Lilach Agemy

Coadministration of a Tumor-Penetrating Peptide Enhances the Efficacy of Cancer Drugs

Penetrating Attack on Tumors While considerable research effort in oncology is focused on the design of new cancer drugs, an important but relatively understudied research area is the development of methods that optimize the delivery and tumor penetration of existing cancer drugs. Previous work has characterized a peptide (iRGD) that selectively targets and penetrates tumor tissue by virtue of its specific interaction with tumor blood vessels. Now, studying mouse models, Sugahara et al. (p. 1031, see the cover) show that coinjection of the iRGD peptide increases the tumor penetration and antitumor activity of several cancer drugs, including the cytotoxic agent doxorubicin and the therapeutic antibody trastuzumab (Herceptin), without increasing their harmful effects on healthy tissue. Importantly, these effects did not require chemical conjugation of the cancer drugs to the peptide. Anticancer drugs are more effective in mice when they are injected with a peptide that helps the drugs penetrate the tumor. Poor penetration of anticancer drugs into tumors can be an important factor limiting their efficacy. We studied mouse tumor models to show that a previously characterized tumor-penetrating peptide, iRGD, increased vascular and tissue permeability in a tumor-specific and neuropilin-1–dependent manner, allowing coadministered drugs to penetrate into extravascular tumor tissue. Importantly, this effect did not require the drugs to be chemically conjugated to the peptide. Systemic injection with iRGD improved the therapeutic index of drugs of various compositions, including a small molecule (doxorubicin), nanoparticles (nab-paclitaxel and doxorubicin liposomes), and a monoclonal antibody (trastuzumab). Thus, coadministration of iRGD may be a valuable way to enhance the efficacy of anticancer drugs while reducing their side effects, a primary goal of cancer therapy research.

Tissue-penetrating delivery of compounds and nanoparticles into tumors

Poor penetration of drugs into tumors is a major obstacle in tumor treatment. We describe a strategy for peptide-mediated delivery of compounds deep into the tumor parenchyma that uses a tumor-homing peptide, iRGD (CRGDK/RGPD/EC). Intravenously injected compounds coupled to iRGD bound to tumor vessels and spread into the extravascular tumor parenchyma, whereas conventional RGD peptides only delivered the cargo to the blood vessels. iRGD homes to tumors through a three-step process: the RGD motif mediates binding to alphav integrins on tumor endothelium and a proteolytic cleavage then exposes a binding motif for neuropilin-1, which mediates penetration into tissue and cells. Conjugation to iRGD significantly improved the sensitivity of tumor-imaging agents and enhanced the activity of an antitumor drug.

Targeted nanoparticle enhanced proapoptotic peptide as potential therapy for glioblastoma

Antiangiogenic therapy can produce transient tumor regression in glioblastoma (GBM), but no prolongation in patient survival has been achieved. We have constructed a nanosystem targeted to tumor vasculature that incorporates three elements: (i) a tumor-homing peptide that specifically delivers its payload to the mitochondria of tumor endothelial cells and tumor cells, (ii) conjugation of this homing peptide with a proapoptotic peptide that acts on mitochondria, and (iii) multivalent presentation on iron oxide nanoparticles, which enhances the proapoptotic activity. The iron oxide component of the nanoparticles enabled imaging of GBM tumors in mice. Systemic treatment of GBM-bearing mice with the nanoparticles eradicated most tumors in one GBM mouse model and significantly delayed tumor development in another. Coinjecting the nanoparticles with a tumor-penetrating peptide further enhanced the therapeutic effect. Both models used have proven completely resistant to other therapies, suggesting clinical potential of our nanosystem.

Transtumoral targeting enabled by a novel neuropilin-binding peptide

We have recently described a class of peptides that improve drug delivery by increasing penetration of drugs into solid tumors. These peptides contain a C-terminal C-end Rule (CendR) sequence motif (R/K)XX(R/K), which is responsible for cell internalization and tissue-penetration activity. Tumor-specific CendR peptides contain both a tumor-homing motif and a cryptic CendR motif that is proteolytically unmasked in tumor tissue. A previously described cyclic tumor-homing peptide, LyP-1 (sequence: CGNKRTRGC), contains a CendR element and is capable of tissue penetration. We use here the truncated form of LyP-1, in which the CendR motif is exposed (CGNKRTR; tLyP-1), and show that both LyP-1 and tLyP-1 internalize into cells through the neuropilin-1-dependent CendR internalization pathway. Moreover, we show that neuropilin-2 also binds tLyP-1 and that this binding equally activates the CendR pathway. Fluorescein-labeled tLyP-1 peptide and tLyP-1-conjugated nanoparticles show robust and selective homing to tumors, penetrating from the blood vessels into the tumor parenchyma. The truncated peptide is more potent in this regard than the parent peptide LyP-1. tLyP-1 furthermore improves extravasation of a co-injected nanoparticle into the tumor tissue. These properties make tLyP-1 a promising tool for targeted delivery of therapeutic and diagnostic agents to breast cancers and perhaps other types of tumors.

Nanoparticle-induced vascular blockade in human prostate cancer

The tumor-homing pentapeptide CREKA (Cys-Arg-Glu-Lys-Ala) specifically homes to tumors by binding to fibrin and fibrin-associated clotted plasma proteins in tumor vessels. Previous results show that CREKA-coated superparamagnetic iron oxide particles can cause additional clotting in tumor vessels, which creates more binding sites for the peptide. We have used this self-amplifying homing system to develop theranostic nanoparticles that simultaneously serve as an imaging agent and inhibit tumor growth by obstructing tumor circulation through blood clotting. The CREKA nanoparticles were combined with nanoparticles coated with another tumor-homing peptide, CRKDKC, and nanoparticles with an elongated shape (nanoworms) were used for improved binding efficacy. The efficacy of the CREKA peptide was then increased by replacing some residues with nonproteinogenic counterparts, which increased the stability of the peptide in the circulation. Treatment of mice bearing orthotopic human prostate cancer tumors with the targeted nanoworms caused extensive clotting in tumor vessels, whereas no clotting was observed in the vessels of normal tissues. Optical and magnetic resonance imaging confirmed tumor-specific targeting of the nanoworms, and ultrasound imaging showed reduced blood flow in tumor vessels. Treatment of mice with prostate cancer with multiple doses of the nanoworms induced tumor necrosis and a highly significant reduction in tumor growth.

Specific penetration and accumulation of a homing peptide within atherosclerotic plaques of apolipoprotein E-deficient mice

The ability to selectively deliver compounds into atherosclerotic plaques would greatly benefit the detection and treatment of atherosclerotic disease. We describe such a delivery system based on a 9-amino acid cyclic peptide, LyP-1. LyP-1 was originally identified as a tumor-homing peptide that specifically recognizes tumor cells, tumor lymphatics, and tumor-associated macrophages. As the receptor for LyP-1, p32, is expressed in atherosclerotic plaques, we tested the ability of LyP-1 to home to plaques. Fluorescein-labeled LyP-1 was intravenously injected into apolipoprotein E (ApoE)-null mice that had been maintained on a high-fat diet to induce atherosclerosis. LyP-1 accumulated in the plaque interior, predominantly in macrophages. More than 60% of cells released from plaques were positive for LyP-1 fluorescence. Another plaque-homing peptide, CREKA, which binds to fibrin-fibronectin clots and accumulates at the surface of plaques, yielded fewer positive cells. Tissues that did not contain plaque yielded only traces of LyP-1+ cells. LyP-1 was capable of delivering intravenously injected nanoparticles to plaques; we observed abundant accumulation of LyP-1–coated superparamagnetic iron oxide nanoparticles in the plaque interior, whereas CREKA-nanoworms remained at the surface of the plaques. Intravenous injection of 4-[18F]fluorobenzoic acid ([18F]FBA)-conjugated LyP-1 showed a four- to sixfold increase in peak PET activity in aortas containing plaques (0.31% ID/g) compared with aortas from normal mice injected with [18F]FBA-LyP-1(0.08% ID/g, P < 0.01) or aortas from atherosclerotic ApoE mice injected with [18F]FBA-labeled control peptide (0.05% ID/g, P < 0.001). These results indicate that LyP-1 is a promising agent for the targeting of atherosclerotic lesions.

Proapoptotic Peptide-Mediated Cancer Therapy Targeted to Cell Surface p32

Antiangiogenic therapy is a promising new treatment modality for cancer, but it generally produces only transient tumor regression. We have previously devised a tumor-targeted nanosystem, in which a pentapeptide, CGKRK, delivers a proapoptotic peptide into the mitochondria of tumor blood vessel endothelial cells and tumor cells. The treatment was highly effective in glioblastoma mouse models completely refractory to other antiangiogenic treatments. Here, we identify p32/gC1qR/HABP, a mitochondrial protein that is also expressed at the cell surface of activated (angiogenic) endothelial cells and tumor cells, as a receptor for the CGKRK peptide. The results demonstrate the ability of p32 to cause internalization of a payload bound to p32 into the cytoplasm. We also show that nardilysin, a protease capable of cleaving CGKRK, plays a role in the internalization of a p32-bound payload. As p32 is overexpressed and surface displayed in breast cancers, we studied the efficacy of the nanosystem in this cancer. We show highly significant treatment results in an orthotopic model of breast cancer. The specificity of cell surface p32 for tumor-associated cells, its ability to carry payloads to mitochondria, and the efficacy of the system in important types of cancer make the nanosystem a promising candidate for further development.

Tumor-homing peptides as tools for targeted delivery of payloads to the placenta

Tumor-homing peptides have been exploited to create nanocarriers for targeted delivery of therapeutic agents to the placenta. The availability of therapeutics to treat pregnancy complications is severely lacking mainly because of the risk of causing harm to the fetus. As enhancement of placental growth and function can alleviate maternal symptoms and improve fetal growth in animal models, we have developed a method for targeted delivery of payloads to the placenta. We show that the tumor-homing peptide sequences CGKRK and iRGD bind selectively to the placental surface of humans and mice and do not interfere with normal development. Peptide-coated nanoparticles intravenously injected into pregnant mice accumulated within the mouse placenta, whereas control nanoparticles exhibited reduced binding and/or fetal transfer. We used targeted liposomes to efficiently deliver cargoes of carboxyfluorescein and insulin-like growth factor 2 to the mouse placenta; the latter significantly increased mean placental weight when administered to healthy animals and significantly improved fetal weight distribution in a well-characterized model of fetal growth restriction. These data provide proof of principle for targeted delivery of drugs to the placenta and provide a novel platform for the development of placenta-specific therapeutics.

Increasing Tumor Accessibility with Conjugatable Disulfide-Bridged Tumor-Penetrating Peptides for Cancer Diagnosis and Treatment

Tumor-homing peptides with tissue-penetrating properties increase the efficacy of targeted cancer therapy by delivering an anticancer agent to the tumor interior. LyP-1 (CGNKRTRGC) and iRGD (CRGDKGPDC) are founding members of this class of peptides. The presence of the cysteines forming the cyclizing disulfide bond complicates conjugation of these peptides with other molecules, such as drugs. Here, we report the synthesis of conjugatable disulfide-bridged peptides and their conjugation to biologically important molecules. We have synthesized the LyP-1, iRGD, and CRGDC (GACRGDCLGA) peptides with a cysteine or maleimidohexanoic acid added externally at N-terminus of the sequences. Subsequent conjugation to payloads yielded stable compounds in which the tumor-homing properties of the peptide and the biological activity of the payload were retained.

Abstract 5415: Targeted nanoparticle based therapy for the treatment of glioblastoma multiforme: Unprecedented anti-cancer activity in highly drug resistant/refractory mouse models of glioblastoma with novel apoptotic nanoparticles which hone to a cell-surface protein f

Glioblastoma multiforme (GBM) or “glioblastoma,” is the most common and aggressive malignant primary brain tumor in humans. An effective treatment that increases patient survival would represent a significant advance in the field, and provide a desperately needed new therapy for this deadly disease. Herein we disclose the identification of the target of the tumor homing peptide which, when conjugated to ‘nanoworms’, deliver and carry the pro-apoptotic nanoparticle system selectively to the tumor target tissue. This nanosystem has shown unprecedented anti-cancer activity in highly drug resistant/refractory mouse models of glioblastoma, which closely mimic the human disease and thus provide an exciting new opportunity for the treatment of GBM. Plans for further development of this Nanosystem will also be discussed. Citation Format: Sarah Hudson, Lilach Agemy, Venkata Ramana Kotamraju, Shweta Sharma, Erkki Ruoslahti, Florence Brunel, Ann MacLaren, John Muraski, Deborah Slee. Targeted nanoparticle based therapy for the treatment of glioblastoma multiforme: Unprecedented anti-cancer activity in highly drug resistant/refractory mouse models of glioblastoma with novel apoptotic nanoparticles which hone to a cell-surface protein f [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 5415. doi:10.1158/1538-7445.AM2014-5415