Radu O. Minea

Development of a Novel Recombinant Disintegrin, Contortrostatin, as an Effective Anti-Tumor and Anti-Angiogenic Agent

Contortrostatin (CN) (Mr 13,500 Da) is a novel homodimeric disintegrin isolated from the venom of Agkistrodon contortrix contortrix (Southern Copperhead) snake and displays two RGD motifs (one on each chain), which modulate its interaction with integrins on tumor cells and angiogenic vascular endothelial cells. In previous studies, we have shown that native CN administered in a liposomal formulation exhibits potent anti-angiogenic and tumor growth inhibitory activities. Current isolation of the protein from crude venom is difficult and prohibitively expensive for translation into the clinic. In this report, we describe a method amenable to large-scale production of a soluble monomeric form of recombinant CN with biologic activity; the protein is expressed directly in the cytoplasm of an engineered bacterial system with an expression yield of approximately 20 mg/l of culture. We present here the in vitro assays as well as the anti-tumor and anti-angiogenic evaluation of liposomal recombinant CN in an orthotopic, xenograft model of human breast cancer.

Vicrostatin – An Anti-Invasive Multi-Integrin Targeting Chimeric Disintegrin with Tumor Anti-Angiogenic and Pro-Apoptotic Activities

Similar to other integrin-targeting strategies, disintegrins have previously shown good efficacy in animal cancer models with favorable pharmacological attributes and translational potential. Nonetheless, these polypeptides are notoriously difficult to produce recombinantly due to their particular structure requiring the correct pairing of multiple disulfide bonds for biological activity. Here, we show that a sequence-engineered disintegrin (called vicrostatin or VCN) can be reliably produced in large scale amounts directly in the oxidative cytoplasm of Origami B E. coli. Through multiple integrin ligation (i.e., αvβ3, αvβ5, and α5β1), VCN targets both endothelial and cancer cells significantly inhibiting their motility through a reconstituted basement membrane. Interestingly, in a manner distinct from other integrin ligands but reminiscent of some ECM-derived endogenous anti-angiogenic fragments previously described in the literature, VCN profoundly disrupts the actin cytoskeleton of endothelial cells (EC) inducing a rapid disassembly of stress fibers and actin reorganization, ultimately interfering with ECs ability to invade and form tubes (tubulogenesis). Moreover, here we show for the first time that the addition of a disintegrin to tubulogenic EC sandwiched in vitro between two Matrigel layers negatively impacts their survival despite the presence of abundant haptotactic cues. A liposomal formulation of VCN (LVCN) was further evaluated in vivo in two animal cancer models with different growth characteristics. Our data demonstrate that LVCN is well tolerated while exerting a significant delay in tumor growth and an increase in the survival of treated animals. These results can be partially explained by potent tumor anti-angiogenic and pro-apoptotic effects induced by LVCN.

Development of a chimeric recombinant disintegrin as a cost-effective anti-cancer agent with promising translational potential.

Vicrostatin (VCN) is a chimeric recombinant disintegrin generated in Origami B (DE3) Escherichia coli as a genetic fusion between the C-terminal tail of a viperid disintegrin echistatin and crotalid disintegrin contortrostatin (CN). The therapeutic modulation of multiple integrin pathways via soluble disintegrins was previously shown by us and others to elicit potent anti-angiogenic and anti-metastatic effects in several animal cancer models. Despite these favorable attributes, these polypeptides are notoriously difficult to produce recombinantly in significant quantity due to their structure which requires the correct pairing of multiple disulfide bonds for biological activity. In this report, we show that VCN can be reliably produced in large amounts (yields in excess of 200 mg of active purified disintegrin per liter of bacterial culture) in Origami B (DE3), an E. coli expression strain engineered to support the folding of disulfide-rich heterologous proteins directly in its oxidative cytoplasmic compartment. VCN retains the integrin binding specificity of both parental molecules it was derived from, but with a different binding affinity profile. While competing for the same integrin receptors that are preferentially upregulated in the tumor microenvironment, VCN exerts a potent inhibitory effect on endothelial cell (EC) migration and tube formation in a dose-dependent manner, by forcing these cells to undergo significant actin cytoskeleton reorganization when exposed to this agent in vitro. Moreover, VCN has a direct effect on breast cancer cells inhibiting their in vitro motility. In an effort to address our main goal of developing a clinically relevant delivery method for recombinant disintegrins, VCN was efficiently packaged in liposomes (LVCN) and evaluated in vivo in an animal breast cancer model. Our data demonstrate that LVCN is well tolerated, its intravenous administration inducing a significant delay in tumor growth and an increase in animal survival, results that can be partially explained by potent tumor apoptotic effects.

Multimeric disintegrin protein polymer fusions that target tumor vasculature.

Recombinant protein therapeutics have increased in number and frequency since the introduction of human insulin, 25 years ago. Presently, proteins and peptides are commonly used in the clinic. However, the incorporation of peptides into clinically approved nanomedicines has been limited. Reasons for this include the challenges of decorating pharmaceutical-grade nanoparticles with proteins by a process that is robust, scalable, and cost-effective. As an alternative to covalent bioconjugation between a protein and nanoparticle, we report that biologically active proteins may themselves mediate the formation of small multimers through steric stabilization by large protein polymers. Unlike multistep purification and bioconjugation, this approach is completed during biosynthesis. As proof-of-principle, the disintegrin protein called vicrostatin (VCN) was fused to an elastin-like polypeptide (A192). A significant fraction of fusion proteins self-assembled into multimers with a hydrodynamic radius of 15.9 nm. The A192-VCN fusion proteins compete specifically for cell-surface integrins on human umbilical vein endothelial cells (HUVECs) and two breast cancer cell lines, MDA-MB-231 and MDA-MB-435. Confocal microscopy revealed that, unlike linear RGD-containing protein polymers, the disintegrin fusion protein undergoes rapid cellular internalization. To explore their potential clinical applications, fusion proteins were characterized using small animal positron emission tomography (microPET). Passive tumor accumulation was observed for control protein polymers; however, the tumor accumulation of A192-VCN was saturable, which is consistent with integrin-mediated binding. The fusion of a protein polymer and disintegrin results in a higher intratumoral contrast compared to free VCN or A192 alone. Given the diversity of disintegrin proteins with specificity for various cell-surface integrins, disintegrin fusions are a new source of biomaterials with potential diagnostic and therapeutic applications.

The use of pepsin in receptor internalization assays

For internalization experiments that use fluorescent antibody (Ab) staining to distinguish between inside versus outside cellular localization of various receptor targeting ligands, it is critical that there be efficient removal of all residual surface-bound fluorescent Ab. To achieve this, a fluorescent Ab removal technique is commonly employed in receptor internalization assays that utilizes low pH glycine-based buffers to wash off the residual non-internalized fluorescent Ab retained on cell surfaces. In this study, we highlight the shortcomings of this technique and propose an alternative in situ proteolytic approach that we found to be non-deleterious to the cells and significantly more effective in removing the residual fluorescence resulting from non-internalized surface-bound Ab.

A SNAKE VENOM DISINTEGRIN WITH POTENT ANTITUMOR AND ANTIANGIOGENIC ACTIVITY

In this report we describe an effective method for delivery of a novel snake venom disintegrin, contortrostatin (CN), in an orthotopic, xenograft model of human mammary cancer in immunodeficient mice. Contortrostatin (Mr 13,500) is a homodimeric disintegrin isolated from venom of the southern copperhead snake. Contortrostatin possesses two RGD sites, one in each chain, which modulate its interaction with integrins on tumor cells and angiogenic vascular endothelial cells. Although our laboratory has previously described the antitumor activity of CN in a mouse model of human mammary cancer, the method of delivery, daily intratumor injection, was not translatable to clinical application. We now describe a clinically relevant method of administering CN, liposomal delivery (LCN). A unique liposomal system has been designed for intravenous (IV) administration of a biologically active protein with full retention of biological activity. Pharmacokinetics, biodistribution, platelet reactivity, and immunogenicity of LCN were examined and compared with similar characteristics of native, unencapsulated CN. There are several advantages to liposomal delivery of CN: (i) LCN has a significantly prolonged circulatory half-life compared to native CN; (ii) LCN is passively accumulated in the tumor; (iii) LCN has no platelet reactivity; and (iv) LCN is not recognized by the immune system. We have previously demonstrated that antiangiogenic activity is an important component of CNs mechanism of antitumor action. In the present communication we demonstrate that IV delivery of LCN leads to potent antitumor and antiangiogenic activity in the orthotopic, xenograft human mammary cancer model.

Anti-Angiogenesis and Disintegrins

Angiogenesis is a critical process in tumor and disease progression. A number of features are central to both tumor growth and development, and the recruitment and invasion of a growing vascular network supplying the tumor with nutrients and a mechanism of escape to allow meastatic growth. One class of molecules important to both tumor growth and blood vessel recruitment are a family of cell surface receptors identified as integrins. Integrins are α/β heterodimeric glycoproteins in which the different α subunits combine with distinct β subunits resulting in a range of specificities toward various extracellular matrix (ECM) proteins. The RGD sequence found in a number of ECM proteins is recognized by several classes of integrins, allowing for linkage of cytoskeletal proteins associated with the integrins to the ECM which leads to involvement in bi-directional signaling that displays profound effects on cellular functions. Among these integrin mediated interactions are the adhesion of both endothelial cells and cancer cells to ECM proteins, an interaction that is integral to metastasis, tumor growth and angiogensis. Antibodies targeted to integrins have been shown to retard tumor growth and subsequent tumor induced angiogenesis. One concern with this approach is that the antibody targets a single integrin, which may allow the tumor to utilize other non-targeted integrins to circumvent this type of blockage. A more broad spectrum agent is available that binds to and blocks the function of several different integrins at a time, this agents is identified as a disintegrin. Originally purified from the venom of Viperidae family of snakes, a disintegrins role in nature is presumably to block platelet aggregation following envenomation based on interaction of an integrin on the activated platelet surface with an RGD sequence in the disintegrin. It has been observed that integrins overexpressed on some tumor types and angiogenic vasculature have similar affinity for RGD motifs found in ECM proteins. Based on disintegrin structure we have developed a recombinant form of a snake venom disintegrin, which we call vicrostatin (VCN). VCN is a potent anti-angiogenic/anti-tumor agent in in vitro and in vivo laboratory studies. Further development of the recombinant venom derived disintegrin along with new technology looking at additional disintegrin-like proteins may offer a novel therapeutic approach in targeting tumor induced angiogenesis.

Rare Stochastic Expression of O6-Methylguanine- DNA Methyltransferase (MGMT) in MGMT-Negative Melanoma Cells Determines Immediate Emergence of Drug-Resistant Populations upon Treatment with Temozolomide In Vitro and In Vivo

The chemotherapeutic agent temozolomide (TMZ) kills tumor cells preferentially via alkylation of the O6-position of guanine. However, cells that express the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), or harbor deficient DNA mismatch repair (MMR) function, are profoundly resistant to this drug. TMZ is in clinical use for melanoma, but objective response rates are low, even when TMZ is combined with O6-benzylguanine (O6BG), a potent MGMT inhibitor. We used in vitro and in vivo models of melanoma to characterize the early events leading to cellular TMZ resistance. Melanoma cell lines were exposed to a single treatment with TMZ, at physiologically relevant concentrations, in the absence or presence of O6BG. Surviving clones and mass cultures were analyzed by Western blot, colony formation assays, and DNA methylation studies. Mice with melanoma xenografts received TMZ treatment, and tumor tissue was analyzed by immunohistochemistry. We found that MGMT-negative melanoma cell cultures, before any drug treatment, already harbored a small fraction of MGMT-positive cells, which survived TMZ treatment and promptly became the dominant cell type within the surviving population. The MGMT-negative status in individual cells was not stable, as clonal selection of MGMT-negative cells again resulted in a mixed population harboring MGMT-positive, TMZ-resistant cells. Blocking the survival advantage of MGMT via the addition of O6BG still resulted in surviving clones, although at much lower frequency and independent of MGMT, and the resistance mechanism of these clones was based on a common lack of expression of MSH6, a key MMR enzyme. TMZ treatment of mice implanted with MGMT-negative melanoma cells resulted in effective tumor growth delay, but eventually tumor growth resumed, with tumor tissue having become MGMT positive. Altogether, these data reveal stochastic expression of MGMT as a pre-existing, key determinant of TMZ resistance in melanoma cell lines. Although MGMT activity can effectively be eliminated by pharmacologic intervention with O6BG, additional layers of TMZ resistance, although considerably rarer, are present as well and minimize the cytotoxic impact of TMZ/O6BG combination treatment. Our results provide rational explanations regarding clinical observations, where the TMZ/O6BG regimen has yielded mostly disappointing outcomes in melanoma patients.

Abstract POSTER-THER-1432: Intraperitoneal delivery of the disintegrin vicrostatin (VCN) effectively limits ovarian cancer growth and progression

Abstracts: 10th Biennial Ovarian Cancer Research Symposium; September 8-9, 2014; Seattle, WA Although prevention and early detection are the ultimate goals for ovarian cancer, the fact cannot be ignored that in 2014 21,980 women will be diagnosed with ovarian cancer in the U.S. 75% of these women will be confronted with a high stage tumor and a five-year survival rate of 45.5% (American Cancer Society, Atlanta). For these patients, improved treatment for residual disease following surgery, reduced complications during therapy as well as a decreased risk of cancer recurrence, would reduce mortality and improve their quality of life significantly. After surgery and first-line chemotherapy, 50% to 75% of responders will relapse within approximately 18 months and require further systemic therapy. Approximately 50% of women die due to carcinomatosis. Exfoliated ovarian tumor cells are carried via peritoneal fluid to secondary sites in the abdominal cavity, where they attach, invade the submesothelial connective tissue and proliferate to create peritoneal micrometastasis. These cancer cells in effusions are not amenable to surgical removal, failure of their eradication is one of the main causes for unsuccessful treatment and recurrence. The strategy of localized intraperitoneal (IP) chemotherapy targets these cells in particular and is therefore highly effective in preventing recurrence. According to the Cochrane database, IP chemotherapy successfully increased overall survival and progression free-survival, resulting in a 21.6% decrease in the risk of death. Therefore, the U.S. National Cancer Institute recommended consideration of IP chemotherapy for patients with advanced stage III ovarian cancer after optimal surgical debulking. Integrins are heterodimeric receptors that evolved to mediate complex cell-ECM interactions. These interactions regulate the ability of cells to mechanically sense their environment by integrating multiple signaling pathways initiated by extracellular cues with the cell’s cytoskeleton. Integrins (including αvβ3, αvβ5, α5β1 and αvβ6) have important roles in OC cell attachment, survival, migration, invasion and angiogenesis. However, the precise roles played by different integrin subunits in various aspects of tumor progression and why some integrins appear to be especially supportive of tumor progression are still not fully understood. Due to their pivotal roles in OC biology, integrins represent an attractive target for a novel form of OC therapy. Disintegrins are among the most potent soluble ligands of integrins representing a class of cysteine-rich polypeptides originally isolated from snake venom, many of which contain a cyclic-Arg-Gly-Asp (c-RGD) motif. These polypeptides hold a significant translational potential as anti-cancer therapeutic agents based on their high affinity interaction with integrins as well as their excellent pharmacological properties. However, these polypeptides are extremely difficult to produce recombinantly. Previously the Markland group reported that a sequence-engineered RGD-disintegrin (called vicrostatin or VCN) can be reliably produced in large quantity. Through multiple integrin ligation (i.e., αvβ3, αvβ5 and α5β1), VCN targets both endothelial and OC cells. The use of small RGD peptides or multimeric cyclic RGD peptides for targeting of integrins for both therapy and imaging of tumors has been widely reported but there is a distinct advantage to the use of disintegrins such as VCN. VCN not only acts as an antagonist (in a manner identical to RGD peptides), VCN also elicits signaling responses through direct interaction with integrins. Importantly, integrin αvβ3 is expressed at very low level on epithelial cells and mature endothelial cells, but is overexpressed on the endothelial cells of the tumor neovasculature and on tumor cells. Therefore, this integrin presents an attractive and tumor specific therapeutic target for rapidly growing solid tumors. In the present studies we delivered VCN formulated in a novel carboxymethyl cellulose (CMC) polyethyleneoxide (PEO) gel (Oxiplex™) through IP delivery. In studies described here we show that IP VCN delivery via Oxiplex™, following implantation of SKOV-3 sphereoids and confirmation of tumor growth by bioluminescent imaging, significantly reduced tumor growth and dissemination. In addition we have evaluated the dose of VCN delivered and the timing of the administrations and shown that a clinically relevant dosing schedule can be achieved. Citation Format: S. Swenson, R. Minea, J. Moore, M. Mikhail, Z. Kirakosyan & Francis S. Markland Jr. Intraperitoneal delivery of the disintegrin vicrostatin (VCN) effectively limits ovarian cancer growth and progression [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1432.

Abstract 5248: A novel ovarian cancer imaging agent based on integrin ligation

Ovarian cancer diagnosis and subsequent evaluation of disease progression is difficult due to the location of tumor mass and access to the organs affected. Ultrasound and surgical exposure are required to confirm disease and response to treatment. In an effort to develop a non-invasive radiologic method for both diagnostic assessment and to follow disease progression we have produced a integrin ligand, vicrostatin (VCN) that can be radiolabeled and imaged upon binding to active integrins over-expressed on the tumor. VCN displays a high affinity for integrins which are active and pivotal in tumor growth and progression. In the present studies we utilized an animal model employing the luciferase expressing cell line OVCAR-3 luc . This procedure allows us to observe both the tumor itself in a living animal, and also co-register the PET image with the optical image enabling us to detect tumors in the intraperitoneal (IP) space. For these experiments we injected OVCAR-3 luc cells IP and allowed the tumors to age for 28 days. At this point, with no obvious sign of disease, we imaged the tumors using the optical imaging system to detect the presence of luciferase. Since luciferase is only found in the tumor, this enables us to specifically image the tumor. Following these images 64 Cu-DOTA-VCN is injected into the tail vein of the study animals and allowed to circulate for 90 minutes. At this time the animal is placed onto the mounting tray of the PET imaging instrument. There is a specific accumulation of 64 Cu-DOTA-VCN in an area in the lower abdomen that correlates with a location of luciferase activity seen in the optical image. Both of these positions were confirmed by dissection and exposure of the tumor in the sacrificed animal. Experiments in which a larger number of cells are injected IP produced unusable optical images, as the tumors grew very large and coated much of the surface in the IP space yielding bright and indistinguishable images in the mouse abdomen. One problem of note is the high level of uptake by the kidneys at the 90 minute time point. The kidneys shield the ability to view an ovarian tumor via a dorsal or ventral total slice view. When viewed transversely, there is clear separation between the kidneys and the tumor mass which binds the 64 Cu-DOTA-VCN. A three dimensional reconstruction yields a three dimensional model that can be rotated and rendered to observe the position of the tumor in relationship to the kidneys in space. These studies show that VCN can be utilized to image early stage OC and could possibly be used to monitor residual disease after surgical or therapeutic intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5248. doi:1538-7445.AM2012-5248