Targeting of Tumor Neovasculature with GrB/VEGF121, a Novel Cytotoxic Fusion Protein

Angiogenesis is a critical process in numerous diseases, and intervention in neovascularization has therapeutic value in several disease settings, including ocular diseases, arthritis, and in tumor progression and metastatic spread. Various vascular targeting agents have been developed, including those that inhibit growth factor receptor tyrosine kinases, blocking antibodies that interfere with receptor signal transduction, and strategies that trap growth factor ligands. Limited anti-tumor efficacy studies have suggested that the targeted delivery of the human pro-apoptotic molecule Granzyme B to tumor cells has significant potential for cancer treatment. Here, we review biological vascular targeting agents, and describe a unique vascular targeting agent composed of Granzyme B and the VEGF receptor ligand VEGF121. The fusion protein GrB/VEGF121 demonstrates cytotoxicity at nanomolar or sub-nanomolar levels, excellent pharmacokinetic and efficacy profiles, and has significant therapeutic potential targeting tumor vasculature.


Angiogenesis and Vascular Targeting Agents in the Clinic
Neovascularization is a normal process that occurs during growth and development, and critical during processes such as wound healing. It is also an important process in several disease states, such as tumor maturation and during metastatic spread, and interfering with this process has been shown to have therapeutic benefits [1][2][3][4]. As such, various elements of tumor neovascularization have been the focus of drug development strategies, and interventions at different points have been developed with varying levels of success in pre-clinical and clinical settings. Some such strategies include: inhibiting the downstream signaling of growth factor receptor tyrosine kinases [5][6][7], the development of blocking antibodies to attenuate receptor signal transduction [8][9][10][11], entrapment of growth factor ligands to prevent receptor activation [12][13][14], and the use of vascular-targeted photodynamic therapy [15]. Vascular targeting agents have also been tested in combination with other therapies, such as radiotherapy [16].

VEGF Receptor Targeting to Inhibit Angiogenesis
Numerous factors have been identified as playing significant roles during tumor neovascularization. Indeed, the complexity of this process is underscored by the fact that molecules that play a role in driving critical events continue to be identified. One key factor is vascular endothelial growth factor-A (VEGF-A) which, along with its receptors, is exceptionally important in many aspects of neovascularization [7,[17][18][19]. VEGFR-1 and VEGFR-2 are generally over-expressed in tumor neovasculature and normalize as the vasculature matures [20]. Indeed, of the over 1600 clinical trials on oncology interventions against pathways related to tumor vasculature reported worldwide

Advantages of GrB-Based Constructs over Other Immunotoxins or ADCs
Creation of an immunotoxin requires several factors to be taken into consideration. Among the key criteria are the potential of the therapeutic to trigger an immunogenic response. In addition, the resulting product cannot be too toxic or lack potency, and the linkers must be sufficiently stable in circulation. The development of immunotoxins composed entirely of human sequences is a significant advantage in mitigating against the possible development of immunogenicity. Bacterial and plant toxin-based immunotoxins have demonstrated remarkable potency and specificity, but a number of obstacles limit their clinical application [39,40]. For example, off-target binding to normal vasculature of toxins such as DT and ricin A chain (RTA) has resulted in vascular damage, leading to the loss of vascular integrity (vascular leak syndrome, VLS), a potentially life-threatening condition that can result in organ failure [41,42]. In addition, instances of immunogenic responses have also been identified in the case of DT as well as other bacterial and plant toxins [43], which limits the number of therapeutic regimens and their potential value in long-term treatment [44]. Immune responses to the toxins in patients also result in the rapid clearance of subsequent courses of therapy, and limit the number of treatment cycles [45,46]. Toxin immunogenicity is being addressed by engineering B-cell epitopes on the structure, but these molecules may be difficult to humanize completely [35,47,48].
There are now a number of clinically effective ADCs demonstrating remarkable activity, and many of these constructs were driven by the impressive success of the Trastuzumab-DM1 (T-DM1) conjugate [49,50]. On the other hand, there are limitations with antibody-drug conjugates, such as facile aggregation, off-target toxicity and potential development of cells with multidrug resistance (MDR) [51,52].
Granzyme B-based fusion proteins, on the other hand, exert a multi-modal and well-known mechanism of cytotoxic action. It is of significance that inhibitors of caspase activation have little impact on the overall cytotoxicity of some GrB constructs, which attests to the presence of multiple, redundant, pro-apoptotic pathways activated by this molecule [53][54][55]. Limited studies against MDR + tumor cells show that the expression of MDR does not seem to result in cross-resistance to GrB-based fusion constructs, which suggests that the emergence of resistance to this class of agents may be difficult from a biological perspective.
A key advantage of Granzyme B as a cytotoxic payload is its extremely limited ability to enter the cell without a carrier (such as a targeting molecule) or via the generation of perforin-mediated pores in the cell membrane, and its relative inertness outside of the cell. In addition, off-target in vivo toxicity is limited, due to the presence of stable amino acid linkers between the targeting molecule and the cytotoxic payload. To date, studies in mice with doses as high as 40 mg/kg have demonstrated a complete lack of toxicity (Zhou, Mohamedali et al. 2014 [55]. Finally, while the upregulation of resistance mechanisms has been observed in cells in response to some chemotherapeutic agents as well as to ADCs, our studies with a limited number of GrB-based constructs have indicated that the development of this resistance does not correlate to resistance to GrB-based targeting [53,[55][56][57].
Because of the many advantages of GrB, as well as to mitigate for the possible development of immunogenicity that has been observed in patients treated with ADCs and immunotoxin-based therapies, several laboratories, including ours, initiated the development of recombinant cell death-inducing fusion proteins, with human GrB as the cytotoxic payload [53][54][55][58][59][60][61][62][63][64][65]. We and others have shown that fusion constructs delivering GrB by either antibody or ligand based targeting have highly selective cytotoxic effects when delivered to the cytoplasm.
Below, we review some of the unique features, including specific cytotoxicity, mechanism of action, and in vivo studies of a unique vascular targeting agent, GrB/VEGF 121 , a novel pro-apoptotic agent targeting tumor vasculature.

Construction, Expression and Purification of GrB/VEGF 121
Mammalian expression of the Granzyme B/VEGF 121 construct was initiated by fusing the GrB/VEGF 121 DNA cassette into the pSecTag vector ( Figure 1A). We have expressed GrB/VEGF 121 using HEK-293T, HEK-293E, and CHO-S cells, and harvested the secreted fusion protein under serum-free conditions ( Figure 1B). The construct is expressed as an inactive pro-GrB/VEGF 121 form. Removal of the N-terminus histidine tag results in an 80 kDa dimer. The expressed protein is enzymatically active, as determined by a chromogenic assay measuring the rate of substrate turnover ( Figure 1C).

Construction, Expression and Purification of GrB/VEGF121
Mammalian expression of the Granzyme B/VEGF121 construct was initiated by fusing the GrB/VEGF121 DNA cassette into the pSecTag vector ( Figure 1A). We have expressed GrB/VEGF121 using HEK-293T, HEK-293E, and CHO-S cells, and harvested the secreted fusion protein under serum-free conditions ( Figure 1B). The construct is expressed as an inactive pro-GrB/VEGF121 form. Removal of the N-terminus histidine tag results in an 80 kDa dimer. The expressed protein is enzymatically active, as determined by a chromogenic assay measuring the rate of substrate turnover ( Figure 1C).

In Vitro Internalization and Cytotoxic Activity
To ensure that toxicity of GrB/VEGF121 is both targeted and specific, we evaluated a panel of endothelial and tumor cells lines with varying levels of VEGFR-1 and VEGFR-2 on their cell surface. Porcine cell lines over-expressing human VEGFR-1 or VEGFR-2 are considered to model tumor neovasculature, and have been used as in vitro models of angiogenesis [66]. Other cell lines were selected based on our previously established cytotoxic profiles with VEGF121/rGel, as well as cell surface VEGFR-1 and VEGFR-2 levels, as established by flow cytometry. In vitro cytotoxicity studies over 72 h showed varying levels of sensitivity to GrB/VEGF121 that correlated closely to total VEGFR-2 expression. IC50 levels were found to be in the nanomolar range ( Figure 2A,B). The cytotoxicity was determined to be VEGFR-driven, as pre-incubation of VEGFR-2 + cells with 1 µM VEGF121 significantly reduced the cytotoxicity of GrB/VEGF121, but had no impact on the cytotoxicity of GrB alone ( Figure  2C). In VEGFR-2 positive endothelial cells, GrB/VEGF121 showed rapid internalization into the entire cytoplasmic space within 24 h, while the internalization into VEGFR-1-expressing cells was significantly reduced (Figure 3), further confirming the VEGFR-driven targeting.

In Vitro Internalization and Cytotoxic Activity
To ensure that toxicity of GrB/VEGF 121 is both targeted and specific, we evaluated a panel of endothelial and tumor cells lines with varying levels of VEGFR-1 and VEGFR-2 on their cell surface. Porcine cell lines over-expressing human VEGFR-1 or VEGFR-2 are considered to model tumor neovasculature, and have been used as in vitro models of angiogenesis [66]. Other cell lines were selected based on our previously established cytotoxic profiles with VEGF 121 /rGel, as well as cell surface VEGFR-1 and VEGFR-2 levels, as established by flow cytometry. In vitro cytotoxicity studies over 72 h showed varying levels of sensitivity to GrB/VEGF 121 that correlated closely to total VEGFR-2 expression. IC 50 levels were found to be in the nanomolar range (Figure 2A,B). The cytotoxicity was determined to be VEGFR-driven, as pre-incubation of VEGFR-2 + cells with 1 µM VEGF 121 significantly reduced the cytotoxicity of GrB/VEGF 121 , but had no impact on the cytotoxicity of GrB alone ( Figure 2C). In VEGFR-2 positive endothelial cells, GrB/VEGF 121 showed rapid internalization into the entire cytoplasmic space within 24 h, while the internalization into VEGFR-1-expressing cells was significantly reduced (

Pro-Apoptotic Activity of GrB/VEGF121
While Granzyme B has well known caspase-dependent and independent mechanisms of apoptosis, it was important to examine whether the fusion protein itself elicited cytotoxicity by these pathways. We first evaluated the physical method of cell death of cells treated with GrB/VEGF121. Over a 24 h period, 35% of GrB/VEGF121-treated VEGFR-2 + cells mobilized into early apoptosis, compared to 4% of control cells. The impact on VEGFR-1 + endothelial cells and on the breast cancer cell line MDA-MB-231 cells was minimal ( Figure 4A). When compared over a 72 h period, the percent of VEGFR-2 + endothelial cells undergoing apoptosis continued to increase compared to cells treated with Granzyme B alone ( Figure 4B).

Pro-Apoptotic Activity of GrB/VEGF 121
While Granzyme B has well known caspase-dependent and independent mechanisms of apoptosis, it was important to examine whether the fusion protein itself elicited cytotoxicity by these pathways. We first evaluated the physical method of cell death of cells treated with GrB/VEGF 121 . Over a 24 h period, 35% of GrB/VEGF 121 -treated VEGFR-2 + cells mobilized into early apoptosis, compared to 4% of control cells. The impact on VEGFR-1 + endothelial cells and on the breast cancer cell line MDA-MB-231 cells was minimal ( Figure 4A). When compared over a 72 h period, the percent of VEGFR-2 + endothelial cells undergoing apoptosis continued to increase compared to cells treated with Granzyme B alone ( Figure 4B). To review whether apoptosis was triggered through well-characterized pathways, we analyzed the cell pathways by which apoptosis occurred. Forty percent of VEGFR-2 + cells underwent mitochondrial depolarization within 24 h of exposure to GrB/VEGF 121 , compared to 13% of controls, while VEGFR-1 + cells seemed largely unaffected ( Figure 5A). Over this same time period, both Granzyme B and GrB/VEGF 121 triggered caspase-3 activation in the VEGFR-2 + cells ( Figure 5B). Caspase-9 activation was also observed, with GrB/VEGF 121 apparently more efficient in activating this target than Granzyme B ( Figure 5C). Finally, complete cleavage of Poly (ADP-ribose) polymerase-1 (PARP-1) was observed after 24h in VEGFR-2 + cells, whereas minimal cleavage was seen in VEGFR-1 + cells ( Figure 5D). Notably, there was no demonstrated impact of a pan-caspase inhibitor on GrB/VEGF 121 -mediated cytotoxicity, while cleavage inhibition of both caspase-3 and caspase-9 showed. Thus, various downstream mediators of apoptosis have been shown to be engaged and activated upon treatment with GrB/VEGF 121 in both a caspase-dependent and caspase-independent manner.  To review whether apoptosis was triggered through well-characterized pathways, we analyzed the cell pathways by which apoptosis occurred. Forty percent of VEGFR-2 + cells underwent mitochondrial depolarization within 24 h of exposure to GrB/VEGF121, compared to 13% of controls, while VEGFR-1 + cells seemed largely unaffected ( Figure 5A). Over this same time period, both Granzyme B and GrB/VEGF121 triggered caspase-3 activation in the VEGFR-2 + cells ( Figure 5B). Caspase-9 activation was also observed, with GrB/VEGF121 apparently more efficient in activating this target than Granzyme B ( Figure 5C). Finally, complete cleavage of Poly (ADP-ribose) polymerase-1 (PARP-1) was observed after 24h in VEGFR-2 + cells, whereas minimal cleavage was seen in VEGFR-1 + cells ( Figure 5D). Notably, there was no demonstrated impact of a pan-caspase inhibitor on GrB/VEGF121-mediated cytotoxicity, while cleavage inhibition of both caspase-3 and caspase-9 showed. Thus, various downstream mediators of apoptosis have been shown to be engaged and activated upon treatment with GrB/VEGF121 in both a caspase-dependent and caspaseindependent manner. (A)

Localization of GrB/VEGF 121 into Tumor Tissue
We investigated the ability of GrB/VEGF 121 to localize into primary tumors by the subcutaneous placement of human prostate PC-3 tumors into male nude mice. This cell line is insensitive to VEGF 121 -mediated targeting in vitro, suggesting that it expresses insufficient receptor levels for targeted cytotoxicity. However, VEGF 121 -mediated targeting, in vivo with other cytotoxic payloads, has resulted in a significant anti-tumor therapeutic effect, presumably due to the highly vascularized nature of these tumors. Following tumor placement, mice were injected intravenously (tail vein) with GrB/VEGF 121 or GrB at molar-equivalent doses. Four hours after administration, tissues were removed and snap frozen. Immunofluorescence staining revealed GrB/VEGF 121 in tumor vessels as well as in in perivascular tumor areas adjacent to tumor microvessels ( Figure 6). Normal organs did not show GrB/VEGF 121 localization, nor did organs of mice treated with free Granzyme B, indicating the specific localization of GrB/VEGF 121 into tumor tissue. Similar results have been observed with MDA-MB-231/Luc tumors [67].

Localization of GrB/VEGF121 into Tumor Tissue
We investigated the ability of GrB/VEGF121 to localize into primary tumors by the subcutaneous placement of human prostate PC-3 tumors into male nude mice. This cell line is insensitive to VEGF121-mediated targeting in vitro, suggesting that it expresses insufficient receptor levels for targeted cytotoxicity. However, VEGF121-mediated targeting, in vivo with other cytotoxic payloads, has resulted in a significant anti-tumor therapeutic effect, presumably due to the highly vascularized nature of these tumors. Following tumor placement, mice were injected intravenously (tail vein) with GrB/VEGF121 or GrB at molar-equivalent doses. Four hours after administration, tissues were removed and snap frozen. Immunofluorescence staining revealed GrB/VEGF121 in tumor vessels as well as in in perivascular tumor areas adjacent to tumor microvessels ( Figure 6). Normal organs did not show GrB/VEGF121 localization, nor did organs of mice treated with free Granzyme B, indicating the specific localization of GrB/VEGF121 into tumor tissue. Similar results have been observed with MDA-MB-231/Luc tumors [67].

In Vivo Efficacy of GrB/VEGF121.
The efficacy of GrB/VEGF121 was also evaluated in a PC-3 subcutaneous tumor model. Mice received intravenous injections of saline, Granzyme B or GrB/VEGF121 every other day for a total of six treatments, and tumor volume was monitored twice weekly. Mice treated with GrB/VEGF121 at 27 mg/mg had very limited tumor growth, whereas those treated with a lower dose of GrB/VEGF121 (11 mg/kg) showed tumor growth of about threefold over 30 days. In contrast, tumors of saline-or Granzyme B-treated mice grew over 15-fold over the same period of time ( Figure 7A). The reduced growth rate of GrB/VEGF121-treated mice compared to vehicle was confirmed by determining the number of cycling tumor cells in lesions, and found to be reduced by over 50% (Figure 7B). The impact of tumor vasculature was assessed by histopathologic analysis of the vascular area, and found to be significantly lower in GrB-VEGF121-treated mice compared to controls ( Figure 7C).

In Vivo Efficacy of GrB/VEGF 121
The efficacy of GrB/VEGF 121 was also evaluated in a PC-3 subcutaneous tumor model. Mice received intravenous injections of saline, Granzyme B or GrB/VEGF 121 every other day for a total of six treatments, and tumor volume was monitored twice weekly. Mice treated with GrB/VEGF 121 at 27 mg/mg had very limited tumor growth, whereas those treated with a lower dose of GrB/VEGF 121 (11 mg/kg) showed tumor growth of about threefold over 30 days. In contrast, tumors of saline-or Granzyme B-treated mice grew over 15-fold over the same period of time ( Figure 7A). The reduced growth rate of GrB/VEGF 121 -treated mice compared to vehicle was confirmed by determining the number of cycling tumor cells in lesions, and found to be reduced by over 50% (Figure 7B). The impact of tumor vasculature was assessed by histopathologic analysis of the vascular area, and found to be significantly lower in GrB-VEGF 121 -treated mice compared to controls ( Figure 7C).

Conclusions
The serine protease GrB appears to be an ideal payload for targeted therapy, in part because GrB exerts a multimodal and well-known mechanism of cytotoxic action. Fusion constructs targeting tumor cells or tumor vasculature and containing GrB are generally active in the low nanomolar range, and studies in tumor xenograft models demonstrate excellent antitumor efficacy. Concerns regarding immunogenicity should be mitigated by designing completely human constructs containing GrB, and the lack of toxicity in animal model studies of GrB-based fusion proteins thus far is encouraging. Expression of VEGFR on the cell surface of other potential targets, such as bone marrow-derived cells that are involved in local or distant tumor development, suggest that this construct may have promise in a multi-targeted approach. Indeed, the potential of other VEGFR-targeting constructs in inhibiting tumor growth in this manner has previously been demonstrated [28]. In addition, the relative lack of systemic toxicity of this agent points to potential utility in combination therapy with standard chemotherapeutic agents. Overall, Granzyme B delivered directly to tumor cells or tumor neovasculature appears to be a promising and exciting opportunity in the field of targeted therapy, with significant therapeutic potential in primary and metastatic disease.