RESUMEN
Mucopolysaccharidosis type II (MPS II), or Hunter syndrome, is a rare X-linked recessive lysosomal storage disorder due to a mutation in the lysosomal enzyme iduronate-2-sulfatase (IDS) gene. IDS deficiency leads to a progressive, multisystem accumulation of glycosaminoglycans (GAGs) and results in central nervous system (CNS) manifestations in the severe form. We developed up to clinical readiness a new hematopoietic stem cell (HSC) gene therapy approach for MPS II that benefits from a novel highly effective transduction protocol. We first provided proof of concept of efficacy of our approach aimed at enhanced IDS enzyme delivery to the CNS in a murine study of immediate translational value, employing a lentiviral vector (LV) encoding a codon-optimized human IDS cDNA. Then the therapeutic LV was tested for its ability to efficiently and safely transduce bona fide human HSCs in clinically relevant conditions according to a standard vs. a novel protocol that demonstrated superior ability to transduce bona fide long-term repopulating HSCs. Overall, these results provide strong proof of concept for the clinical translation of this approach for the treatment of Hunter syndrome.
Asunto(s)
Iduronato Sulfatasa , Mucopolisacaridosis II , Humanos , Animales , Ratones , Mucopolisacaridosis II/terapia , Mucopolisacaridosis II/tratamiento farmacológico , Iduronato Sulfatasa/genética , Iduronato Sulfatasa/metabolismo , Terapia Genética , Sistema Nervioso Central/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Células Madre Hematopoyéticas/metabolismoRESUMEN
NGR-hTNF is a therapeutic agent for a solid tumor that specifically targets angiogenic tumor blood vessels, through the NGR motif. Its activity has been assessed in several clinical studies encompassing tumors of different histological types. The drug's activity is based on an improved permeabilization of newly formed tumor vasculature, which favors intratumor penetration of chemotherapeutic agents and leukocyte trafficking. This work investigated the binding and the signaling properties of the NGR-hTNF, to elucidate its mechanism of action. The crystal structure of NGR-hTNF and modeling of its interaction with TNFR suggested that the NGR region is available for binding to a specific receptor. Using 2D TR-NOESY experiments, this study confirmed that the NGR-peptides binds to a specific CD13 isoform, whose expression is restricted to tumor vasculature cells, and to some tumor cell lines. The interaction between hTNF or NGR-hTNF with immobilized TNFRs showed similar kinetic parameters, whereas the competition experiments performed on the cells expressing both TNFR and CD13 showed that NGR-hTNF had a higher binding affinity than hTNF. The analysis of the NGR-hTNF-triggered signal transduction events showed a specific impairment in the activation of pro-survival pathways (Ras, Erk and Akt), compared to hTNF. Since a signaling pattern identical to NGR-hTNF was obtained with hTNF and NGR-sequence given as distinct molecules, the inhibition observed on the survival pathways was presumably due to a direct effect of the NGR-CD13 engagement on the TNFR signaling pathway. The reduced activation of the pro survival pathways induced by NGR-hTNF correlated with the increased caspases activation and reduced cell survival. This study demonstrates that the binding of the NGR-motif to CD13 determines not only the homing of NGR-hTNF to tumor vessels, but also the increase in its antiangiogenic activity.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Neoplasias/irrigación sanguínea , Oligopéptidos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Inhibidores de la Angiogénesis/química , Línea Celular Tumoral , Cristalografía por Rayos X , Células Endoteliales de la Vena Umbilical Humana , Humanos , Modelos Moleculares , Oligopéptidos/química , Proteínas Recombinantes de Fusión/química , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/químicaRESUMEN
The long-term expression and the ability of a therapeutic gene to confer survival advantage to transduced cells are mandatory requirements for successful anti-HIV gene therapy. In this context, we developed lentiviral vectors (LVs) expressing the F12-viral infectivity factor (Vif) derivative Chim3. We recently showed that Chim3 inhibits HIV-1 replication in primary cells by both blocking the accumulation of retrotranscripts, independently of either human APOBEC3G (hA3G) or Vif, and by preserving the antiviral function of hA3G. These results were predictive of long-lasting survival of Chim3(+) cells after HIV-1 infection. Furthermore, Vif, like Vpr, deregulates cell-cycle progression by inducing a delay in G(2) phase. Thus, the aim of this study was to investigate the role of Chim3 on both cell survival and cell-cycle regulation after HIV-1 infection. Here, we provide evidence that infected Chim3(+) T cells prevail over either mock- or empty-LV engineered cells, show reduced G(2) accumulation, and, as a consequence, ultimately extend their lifespan. Based on these findings, Chim3 rightly belongs to the most efficacious class of antiviral genes. In conclusion, Chim3 usage in anti-HIV gene therapy based on hematopoietic stem cell (HSC) modification has to be considered as a promising therapeutic intervention to eventually cope with HIV-1 infection.
Asunto(s)
Linfocitos T CD4-Positivos/fisiología , ADN Viral/genética , Fase G2/fisiología , Terapia Genética , VIH-1/fisiología , Integración Viral , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/fisiología , Southern Blotting , Linfocitos T CD4-Positivos/virología , Supervivencia Celular , Células Cultivadas , ADN Viral/metabolismo , Células Madre Hematopoyéticas , Humanos , Inmunoprecipitación , Replicación Viral , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/fisiologíaRESUMEN
Ain't got that swing(-out): The cyclopeptide isoDGR is emerging as a new αvß3 integrin binding motif. Agreement between the results of computational and biochemical studies reveals that isoDGR-containing cyclopeptides are true αvß3 integrin antagonists that block αvß3 in its inactive conformation (see scheme). isoDGR-based ligands may give αvß3 antagonists without paradoxical effects.
Asunto(s)
Integrina alfaVbeta3/antagonistas & inhibidores , Simulación de Dinámica Molecular , Oligopéptidos/farmacología , Péptidos Cíclicos/farmacología , Regulación Alostérica , Integrina alfaVbeta3/metabolismo , Modelos Moleculares , Oligopéptidos/química , Péptidos Cíclicos/químicaRESUMEN
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 alphavbeta3-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 alpha-amino group, which generate non-functional six/seven-membered ring compounds, unable to bind alphavbeta3, and small amount of isoDGR. Structure-activity studies showed that cyclic isoDGR could bind alphavbeta3 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 (alphavbeta5, alphavbeta6, alphavbeta8, and alpha5beta1), although with 10-100-fold lower affinity. Peptide linearization caused loss of affinity for all integrins and loss of specificity, whereas alpha-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.
Asunto(s)
Antígenos CD13/metabolismo , Integrinas/metabolismo , Oligopéptidos/química , Animales , Antineoplásicos/farmacología , Adhesión Celular , Disulfuros/química , Células Endoteliales/citología , Humanos , Ácido Isoaspártico/química , Ratones , Ratones Endogámicos BALB C , Unión Proteica , Proteínas Recombinantes/química , Relación Estructura-ActividadRESUMEN
The viral infectivity factor (Vif) is essential for HIV-1 infectivity and hence is an ideal target for promising anti-HIV-1/AIDS gene therapy. We previously demonstrated that F12-Vif mutant inhibits HIV-1 replication in CD4(+) T lymphocytes. Despite macrophage relevance to HIV-1 pathogenesis, most gene therapy studies do not investigate macrophages because of their natural resistance to genetic manipulation. Here, we confirm the F12-Vif antiviral activity also in macrophages differentiated in vitro from transduced CD34(+) human stem cells (HSCs). Moreover, we identified the 126- to 170-amino-acid region in the C-terminal half of F12-Vif as responsible for its antiviral function. Indeed, Chim3 protein, containing this 45-amino-acid region embedded in a WT-Vif backbone, is as lethal as F12-Vif against HIV-1. Of major relevance, we demonstrated a dual mechanism of action for Chim3. First, Chim3 functions as a transdominant factor that preserves the antiviral function of the natural restriction factor APOBEC3G (hA3G). Second, Chim3 blocks the early HIV-1 retrotranscript accumulation and thereby HIV-1 DNA integration regardless of the presence of WT-Vif and hA3G. In conclusion, by impairing the early steps of HIV-1 life cycle, Chim3 conceivably endows engineered cells with survival advantage, which is required for the efficient immune reconstitution of patients living with HIV/AIDS.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Terapia Genética/métodos , Infecciones por VIH/terapia , VIH-1/crecimiento & desarrollo , Macrófagos/virología , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Antígenos CD34/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/fisiología , Diferenciación Celular/inmunología , Línea Celular , Sangre Fetal/citología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/genética , VIH-1/inmunología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Riñón/citología , Macrófagos/citología , Macrófagos/fisiología , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Transducción Genética , Integración Viral , Replicación Viral , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
Targeted delivery of IFNgamma to tumors has been achieved by fusing this cytokine with GCNGRC, a tumor neovasculature homing peptide. Although the therapeutic efficacy of this protein (called IFNgamma-NGR) in animal models is greater than that of IFNgamma, frequent administrations of IFNgamma-NGR may result in lower efficacy and tumor resistance. We investigated the role of indoleamine 2,3-dioxygenase (IDO), an IFNgamma-inducible enzyme that may down-regulate T cells by affecting local tryptophan catabolism in tumor resistance to repeated treatments with IFNgamma-NGR. The study was carried out in immunocompetent mice and in nu/nu mice bearing RMA lymphoma, B16F melanoma, or WEHI-164 fibrosarcoma and in vitro using cultured tumor cells. IDO activity was increased in lymphoma homogenates after multiple treatments with IFNgamma-NGR but not after a single treatment. Coadministration of 1-methyl-tryptophan, an inhibitor of IDO, increased tumor responses to multiple treatments in the lymphoma, melanoma, and fibrosarcoma models. No synergism between IFNgamma-NGR and 1-methyl-tryptophan was observed in vitro in tumor cell proliferation assays or in nu/nu tumor-bearing mice, suggesting that the antitumor effect was host mediated. We conclude that IDO is critically involved in tumor resistance to repeated treatments with IFNgamma-NGR, likely causing excessive stimulation of tryptophan catabolism and inhibiting antitumor immune mechanisms. Coadministration of IFNgamma-NGR with IDO inhibitors could represent a new strategy for increasing its antitumor activity.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Indolamina-Pirrol 2,3,-Dioxigenasa/farmacología , Interferón gamma/fisiología , Neoplasias/tratamiento farmacológico , Proteínas Recombinantes de Fusión/farmacología , Animales , Línea Celular Tumoral , Humanos , Interferón gamma/metabolismo , Melanoma Experimental , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Neoplasias/patología , Proteínas Recombinantes de Fusión/química , Triptófano/análogos & derivados , Triptófano/farmacologíaRESUMEN
In this study we have characterized intra-patient length polymorphism in V4 by cloning and sequencing a C2-C4 fragment from HIV plasma RNA in patients at different stages of HIV disease. Clonal analysis of clade B, G, and CRF02 isolates during early infection shows extensive intra-patient V4 variability, due to the presence of indel-associated polymorphism. Indels, coupled to amino acid substitution events, affect the number and distribution of potential N-glycosylation sites, resulting in the coexistence, within the same patient, of V4 subsets, each characterized by different sizes, amino acid sequences, and potential N-glycosylation patterns. In contrast, V3 appears to be relatively homogeneous, with similar V3 associated to significantly different V4 within the same clinical specimen. Based on these data, we propose that during early chronic infection V4 is present as a highly divergent quasispecies, enabling the virus to adopt different conformational structures according to immune constrains and other selective pressures.
Asunto(s)
Evolución Molecular , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/genética , Mutación INDEL , Fragmentos de Péptidos/genética , Enfermedad Aguda , Secuencia de Aminoácidos , Fármacos Anti-VIH/uso terapéutico , Enfermedad Crónica , Glicosilación , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/metabolismo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Filogenia , Polimorfismo GenéticoRESUMEN
Ex vivo transduction of human CD34+ hematopoietic stem/progenitor cells (hCD34+ HSPCs) and T lymphocytes is a key process that requires high efficiency and low toxicity to achieve effective clinical results. So far, several enhancers have been used to improve this process. Among them, Retronectin highly meliorates VSV-G and RD114-TR pseudotyped lentiviral vector delivery in hCD34+ HSPCs and T lymphocytes. However, Retronectin is expensive and requires pre-coating of culture dishes or bags before cell seeding, resulting in a cumbersome procedure. Recently, an alternative transduction adjuvant has been developed, named Vectofusin-1, whose effect has been demonstrated on gene delivery to cell lines and primary hCD34+ HSPCs by lentiviral vectors pseudotyped with different envelope glycoproteins. In this study, we have focused our analysis on the effect of Vectofusin-1 on the transduction of hCD34+ HSPCs and T lymphocytes by using mostly RD114-TR pseudotyped lentivectors and clinical transduction protocols. Here, we have proved that Vectofusin-1 reproducibly enhances gene delivery to hCD34+ HSPCs and activated T cells without cell toxicity and with efficacy comparable to that of Retronectin. The use of Vectofusin-1 will therefore help to shorten and simplify clinical cell manipulation, especially if automated systems are planned for transducing large-scale clinical lots.
RESUMEN
Lentiviral vectors (LVs) are a highly valuable tool for gene transfer currently exploited in basic, applied, and clinical studies. Their optimization is therefore very important for the field of vectorology and gene therapy. A key molecule for LV function is the envelope because it guides cell entry. The most commonly used in transiently produced LVs is the vesicular stomatitis virus glycoprotein (VSV-G) envelope, whose continuous expression is, however, toxic for stable LV producer cells. In contrast, the feline endogenous retroviral RD114-TR envelope is suitable for stable LV manufacturing, being well tolerated by producer cells under constitutive expression. We have previously reported successful, transient and stable production of LVs pseudotyped with RD114-TR for good transduction of T lymphocytes and CD34+ cells. To further improve RD114-TR-pseudotyped LV cell entry by increasing envelope expression, we codon-optimized the RD114-TR open reading frame (ORF). Here we show that, despite the RD114-TRco precursor being produced at a higher level than the wild-type counterpart, it is unexpectedly not duly glycosylated, exported to the cytosol, and processed. Correct cleavage of the precursor in the functional surface and transmembrane subunits is prevented in vivo, and, consequently, the unprocessed precursor is incorporated into LVs, making them inactive.
RESUMEN
To date, gene therapy with transiently derived lentivectors has been very successful to cure rare infant genetic diseases. However, transient manufacturing is unfeasible to treat adult malignancies because large vector lots are required. By contrast, stable manufacturing is the best option for high-incidence diseases since it reduces the production cost, which is the major current limitation to scale up the transient methods. We have previously developed the proprietary RD2-MolPack technology for the stable production of second-generation lentivectors, based on the RD114-TR envelope. Of note, opposite to vesicular stomatitis virus glycoprotein (VSV-G) envelope, RD114-TR does not need inducible expression thanks to lack of toxicity. Here, we present the construction of RD2- and RD3-MolPack cells for the production of self-inactivating lentivectors expressing green fluorescent protein (GFP) as a proof-of-concept of the feasibility and safety of this technology before its later therapeutic exploitation. We report that human T lymphocytes transduced with self-inactivating lentivectors derived from RD3-MolPack cells or with self-inactivating VSV-G pseudotyped lentivectors derived from transient transfection show identical T-cell memory differentiation phenotype and comparable transduction efficiency in all T-cell subsets. RD-MolPack technology represents, therefore, a straightforward tool to simplify and standardize lentivector manufacturing to engineer T-cells for frontline immunotherapy applications.
Asunto(s)
Antígenos CD13/análisis , Integrina alfaVbeta3/análisis , Resonancia Magnética Nuclear Biomolecular/métodos , Antígenos CD13/química , Antígenos CD13/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Humanos , Integrina alfaVbeta3/química , Integrina alfaVbeta3/metabolismo , Unión ProteicaRESUMEN
Tumor vessels are an attractive target for cancer therapy, including metastasis treatment. Angiogenesis inhibitors targeting the VEGF signalling pathway have proven to be efficacious in preclinical cancer models and in clinical trials. However, angiogenesis inhibition concomitantly elicits tumor adaptation and progression to stages of greater malignancy, with heightened invasiveness and in some cases increased distant metastasis. Here, we investigated whether NGR-TNF, a vascular targeting agent in phase III clinical development, coupling the CNGRCG angiogenic vessel-homing peptide with TNF-α, has an effect on metastasis in a model of murine breast cancer, which spontaneously metastasize to lungs, and on the growth of experimental melanoma lung metastasis. We report that NGR-TNF does not increase cancer invasiveness, as other antiangiogenics agents do, but controls metastatic growth in both models, both when administered as primary treatment and in adjuvant settings, improving the overall survival of metastasis-bearing mice.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Mamarias Animales/tratamiento farmacológico , Melanoma Experimental/tratamiento farmacológico , Neovascularización Patológica/prevención & control , Proteínas Recombinantes de Fusión/uso terapéutico , Factor de Necrosis Tumoral alfa/uso terapéutico , Animales , Femenino , Citometría de Flujo , Técnicas para Inmunoenzimas , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Animales/mortalidad , Neoplasias Mamarias Animales/patología , Melanoma Experimental/mortalidad , Melanoma Experimental/secundario , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
NGR-TNF is a vascular targeting agent in advanced clinical development, coupling tumor necrosis factor-α (TNF) with the CNGRCG peptide, which targets a CD13 isoform specifically expressed by angiogenic vessels. Antitumor efficacy of NGR-TNF has been described in different transplantation tumor models. Nevertheless, the mechanism underlying its activity is not fully understood. In the wild type and in the immunodeficient (RAG-/-) RIP1-Tag2 models of multistage pancreatic carcinogenesis, we demonstrate that CD13 is highly expressed on endothelial cells of hyperplastic and angiogenic islets, whereas its expression is down regulated in tumors where it partially colocalize with pericytes. In vivo CNGRCG peptides coupled to fluorescent nanoparticles (quantum dots) bind to CD13 and colocalize with anti-CD31, in pancreatic islets. At early stage, low doses of NGR-murine (m)TNF have a direct cytotoxic effect inducing endothelial cell apoptosis, reducing vessel density and eventually inhibiting the development of angiogenic islets. At a later stage, NGR-mTNF is able to reduce tumor growth inducing vascular normalization, exclusively when treatment is carried out in the immunocompetent mice. Interestingly, NGR-mTNF-treated tumors from these mice are characterized by CD8+ T cell infiltration. At molecular level, overexpression of genes involved in vessels normalization was detected only in NGR-mTNF-treated tumors from immunocompetent mice. These findings identified a new mechanism of action of NGR-mTNF, providing support for the development of new therapeutic strategies combining chemotherapy or active/adoptive immunotherapies to low dose NGR-TNF treatment.
RESUMEN
Over the last two decades, several attempts to generate packaging cells for lentiviral vectors (LV) have been made. Despite different technologies, no packaging clone is currently employed in clinical trials. We developed a new strategy for LV stable production based on the HEK-293T progenitor cells; the sequential insertion of the viral genes by integrating vectors; the constitutive expression of the viral components; and the RD114-TR envelope pseudotyping. We generated the intermediate clone PK-7 expressing constitutively gag/pol and rev genes and, by adding tat and rd114-tr genes, the stable packaging cell line RD2-MolPack, which can produce LV carrying any transfer vector (TV). Finally, we obtained the RD2-MolPack-Chim3 producer clone by transducing RD2-MolPack cells with the TV expressing the anti-HIV transgene Chim3. Remarkably, RD114-TR pseudovirions have much higher potency when produced by stable compared with transient technology. Most importantly, comparable transduction efficiency in hematopoietic stem cells (HSC) is obtained with 2-logs less physical particles respect to VSV-G pseudovirions produced by transient transfection. Altogether, RD2-MolPack technology should be considered a valid option for large-scale production of LV to be used in gene therapy protocols employing HSC, resulting in the possibility of downsizing the manufacturing scale by about 10-fold in respect to transient technology.
Asunto(s)
Terapia Genética/métodos , Vectores Genéticos/genética , Lentivirus/genética , Transducción Genética/métodos , Ensamble de Virus , Animales , Proteínas de Fusión gag-pol/genética , Proteínas de Fusión gag-pol/metabolismo , Productos del Gen rev/genética , Productos del Gen rev/metabolismo , Vectores Genéticos/metabolismo , Células HEK293 , Infecciones por VIH/terapia , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/virología , Humanos , Lentivirus/metabolismo , Lentivirus/fisiología , Células Sf9 , Spodoptera , Transgenes/genéticaRESUMEN
Poor penetration of antitumor drugs into the extravascular tumor tissue is often a major factor limiting the efficacy of cancer treatments. Our group has recently described a strategy to enhance tumor penetration of chemotherapeutic drugs through use of iRGD peptide (CRGDK/RGPDC). This peptide comprises two sequence motifs: RGD, which binds to αvß3/5 integrins on tumor endothelia and tumor cells, and a cryptic CendR motif (R/KXXR/K-OH). Once integrin binding has brought iRGD to the tumor, the peptide is proteolytically cleaved to expose the cryptic CendR motif. The truncated peptide loses affinity for its primary receptor and binds to neuropilin-1, activating a tissue penetration pathway that delivers the peptide along with attached or co-administered payload into the tumor mass. Here, we describe the design of a new tumor-penetrating peptide based on the current knowledge of homing sequences and internalizing receptors. The tumor-homing motif in the new peptide is the NGR sequence, which binds to endothelial CD13. The NGR sequence was placed in the context of a CendR motif (RNGR), and this sequence was embedded in the iRGD framework. The resulting peptide (CRNGRGPDC, iNGR) homed to tumor vessels and penetrated into tumor tissue more effectively than the standard NGR peptide. iNGR induced greater tumor penetration of coupled nanoparticles and co-administered compounds than NGR. Doxorubicin given together with iNGR was significantly more efficacious than the drug alone. These results show that a tumor-specific, tissue-penetrating peptide can be constructed from known sequence elements. This principle may be useful in designing tissue-penetrating peptides for other diseases.
Asunto(s)
Antineoplásicos/uso terapéutico , Sistemas de Liberación de Medicamentos/métodos , Neoplasias/tratamiento farmacológico , Oligopéptidos/uso terapéutico , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Diseño de Fármacos , Humanos , Ratones , Unión ProteicaRESUMEN
Aminopeptidase-N (CD13) is an important target of tumor vasculature-targeting drugs. The authors investigated its expression by immunohistochemistry with three anti-CD13 monoclonal antibodies (WM15, 3D8, and BF10) in normal and pathological human tissues, including 58 normal, 32 inflammatory, and 149 tumor tissue specimens. The three antibodies stained vessels in most neoplastic tissues, interestingly with different patterns. As a matter of fact, WM15 stained almost all intratumor and peritumor capillaries and only partially large vessels, whereas BF10 and 3D8 reacted with arteries and venules and to a lesser extent with capillaries. These antibodies also stained the stroma in about half of neoplastic tissues. In inflammatory lesions, the three antibodies stained vessels and stroma, whereas in normal tissues, they stained a small percentage of blood vessels. Finally, the three antibodies failed to stain endothelial cells of normal colon, whereas they reacted with activated human umbilical vein endothelial cells and with endothelial cells of colon adenocarcinoma vessels. Overall, WM15 was the most specific antibody for angiogenic tumor vessels, suggesting that it may be a good tool for detecting the CD13 form associated with the tumor vasculature. This finding may be relevant for CD13-mediated vascular targeting therapies.
Asunto(s)
Vasos Sanguíneos/metabolismo , Antígenos CD13/metabolismo , Regulación Neoplásica de la Expresión Génica , Inflamación/patología , Inflamación/fisiopatología , Neoplasias/irrigación sanguínea , Neoplasias/patología , Anticuerpos Monoclonales/inmunología , Antígenos CD13/inmunología , Epitelio/metabolismo , Humanos , Inmunohistoquímica , Inflamación/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Transporte de ProteínasRESUMEN
BACKGROUND: NGR-hTNF consists of human tumour necrosis factor (hTNF) fused with the tumour-homing peptide Asp-Gly-Arg (NGR), which is able to selectively bind an aminopeptidase N overexpressed on tumour blood vessels. Preclinical antitumour activity was observed even at low doses. We evaluated the activity and safety of low-dose NGR-hTNF in colorectal cancer (CRC) patients failing standard therapies. PATIENTS AND METHODS: Thirty-three patients with progressive disease at study entry received NGR-hTNF 0.8 µg/m(2) given intravenously every 3 weeks. The median number of prior treatment regimens was three (range, 2-5). One-quarter of patients had previously received four or more regimens and two-thirds targeted agents. Progression-free survival (PFS) was the primary study objective. RESULTS: NGR-hTNF was well tolerated. No treatment-related grade 3 to 4 toxicities were detected, most common grade 1 to 2 adverse events being short-lived, infusion-time related chills (50.0%). One partial response and 12 stable diseases were observed, yielding a disease control rate of 39.4% (95% CI, 22.9-57.8%). Median PFS and overall survival were 2.5 months (95% CI, 2.1-2.8) and 13.1 months (95% CI, 8.9-17.3), respectively; whereas in patients who achieved disease control the median PFS and overall survival were 3.8 and 15.4 months, respectively. In an additional cohort of 13 patients treated at same dose with a weekly schedule, there was no increased toxicity and 2 patients experienced PFS longer than 10 months. CONCLUSION: Based on tolerability and preliminary evidence of disease control in heavily pretreated CRC patients, NGR-hTNF deserves further evaluation in combination with standard chemotherapy.