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. 2024 Apr 20:19:3623-3639.
doi: 10.2147/IJN.S450534. eCollection 2024.

EGFR Targeting of Liposomal Doxorubicin Improves Recognition and Suppression of Non-Small Cell Lung Cancer

Ernest Moles  1   2   3   4 , David W Chang  1   2   3 , Friederike M Mansfeld  1   2   3 , Alastair Duly  1   2 , Kathleen Kimpton  1 , Amy Logan  1   2   3   4 , Christopher B Howard  5 , Kristofer J Thurecht  5   6 , Maria Kavallaris  1   2   3   4
Affiliations

EGFR Targeting of Liposomal Doxorubicin Improves Recognition and Suppression of Non-Small Cell Lung Cancer

Ernest Moles et al. Int J Nanomedicine. .

Abstract

Introduction: Despite improvements in chemotherapy and molecularly targeted therapies, the life expectancy of patients with advanced non-small cell lung cancer (NSCLC) remains less than 1 year. There is thus a major global need to advance new treatment strategies that are more effective for NSCLC. Drug delivery using liposomal particles has shown success at improving the biodistribution and bioavailability of chemotherapy. Nevertheless, liposomal drugs lack selectivity for the cancer cells and have a limited ability to penetrate the tumor site, which severely limits their therapeutic potential. Epidermal growth factor receptor (EGFR) is overexpressed in NSCLC tumors in about 80% of patients, thus representing a promising NSCLC-specific target for redirecting liposome-embedded chemotherapy to the tumor site.

Methods: Herein, we investigated the targeting of PEGylated liposomal doxorubicin (Caelyx), a powerful off-the-shelf antitumoral liposomal drug, to EGFR as a therapeutic strategy to improve the specific delivery and intratumoral accumulation of chemotherapy in NSCLC. EGFR-targeting of Caelyx was enabled through its complexing with a polyethylene glycol (PEG)/EGFR bispecific antibody fragment. Tumor targeting and therapeutic potency of our treatment approach were investigated in vitro using a panel of NSCLC cell lines and 3D tumoroid models, and in vivo in a cell line-derived tumor xenograft model.

Results: Combining Caelyx with our bispecific antibody generated uniform EGFR-targeted particles with improved binding and cytotoxic efficacy toward NSCLC cells. Effects were exclusive to cancer cells expressing EGFR, and increments in efficacy positively correlated with EGFR density on the cancer cell surface. The approach demonstrated increased penetration within 3D spheroids and was effective at targeting and suppressing the growth of NSCLC tumors in vivo while reducing drug delivery to the heart.

Conclusion: EGFR targeting represents a successful approach to enhance the selectivity and therapeutic potency of liposomal chemotherapy toward NSCLC.

Keywords: EGFR targeting; PEGylated liposomal doxorubicin; bispecific antibodies; non-small cell lung cancer; targeted drug delivery.

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Conflict of interest statement

Professor Kristofer Thurecht reports the bispecific antibody technology has been awarded patent number WWO2016123675A1. The authors declare that they have no other competing interests in this work.

Figures

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Graphical abstract
Figure 1
Figure 1
Bidirectional binding of αEGFR mediates the delivery of Caelyx to NSCLC cells in vitro. (A) Schematic illustration showing αEGFR BsAb construct, and Caelyx complexing step with αEGFR to form αEGFR-Caelyx. (B) Percentage of H460 or Calu-6 cells targeted by αEGFR-Caelyx complexes (Caelyx+ cells) formed with increasing densities of αEGFR. (C–E) Dynamic and Electrophoretic Light Scattering (DLS) measurement of Caelyx and αEGFR-Caelyx (prepared at 64:1 BsAb-to-Caelyx molar ratio), including Z-average size (C), polydispersity (D), and size distribution (E). (F) Percentage of H1299 cells targeted by αEGFR-Caelyx (Caelyx+ cells) mixed with anti-EGFR mAb panitumumab or IsoAb at increasing Ab-to-BsAb densities. (G) Representative flow cytometry plot (Caelyx fluorescence versus side scatter area or SCC indicating cell complexity) showing Caelyx+ H1299 cells after exposure to Caelyx alone, αEGFR-Caelyx, or αEGFR-Caelyx mixed with IsoAb or panibumumab (5:1 Ab-to-BsAb molar ratio). Histograms are shown for respective Caelyx and SSC channels. (H) Live cell confocal microscopy imaging of cancer cells after a 3-hour exposure to Caelyx alone or αEGFR-Caelyx. Nuclei and plasma membrane of cells were visualized using Hoechst 33,342 (blue), and CellMask Plasma Membrane Stain (green), respectively. Scale bars, 20 µm. In (B, F, and G), Caelyx+ cells were determined by flow cytometry analysis of Caelyx fluorescence in treated compared to untreated cells. Data in (B–F) are presented as means ± SDs of three (B–F) and five (C–E) independent experiments.
Figure 2
Figure 2
In vitro NSCLC cell targeting analysis of αEGFR-Caelyx. (A) Illustration showing cell targeting analysis of αEGFR-Caelyx and Caelyx alone. Percentage of cells targeted by αEGFR-Caelyx or Caelyx alone were determined by flow cytometry analysis, comparing Caelyx fluorescence in treated cells to cells receiving vector (autofluorescence). (B and C) Flow cytometry measurement of EGFR cell surface expression in cancer cells using a tandem of anti-human EGFR mAb or IsoAb plus a secondary Alexa Fluor (AF) 647-labelled IgG, including histograms (b) and AF 647 median fluorescence intensity (MFI) (C). (D) Dose–response curves showing percentage of targeted cancer cells (Caelyx+ cells) at increasing concentrations of αEGFR-Caelyx or untargeted Caelyx in a representative experiment. EC50 indicates drug concentration leading to 50% targeted cells. Data are means ± SDs of three experimental replicates. (E) Plots comparing EC50 of Caelyx alone versus αEGFR-Caelyx from three independent experiments. EC50 fold changes (Caelyx alone/αEGFR-Caelyx) were determined for individual experiments and plotted as means ± SDs. P values were computed using ratio paired t-test. (F) Correlation plot comparing EC50 fold change (Caelyx alone/αEGFR-Caelyx) against EGFR cell surface expression (AF 647 MFI). Pearson’s correlation coefficient, r. Error lines indicate 95% confidence bands of the best-fit line. Cutoff value (green) of twofold is defined to establish relevant changes in EC50. EGFR expression. In (C and F), data are presented as means (F) or means ± SDs (C) of three independent experiments.
Figure 3
Figure 3
Investigating cytotoxicity of αEGFR-Caelyx against NSCLC cells in vitro. (A) Illustration depicting cytotoxicity analysis αEGFR-Caelyx and Caelyx alone. Viable cells were defined by flow cytometry analysis as those double-negative for Annexin V-allophycocyanin (APC) and Sytox green markers of early apoptosis and cell death, respectively, and counted using flow cytometry counting beads. Flow cytometry plots indicate cell counts (cells/mL) in a representative experiment. (B) Percentage of viable, apoptotic, dead, and necrotic cells (clustered stacked chart; left), and total cell count (relative to vehicle; right) of 5 µM (Calu-6), 10 µM (H460) or 20 µM (A549 and H1299) Caelyx-treated cells compared to vehicle (PBS)-treated cells. *P < 0.05, **P < 0.01, and ***P < 0.001 (one-way ANOVA followed by Dunnett’s multiple comparisons test) comparing percentage of viable cells or total cell count after treatment with αEGFR-Caelyx versus Caelyx alone. (C) Dose–response curves show viable cell counts (relative to vehicle) after treatment with increasing concentrations of EGFR-Caelyx or Caelyx alone (left) in the first of three repeated experiments; second and third replicates are shown in Figure S3. Data are means ± SDs of three experimental replicates. (D) Plots comparing IC50 of Caelyx alone versus αEGFR-Caelyx from three independent experiments. IC50 fold changes (Caelyx alone/αEGFR-Caelyx) were determined for individual experiments and plotted as means ± SDs. P values were computed using ratio paired t-test. (E) Correlation plot comparing IC50 fold change (Caelyx alone/αEGFR-Caelyx) against EGFR cell surface expression (AF 647 MFI). Pearson’s correlation coefficient, r. Error lines indicate 95% confidence bands of the best-fit line. Cutoff value (green) of twofold change is defined to establish relevant changes in EC50 and IC50. In (B and E), data are presented as means (E) or means ± SDs (B) of three independent experiments.
Figure 4
Figure 4
Studying internalization of αEGFR-Caelyx into 3D spheroid models. (A) Illustration depicting the internalization analysis of αEGFR-Caelyx and Caelyx alone. We draw 20 radii (0 to 100 µm below spheroids surface) distributed across the spheroids equatorial plane and measured Caelyx fluorescence in them using live cell confocal microscopy. Plot shows grouped fluorescence intensity over distance of 20 radii in a representative spheroid; data are means +SDs. (B) Live confocal microscopy images of spheroids equatorial plane (scale bars, 125 µm); Caelyx fluorescence is shown in red. Images are representative of three independent experiments. An enlarged image (i) is also shown for spheroids exposed to αEGFR-Caelyx or Caelyx alone (scale bars, 50 µm). (C and D) Plots (C) and respective area under curves (AUCs) (D) show fluorescence intensity of Caelyx at different distances below the spheroids surface. Data represent means ± SDs of 6 spheroids per condition pooled from three independent experiments. P values comparing αEGFR-Caelyx versus Caelyx alone were computed using one-way ANOVA followed by Dunnett’s multiple comparisons test.
Figure 5
Figure 5
αEGFR-Caelyx accumulates in NSCLC tumors in vivo with minimal accumulation in the heart. (A) Timeline of biodistribution experiment. SCID-beige mice bearing subcutaneous H460 NSCLC tumors received an intravenous injection of αEGFR-Caelyx (1 mg/kg; n = 4 mice) or Caelyx alone (1 mg/kg; n = 4 mice) and drug amounts (nmol drug/g tissue) in tumor, liver, and heart were measured ex vivo at 4, 24 or 48 hours after injection. (B–D) Plots show drug amounts (median plus interquartile range (IQR); 4 animals per condition) in tumor (B), heart (C), and liver (D). P values were computed using Mann–Whitney U-test; *P < 0.05.
Figure 6
Figure 6
αEGFR-Caelyx significantly suppresses NSCLC progression in a tumor-xenograft model. (A) Timeline and treatment course of therapeutic experiments. SCID-beige mice engrafted with H460 cells were intravenously injected with vehicle (PBS; n = 8 mice), Caelyx alone (1 mg/kg; n = 8 mice), αCD19-Caelyx (1 mg/kg; n = 8 mice), or αEGFR-Caelyx (1 mg/kg; n = 7 mice). (B and C) Tumor size of treated mice over time; data are shown for individual (B) or grouped (C) mice. (D)Tumor size on day 21 (completion of the study). (E) Body weight of treated animals over time. Arrows in (A–C) and (E) indicate days of injection. Dash line in (B–D) indicates tumor size defined as event (1,000 mm3). In (B and D), (†) indicates individual mice that reached event on day 18. In (C and D), data are presented as medians plus IQR. P values comparing tumor size injection of Caelyx alone, αCD19-Caelyx, or αEGFR-Caelyx versus vehicle were computed using Kruskal–Wallis test followed by Dunn’s multiple comparisons test; ***P < 0.001.
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Grants and funding

This work was funded in part from the Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology (CE140100036, to M.K. and K.J.T.); ARC Training Centre for Innovation in Biomedical Imaging Technologies (IC170100035, to K.J.T.); National Health and Medical Research Council Investigator Grant (#2016464 to MK) and a Tour de Cure Grant (to MK). K.J.T. acknowledges the award of a Career Development Fellowship (APP1148582).
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