Jessica Sims

A Mechanistic Study of Tumor-Targeted Corrole Toxicity

HerGa is a self-assembled tumor-targeted particle that bears both tumor detection and elimination activities in a single, two-component complex (Agadjanian et al. Proc. Natl. Acad. Sci. U.S.A.2009, 106, 6105-6110). Given its multifunctionality, HerGa (composed of the fluorescent cytotoxic corrole macrocycle, S2Ga, noncovalently bound to the tumor-targeted cell penetration protein, HerPBK10) has the potential for high clinical impact, but its mechanism of cell killing remains to be elucidated, and hence is the focus of the present study. Here we show that HerGa requires HerPBK10-mediated cell entry to induce toxicity. HerGa (but not HerPBK10 or S2Ga alone) induced mitochondrial membrane potential disruption and superoxide elevation, which were both prevented by endosomolytic-deficient mutants, indicating that cytosolic exposure is necessary for corrole-mediated cell death. A novel property discovered here is that corrole fluorescence lifetime acts as a pH indicator, broadcasting the intracellular microenvironmental pH during uptake in live cells. This feature in combination with two-photon imaging shows that HerGa undergoes early endosome escape during uptake, avoiding compartments of pH < 6.5. Cytoskeletal disruption accompanied HerGa-mediated mitochondrial changes whereas oxygen scavenging reduced both events. Paclitaxel treatment indicated that HerGa uptake requires dynamic microtubules. Unexpectedly, low pH is insufficient to induce release of the corrole from HerPBK10. Altogether, these studies identify a mechanistic pathway in which early endosomal escape enables HerGa-induced superoxide generation leading to cytoskeletal and mitochondrial damage, thus triggering downstream cell death.

Photoexcitation of tumor-targeted corroles induces singlet oxygen-mediated augmentation of cytotoxicity.

The tumor-targeted corrole particle, HerGa, displays preferential toxicity to tumors in vivo and can be tracked via fluorescence for simultaneous detection, imaging, and treatment. We have recently uncovered an additional feature of HerGa in that its cytotoxicity is enhanced by light irradiation. In the present study, we have elucidated the cellular mechanisms for HerGa photoexcitation-mediated cell damage using fluorescence optical imaging. In particular, we found that light irradiation of HerGa produces singlet oxygen, causing mitochondrial damage and cytochrome c release, thus promoting apoptotic cell death. An understanding of the mechanisms of cell death induced by HerGa, particularly under conditions of light-mediated excitation, may direct future efforts in further customizing this nanoparticle for additional therapeutic applications and enhanced potency.

Investigating photoexcitation-induced mitochondrial damage by chemotherapeutic corroles using multimode optical imaging

We recently reported that a targeted, brightly fluorescent gallium corrole (HerGa) is highly effective for breast tumor detection and treatment. Unlike structurally similar porphryins, HerGa exhibits tumor-targeted toxicity without the need for photoexcitation. We have now examined whether photoexcitation further modulates HerGa toxicity, using multimode optical imaging of live cells, including two-photon excited fluorescence, differential interference contrast (DIC), spectral, and lifetime imaging. Using two-photon excited fluorescence imaging, we observed that light at specific wavelengths augments the HerGa-mediated mitochondrial membrane potential disruption of breast cancer cells in situ. In addition, DIC, spectral, and fluorescence lifetime imaging enabled us to both validate cell damage by HerGa photoexcitation and investigate HerGa internalization, thus allowing optimization of light dose and timing. Our demonstration of HerGa phototoxicity opens the way for development of new methods of cancer intervention using tumor-targeted corroles.

A corrole nanobiologic elicits tissue-activated MRI contrast enhancement and tumor-targeted toxicity

Water-soluble corroles with inherent fluorescence can form stable self-assemblies with tumor-targeted cell penetration proteins, and have been explored as agents for optical imaging and photosensitization of tumors in pre-clinical studies. However, the limited tissue-depth of excitation wavelengths limits their clinical applicability. To examine their utility in more clinically-relevant imaging and therapeutic modalities, here we have explored the use of corroles as contrast enhancing agents for magnetic resonance imaging (MRI), and evaluated their potential for tumor-selective delivery when encapsulated by a tumor-targeted polypeptide. We have found that a manganese-metallated corrole exhibits significant T1 relaxation shortening and MRI contrast enhancement that is blocked by particle formation in solution but yields considerable MRI contrast after tissue uptake. Cell entry but not low pH enables this. Additionally, the corrole elicited tumor-toxicity through the loss of mitochondrial membrane potential and cytoskeletal breakdown when delivered by the targeted polypeptide. The protein-corrole particle (which we call HerMn) exhibited improved therapeutic efficacy compared to current targeted therapies used in the clinic. Taken together with its tumor-preferential biodistribution, our findings indicate that HerMn can facilitate tumor-targeted toxicity after systemic delivery and tumor-selective MR imaging activatable by internalization.

Abstract 4487: Targeting trastuzumab-resistant HER2+ breast cancer with a HER3-targeting nanoparticle

HER2-positive breast cancers represent almost a quarter of invasive breast cancers and are indicative of poor patient survival. Although many patients with HER2-positive breast cancer initially respond to anti-HER2 treatments, such as trastuzumab, a significant portion of them develop resistance to these therapies. Consequently, there is a great need to develop new drugs that are effective against these HER2+ tumors that are non-responsive or have become resistant to these therapies. Recently, it has been shown that another member of the HER family, HER3, is commonly upregulated in these drug-resistant cancers. This observation led us to develop a novel drug delivery protein, called HerPBK10, that specifically targets another member of the HER receptor family, HER3. HerPBK10, once it has bound to the HER3 receptor, triggers rapid endocytosis and endosomal penetration, enabling it to deliver a toxic payload to the cell, resulting in cell death. We hypothesized that cytotoxic drugs delivered by HerPBK10 would induce significant targeted cell death in trastuzumab-resistant HER2+ breast cancers due to the high levels of surface HER3 and would therefore provide an effective treatment for patients who have developed resistance to traditional therapies. First, we verified that cell surface levels of HER3 are elevated in trastuzumab-resistant cell lines compared to trastuzumab-sensitive cells. We then demonstrated, through competitive inhibition with free HER3 ligand, that HerPBK10 binds specifically to HER3 on multiple HER2+ cell lines. We assembled our targeted molecule, HerPBK10 with the chemotherapeutic doxorubicin. The resulting nanoparticle, called HerDox, was used to treat HER2+ breast cancer cell lines that were either inherently resistant to trastuzumab or had acquired resistance to trastuzumab. We demonstrated that HerDox caused significant cell death in both types of resistant cells. We also compared the effect of the HerDox nanoparticle to trastuzumab, pertuzumab, and the two drugs together and showed that it caused superior cell death in all three instances. In addition, we combined our nanoparticle with trastuzumab, and showed that together, they have an additive effect on cell death. These results indicate that our HER3 targeting nanoparticle, HerDox, efficiently targets and kills cancer cells that have become resistant to trastuzumab, and has the potential to be used either as a single drug or as part of a combinatorial therapy in eliminating drug-resistant HER2+ breast cancers. We are in the process of verifying these findings in vivo in order to demonstrate the potential of HerDox as a treatment for patients who have become non-responsive to traditional anti-HER2 therapies. Citation Format: Jessica Sims, Michael Taguaim, Chris Hanson, Xiaojiang Cui, Lali K. Medina-Kauwe. Targeting trastuzumab-resistant HER2+ breast cancer with a HER3-targeting nanoparticle. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4487. doi:10.1158/1538-7445.AM2014-4487

Abstract P5-08-08: A human epidermal growth factor receptor 3 (HER3)-binding nanoparticle targets and kills Herceptin®-resistant human epidermal growth factor receptor 2 (HER2)-positive breast cancer

The human epidermal growth factor receptor subunit (HER2), or ErbB2, is a receptor tyrosine kinase that is amplified in approximately 20-25% of invasive breast cancers. Anti-HER2 therapies such as trastuzumab (Herceptin®) have become important in the management of aggressive and metastatic breast cancer. Although many patients with HER2-positive breast cancer initially respond to anti-HER2 treatments, such as Herceptin, a significant portion of them develop resistance to these therapies. Consequently, there is a great need to develop therapies that will treat these tumors once they become resistant. Recently it has been shown that another member of the HER family, HER3, is commonly upregulated in these tumors. This led us to develop a unique drug delivery protein (HerPBK10) that specifically targets the cell surface receptor, HER3. HerPBK10, once it has bound to the HER3 receptor, triggers rapid endocytosis and endosomal penetration, enabling it to deliver a toxic payload to the cell, resulting in cell death. We hypothesized that cytotoxic drugs delivered by HerPBK10 would induce significant targeted cell death in Herceptin-resistant, HER2+ breast cancers and would provide an effective treatment for patients whose tumors have become resistant to traditional therapies. We have demonstrated that HerPBK10 binds to the cell surface of three different HER2+ breast cancer cell lines and that this binding can be competitively inhibited by free HER3 ligand, indicating that HerPBK10 binds specifically to HER3. Next, we showed in multiple Herceptin-resistant cell lines that HER3 receptor levels are significantly increased in drug-resistant cells, confirming the results of other researchers. We then assembled our targeted molecule, HerPBK10 with the chemotherapeutic doxorubicin. The resulting nanoparticle, called HerDox, was used to treat two different HER2+ breast cancer cell lines that are susceptible to Herceptin treatment and two that had acquired Herceptin resistance. We demonstrated that HerDox caused cell death in all cell lines, but at a greater level and at a lower dosage in the drug-resistant lines. We also compared the effect of the HerDox nanoparticle to Herceptin and showed that it caused greater overall cell death. In addition, we combined our nanoparticle with Herceptin, and showed that together, they induced even greater cell death. These results indicate that our HER3 targeting nanoparticle, HerDox, efficiently targets and kills cancer cells that have become resistant to Herceptin, and has the potential to be used either as a single drug or as part of a combinatorial therapy in eliminating drug-resistant HER2+ breast cancers. We are in the process of verifying these findings in vivo in order to demonstrate the potential of HerDox as a treatment for patients who have become non-responsive to traditional anti-HER2 therapies. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-08.

Resistance to receptor-blocking therapies primes tumors as targets for HER3-homing nanobiologics

ABSTRACT Resistance to anti‐tumor therapeutics is an important clinical problem. Tumor‐targeted therapies currently used in the clinic are derived from antibodies or small molecules that mitigate growth factor activity. These have improved therapeutic efficacy and safety compared to traditional treatment modalities but resistance arises in the majority of clinical cases. Targeting such resistance could improve tumor abatement and patient survival. A growing number of such tumors are characterized by prominent expression of the human epidermal growth factor receptor 3 (HER3) on the cell surface. This study presents a “Trojan‐Horse” approach to combating these tumors by using a receptor‐targeted biocarrier that exploits the HER3 cell surface protein as a portal to sneak therapeutics into tumor cells by mimicking an essential ligand. The biocarrier used here combines several functions within a single fusion protein for mediating targeted cell penetration and non‐covalent self‐assembly with therapeutic cargo, forming HER3‐homing nanobiologics. Importantly, we demonstrate here that these nanobiologics are therapeutically effective in several scenarios of resistance to clinically approved targeted inhibitors of the human EGF receptor family. We also show that such inhibitors heighten efficacy of our nanobiologics on naïve tumors by augmenting HER3 expression. This approach takes advantage of a current clinical problem (i.e. resistance to growth factor inhibition) and uses it to make tumors more susceptible to HER3 nanobiologic treatment. Moreover, we demonstrate a novel approach in addressing drug resistance by taking inhibitors against which resistance arises and re‐introducing these as adjuvants, sensitizing tumors to the HER3 nanobiologics described here. Graphical abstract Figure. Tumor cells with resistance to HER2 inhibitors can be prime targets for HER3‐directed nanobiologics. Schematic summarizes several scenarios that may explain how drug‐resistant tumor cells can be targeted by HER3‐nanobiologics. In the first scenario, tumor cells with acquired resistance to HER2‐targeted inhibitors are characterized by increased cell surface display of HER3, cornering these resistant cells for attack by the HER3‐nanobiologics described here. These studies also suggest that pre‐treatment of sensitive or naïve tumors may yield the same result by shifting naïve tumor cells to a HER3‐elevated phenotype. In the second scenario, HER2‐sensitive tumor cells in a heterogenous population may be eliminated, leaving resistant cells that could be vulnerable to nanobiologic attack due to increased cell surface display of HER3. The studies described here also suggest that such tumors with inherent or pre‐existing resistance to HER2 inhibitors may likely have increased cell surface HER3, thus making them vulnerable to nanobiologic attack.

Abstract P6-17-05: A corrole nanobiologic crosses the blood-brain-barrier and recognizes triple negative breast cancer: Implications for targeting brain metastases

Patients with breast cancer metastases to the brain on average survive less than one year. These tumors tend to be resistant to current therapies, and the majority of targeted therapeutics are unable to breach the blood brain barrier (BBB) to reach these tumors, thus improved alternatives are urgently needed.

Abstract 4490: A novel tumor-targeting construct aimed at c-Met

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA C-Met is a receptor tyrosine kinase (RTK) that is activated by the binding of its ligand, hepatocyte growth factor (HGF). The HGF/c-Met pathway contributes to embryonic development, wound healing, cell proliferation and survival, motility and morphogenesis. It is also one of the most frequently dysregulated pathways in a variety of human cancers, including breast, ovarian, prostate, lung, and pancreatic. Importantly, cell surface elevation of c-Met has been associated with drug-resistance, including acquired resistance to current signal-blocking therapies. Tumor-targeting strategies that do not require signal inhibition may prove more effective on c-Met positive cancer cells. This may be addressed by ligands that recognize c-Met to trigger cell uptake of attached therapeutics, thus bypassing the need to block signaling. HGF has the potential to accomplish this but its requirement for tetramerization and disulfide bonding presents technical complications for therapeutics development. An alternative, and potentially superior ligand for c-Met targeting may possibly be derived from a bacterium that causes food-poisoning. The human pathogen, Listeria monocytogenes, binds c-Met to invade host cells through its surface proteins called Internalins. Specifically, Internalin B (InlB) triggers receptor-mediated endocytosis after c-Met binding. InlB and HGF recognize different regions of c-Met, and InlB does not require tetramerization or disulfide bonds for binding. Consequently, InlB is more suitable for recombinant protein production than HGF. We have previously shown that PBK10, a recombinant protein derived from the adenovirus capsid penton base, can mediate gene and drug delivery into cells through the membrane penetrating activity of the penton base. Moreover, PBK10 can be targeted to tumor cells via recombinant fusion to tumor-homing ligands. Here, we explore the possibility of fusing the receptor-binding site of InlB to PBK10 for targeting attached cytotoxic agents to c-Met positive cancer cells, and transporting such agents directly into these cells via receptor-mediated endocytosis and membrane penetration. Citation Format: mitra mastali, Jessica D Sims, Jan M Taguiam, Chris Hanson, Felix Alonso Valenteen, Lali K. Medina-Kauwe. A novel tumor-targeting construct aimed at c-Met. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4490. doi:10.1158/1538-7445.AM2014-4490

Abstract A101: Treating trastuzumab-resistant HER2+ breast cancers with a HER3-targeted nanoparticle

The human epidermal growth factor receptor subunit (HER2), or ErbB2, is receptor tyrosine kinase that is amplified in approximately 20-25% of invasive breast cancers. Anti-HER2 therapies such as trastuzumab (Herceptin®) have become important in the management of aggressive and metastatic breast cancer. Although many patients with HER2-positive breast cancer initially respond to anti-HER2 treatments, such as trastuzumab, a significant portion of them develop resistance to these therapies. Consequently, there is a great need to develop therapies that will treat these tumors once they become resistant. We have developed a unique drug delivery protein (HerPBK10) that specifically targets the cell surface receptor HER3, which has been shown to be elevated in trastuzumab-resistant HER2+ breast cancers. HerPBK10, once it has bound to the HER3 receptor subunit, triggers rapid endocytosis and endosomal penetration, enabling it to deliver a toxic payload to the cell, resulting in cell death. We hypothesized that cytotoxic drugs delivered by HerPBK10 would induce significant targeted cell death in trastuzumab-resistant Her2+ breast cancers and will provide an effective treatment for patients who have developed these resistant tumors. We have demonstrated that HerPBK10 binds specifically to HER3 in vitro and binds to the cell surface of three different breast cancer cell lines. Importantly, this binding can be competitively inhibited by free HER3 ligand, indicating that HerPBK10 binds specifically to HER3. We also examined whether cells that have become resistant to trastuzumab have altered levels of HER receptors on their surface when compared to parental, trastuzumab-responsive, cancer cells. We demonstrated in multiple trastuzumab-resistant cell lines that HER3 receptor levels are significantly increased. We then assembled HerPBK10 with a cytotoxic gallium corrole, which we have previously demonstrated to be toxic once taken up into a cell. The resulting nanoparticle, called HerGa, was used to treat three different aggressive breast cancer cell lines (one that is susceptible to trastuzumab treatment and two that have become resistant to trastuzumab) and demonstrated that HerGa caused cell death in all three cell lines, but at a greater level and at a lower dosage in the resistant cell lines. We also compared the effect of the HerGa nanoparticle to trastuzumab and showed that it caused greater overall cell death. Together, these results indicate that our HER3-targeting nanoparticle, HerGa, efficiently targets and kills cancer cells that have become resistant to trastuzumab, and warrants further in vivo testing to investigate its potential as a treatment for patients who have become non-responsive to traditional anti-HER2 therapies. Citation Format: Jessica D. Sims, Michael Taguaim, Chris Hanson, Xiaojiang Cui, Lali K. Medina-Kauwe. Treating trastuzumab-resistant HER2+ breast cancers with a HER3-targeted nanoparticle. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A101.