Elena V. Rosca

Anti-angiogenic peptides for cancer therapeutics

Peptides have emerged as important therapeutics that are being rigorously tested in angiogenesis-dependent diseases due to their low toxicity and high specificity. Since the discovery of endogenous proteins and protein fragments that inhibit microvessel formation (thrombospondin, endostatin) several peptides have shown promise in pre-clinical and clinical studies for cancer. Peptides have been derived from thrombospondin, collagens, chemokines, coagulation cascade proteins, growth factors, and other classes of proteins and target different receptors. Here we survey recent developments for anti-angiogenic peptides with length not exceeding 50 amino acid residues that have shown activity in pre-clinical models of cancer or have been tested in clinical trials; some of the peptides have been modified and optimized, e.g., through L-to-D and non-natural amino acid substitutions. We highlight technological advances in peptide discovery and optimization including computational and bioinformatics tools and novel experimental techniques.

Small peptides derived from somatotropin domain-containing proteins inhibit blood and lymphatic endothelial cell proliferation, migration, adhesion and tube formation

Angiogenesis is thoroughly balanced and regulated in health; however, it is dysregulated in many diseases including cancer, age-related macular degeneration, cardiovascular diseases such as coronary and peripheral artery diseases and stroke, abnormal embryonic development, and abnormal wound healing. In addition to angiogenesis, lymphangiogenesis is pivotal for maintaining the immune system, homeostasis of body fluids and lymphoid organs; dysregulated lymphangiogenesis may cause inflammatory diseases and lymph node mediated tumor metastasis. Anti-angiogenic or anti-lymphangiogenic small peptides may play an important role as therapeutic agents normalizing angiogenesis or lymphangiogenesis in disease conditions. Several novel endogenous peptides derived from proteins containing a conserved somatotropin domain have been previously identified with the help of our bioinformatics-based methodology. These somatotropin peptides were screened for inhibition of angiogenesis and lymphangiogenesis using in vitro proliferation, migration, adhesion and tube formation assays with blood and lymphatic endothelial cells. We found that the peptides have the potential for inhibiting both angiogenesis and lymphangiogenesis. Focusing the study on the inhibition of lymphangiogenesis, we found that a peptide derived from the somatotropin conserved domain of transmembrane protein 45A human was the most potent lymphangiogenesis inhibitor, blocking lymphatic endothelial cell migration, adhesion, and tube formation.

Synergy between a collagen IV mimetic peptide and a somatotropin-domain derived peptide as angiogenesis and lymphangiogenesis inhibitors

Angiogenesis is central to many physiological and pathological processes. Here we show two potent bioinformatically-identified peptides, one derived from collagen IV and translationally optimized, and one from a somatotropin domain-containing protein, synergize in angiogenesis and lymphangiogenesis assays including cell adhesion, migration and in vivo Matrigel plugs. Peptide-peptide combination therapies have recently been applied to diseases such as human immunodeficiency virus (HIV), but remain uncommon thus far in cancer, age-related macular degeneration and other angiogenesis-dependent diseases. Previous work from our group has shown that the collagen IV-derived peptide primarily binds β1 integrins, while the receptor for the somatotropin-derived peptide remains unknown. We investigate these peptides’ mechanisms of action and find both peptides affect the vascular endothelial growth factor (VEGF) pathway as well as focal adhesion kinase (FAK) by changes in phosphorylation level and total protein content. Blocking of FAK both through binding of β1 integrins and through inhibition of VEGFR2 accounts for the synergy we observe. Since resistance through activation of multiple signaling pathways is a central problem of anti-angiogenic therapies in diseases such as cancer, we suggest that peptide combinations such as these are an approach that should be considered as a means to sustain anti-angiogenic and anti-lymphangiogenic therapy and improve efficacy of treatment.

Novel peptide-specific quantitative structure-activity relationship (QSAR) analysis applied to collagen IV peptides with antiangiogenic activity

Angiogenesis is the growth of new blood vessels from existing vasculature. Excessive vascularization is associated with a number of diseases including cancer. Antiangiogenic therapies have the potential to stunt cancer progression. Peptides derived from type IV collagen are potent inhibitors of angiogenesis. We wanted to gain a better understanding of collagen IV structure-activity relationships using a ligand-based approach. We developed novel peptide-specific QSAR models to study the activity of the peptides in endothelial cell proliferation, migration, and adhesion inhibition assays. We found that the models produced quantitatively accurate predictions of activity and provided insight into collagen IV derived peptide structure-activity relationships.

Development of a biomimetic peptide derived from collagen IV with anti-angiogenic activity in breast cancer

Breast cancer is one of the most commonly diagnosed malignancies in women. Despite the remarkable success of mammography screening and use of adjuvant systemic therapy, it is estimated that approximately 200,000 new diagnoses will be made this year and 40,000 deaths will occur due to this disease (American Cancer Society). Angiogenesis, the growth of vessels from pre-existing microvasculature, is an essential component of tumor progression and has emerged as a therapeutic modality for anti-angiogenic therapies in cancer. Here we report in vitro and in vivo findings with a 20 amino acid peptide belonging to the collagen IV family, modified to facilitate possible translation to clinical applications. The two cysteines in its natural peptide progenitor were replaced by L-α-amino-n-butyric acid, a non-natural amino acid. The modified peptide was tested in vitro using endothelial cells and in vivo using mouse orthotopic breast cancer xenograft model with MDA-MB-231 human breast cancer cells. This modified peptide demonstrated no significant changes in activity from the parent peptide; however, because it lacks cysteines, it is more suitable for clinical translation. We also investigated its efficacy in combination with a commonly used chemotherapeutic agent paclitaxel; the inhibition of tumor growth by the peptide was similar to that of paclitaxel alone, but the combination did not exhibit any additional inhibition. We have performed further characterization of the mechanism of action (MOA) for this peptide to identify its target receptors, enhancing its translation potential as an anti-angiogenic, non-vascular endothelial growth factor (VEGF) targeting agent for therapy in breast cancer.

Collagen IV and CXC chemokine-derived antiangiogenic peptides suppress glioma xenograft growth.

Peptides are receiving increasing attention as therapeutic agents due to their high binding specificity and versatility to be modified as targeting or carrier molecules. Particularly, peptides with antiangiogenic activity are of high interest because of their applicability to a wide range of cancers. In this study, we investigate the biological activity of two novel antiangiogenic peptides in preclinical glioma models. One peptide SP2000 is derived from collagen IV and the other peptide SP3019 belongs to the CXC family. We have previously characterized the capacity of SP2000 and SP3019 to inhibit multiple biological endpoints linked to angiogenesis in human endothelial cells in several assays. Here, we report additional studies using endothelial cells and focus on the activity of these peptides against human glioma cell growth, migration and adhesion in vitro, and growth as tumor xenografts in vivo. We found that SP2000 completely inhibits migration of the glioma cells at 50 µmol/l and SP3019 produced 50% inhibition at 100 µmol/l. Their relative antiadhesion activities were similar, with SP2000 and SP3019 generating 50% adhesion inhibition at 4.9±0.82 and 21.3±5.92 µmol/l, respectively. In-vivo glioma growth inhibition was 63% for SP2000 and 76% for SP3019 after 2 weeks of administration at daily doses of 10 and 20 mg/kg, respectively. The direct activity of these peptides against glioma cells in conjunction with their antiangiogenic activities warrants their further development as either stand-alone agents or in combination with standard cytotoxic or emerging targeted therapies in malignant brain tumors.

Structure–Activity Relationship Study of Collagen-Derived Anti-Angiogenic Biomimetic Peptides

Structure–activity relationship (SAR) studies are essential in the generation of peptides with enhanced activity and efficacy as therapeutic agents. In this study, we report a Structure–activity relationship study for a family of mimetic peptides derived from type IV collagen with potent anti‐angiogenic properties. The Structure–activity relationship study was conducted using a number of validated in vitro assays including cell proliferation, adhesion, migration, and tubule formation. We report a critical sequence (NINNV) within this peptide series, which is required for the potent anti‐angiogenic activity. Detailed amino acid substitutions resulted in peptides with superior efficacy. Specifically, substitutions with isoleucine at positions 12 and 18 along with the substitution of the methionine at position 10 with the non‐natural amino acid d‐alanine led to an increase in potency by two orders of magnitude over the parent peptide. Several mimetic peptides in this series exhibit a significant improvement of activity over the parent peptide. This improved in vitro activity is expected to correlate with an increase in in vivo activity leading to effective peptides for anti‐angiogenic therapy for different disease applications including cancer and age‐related macular degeneration.

A biomimetic collagen derived peptide exhibits anti-angiogenic activity in triple negative breast cancer.

We investigated the application of a mimetic 20 amino acid peptide derived from type IV collagen for treatment of breast cancer. We showed that the peptide induced a decrease of proliferation, adhesion, and migration of endothelial and tumor cells in vitro. We also observed an inhibition of triple negative MDA-MB-231 xenograft growth by 75% relative to control when administered intraperitoneally for 27 days at 10 mg/kg. We monitored in vivo the changes in vascular properties throughout the treatment using MRI and found that the vascular volume and permeability surface area product decreased significantly. The treatment also resulted in an increase of caspase-3 activity and in a reduction of microvascular density. The multiple mode of action of this peptide, i.e., anti-angiogenic, and anti-tumorigenic, makes it a viable candidate as a therapeutic agent as a monotherapy or in combination with other compounds.

Abstract 4267: Biomimetic anti-angiogenic peptide as therapeutic agent for breast cancer

Despite the continuous research in novel therapeutics, globally each year more people succumb to cancer than HIV, TB and malaria combined. Angiogenesis, the development of neovasculature, has been a major focus of cancer research, and it resulted in the development, approval and clinical use of bevacizumab, an anti-VEGF monoclonal antibody. However, in the case of breast cancer, the results show gains in progression-free survival but not in overall survival. Other agents targeting VEGF receptors are under investigation with the premise that a cocktail of agents could be more effective. Recently, there has been great interest in the development of peptides as therapeutic agents due to their high specificity and low toxicity, which are conducive for the development of new therapeutic cocktails. Identification of pharmacophores (minimal information content) allows for the introduction of non-natural amino acids in the peptide sequence to increase its stability while maintaining or improving its activity, thus creating optimized peptides for clinical applications. We present the application of a 20-mer biomimetic peptide derived from the α5 fibril of collagen IV in which a cysteine is replaced by a non-natural amino acid, i.e., amino-butyric acid, and investigate its mechanism of action as an anti-angiogenic agent in treatment of breast cancer. We demonstrated its activity in suppressing tumor growth in an orthotopic triple negative breast tumor model. In vivo magnetic resonance imaging (MRI) demonstrated reduction in vascular permeability and vascular volume. In vitro studies of receptor pulldown and cellular surface proximity staining demonstrate that this peptide interacts specifically with the αVβ1 integrin. This biomimetic stable peptide is a novel non-VEGF targeting agent active as a single agent, or that could be used in combination with chemotherapy or other therapeutic peptide-based cocktails. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4267. doi:10.1158/1538-7445.AM2011-4267

Abstract 1365: Novel anti-angiogenic peptides as potential therapeutic agents in combination with chemotherapy for breast cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Breast cancer is one of the most commonly diagnosed malignancies in women. Despite the intensive ongoing research it is estimated that approximately 200,000 new diagnoses will be made in 2009 and 40,000 deaths will occur due to this disease. Angiogenesis, the development of vessels from pre-existing microvasculature, is an essential component of tumor progression and has emerged as a therapeutic target for anti-angiogenic therapies in cancer. The process is controlled by a balance between endogenous pro- and anti-angiogenic factors, and involves numerous interactions among several cell types including proliferation, invasion, and migration. Bevacizumab (Avastin, Roche/Genentech), an anti-VEGF monoclonal antibody, has been approved by the FDA for several types of cancer; however, in the case of breast cancer its effect on the overall survival is only marginal. Our laboratory has established a systematic bioinformatics approach to identify short novel anti-angiogenic peptides from natural proteins. These peptides belong to different classes (i.e. thrombospondin containing domain, collagen IV, chemokines etc) and in vitro experiments demonstrated their anti-angiogenic potential through inhibition of proliferation and migration of human endothelial cells. Moreover, several peptides have demonstrated efficacy in suppressing tumor growth when delivered as daily intraperitoneal injections in a mouse orthotopic breast cancer model. The repression was associated with a decrease in tumor microvasculature and an increase in necrotic core of the tumors (Koskimaki et al., 2009). The top performing candidate (a 24 amino acid peptide belonging to the collagen IV family) was further modified to find the optimal sequence for possible translation to clinical applications. Several truncations and substitutions of cystines by amino alpha-butyric acid were developed. The candidates were screened in in vitro proliferation assays and the top performer (full sequence length with substituted cystines) was tested in vivo using the orthotopic breast cancer model. This candidate demonstrated no significant changes in activity from the parent peptide, however making it is more conducive for clinical translation. We also investigate its application in combination with a typical chemotherapeutic agent (paclitaxel) and confirmed no decrease in activity in comparison to single agent therapy. We have performed further characterization of the mode of action for this peptide to identify its target receptors; this enhances the translation potential of this candidate as an anti-angiogenic, non-VEGF targeting agent for therapy in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1365.