained nuclei are displayed in blue. ns, not significant. ***P < 0.001, ****P < 0.0001

Fig. 2

In vitro binding competition of DARPin-800CW conjugates on HT-29 and COLO-320 cells. Cells were preincubated with PBS (control), unconjugated Ac2, Ec4.1, or Off7 (each at 200 nM), followed by incubation with DARPin-800CW conjugates (100 nM). a.u, arbitrary units; MFI, mean fluorescence intensity; ns, not significant. **P < 0.01, ***P < 0.001. Experiments were performed in triplicate

Fig. 3

In vivo dose and time window optimization of DARPin-800CW conjugates. A Tumor MFIs and TBRs as a function of time after intravenous administration of 3, 6, or 9 nmol Ac2-800CW or Ec4.1-800CW in subcutaneous HT-29_luc2 tumor-bearing mice. B TBRs as a function of time after intravenous administration of 6 nmol Ac2-800CW, Ec4.1-800CW, and negative control tracer Off7-800CW in subcutaneous HT-29_luc2 tumor-bearing mice. C NIRF-color merge and NIRF images of subcutaneous HT-29_luc2 tumor-bearing mice at 24 h post-injection of Ac2-800CW, Ec4.1-800CW, or Off7-800CW. Images were captured using the clinical Artemis NIRF imager at a similar exposure time of 150 ms, allowing real-time imaging. White arrows indicate an example of a representative tumor. NIRF, near-infrared fluorescence; p.i., post-injection; TBR, tumor-to-background ratio. **P < 0.01

Fig. 4

In vivo NIRF imaging and PA imaging using DARPin-800CW conjugates. A NIRF-color merge and NIRF images of orthotopic HT-29_luc2 tumor-bearing mice at 24 h post-injection of Ac2-800CW or Ec4.1-800CW. Images were captured using the clinical Artemis NIRF imager at an exposure time of 150 ms. “T” indicates the tumor localization, while “Cae’” indicates the corresponding background tissue (caecum). Mouse-specific TBRs are indicated in white in the right-upper quadrant of the NIRF images. B TBRs of orthotopic HT-29_luc2 tumors 24 h after intravenous administration of 6 nmol Ac2-800CW or Ec4.1-800CW as measured using the clinical Artemis NIRF imager. Mean TBRs are represented by the horizontal line together with their error bars representing the standard deviation. C Representative US and PA images of orthotopic HT-29_luc2 tumor-bearing mice at 24 h post-injection of Ac2-800CW or Ec4.1-800CW. Images were captured using a penetration depth of approximately 1.5 cm. Tumors are delineated with a green line. D Biodistribution in orthotopic HT-29_luc2 tumors and healthy organs of mice at 24 h post-injection of Ac2-800CW or Ec4.1-800CW. 1: lungs, 2: heart, 3: pancreas, 4: spleen, 5: stomach, 6: small intestine, 7: caecum, 8: rectum, 9: muscle, 10: brain, 11: skin, 12: liver, 13: kidneys, and 14: tumor. E Macroscopic fluorescence biodistribution of orthotopic HT-29_luc2 tumors and healthy organs at 24 h post-injection of Ac2-800CW or Ec4.1-800CW (Pearl imager). F HE staining, 800 nm heatmap and merge, as well as cytokeratin and EpCAM stainings of sequential tissue sections derived from orthotopic HT-29_luc2 tumors at 24 h post-injection of Ac2-800CW or Ec4.1-800CW. Tumors are delineated by dashed white lines. HE-NIRF and cytokeratin-EpCAM images are taken at × 2 and × 15 magnification, respectively. Scale bars represent 100 µm. a.u, arbitrary units; HE, hematoxylin-eosin; MFI, mean fluorescence intensity; NIRF, near-infrared fluorescence; PA, photoacoustic; TBR, tumor-to-background ratio; US, ultrasound