The functionalized human serine protease granzyme B/VEGF₁₂₁ targets tumor vasculature and ablates tumor growth

Abstract

The serine protease granzyme B (GrB) induces apoptosis through both caspase-dependent and -independent multiple-cascade mechanisms. VEGF₁₂₁ binds to both VEGF receptor (VEGFR)-1 and VEGFR-2 receptors. We engineered a unique GrB/VEGF₁₂₁ fusion protein and characterized its properties in vitro and in vivo. Endothelial and tumor cell lines showed varying levels of sensitivity to GrB/VEGF₁₂₁ that correlated closely to total VEGFR-2 expression. GrB/VEGF₁₂₁ localized efficiently into VEGFR-2-expressing cells, whereas the internalization into VEGFR-1-expressing cells was significantly reduced. Treatment of VEGFR-2(+) cells caused mitochondrial depolarization in 48% of cells by 48 hours. Exposure to GrB/VEGF₁₂₁ induced apoptosis in VEGFR-2(+), but not in VEGFR-1(+), cells and rapid caspase activation was observed that could not be inhibited by treatment with a pan-caspase inhibitor. In vivo, GrB/VEGF₁₂₁ localized in perivascular tumor areas adjacent to microvessels and in other areas in the tumor less well vascularized, whereas free GrB did not specifically localize to tumor tissue. Administration (intravenous) of GrB/VEGF₁₂₁ to mice at doses up to 40 mg/kg showed no toxicity. Treatment of mice bearing established PC-3 tumor xenografts with GrB/VEGF₁₂₁ showed significant antitumor effect versus treatment with GrB or saline. Treatment with GrB/VEGF₁₂₁ at 27 mg/kg resulted in the regression of four of five tumors in this group. Tumors showed a two-fold lower Ki-67-labeling index compared with controls. Our results show that targeted delivery of GrB to tumor vascular endothelial cells or to tumor cells activates apoptotic cascades and this completely human construct may have significant therapeutic potential.

Figure 1

Construction, expression and purification of GrB/VEGF₁₂₁. (A) The Granzyme B/VEGF₁₂₁ cassette encoding the hexa-histidine tag and an enterokinase cleavage site (H₆D₄K) was cloned in frame 3′ to the secretion leader peptide in the 5.2 kb pSecTag plasmid. LS, Igκ leader sequence and Myc tag with the following amino acid sequence: METDTLLLWVLLLWVPGSTGDAAQPAEQKLISEEDLNSAVDGSGSGHM. (B) Coomassie Blue staining showing purification of pro-Granzyme B/VEGF₁₂₁ from transiently transfected HEK-293T cells using Cobalt-IMAC. The activated GrB/VEGF₁₂₁ was purified as an 80 kDa homodimer. (C) Granzyme B enzymatic activity assay indicating that the granzyme B portion of the fusion construct was comparable to commercially prepared granzyme B.

Figure 2

GrB/VEGF₁₂₁ internalization into PAE/VEGFR-2 was driven by VEGF₁₂₁. Cells were (A) untreated or (B) treated with 20 nM GrB/VEGF₁₂₁ for 24 h, followed by detection of GrB. Nuclei were stained with propidium iodide. Only nuclei were visible in untreated PAE/VEGFR-2 cells whereas fluorescent GrB staining was observed in the cytoplasm of GrB/VEGF₁₂₁-treated cells.

Figure 3

GrB/VEGF₁₂₁ triggers apoptosis in target cells via mitochondrial depolarization. (A–B). Mitochondrial depolarization of PAE/VEGFR-2 and PAE/VEGFR-1 cells following treatment with GrB or GrB/VEGF₁₂₁. Granzyme B treatment (20 nM) did not trigger mitochondrial depolarization in either cell. GrB/VEGF₁₂₁ treatment resulted in significant levels of mitochondrial depolarization in PAE/VEGFR-2 cells within 4 h of treatment. (C–D) Detection of apoptosis in VEGFR-2⁺ cells. Cells were treated with GrB or GrB/VEGF₁₂₁ for various times. Apoptosis was detected by flow cytometry following incubation with Annexin V and propidium iodide. N.D., not done. NT, no treatment.

Figure 4. PARP-1 cleavage by GrB/VEGF₁₂₁ is caspase-dependent and –independent

(A–B) GrB/VEGF₁₂₁ activates caspase-3 (4A) and -9 (4B) in whole cell lysates. Chromogenic substrates for caspase-3 and caspase-9 were incubated in PAE/KDR whole cell lysates with or without GrB or GrB/VEGF₁₂₁. Activation of the caspases was indicated by an increase in absorbance of the cleaved substrate. (C) Pre-incubation of PAE/VEGFR-2 cells with z-VAD-fmk (20 or 100 μM) for 1 h prior to GrB/VEGF₁₂₁ treatment for 2 h significantly blocked caspase-3 activation. (D–E) GrB/VEGF₁₂₁-mediated cytotoxicity on PAE/VEGFR-2 and PAE/VEGFR-1 cells over 72 h is unchanged despite co-incubation with various concentrations of z-VAD-fmk. (F) Granzyme B internalization results in caspase-3 cleavage in vitro. PAE/VEGFR-2 cells were pre-treated with up to 100 μM z-VAD-fmk prior to addition of 20 nM GrB/VEGF₁₂₁ for one hour. GrB/VEGF₁₂₁-mediated cleavage of caspase-3 cleavage occurred in the presence of up to 100 μM z-VAD-fmk. (G–H) Caspase-independent PARP cleavage. PAE/VEGFR-2 and PAE/VEGFR-1 cells were treated with up to 48 with 20 nM GrB/VEGF₁₂₁ in the presence or absence of 20 μM z-VAD-fmk. PARP cleavage was observed in PAE/VEGFR-2, but not PAE/VEGFR-1, cells.

Figure 5. Specific localization of GrB/VEGF₁₂₁ to PC-3 tumors

Nu/nu mice bearing human prostate PC-3 tumors were injected i.v. with GrB/VEGF₁₂₁ or GrB at molar equivalent doses. Four hours after administration, tissues were removed and snap frozen. Sections were stained with immunofluorescent reagents to detect murine blood vessels (CD31, red), nuclei (Hoechst, blue) and granzyme B (green). Co-localization of GrB into CD31⁺ tumor vessels appear yellow (representative areas indicated with white arrows). (A–D) GrB/VEGF₁₂₁ localized to the tumor tissue, whereas (E–F) GrB did not. Robust GrB staining was observed in the GrB/VEGF₁₂₁-treated tumor core (B–C) as well as tumor vessels (D). In contrast, no GrB was detected in GrB-treated tumors. Vessels in all normal organs were unstained by GrB/VEGF₁₂₁ or GrB.

Figure 6. Anti-tumor efficacy of GrB/VEGF₁₂₁

(A) Mice with PC-3 tumor xenografts were intravenously injected with saline, GrB or GrB/VEGF₁₂₁ at the indicated times (arrows). Mean tumor volume was calculated as W × H × L as measured with digital calipers. (B) Representative immunofluorescent CD31 staining of saline- and GrB/VEGF₁₂₁-treated tumors. Quantification of percent CD31⁺ area revealed that GrB/VEGF₁₂₁ significantly reduced the overall vascular area compared to the saline treatment. (C) Quantification of the growth rate of PC-3 tumor cells by Ki-67. A minimum of 5 fields were assessed per slide. GrB/VEGF₁₂₁ treatment reduced the number of cycling cells by 50% compared to tumors from Saline-treated mice.