Angelina Sacchi

Enhancement of tumor necrosis factor alpha antitumor immunotherapeutic properties by targeted delivery to aminopeptidase N (CD13).

The clinical use of tumor necrosis factor α (TNF) as an anticancer drug is limited to local treatments because of its dose-limiting systemic toxicity. We show here that murine TNF fused with CNGRC peptide (NGR-TNF), an aminopeptidase N (CD13) ligand that targets activated blood vessels in tumors, is 12–15 times more efficient than murine TNF in decreasing the tumor burden in lymphoma and melanoma animal models, whereas its toxicity is similar. Similarly, human NGR-TNF induced stronger antitumor effects than human TNF, even with 30 times lower doses. Coadministration of murine NGR-TNF with a CNGRC peptide or an anti-CD13 antibody markedly decreased its antitumor effects. Tumor regression, induced by doses of murine NGR-TNF lower than the LD50, was accompanied by protective immunity. In contrast, no cure was induced by TNF at any dose. These results suggest that targeted delivery of TNF to CD13 may enhance its immunotherapeutic properties. Moreover, these findings reveal the potential of tumor homing peptides to generate a new class of recombinant cytokines that compared to immunocytokines have a simpler structure, could be easier to produce and are potentially less immunogenic.

Improving chemotherapeutic drug penetration in tumors by vascular targeting and barrier alteration.

Drug delivery and penetration into neoplastic cells distant from tumor vessels are critical for the effectiveness of solid-tumor chemotherapy. We have found that targeted delivery to tumor vessels of picogram doses of TNF-alpha (TNF), a cytokine able to alter endothelial barrier function and tumor interstitial pressure, enhances the penetration of doxorubicin in tumors in murine models. Vascular targeting was achieved by coupling TNF with CNGRC, a peptide that targets the tumor neovasculature. This treatment enhanced eight- to tenfold the therapeutic efficacy of doxorubicin, with no evidence of increased toxicity. Similarly, vascular targeting enhanced the efficacy of melphalan, a different chemotherapeutic drug. Synergy with chemotherapy was observed with 3-5 ng/kg of targeted TNF (intraperitoneally), about 10(6)-fold lower than the LD(50) and 10(5)-fold lower than the dose required for nontargeted TNF. In addition, we have also found that targeted delivery of low doses of TNF to tumor vessels does not induce the release of soluble TNF receptors into the circulation. The delivery of minute amounts of TNF to tumor vessels represents a new approach for avoiding negative feedback mechanisms and preserving its ability to alter drug-penetration barriers. Vascular targeting could be a novel strategy for increasing the therapeutic index of chemotherapeutic drugs.

Coupling Tumor Necrosis Factor-α with αV Integrin Ligands Improves Its Antineoplastic Activity

Despite the impressive results obtained in animal models, the clinical use of tumor necrosis factor-α (TNF) as an anticancer drug is limited by severe toxicity. We have shown previously that targeted delivery of TNF to aminopeptidase N (CD13), a marker of angiogenic vessels, improved the therapeutic index of this cytokine in tumor-bearing mice. To assess whether the vascular-targeting approach could be extended to other markers of tumor blood vessels, in this work, we have fused TNF with the ACDCRGDCFCG peptide, a ligand of αV integrins by recombinant DNA technology. We have found that subnanogram doses of this conjugate are sufficient to induce antitumor effects in tumor-bearing mice when combined with melphalan, a chemotherapeutic drug. Cell adhesion assays and competitive binding experiments with anti-integrin antibodies showed that the Arg-Gly-Asp moiety interacts with cell adhesion receptors, including αVβ3 integrin, as originally postulated. In addition, ACGDRGDCFCG-mouse TNF conjugate induced cytotoxic effects in standard cytolytic assays, implying that ACGDRGDCFCG-mouse TNF conjugate can also bind TNF receptors and trigger death signals. These results indicate that coupling TNF with αV integrin ligands improves its antineoplastic activity and supports the concept that vascular targeting is a strategy potentially applicable to different endothelial markers, not limited to CD13.

NGR-tagged nano-gold: A new CD13-selective carrier for cytokine delivery to tumors

Colloidal gold (Au), a well-tolerated nanomaterial, is currently exploited for several applications in nanomedicine. We show that gold nanoparticles tagged with a novel tumor-homing peptide containing Asn-Gly-Arg (NGR), a ligand of CD13 expressed by the tumor neovasculature, can be exploited as carriers for cytokine delivery to tumors. Biochemical and functional studies showed that the NGR molecular scaffold/linker used for gold functionalization is critical for CD13 recognition. Using fibrosarcoma-bearing mice, NGR-tagged nanodrugs could deliver extremely low, yet pharmacologically active doses of tumor necrosis factor (TNF), an anticancer cytokine, to tumors with no evidence of toxicity. Mechanistic studies confirmed that CD13 targeting was a primary mechanism of drug delivery and excluded a major role of integrin targeting consequent to NGR deamidation, a degradation reaction that generates the isoAsp-Gly-Arg (isoDGR) integrin ligand. NGR-tagged gold nanoparticles can be used, in principle, as a novel platform for single- or multi-cytokine delivery to tumor endothelial cells for cancer therapy.

Synergistic Antitumor Activity of Cisplatin, Paclitaxel, and Gemcitabine with Tumor Vasculature-Targeted Tumor Necrosis Factor-α

Purpose: Subnanogram doses of NGR-tumor necrosis factor (TNF), a TNF-α derivative able to target tumor neovessels, can enhance the antitumor activity of doxorubicin and melphalan in murine models. We have examined the antitumor activity of NGR-TNF in combination with various chemotherapeutic drugs acting via different mechanisms, including, besides doxorubicin and melphalan, cisplatin, paclitaxel, and gemcitabine. Experimental Design: Chemotherapeutic drugs were tested alone and in combination with NGR-TNF (0.1 ng) in murine lymphoma, fibrosarcoma, and mammary adenocarcinoma models. Different administration schedules have been tested and the effects on tumor growth, animal weight, tumor perfusion, and cell cytotoxicity have been investigated. Results: Pretreatment with NGR-TNF enhanced the response to all these drugs although to a different extent. The increased efficacy was not accompanied by increased toxicity at least as judged from the loss of animal weight. The synergistic effect was transient, maximal synergism being observed with a 2-hour delay between NGR-TNF and drug administrations in all models and with all drugs tested. NGR-TNF did not increase the in vitro cytotoxicity of chemotherapeutic drugs against tumor cells, suggesting that the in vivo synergism depends on NGR-TNF effects on host cells rather than on tumor cells. Conclusions: Targeted delivery of low doses of NGR-TNF to the tumor vasculature can increase the efficacy of various drugs acting via different mechanisms. Optimal administration schedule requires 2 hours of pretreatment with NGR-TNF independently from the mechanism of drug cytotoxicity. This work could provide important information for designing clinical studies with NGR-TNF in combination with chemotherapeutic drugs.

Targeted Delivery of IFNγ to Tumor Vessels Uncouples Antitumor from Counterregulatory Mechanisms

Because of its immunomodulatory and anticancer activities, IFNgamma has been used as an anticancer drug in several clinical studies, unfortunately with modest results. Attempts to increase the response by increasing the dose or by repeated continuous injection often resulted in lower efficacy, likely due to counterregulatory effects. We show here that targeted delivery of low doses of IFNgamma to CD13, a marker of angiogenic vessels, can overcome major counterregulatory mechanisms and delay tumor growth in two murine models that respond poorly to IFNgamma. Tumor vascular targeting was achieved by coupling IFNgamma to GCNGRC, a CD13 ligand, by genetic engineering technology. The dose-response curve was bell-shaped. Maximal effects were induced with a dose of 0.005 microg/kg, about 500-fold lower than the dose used in patients. Nontargeted IFNgamma induced little or no effects over a range of 0.003 to 250 microg/kg. Studies on the mechanism of action showed that low doses of targeted IFNgamma could activate tumor necrosis factor (TNF)-dependent antitumor mechanisms, whereas high doses of either targeted or nontargeted IFNgamma induced soluble TNF-receptor shedding in circulation, a known counterregulatory mechanism of TNF activity. These findings suggest that antitumor activity and counterregulatory mechanisms could be uncoupled by tumor vascular targeting with extremely low doses of IFNgamma.

Isoaspartate-Glycine-Arginine: A New Tumor Vasculature–Targeting Motif

Asparagine deamidation in peptides or in fibronectin fragments containing the asparagine-glycine-arginine sequence generates isoaspartate-glycine-arginine (isoDGR), a new alphavbeta3 integrin-binding motif. Because alphavbeta3 is expressed in angiogenic vessels, we hypothesized that isoDGR-containing peptides could be exploited as ligands for targeted delivery of drugs to tumor neovasculature. We found that a cyclic CisoDGRC peptide coupled to fluorescent nanoparticles (quantum dots) could bind alphavbeta3 integrin and colocalize with anti-CD31, anti-alphavbeta3, and anti-alpha5beta1 antibodies in human renal cell carcinoma tissue sections, indicating that this peptide could efficiently recognize endothelial cells of angiogenic vessels. Using CisoDGRC fused to tumor necrosis factor alpha (TNF) we observed that ultralow doses (1-10 pg) of this product (called isoDGR-TNF), but not of TNF or CDGRC-TNF fusion protein, were sufficient to induce antitumor effects when administered alone or in combination with chemotherapy to tumor-bearing mice. The antitumor activity of isoDGR-TNF was efficiently inhibited by coadministration with an excess of free CisoDGRC, as expected for ligand-directed targeting mechanisms. These results suggest that isoDGR is a novel tumor vasculature-targeting motif. Peptides containing isoDGR could be exploited as ligands for targeted delivery of drugs, imaging agents, or other compounds to tumor vasculature.

Critical Role of Flanking Residues in NGR-to-isoDGR Transition and CD13/Integrin Receptor Switching

Various NGR-containing peptides have been exploited for targeted delivery of drugs to CD13-positive tumor neovasculature. Recent studies have shown that compounds containing this motif can rapidly deamidate and generate isoaspartate-glycine-arginine (isoDGR), a ligand of αvβ3-integrin that can be also exploited for drug delivery to tumors. We have investigated the role of NGR and isoDGR peptide scaffolds on their biochemical and biological properties. Peptides containing the cyclic CNGRC sequence could bind CD13-positive endothelial cells more efficiently than those containing linear GNGRG. Peptide degradation studies showed that cyclic peptides mostly undergo NGR-to-isoDGR transition and CD13/integrin switching, whereas linear peptides mainly undergo degradation reactions involving the α-amino group, which generate non-functional six/seven-membered ring compounds, unable to bind αvβ3, and small amount of isoDGR. Structure-activity studies showed that cyclic isoDGR could bind αvβ3 with an affinity >100-fold higher than that of linear isoDGR and inhibited endothelial cell adhesion and tumor growth more efficiently. Cyclic isoDGR could also bind other integrins (αvβ5, αvβ6, αvβ8, and α5β1), although with 10–100-fold lower affinity. Peptide linearization caused loss of affinity for all integrins and loss of specificity, whereas α-amino group acetylation increased the affinity for all tested integrins, but caused loss of specificity. These results highlight the critical role of molecular scaffold on the biological properties of NGR/isoDGR peptides. These findings may have important implications for the design and development of anticancer drugs or tumor neovasculature-imaging compounds, and for the potential function of different NGR/isoDGR sites in natural proteins.

Crucial Role for Interferon γ in the Synergism between Tumor Vasculature-Targeted Tumor Necrosis Factor α (NGR-TNF) and Doxorubicin

NGR-TNF is a derivative of TNF-α, consisting of TNF fused to CNGRCG, a tumor vasculature-targeting peptide. Previous studies showed that NGR-TNF can exert synergistic antitumor effects with doxorubicin and with other chemotherapeutic drugs in murine models. In this study, we have investigated the role of endogenous IFN-γ on the antitumor activity of NGR-TNF in combination with doxorubicin. The study was carried out using murine B16F1 melanoma and TS/A mammary adenocarcinoma implanted subcutaneously in (a) immunocompetent mice, (b) athymic nude mice, and (c) IFN-γ–knockout mice. Synergism between NGR-TNF and doxorubicin was observed in immunocompetent mice but not in nude or IFN-γ–knockout mice. Preadministration of a neutralizing anti-IFN-γ antibody to immunocompetent mice inhibited the NGR-TNF/doxorubicin synergism, whereas administration of IFN-γ to nude and to IFN-γ–knockout mice restored the synergistic activity. The synergism in nude mice was restored also by transfecting tumor cells with the IFN-γ cDNA. Administration of NGR-TNF in combination with IFN-γ to nude mice, but not of NGR-TNF alone, doubled the penetration of doxorubicin in TS/A tumors. These findings point to a crucial role for locally produced IFN-γ in tumor vascular targeting with NGR-TNF and doxorubicin. Finally, addition of IFN-γ to the treatment of immunocompetent mice with NGR-TNF/doxorubicin induced only modest improvement in response, suggesting that exogenous IFN-γ can improve the therapeutic activity of these drugs only in case of suboptimal production of endogenous IFN-γ.

Synergistic Damage of Tumor Vessels with Ultra Low-Dose Endothelial-Monocyte Activating Polypeptide-II and Neovasculature-Targeted Tumor Necrosis Factor-α

High-dose endothelial-monocyte activating polypeptide II (EMAP-II), a tumor-derived antiangiogenic cytokine, can sensitize tumor vasculature to the damaging activity of high-dose tumor necrosis factor (TNF)-alpha. However, this combination cannot be used for systemic treatment of patients because of prohibitive toxicity. We have found that this limitation can be overcome by combining a TNF-targeting strategy with the use of ultra low-dose EMAP-II. Coadministration of 0.1 ng of EMAP-II and 0.1 ng of CNGRCG-TNF (NGR-TNF), a peptide-TNF conjugate able to target tumor blood vessels, inhibited lymphoma and melanoma growth in mice, with no evidence of toxicity. This drug combination induced endothelial cell apoptosis in vivo and, at later time points, caused reduction of vessel density and massive apoptosis of tumor cells. Ligand-directed targeting of TNF was critical because the combination of nontargeted TNF with EMAP-II was inactive in these murine models. The synergism was progressively lost when the dose of EMAP-II was increased in the nanogram to microgram range, supporting the concept that the use of low-dose EMAP-II is critical. Studies on the mechanism of this paradoxical behavior showed that EMAP-II doses >1 ng induce the release of soluble TNF receptor 1 in circulation, a strong counter-regulatory inhibitor of TNF. Tumor vascular targeting with extremely low amounts of these cytokines may represent a new strategy for cancer treatment.