Antitumor activity of a humanized, bivalent immunotoxin targeting fn14-positive solid tumors

Abstract

The TNF-like weak inducer of apoptosis (TWEAK; TNFSF12) receptor Fn14 (TNFRSF12A) is expressed at low levels in normal tissues but frequently highly expressed in a wide range of tumor types such as lung, melanoma, and breast, and therefore it is a potentially unique therapeutic target for these diverse tumor types. We have generated a recombinant protein containing a humanized, dimeric single-chain anti-fibroblast growth factor-inducible 14-kDa protein (Fn14) antibody fused to recombinant gelonin toxin as a potential therapeutic agent (designated hSGZ). The hSGZ immunotoxin is a highly potent and selective agent that kills Fn14-positive (Fn14(+)) tumor cells in vitro. Treatment of cells expressing the MDR protein MDR1 (ABCB1B) showed no cross-resistance to hSGZ. Induced overexpression of Fn14 levels in MCF7 cells through HER2 (ERBB2) signaling translated to an improved therapeutic index of hSGZ treatment. In combination with trastuzumab, hSGZ showed an additive or synergistic cytotoxic effect on HER2(+)/Fn14(+) breast cancer cell lines. Also, hSGZ treatment inhibited Erb3/Akt signaling in HER2-overexpressing breast cancer cells. Pharmacokinetic studies in mice revealed that hSGZ exhibited a biexponential clearance from plasma with a rapid initial clearance (t1/2α = 1.26 hours) followed by a seven-fold longer plasma half-life (t1/2β = 7.29 hours). At 24, 48, and 72 hours after injection, uptake of the hSGZ into tumors was 5.1, 4.8, and 4.7%ID/g, with a tumor-to-muscle ratio of 5.6, 6.2, and 9.0, respectively. Therapeutic efficacy studies showed significant tumor inhibition effects using an MDA-MB-231/Luc breast cancer xenograft model. Our findings show that hSGZ is an effective anticancer agent and a potential candidate for clinical studies.

Figure 1

Effect of the humanized, dimeric single-chain immunotoxin hSGZ on MDA-MB-231 cells. A, cells with either left untreated or treated with 50 nmol/L rGel and 50 or 100 nmol/L hSGZ for 4 h. The cells were fixed, acid washed to remove surface-bound material, permeabilized, and immunostained for the presence of rGel (green). The cells were counterstained with propidium iodide (red) to identify nuclei and visualized using a confocal microscope. Bar=50 µm. B, cells were treated with indicated concentration of rGel, ITEM4-rGel or hSGZ for 72 h. Additionally, cells were pretreated with 1 µmol/L ITEM-4 for 2 h and then coincubated with different concentration of hSGZ for another 72 h. Cell viability was assessed by crystal violet staining. C, cells were plated and then exposed to hSGZ at 0.3 nmol/L for indicated periods of time. Cell viability was assessed at 72 h as described above. D, cells were treated with different concentrations of hSGZ for 24 or 48 h and LDH release was measured. Treatment of cells with Triton X-100 served as a positive control causing maximum LDH release. Results represent mean ± SD, n = 3.

Figure 2

Effect of hSGZ treatment on MCF-7 and MCF-7/HER2 cells. A, parental MCF-7 cells and MCF-7 cells that stably overexpress HER2 (MCF-7/HER2) were analyzed for Fn14 cell surface expression by flow cytometry. B, cytotoxic effects of rGel, ITEM4-rGel and hSGZ were analyzed on MCF-7 and MCF-7/HER2 cells. Student’s t test was used to calculate statistical significance. Data are representative of three independent experiments. C and D, MCF-7 and MCF-7/HER2 cells were treated with indicated concentration of hSGZ for 48 h and whole cell lysates were analyzed by Western blot with the indicated antibodies.

Figure 3

Effect of hSGZ in combination with trastuzumab (T) in breast cancer cell lines. A, MCF-7 and MCF-7/HER2 cells were treated with hSGZ (at IC₂₅ dose), with or without trastuzumab (50 or 100 µg/ml) for 72 h. B, SKBR3 cells were first treated with hSGZ (at IC₂₅ dose) for 6 h, and then 50 µg/ml trastuzumab were added for 72 h (sequence I). Alternatively, cells were pretreated with 50 µg/ml trastuzumab for 6 h, followed by the addition of hSGZ (IC₂₅). The cells were then incubated for a total of 72 h (sequence II). Coexposure hSGZ (at IC₂₅ dose) and 50 µg/ml trastuzumab to the cells for 72 h (sequence III). Cell viability was assessed by crystal violet staining. Statistical significance was calculated using a two-tailed, independent samples t test. n.s., not significant (p>0.05).

Figure 4

Pharmacokinetics and biodistibution of IRDye 800CW-labeled hSGZ (IR-hSGZ) in mice. A, pharmacokinetic of IR-hSGZ. The IR-hSGZ was injected intravenously into BALB/c mice. Groups of mice (3 mice per group) were sacrificed at various time points after injection. The fluorescent activity in plasma was assessed, and the mean blood concentration–time profile of IR-hSGZ generated using a least squares nonlinear regression. t _1/2α and t _1/2β represent half-lives in initial distribution phase and terminal elimination phase, respectively; C₀ is the estimated initial drug concentration in the blood; Vd is the volume of distribution of central compartment; Vss is volume of distribution at steady state; AUC_0-∞ is the area under the blood concentration versus time curve; CL is total body clearance; and MRT is mean resident time. B, whole body imaging results of the nude mice bearing MDA-MB-231/Luc tumors intravenously injected with IR-hSGZ and imaged at 24, 48 and 72 h. The BLI images show only tumor burden. NIR, Near-infrared imaging; BLI, Bioluminescent imaging. Arrow indicates tumor burden. C, biodistribution of IR-hSGZ at 24, 48 and 72 h after intravenous injection of IR-hSGZ in nude mice bearing MDA-MB-231/Luc tumors. Data are presented as percentage of injected dose per gram of tissues (%ID/g tissue), represented as mean ± SD (n=5).

Figure 5

Both ITEM4-rGel and hSGZ inhibit tumor growth and prolong survival in a MDA-MB-231/Luc breast tumor xenograft model. MDA-MB-231/luc cells were implanted subcutaneously and groups of mice (n = 5) were treated (i.v. via tail vein) with saline, ITEM-4 plus rGel, ITEM4-rGel (36 mg/kg), and hSGZ (36 mg/kg and 25 mg/kg) every 6 days starting when the tumors were approximately 100 mm³. Arrow indicates dosing days. A, efficacy data are plotted as mean tumor volume (in mm³) ± SEM. Tumor size assessed by direct caliper measurement. B, survival data are plotted as percent of animals surviving in each group using a predefined cutoff volume of 1,200 mm³ as a surrogate for survival. C, the BLI images of mice on selected days were shown. D, percent change in body weight of each group of mice is plotted as a function of time. E, Tumor tissues from the xenograft experiment in (A) were analyzed for Fn14 and GAPDH expression by Western blot.