Targeting Of Tumor Neovasculature Research Articles

IRGD-targeted Physalis Mottle Virus-like Nanoparticles for Targeted Cancer Delivery.

Nanomedicine provides a promising platform for the molecular treatment of disease. An ongoing challenge in nanomedicine is the targeted delivery of intravenously administered nanoparticles to particular tissues, which is of special interest in cancer. In this study, we show that the conjugation of iRGD peptides, which specifically target tumor neovasculature, to the surface of Physalis mottle virus (PhMV)-like nanoparticles leads to rapid cellular uptake in vitro and tumor homing in vivo. We then show that iRGD-targeted PhMV loaded with the chemotherapeutic doxorubicin shows increased potency in a murine flank xenograft model of cancer. Our results validate that PhMV-like nanoparticles can be targeted to tumors through iRGD-peptide conjugation and suggest that iRGD-PhMV provides a promising platform for the targeted delivery of molecular cargo to tumors.

Palmitoyl Carnitine-Anchored Nanoliposomes for Neovasculature-Specific Delivery of Gemcitabine Elaidate to Treat Pancreatic Cancer

Being the fourth most fatal malignancy worldwide, pancreatic cancer is on track to become the second leading cause of cancer-related deaths in the United States by 2030. Gemcitabine is a first-line chemotherapeutic agent for pancreatic ductal adenocarcinoma (PDAC). Gemcitabine Elaidate (Gem Elaidate) is a lipophilic derivative which allows hENT1-independent intracellular delivery of gemcitabine and better pharmacokinetics and entrapment in a nanocarrier. Cancer cells and neovasculature are negatively charged compared to healthy cells. Palmitoyl-DL-carnitine chloride (PC) is a Protein kinase C (PKC) inhibitor which also provides a cationic surface charge to nanoliposomes for targeting tumor neovasculature and augmented anticancer potency. The objectives of our study are: (a) to develop and characterize a PKC inhibitor-anchored Gem Elaidate-loaded PEGylated nanoliposome (PGPLs) and (b) to investigate the anticancer activity of Gem Elaidate and PGPLs in 2D and 3D models of pancreatic cancer. The optimized PGPLs resulted in a particle size of 80 ± 2.31 nm, a polydispersity index of 0.15 ± 0.05 and a ζ-potential of +31.6 ± 3.54 mV, with a 93.25% encapsulation efficiency of Gem Elaidate in PGPLs. Our results demonstrate higher cellular uptake, inhibition in migration, as well as angiogenesis potential and significant apoptosis induced by PGPLs in 3D multicellular tumor spheroids of pancreatic cancer cells. Hence, PGPLs could be an effective and novel nanoformulation for the neovasculature-specific delivery of Gemcitabine Elaidate to treat PDAC.

Molecular dynamics, MMGBSA, and docking studies of natural products conjugated to tumor-targeted peptide for targeting BRAF V600E and MERTK receptors.

Recent studies have revealed that MERTK and BRAF V600E receptors have been found to be over-expressed in several types of cancers including melanoma, making these receptors targets for drug design. In this study, we have designed novel peptide conjugates with the natural products vanillic acid, thiazole-2-carboxylic acid, cinnamic acid, theanine, and protocatechuic acid. Each of these compounds was conjugated with the tumor targeting peptide sequence TAASGVRSMH, known to bind to NG2 and target tumor neovasculature. We examined their binding affinities and stability with MERTK and BRAF V600E receptors using molecular docking and molecular dynamics studies. Compared to the neat compounds, the peptide conjugates displayed higher binding affinity toward both receptors. In the case of MERTK, the most stable complexes were formed with di-theaninate-peptide, vanillate-peptide, and thiazole-2-amido peptide conjugates and binding occurred in the hinge region. Additionally, it was discovered that the peptide alone also had high binding ability and stability with the MERTK receptor. In the case of BRAF V600E, the peptide conjugates of protocatechuate, vanillate and thiazole-2-amido peptide conjugates showed the formation of the most stable complexes and binding occurred in the ATP binding cleft. Further analysis revealed that the number of hydrogen bonds and hydrophobic interactions played a critical role in enhanced stability of the complexes. Docking studies also revealed that binding affinities for NG2 were similar to MERTK and higher for BRAF V600E. MMGBSA studies of the trajectories revealed that the protocatechuate-peptide conjugate showed the highest binding energy with BRAF V600E while the peptide-TAASGVRSMH showed the highest binding energy with MERTK. ADME studies revealed that each of the compounds showed medium to high permeability toward MDCK cells and were not hERG blockers. Furthermore, the conjugates were not CYP inhibitors or substrates, but they were found to be Pgp substrates. Our results indicated that the protocatechuate-TAASGVRSMH, thiazole-2-amido-TAASGVRSMH, and vanillate-TAASGVRSMH conjugates may be furthered developed for in vitro and in vivo studies as novel tumor targeting compounds for tumor cells over-expressing BRAF V600E, while di-theaninate-amido-TAASGVRSMH and thiazole-2-amido-TAASGVRSMH conjugates may be developed for targeting MERTK receptors. These studies provide insight into the molecular interactions of natural product-peptide conjugates and their potential for binding to and targeting MERTK and BRAF V600E receptors in developing new therapeutics for targeting cancer.

EXTH-32. DEVELOPMENT OF EPHA3 DIRECTED CHIMERIC ANTIGEN RECEPTOR T CELL THERAPY FOR THE TREATMENT OF GLIOBLASTOMA MULTIFORME

Abstract BACKGROUND Efficacy of chimeric antigen receptor T cell (CART) therapy remains limited in solid tumors. Given the heterogeneity of surface receptor expression and immunosuppressive stromal microenvironment, strategies to target tumor neovasculature and tumor stromal cells are needed to help overcome CART inhibition by GBM. Eph receptors are the largest family of receptor tyrosine kinases and are integral to cell adhesion, migration, and axon guidance, during development and homeostasis. EphA3 is a receptor tyrosine kinase which is lowly expressed in adult tissues but is highly expressed in tumor neovasculature and tumor stromal cells in GBM and other solid tumors. EphA3 is over-expressed in up to 40% of GBM samples. We aimed to develop CART cells directed against EphA3 to use in targeting tumor neovasculature and tumor stromal cells in GBM. METHODS we developed a second generation CD28 co-stimulated CAR construct in a third generation lentivirus backbone to generate EphA3 CART cells using the single chain variable fragment of ifabotuzumab, a monoclonal antibody directed against EphA3. Patient derived GBM xenograft cell lines were used in these experiments. RESULTS EphA3 directed CART cells exhibited specific and potent antitumor activity against EphA3+ GBM cell lines with variable transcriptome EphA3 expression indicating its broader applicability in patients with GBM. Killing over 24-hour incubation was significant at low effector: target ratio: 52.5% killing at 1.25:1 against cell lines with 25.05% EphA3 expression and 37.1% killing at 1.25:1and 90% killing at 5:1 ratio against a cell line with 19.28% expression. Conversely, when co-cultured with UTD controls there was significantly lower killing, or growth of tumor cells. CONCLUSION We demonstrate for the first time that targeting EphA3 with CART cells is feasible, efficacious and represents a novel therapeutics strategy to target GBM. Data from in vivo and combinatorial CART experiments will be reported at the meeting.

Abstract 2396: Development of a new generation of dimeric, highly cytotoxic fusion proteins containing active GrB and VEGF targeting tumor neovasculature: Incorporating IgG heavy chain Fc fragments improves cytotoxicity, stability and pharmacokinetics

Abstract VEGF121 is a naturally-occurring splice variant that binds to VEGF receptors R-1 and R-2, which are over-expressed on the endothelium of tumor vasculature but not normal vasculature. The serine protease granzyme B (GrB) is capable of inducing intense cellular apoptosis through both caspase-dependent and caspase-independent multiple-cascade mechanisms. We have previously reported the expression of a fusion protein composed of Granzyme B and VEGF121 (GrB/VEGF121) and its subsequent in vitro and in vivo characterization. Here we report on the development and characterization of GrB-Fc-VEGF, which fuses the two domains via an IgG heavy chain Fc-fragment. We hypothesized that this modification, resulting in a relatively high molecular weight (133 kDa vs 80 kDa) would result in improved in vivo circulation and efficacy. VEGF inter-chain dimerization domains were modified such that dimerization occurred only through the Fc domain. GrB-Fc-VEGF121was expressed in HEK-293E cells under serum-free conditions and purified from the media by immobilized metal affinity chromatography (25 mg/L). Western blotting confirmed incorporation of both VEGF121 and GrB moieties into the construct. Comparison of GrB-Fc-VEGF121 with its expected molecular weight suggests significant glycosylation when produced in HEK293E cells. The enzymatic activity of GrB in GrB-Fc-VEGF121 was comparable to that of human GrB. In vitro studies of GrB-Fc-VEGF121 suggests improved cytotoxicity against cell lines expressing high levels of VEGFR-1 or VEGFR-2, unlike GrB/VEGF121, which targeted VEGFR-2+ cells more efficiently. Thus, the GrB-Fc-VEGF121 produced in HEK293E cells is active and has significant potential as a targeted therapeutic. In vitro cytotoxicity against an expanded panel of cells, internalization, and in vivo pharmacokinetics and efficacy against various tumor models are currently ongoing and will be presented. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Ana Alvarez-Cienfuegos, Michael G. Rosenblum. Development of a new generation of dimeric, highly cytotoxic fusion proteins containing active GrB and VEGF targeting tumor neovasculature: Incorporating IgG heavy chain Fc fragments improves cytotoxicity, stability and pharmacokinetics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2396.

MPEG-g-CS-Modified PLGA Nanoparticle Carrier for the Codelivery of Paclitaxel and Epirubicin for Breast Cancer Synergistic Therapy.

The aim of this study was to investigate a novel double-layer core-shell structure nanoparticle (NP) delivery system, comprising a nanoscale self-assembly mPEG-g-CS envelope coating on a nuclear PLGA NPs. We then constructed the core-shell NPs for paclitaxel (PTX) loading in the outer envelope to target tumor neovasculature and epirubicin (EPI) loading in the core to antitumor cells. We characterized the NPs for physical/chemical properties, controlled drug release kinetics, and finally evaluated in vitro and in vivo antitumor activities. It could be seen that these three blank NPs (core-shell NPs, PLGA NPs, and mPEG-g-CS NPs) had no significant influence on the cell viability of human breast adenocarcinoma cell line (MCF-7) and human umbilical vein endothelial cells (HUVEC) in a NPs concentration range of 0.5-1000 μg/mL for 24 and 48 h, thus it can be inferred that the NPs constructed by mPEG-g-CS and PLGA polymers have good biocompatibility for further application. The confocal images and flow cytometry results showed that the core-shell NPs were efficiently internalized by these cells. After intravenous injection to nude mice bearing MCF-7 breast tumor, core-shell NPs achieved enhanced antitumor and antiangiogenic effects, prolonged retention, increased distribution, and significantly reduced microvessel density (MVD) in tumor tissues compared to both PLGA NPs and mPEG-g-CS NPs. In conclusion, this novel NPs system represent a new way of combining traditional combination chemotherapy and antiangiogenesis therapy for controlled drug delivery applications, and appeared to be a promising combination therapy to treat breast cancer.

Tumor Neovasculature-Targeted APRPG-PEG-PDLLA/MPEG-PDLLA Mixed Micelle Loading Combretastatin A-4 for Breast Cancer Therapy.

Breast cancer has been the first killer among women. In this study, combretastatin A-4 (CA-4) loaded 5-amino acid peptide Ala-Pro-Arg-Pro-Gly (APRPG) modified PEG-PDLLA mixed micelles was developed to target tumor neovasculature for breast cancer therapy. CA-4 is an effective vascular disrupting agent. The APRPG-modified PEG-PDLLA polymer was successfully synthesized and thin-film hydration method was used to prepare APRPG-PEG-PDLLA/MPEG-PDLLA mixed micelles. Drug loading capacity (DL), encapsulation efficiency (EE), and the optimized ratio of APRPG-PEG-PDLLA: MPEG-PDLLA for efficient drug loading was investigated. The particle size, zeta potential, morphology, and the crystallographic study were carried out to characterize the micelles. In vitro release study revealed a sustained release of CA-4 from the mixed micelles while compared to free CA-4. Moreover, the cytotoxicity data of blank and drug loaded mixed micelles suggested that the APRPG-PEG-PDLLA/MPEG-PDLLA mixed micelles were safe drug carriers and the encapsulated CA-4 remained potent antitumor effect. The cellular uptake study and the in vivo imaging and biodistribution study demonstrated that the APRPG peptide modified mixed micelles has the higher cellular uptake efficiency and could significantly facilitate the accumulation at tumor site. Furthermore, the micelles were slowly extravasated from blood vessels and inhibited embryonic angiogenesis in the transgenic zebrafish model. Consequently, the CA-4 loaded APRPG-PEG-PDLLA/MPEG-PDLLA mixed micelles group demonstrated a significant inhibition of tumor growth in 4T1 breast cancer model. In short, the CA-4 loaded APRPG-PEG-PDLLA/MPEG-PDLLA mixed micelles might have great potential for breast cancer therapy.

Targeting of Tumor Neovasculature with GrB/VEGF121, a Novel Cytotoxic Fusion Protein.

Angiogenesis is a critical process in numerous diseases, and intervention in neovascularization has therapeutic value in several disease settings, including ocular diseases, arthritis, and in tumor progression and metastatic spread. Various vascular targeting agents have been developed, including those that inhibit growth factor receptor tyrosine kinases, blocking antibodies that interfere with receptor signal transduction, and strategies that trap growth factor ligands. Limited anti-tumor efficacy studies have suggested that the targeted delivery of the human pro-apoptotic molecule Granzyme B to tumor cells has significant potential for cancer treatment. Here, we review biological vascular targeting agents, and describe a unique vascular targeting agent composed of Granzyme B and the VEGF receptor ligand VEGF121. The fusion protein GrB/VEGF121 demonstrates cytotoxicity at nanomolar or sub-nanomolar levels, excellent pharmacokinetic and efficacy profiles, and has significant therapeutic potential targeting tumor vasculature.

Abstract 1203: Targeting activated tumor vasculature with human Granzyme B variants engineered for improved stability and activity

Abstract Angiogenesis is a critical process in numerous diseases, and targeting tumor neovasculature has therapeutic value in controlling tumor progression and metastatic spread. Vascular endothelial growth factor-A (VEGF-A) and its receptors VEGFR-1 and VEGFR-2 have been implicated as central mediators of normal angiogenesis and tumor neovascularization. VEGF121 is a naturally-occurring VEGF-A splice variant that binds to these receptors, which are over-expressed on the endothelium of activated tumor vasculature or neovasculature but not normal vasculature. Granzyme B (GrB) plays a critical role in the body's defense against viral infection and tumor development by initiating the apoptotic cascade through both caspase-dependent and -independent mechanisms. The fusion protein GrB/VEGF121 expressed in mammalian cells and purified to homogeniety was lethal to tumor and endothelial cells in vitro in a manner that correlated closely to total VEGFR-2 expression. IC50 levels were found to be in the nanomolar range. GrB/VEGF121 internalized rapidly into VEGFR-2 expressing cells while the internalization into VEGFR-1 expressing cells was significantly reduced. We engineered GrB variants for improved protein production and for resistance to serpin B9 (PI-9), a natural inhibitor of Granzyme B found at varying levels in cells and plasma, and compared these GrB/VEGF121 constructs directly to unmodified GrB/VEGF121. Variants took into account the impact of the mutation on substrate specificity, and the ability of the mutated GrB to cleave its various cellular substrates. As expected, mutation of the active site 195serine to alanine (S195A) resulted in a complete loss of GrB enzymatic activity as well as in vitro cytotoxicity against VEGFR-1+ and VEGFR-2+ cells. Enzymatic activity in PBS of the K27E, R28A (“EA-GV”) double mutant was identical to that of GrB/VEGF121. In 50% human serum, however, the enzymatic activity of GrB/VEGF121 steadily declined to less than 10% over 24 hours while the enzymatic activity of EA-GV remained over 40% during that time period. This construct retained higher cytotoxic activity after pre-incubation in serum for 4h, compared to GrB/VEGF121. C-GrB/VEGF121, without an unpaired cysteine, resulted in reduced aggregation and a vastly improved yield with otherwise similar serum stability and in vitro cytotoxicity as unmodified GrB/VEGF121. The pharmacokinetic profile for C-GrB/VEGF121 indicated a t1/2α of 6.6 min and t1/2β of 33.3 h. A limited profile for EA-GV indicates a longer terminal-phase half-life for this construct compared to C-GrB/VEGF121. Our studies suggest that these novel GrB variants may significantly improve in vivo half-life. Further studies are underway to determine whether these modifications impact production of soluble material and in vivo therapeutic efficacy against established tumor xenograft models. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Michael G. Rosenblum. Targeting activated tumor vasculature with human Granzyme B variants engineered for improved stability and activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1203.

Fosbretabulin tromethamine in the treatment of thyroid cancer

ABSTRACTIntroduction: Anaplastic thyroid carcinoma [ATC] is a rare, highly aggressive neoplasm with a disease-specific mortality of nearly 100%. Available treatment options include surgery, radiotherapy, chemotherapy or their combination and, more often recently, participation in clinical trials, as recommended by all available guidelines. However, despite of many attempts with the use of new targeted drugs, to date no remarkable progress in the improvement in patients’ survival has been made. Fosbretabulin (combretastatin A4 phosphate, CA4P) is a vascular disrupting agent, and a new drug that targets tumor neovasculature leading to an acute, reversible reduction in tumor blood flow and tumor central necrosis. Its activity against ATC was evaluated in the FACT trial.Areas covered: Published results of preclinical, phase I and II studies, and other reports evaluating the mechanism of action, efficacy and safety of fosbretabulin alone or combination with other drugs in ATC are evaluated.Expert Opinion: Disappointing results of previous randomized studies, referred to ATC treatment, force us to draw conclusions. Among the reasons leading to the failure of FACT study was an insufficient number of enrolled patients. Since available guidelines emphasized the necessity to treat ATC under clinical trials, inclusion criteria should be reconsidered to make the recruitment process much easier and faster in future.

Use of the Hollow Fiber Assay to Evaluate Agents That Target the Tumor Neovasculature.

In vivo preclinical assays are required to screen potential agents that target the tumor vasculature. Here a hollow fiber-based assay for the quantification of neovasculature in the presence or absence of an agent that potentially targets tumor neovasculature is described. The neovasculature is developed as a consequence of the presence of tumor cells encapsulated in hollow fibers, which are transplanted sub-cutaneously in the dorsal flanks of mice.

Abstract CT413: Lutetium-177-labeled anti-prostate-specific membrane antigen (PSMA) monoclonal antibody J591 (177Lu-J591) for metastatic non-prostate solid tumors

Abstract Introduction: PSMA is a non-secreted cell-surface protein that is expressed by the neovascular endothelium of most solid tumors (but not by vascular endothelium of normal tissue), making it a candidate for vascular-targeted therapy. The purpose of this study was to define the change in tumor perfusion and cellularity after 177Lu-J591 therapy in patients (pts) with advanced non-prostate solid tumors and PSMA expression as determined by radionuclide imaging (with 111In-J591) and/or by immunohistochemistry (IHC). Of note, radiolabeled J591 is hepatically cleared (negating accurate assessment of liver metastases by J591 imaging). Methods: In this pilot study, following confirmation of PSMA expression by 111In-J591 imaging and/or IHC of tumor neovasculature), 14 pts in 2 cohorts received 177Lu-J591 therapy (7 received a single dose at 70 mCi/m2; 7 received 2 fractionated doses at 35 mCi/m2 each). Pts underwent dynamic contrast enhanced (DCE)-MRI and diffusion weighted imaging (DWI) at baseline, then repeated at 1, 4, 8, and 12 wks post treatment to evaluate tumor perfusion (DCE-MRI) and cellularity (Apparent Diffusion Coefficient (ADC) values from DWI). Values were compared with paired t-testing using Wilcoxon signed-rank test for significance. Results: Among the 14 treated pts, tumor types included: lung (5), bladder (2), pancreas (2), colon, GE junction, renal, ovarian carcinomas and melanoma (1 each). Median age was 61.5 with a median of 3 prior therapies including 5 with prior anti-angiogenic agents; 8 pts received prior radiation. Metastatic sites included: bone, lymph nodes, liver, lung, brain, other. 11 of 14 pts had PSMA expression confirmed by imaging, 3 additional by IHC of archival tumor tissue (with liver metastases). DCE-MRI demonstrated that treatment significantly affected tumor perfusion, with decreased perfusion at the 1-wk (n=11, p=0.03) and 4-wk (n=6, p=0.01) post 177Lu-J591 time points. Compared to baseline, DCE slope was lower than baseline at 1 wk (p=0.045) and 4-wk (p=0.02). Week 8 (n=4) was non-significant for both % change and DCE slope and only 2 pts completed the wk 12 time point. In evaluation of cellularity, 5 of 9 pts had an increase in ADC at either the 1- or 4-wk time point (1 stable, 3 decreased). However, paired comparisons at individual time points were non-significant. Conclusion: Anti-PSMA radioimmunotherapy targeting tumor neovasculature is feasible using 177Lu-J591 and can alter tumor perfusion and cellularity, including in those with prior exposure to anti-angiogenic therapy and/or radiation. Refinement in patient selection with improved imaging (anti-PSMA immuno-PET) is ongoing. Citation Format: Orrin Pail, Gurveen Kaur, Jonathan Dyke, Yuliya Jhanwar, Paul Christos, Allyson Ocean, Manish Shah, Tsiporah Shore, Bryan Schneider, Ronald Scheff, Himisha Beltran, Douglas Ballon, Brian Robinson, David M. Nanus, Neil H. Bander, Scott T. Tagawa. Lutetium-177-labeled anti-prostate-specific membrane antigen (PSMA) monoclonal antibody J591 (177Lu-J591) for metastatic non-prostate solid tumors. [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 CT413. doi:10.1158/1538-7445.AM2014-CT413

OP0108 The Antibody-Based Delivery of IL4 to the Neo-Vasculature Cures Mice with Arthritis

BackgroundAntibody-cytokine fusion proteins (“immunocytokines”) are innovative biopharmaceutical agents, which are being considered for the therapy of cancer and chronic inflammatory conditions. Immunomodulatory fusion proteins capable of selective localization at the...

Abstract 1767: Targeting prostate cancer osteoblastic progression with VEGF121/rGel, a single agent targeting osteoblasts, osteoclasts, and tumor neovasculature

Abstract Purpose: A hallmark of prostate cancer (PCa) progression is the development of osteoblastic bone metastases, which respond poorly to available therapies. We previously reported that VEGF121/rGel targets osteoclast precursors and tumor neovasculature. Here we tested the hypothesis that targeting non-tumor cells expressing the receptors for VEGF121, namely VEGFR-1 and VEGFR-2, can inhibit tumor progression in a clinically relevant model of osteoblastic PCa. Experimental Design: We examined the effect of VEGF121/rGel on osteoblast precursors and several PCa cell lines in vitro; on osteoid formation in a mouse calvaria culture assay ex vivo; and on PCa osteoblastic progression in the femurs of mice injected with MDA PCa 118b, a PCa xenograft obtained from a bone metastasis in a patient with castrate-resistant PCa. Results: VEGF121/rGel was cytotoxic in vitro to osteoblast precursor cells. This cytotoxicity was specific as VEGF121/rGel internalization into osteoblasts was VEGF121 receptor driven. Furthermore, VEGF121/rGel significantly inhibited PCa-induced bone formation in a mouse calvaria culture assay. In vivo, systemic administration of VEGF121/rGel significantly inhibited the osteoblastic progression of PCa cells in the femurs of nude mice. Microcomputed tomography analysis revealed that VEGF121/rGel restored the bone volume fraction of tumor-bearing femurs to values similar to those of the contralateral (non-tumor bearing) femurs. VEGF121/rGel significantly reduced the number of tumor-associated osteoclasts but did not change the numbers of peritumoral osteoblasts. Importantly, VEGF121/rGel-treated mice had significantly less tumor burden than control mice did. Our results thus indicate that VEGF121/rGel inhibits osteoblastic tumor progression by targeting angiogenesis, osteoclastogenesis, and bone formation. Conclusions: Targeting VEGFR-1- or VEGFR-2-expressing cells is effective in controlling the osteoblastic progression of PCa in bone. These findings provide the basis for an effective multitargeted approach for metastatic PCa. We believe that VEGF121/rGel in combination with tumor cell-targeting therapies (e.g., chemotherapy) constitutes a novel strategy for advanced PCa. Research conducted, in part, by the Clayton Foundation for Research. 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 1767. doi:10.1158/1538-7445.AM2011-1767

Inhibition of Prostate Cancer Osteoblastic Progression with VEGF121/rGel, a Single Agent Targeting Osteoblasts, Osteoclasts, and Tumor Neovasculature

A hallmark of prostate cancer (PCa) progression is the development of osteoblastic bone metastases, which respond poorly to available therapies. We previously reported that VEGF(121)/rGel targets osteoclast precursors and tumor neovasculature. Here we tested the hypothesis that targeting nontumor cells expressing these receptors can inhibit tumor progression in a clinically relevant model of osteoblastic PCa. Cells from MDA PCa 118b, a PCa xenograft obtained from a bone metastasis in a patient with castrate-resistant PCa, were injected into the femurs of mice. Osteoblastic progression was monitored following systemic administration of VEGF(121)/rGel. VEGF(121)/rGel was cytotoxic in vitro to osteoblast precursor cells. This cytotoxicity was specific as VEGF(121)/rGel internalization into osteoblasts was VEGF(121) receptor driven. Furthermore, VEGF(121)/rGel significantly inhibited PCa-induced bone formation in a mouse calvaria culture assay. In vivo, VEGF(121)/rGel significantly inhibited the osteoblastic progression of PCa cells in the femurs of nude mice. Microcomputed tomographic analysis revealed that VEGF(121)/rGel restored the bone volume fraction of tumor-bearing femurs to values similar to those of the contralateral (non-tumor-bearing) femurs. VEGF(121)/rGel significantly reduced the number of tumor-associated osteoclasts but did not change the numbers of peritumoral osteoblasts. Importantly, VEGF(121)/rGel-treated mice had significantly less tumor burden than control mice. Our results thus indicate that VEGF(121)/rGel inhibits osteoblastic tumor progression by targeting angiogenesis, osteoclastogenesis, and bone formation. Targeting VEGF receptor (VEGFR)-1- or VEGFR-2-expressing cells is effective in controlling the osteoblastic progression of PCa in bone. These findings provide the basis for an effective multitargeted approach for metastatic PCa.

Specific Induction of Tumor Neovasculature Death by Modified Murine PPE-1 Promoter Armed with HSV-TK

Background: One strategy to increase tissue specificity of gene therapy is to use promoters or enhancers. Objectives: (1) To enhance the selectivity of a murine preproendothelin-1 (PPE-1) promoter in tumor angiogenesis by using a positive endothelial transcription-binding element. (2) To test the specificity and efficiency of the modified PPE-1 promoter [PPE-1(3X)] in vitro and in vivo by using reporter genes, and the therapeutic gene herpes simplex virus-thymidine kinase (HSV-TK) in a mouse model of Lewis lung carcinoma (LLC). Results: The modified PPE-1 promoter specifically induced expression in the tumor angiogenic vascular bed with a 35-fold higher expression compared to the normal vasculare bed of the lung. Thus, when the HSV-TK gene controlled by the modified PPE-1 promoter was used systemically, it induced tumor-specific necrosis, apoptosis and mononuclear infiltrates, leading to massive destruction of the neovasculature of the pulmonary metastasis, which suppressed metastasis development. Conclusions: These results show that an adenoviral vector armed with HSV-TK controlled by the endothelial-selective murine PPE-1(3X) promoter is efficient and safe to target tumor neovasculature.

Peptide-Mediated Targeting to Tumor Blood Vessels of Lung Cancer for Drug Delivery

Antiangiogenesis therapies for the treatment of cancers hold the promise of high efficacy and low toxicity. In vivo phage display was used to identify peptides specifically targeting tumor blood vessels. The peptide SP5-52 recognized tumor neovasculature but not normal blood vessels in severe combined immunodeficiency mice bearing human tumors. Synthetic peptide was shown to inhibit the binding of PC5-52 phage particles to the tumor mass in the competitive inhibition assay. Several selected phage clones displayed the consensus motif, proline-serine-proline, and this motif was crucial for peptide binding to the tumor neovasculature. SP5-52 peptides also bound vascular endothelial growth factor-stimulated human umbilical vein endothelial cells and blood vessels of human lung cancer surgical specimens. Furthermore, this targeting phage was shown to home to tumor tissues from eight different types of human tumor xenografts following in vivo phage display experiments. An SP5-52 peptide-linked liposome carrying doxorubicin enhanced the therapeutic efficacy of the drug, markedly decreased tumor blood vessels, and resulted in higher survival rates of human lung and oral cancer-bearing xenograft mice. The current study indicates that ligand-targeted therapy offers improved therapeutic effects over conventional anticancer drug therapy, and that the peptide SP5-52 specifically targets tumor neovasculature and is a good candidate for targeted drug delivery to solid tumors.

Tumor endothelial marker 8 enhances tumor immunity in conjunction with immunization against differentiation Ag

We have previously shown that xenogeneic DNA vaccines encoding rat neu and melanosomal differentiation Ag induce tumor immunity. Others have developed vaccines targeting tumor neovasculature. Tumor endothelial marker 8 (TEM8) is expressed in the neovasculature of human tumors, and in the mouse melanoma B16, but its expression is limited in normal adult tissues. We describe a DNA vaccine combining xenogeneic tumor Ag and TEM8. In-situ hybridization was used to detect TEM8 RNA in mouse tumors. Mice transgenic for the rat neu proto-oncogene were immunized with DNA vaccines encoding TEM8 and the extracellular domain of rat neu and challenged with the 233-VSGA1 breast cancer cell line. In parallel experiments, C57BL/6 mice were immunized with TEM8 and human tyrosinase-related protein 1 (hTYRP1/hgp75) and challenged with B16F10 melanoma. TEM8 was expressed in the stroma of transplantable mouse breast and melanoma tumors. In both model systems, TEM8 DNA had no activity as a single agent but significantly enhanced the anti-tumor immunity of neu and hTYRP1/hgp75 DNA vaccines when given in concert. The observed synergy was dependent upon CD8+ T cells, as depletion of this cell population just prior to tumor challenge obviated the effect of the TEM8 vaccine in both tumor models. A local immune response to TEM8 may increase inflammation or tumor necrosis within the tumor, resulting in improved Ag presentation of HER2/neu and hTYRP1/hgp75. Alternatively, TEM8 expression in host APC may alter T-cell interactions or homing. In this way, TEM8 may act more as an adjuvant than an immunologic target.

Polymer-peptide conjugates for angiogenesis targeted tumor radiotherapy

Introduction New methods of delivering radiotherapy to sites of occult or disseminated cancer are needed to control the disease and address the failure of conventional therapy. Because tumor cells rely on angiogenesis for survival, we assessed the effectiveness of β-emitter radiotherapy delivered by polymer-peptide conjugates that target tumor neovasculature. This molecularly targeted radiation is intended to damage both the endothelial bed and surrounding neoplastic cells. Methods N-(2-Hydroxypropyl) methacrylamide (HPMA), a biocompatible and water-soluble copolymer, was derivatized to incorporate side chains for 99mTc and 90Y chelation and was further conjugated to a α Vβ 3 integrin-targeting peptide (RGD4C). The HPMA copolymer-RGD4C conjugate was characterized by its side-chain contents, in vitro endothelial cell adhesion assay and its biodistribution and antitumor effectiveness in a SCID mouse xenograft model of human prostate carcinoma. Results The conjugate contained about 16 RGD4C moieties per polymer backbone. Tumor accumulation significantly increased ( P<.01) over time from 1.05±0.03 % injected dose (%ID)/g tissue at 1 h to 4.32±0.32% at 72 h. The activity in major normal tissues significantly decreased ( P<.05) during that period. At 21 days, the control tumors increased 442% in volume from baseline. In contrast, a 7% and a 63% decrease of tumor volume were observed for the 100- and 250-μCi 90Y treatment groups, respectively. Histopathological examination revealed increased apoptosis in the treated tumors with no acute signs of radiation-induced toxicity to other organs. Conclusion This copolymer-peptide conjugate targets tumor angiogenic vessels and delivers sufficient radiotherapy to arrest tumor growth.

Adenoviral Vectors Expressing Human Endostatin–Angiostatin and Soluble Tie2: Enhanced Suppression of Tumor Growth and Antiangiogenic Effects in a Prostate Tumor Model

Angiogenesis is essential for prostate cancer development and metastasis. Antiangiogenic therapy targeting tumor neovasculature, therefore, represents a promising approach for prostate cancer treatment. We hypothesized that adenoviral-mediated delivery of a combination of antiangiogenic factors might have an enhanced antitumor response. We developed the adenoviral vectors Ad-hEndo-angio, expressing a unique, chimeric human endostatin–angiostatin fusion protein, and Ad-sTie2, expressing a soluble form of endothelium-specific receptor tyrosine kinase Tie2. Matrigel angiogenesis assays using Ad-hEndo-angio revealed significant inhibition of tubular network formation and endothelial sprouting compared to Ad-sTie2. In vivo studies in a bilateral PC-3 tumor xenograft model following either intratumoral or systemic administration of Ad-hEndo-angio led to enhanced tumor growth suppression compared to Ad-sTie2. A novel finding is that an intratumoral, combination therapy employing one-half the dose of Ad-hEndo-angio as well as Ad-sTie2 led to a complete regression of the injected, as well as the contralateral uninjected, tumor and prolonged the tumor-free survival in 80% of the animals. In addition, a novel, real-time, intravital imaging modality was used to monitor antiangiogenic responses following adenoviral-mediated gene transfer. These results suggest that a combinatorial antiangiogenic gene therapy approach involving Ad-hEndo-angio and Ad-sTie2 could become a novel form of treatment for localized human prostate cancer.