Developing a tumor-targeted cell penetration protein
Studying the self-assembly, cell binding, and cell penetration activities of the proteins that comprise the outer shell of many viruses has guided the engineering of one such protein, the penton base, to target the delivery of therapeutic agents (including genes and drugs) to tumor cells in the absence of the rest of the virus. The penton base, which lies at each vertex of the twelve-sided outer shell of the adenovirus (an upper respiratory virus), mediates several important early steps in the viral infection mechanism. Most notably, the penton base facilitates penetration of the virus through the cell membrane, a major barrier to breach in order to deliver a payload to the cell interior.
|Cell binding and penetration pathway of Adenovirus.|
Dr. Medina-Kauwe first synthesized artificial penton base proteins for the delivery of nucleic acids to tumor cells as a non-viral gene therapy approach to cancer treatment. DNA is unable to breach the cell membrane without facilitation by carrier molecules that can disrupt the double-layer of lipids that comprise this cellular barrier. These synthetic penton base proteins can self-assemble with nucleic acid and recapitulate the early infection steps of the whole adenovirus. She has modified these proteins to specifically home in on and penetrate certain tumor cells. One such protein, HerPBK10, is designed to specifically target and penetrate HER2+ tumors, which display elevated levels of the cell surface protein, HER2. These highly aggressive tumors are resistant to standard forms of therapy, predict a poor prognosis, and comprise a significant subset of breast cancers, as well as ovarian, prostate, brain, and colon tumors.
The ability of HerPBK10 to target and penetrate HER2+ tumor cells is due to the receptor-ligand interaction that takes place at the cell surface upon cell binding. This interaction triggers rapid uptake of the protein into intracellular vesicles, through which HerPBK10 can penetrate via the penton base. Dr. Medina-Kauwe has assembled HerPBK10 with a variety of therapeutic agents for their delivery to the requisite intracellular targets. In contrast to signal-blocking molecules, HerPBK10-mediated delivery of therapeutic molecules circumvents signal inhibition as a means to modulate tumor growth by directly transporting drugs into the cell and killing tumors from within.
Until recently, there has been little appreciation for the different pathways by which molecules must traverse to breach the various cellular barriers to gain access to the cell interior. Understanding how pathogens accomplish this has aided the development of improved delivery vehicles in the Medina-Kauwe lab. To date, the lab has developed HerPBK10 to deliver chemotherapy, novel tumor-toxic imaging compounds, and therapeutic nucleic acids to HER2+ tumors. Meanwhile, the targeting ligand of HerPBK10 is being replaced by other ligands to target other types of tumors.
|Overview of Medina-Kauwe’s tumor-targeting technologies for imaging and treatment.|
HerDox: a chemotherapy smart-bomb
The Medina-Kauwe lab has developed a new and potentially improved method of systemically delivering the chemotherapy drug, doxorubicin, to tumors in a missile-like targeted fashion by exploiting the high efficiency cell targeting and cell penetration properties of HerPBK10. Taking advantage of certain features of the protein and the drug, doxorubicin can be encapsulated by the protein by spontaneous, self-assembly, without chemical modification of the protein or drug. The resulting nanoparticle, HerDox, bears the features of a tumor-targeted molecular missile for chemotherapy delivery.
Recent studies from the Medina-Kauwe lab show that intravenous delivery of HerDox in mice specifically targets and kills HER2+ tumors at over ten-times lower dose compared to untargeted doxorubicin. Moreover, HerDox spared the heart while doxorubicin caused notable damage to heart structure and function during therapeutic treatment. HerDox resisted premature drug release in the blood, yet released the drug after nanoparticle entry into tumor cells, much in the same way that viruses deliver and release their DNA payload during infection. Delivery of a long-established, FDA-approved chemotherapeutic may assist progress toward clinical trials.
|Summary of HerDox delivery, imaging, and therapeutic efficacy.|
HerGa: tumor imaging and tumor-killing in a single nanoparticle
In collaboration with chemists, Drs. Harry Gray and Zeev Gross, and imaging expert, Dr. Daniel Farkas, Dr. Medina-Kauwe has demonstrated that HerPBK10 can be combined with corrole molecules, forming nanoparticles capable of simultaneous tumor-detection and treatment. While HerPBK10 facilitates tumor targeting and cell membrane penetration, the attached corrole molecule enables detection and cytotoxicity. The Medina-Kauwe lab’s studies show that sulfonated, water-soluble corroles require delivery into the cell interior to induce tumor cell toxicity, and are incapable of penetrating through the cell membrane and entering into the cell without facilitation by a membrane-penetrating protein, such as HerPBK10. HerPBK10-mediated delivery of a water-soluble corrole containing a gallium (Ga) metal ion (resulting in the complex, HerGa) can emit an intense red fluorescence to track tumor-targeting while selectively killing HER2+ tumors. Tumor cell killing by HerGa can be further augmented by irradiation with light, which causes excitation of the corrole molecule and release of cell-damaging free radicals. The most recent preliminary studies in the Medina-Kauwe lab have indicated that HerGa can eliminate tumor cells that have become resistant to Herceptin®, suggesting a promising strategy for eradicating drug-resistant HER2+ tumors.
|Summary of HerGa assembly, therapeutic treatment, and imaging.|
To facilitate translation into the clinic, the Medina-Kauwe lab has attached HerPBK10 to a corrole containing manganese (Mn), which has proven to be more conducive for imaging using clinically relevant devices such as MRI. The resulting particle, HerMn, bears sufficient contrast for MRI while sustaining targeted-toxicity to HER2+ tumor cells in vivo.
H2PO and HerSi: targeted gene expression and gene silencing.
The Medina-Kauwe lab has tested HerPBK10 for delivering therapeutic genes that can encode toxic products, as well as for delivering small interfering RNA, or siRNA, to silence cancer growth genes. The former approach was accomplished by the particle, H2PO, formed by the attachment of condensed DNA to HerPBK10. Gene delivery requires entry into the cell nucleus for gene expression, thus necessitating an ability to penetrate several barriers within the tumor cell. Delivery of siRNA, on the other hand, presents fewer cellular obstacles to overcome, and has proven more conducive for nucleic acid delivery by non-viral means. The attachment of siRNA to HerPBK10 forms the particle, HerSi, which can launch a three-pronged attack on HER2+ tumors by combining missile-like targeting with siRNA-mediated gene silencing, and immune-mediated cell death. The latter effect could be accomplished by using siRNA molecules containing modifications that stimulate immune-mediated toxicity against recipient tumor cells. Delivery of a siRNA payload allows the flexibility to strategically silence certain pro-survival genes crucial to tumor growth and drug-resistance whose interference would result in catastrophic cell death when combined with the targeting strategy afforded by HerPBK10.
|Assembly of gene delivery complex, H2PO, and gene transfer to breast cancer cells in vitro.|
All of the described particles (HerDox, HerGa, HerSi, and H2PO) could be formed by self-assembly through the use of naturally occurring, spontaneous intermolecular interactions that similarly govern virus particle assembly. The advantage to self-assembly is that it allows drug potency to remain unaltered during assembly, transport, and release into target cells. This contrasts from strategies in which the drug is chemically linked to a carrier molecule, which can alter the activity and potency of both the drug and the carrier. While stability is a concern regarding self-assembled particles, HerDox, HerGa and HerSi have exhibited stability in storage and in the bloodstream. An additional potential concern is the use of a protein derived from a virus, which has the potential to elicit an adverse immune response. However, unlike many viruses, therapeutic levels of HerPBK10 have not elicited an immune reaction in mice.
This research has been funded by:
- National Institutes of Health
- National Cancer Institute
- CDMRP - Congressionally Directed Medical Research Programs
- American Cancer Society
- Susan G. Komen for the Cure