Targeting pediatric sarcoma with a bispecific ligand immunotoxin targeting urokinase and epidermal growth factor receptors

Abstract

Children with high risk sarcoma have a poor prognosis despite surgical resection, irradiation and chemotherapy. Alternative therapies are urgently needed. Urokinase-type plasminogen activator receptor (uPAR) and epidermal growth factor receptor (EGFR) are surface proteins expressed by some pediatric sarcomas. We show for the first time that a de-immunized bispecific ligand toxin, EGFATFKDEL, directed against EGFR and uPAR, successfully targets pediatric sarcoma. Using flow cytometry, we identified a rhabdomyosarcoma (RMS) cell line, RH30, that expresses both uPAR and EGFR, and a Ewing sarcoma (EWS) cell line, TC-71, that expresses only uPAR. We tested the differential sensitivity of these two sarcoma cell lines to toxin-induced killing, using both in vitro assays and an in vivo murine model. We show that pediatric sarcomas are highly sensitive to EGFATFKDEL (at subnanomolar concentrations) in vitro. In vivo, tumor growth was significantly attenuated after treatment with EGFTFKDEL, compared to untreated controls, in both RH30 and TC-71 tumor bearing mice. In addition, we found that simultaneously targeting both receptors in a dual positive cell line was more effective than targeting a single receptor or antigen, resulting in a greater tumor response, including complete tumor regression in an animal model of bulky disease. Our findings provide support for further exploration of bispecific targeting of pediatric sarcomas with bispecific ligand toxins, such as EGFATFKDEL.

Keywords: Ewing’s sarcoma; epidermal growth factor receptor (EGFR); immunotherapy; rhabdomyosarcoma; urokinase-type plasminogen activator receptor (uPAR).

Figure 1. Surface expression of EGFR and uPAR on RH30 (RMS) and TC-71 (EWS) cell lines by flow cytometry

Cells lines were stained with mAbs against EGFR Brilliant Violet 421 and uPAR APC. Representative experiment is shown with 73.6% and 86.6% of RH30 and TC-71 showing uPAR expression and 99.7% of RH30 showing EGFR expression with 7.23% on TC-71 cells. The x-axis on the right represents uPAR surface expression and on the left represents EGFR surface expression. The dark shaded plot represents the unstained control sample while the lighter shaded plot represents the stained samples for each cell line.

Figure 2. The effect of EGFATFKDEL on RH30 and TC-71 cells in vitro

(A and B) ³H Leucine protein synthesis assay where 1x10⁴ cells/ well were plated in triplicate and allowed to adhere overnight. RH30 (A) or TC-71 (B) cells were then pulsed for 72 hours with increasing doses (0.01-100 nM) of either EGFATFKDEL (circle) vs. BIC3 (triangle, negative control). ³H Leucine uptake was measured and data are reported as percent control response (y-axis). (C and D) shows the percent viability using Annexin V and 7AAD staining of RH30 (C) and TC-71 cells (D) treated with 2.5 μg EGFATFKDEL (open circle), compared to mitomycin positive control (triangle). Negative controls included BIC3 treated (square) and untreated cells (closed circle). Cells were harvested and analyzed at time points ranging from 12-96 hours. Above experiments are representative of 2 individual experiments.

Figure 3. EGFATFKDEL treated RH30 and TC-71 tumor spheres

(A) shows representative GFP images for RH30 (left) or TC-71 (right) tumor spheres, 3 days after treatment with EGFATFKDEL at doses ranging from 0-100 nM of. Data in (B-C) is representative of 2 experiments done in triplicate. (B) shows % change in GFP expression at day 3 for RH30 and TC-71 spheres and (C) shows the % change in GFP expression for day 7. Comparison in percent change of GFP expression between these two cell lines at these time points were significantly different (p <0.0001) with RH30 cells showing a greater response.

Figure 4. In vivo tumor models

(A) Representative RH30 and TC-71 tumor bearing mice treated with EGFATFKDEL compared to untreated control mice. (B) Average radiance of all RH30 (left, n=24) and TC-71 (right, n=34)-bearing mice, as measured by BLI. Mice were treated with stated dose (5μg daily/5μg BID/10μg daily on M, T, Th, F for 4 weeks). (C) Average % change in the average radiance of RH30 (left, n=24) and TC-71 (right, n=34)-bearing mice over 4 weeks of treatment compared to controls.