RESUMO
Cell-based immunotherapy for the treatment of hematologic malignancies, such as leukemia and lymphoma, has seen much success and played an increasingly important role in clinical studies. Nevertheless, the efficacy of immunotherapy in solid tumors still needs improvements due to the immunosuppressive properties of tumor cells and the microenvironment. To overcome these limitations, we prepared a novel tumor-targeting delivery system based on the underlying mechanism of immune-targeted cell death that encapsulated granzyme B protein within a porous polymeric nanocapsule. Methods: A cell-penetrating peptide TAT was attached onto granzyme B (GrB) to enhance its transmembrane transport efficiency and potency to induce cell apoptosis. The endocytosis and internalization pathways of GrB-TAT (GrB-T) were analyzed in comparison with perforin by confocal microscopy and flow cytometry. Furthermore, the positively charged GrB-T was wrapped into nanoparticles by p-2-methacryloyloxy ethyl phosphorylcholine (PMPC)-modified HA (hyaluronic acid). The nanoparticles (called TCiGNPs) were characterized in terms of zeta potential and by transmission electron microscopy (TEM). The in vitro anti-tumor effects of GrB-T were examined by cell apoptosis assay and Western blotting analysis. The in vivo anti-tumor therapeutic efficacy of TCiGNPs was evaluated in a mouse tumor model. Results: The TAT peptide could play a role similar to perforin to mediate direct transmembrane transfer of GrB and improve GrB-induced cell apoptosis. The TCiGNPs were successfully synthesized and accumulated in the solid tumor through enhanced permeability and retention (EPR) effect. In the tumor microenvironment, TCiGNPs could be degraded by hyaluronidase and triggered the release of GrB-T. The TAT peptide enabled the translocation of GrB across the plasma membrane to induce tumor cell apoptosis in vivo.Conclusion: We successfully developed a granzyme B delivery system with a GrB-T core and a PMPC/HA shell that simulated CTL/NK cell-mediated cancer immunotherapy mechanism. The GrB delivery system holds great promise for cancer treatment analogous to the CTL/NK cell-induced immunotherapy.
Assuntos
Granzimas/administração & dosagem , Sistema Imunitário/efeitos dos fármacos , Nanopartículas/administração & dosagem , Neoplasias/imunologia , Neoplasias/terapia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Peptídeos Penetradores de Células/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Humanos , Imunoterapia/métodos , Células Matadoras Naturais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Perforina/administração & dosagem , Linfócitos T Citotóxicos/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacosRESUMO
BACKGROUND: Immunotherapeutic approaches designed to augment T and B cell mediated killing of tumor cells has met with clinical success in recent years suggesting tremendous potential for treatment in a broad spectrum of tumor types. After complex recognition of target cells by T and B cells, delivery of the serine protease granzyme B (GrB) to tumor cells comprises the cytotoxic insult resulting in a well-characterized, multimodal apoptotic cascade. METHODS: We designed a recombinant fusion construct, GrB-Fc-4D5, composed of a humanized anti-HER2 scFv fused to active GrB for recognition of tumor cells and internal delivery of GrB, simulating T and B cell therapy. We assessed the construct's antigen-binding specificity and GrB enzymatic activity, as well as in vitro cytotoxicity and internalization into target and control cells. We also assessed pharmacokinetic and toxicology parameters in vivo. RESULTS: GrB-Fc-4D5 was highly cytotoxic to Her2 positive cells such as SKBR3, MCF7 and MDA-MB-231 with IC50 values of 56, 99 and 27 nM, respectively, and against a panel of HER2+ cell lines regardless of endogenous expression levels of the PI-9 inhibitor. Contemporaneous studies with Kadcyla demonstrated similar levels of in vitro activity against virtually all cells tested. GrB-Fc-4D5 internalized rapidly into target SKOV3 cells within 1 h of exposure rapidly delivering GrB to the cytoplasmic compartment. In keeping with its relatively high molecular weight (160 kDa), the construct demonstrated a terminal-phase serum half-life in mice of 39.2 h. Toxicity studies conducted on BALB/c mice demonstrated no statistically significant changes in SGPT, SGOT or serum LDH. Histopathologic analysis of tissues from treated mice demonstrated no drug-related changes in any tissues examined. CONCLUSION: GrB-Fc-4D5 shows excellent, specific cytotoxicity and demonstrates no significant toxicity in normal, antigen-negative murine models. This construct constitutes a novel approach against HER2-expressing tumors and is an excellent candidate for further development.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Desenvolvimento de Medicamentos , Terapia de Alvo Molecular , Receptor ErbB-2/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Animais , Antígenos de Neoplasias/imunologia , Antineoplásicos Imunológicos/isolamento & purificação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Expressão Gênica , Vetores Genéticos/genética , Granzimas/administração & dosagem , Granzimas/genética , Humanos , Camundongos , Terapia de Alvo Molecular/métodos , Ligação Proteica/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Anticorpos de Cadeia Única/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The clinical use of protein therapeutics with intracellular targets is hampered by its in vivo fragility and low cell permeability. Here, we report that cell-selective penetrating and reduction-responsive polymersomes (CPRPs) mediate high-efficiency targeted delivery of granzyme B (GrB) to orthotopic human lung tumor in vivo. Model protein studies using FITC-labeled cytochrome C (FITC-CC) revealed efficient and high protein loading up to 17.2â¯wt% for CPRPs. FITC-CC-loaded CPRPs exhibited a small size of 82-90â¯nm, reduction-responsive protein release, as well as greatly enhanced internalization and cytoplasmic protein release in A549 lung cancer cells compared with the non-targeted FITC-CC-loaded RPs control. GrB-loaded CPRPs showed a high potency toward A549 lung cancer cells with a half maximal inhibitory concentration (IC50) of 20.7â¯nM. Under the same condition, free GrB was essentially non-toxic. Importantly, installing cell-selective penetrating peptide did not alter the circulation time but did enhance tumor accumulation of RPs. Orthotopic A549-Luc lung tumor-bearing nude mice administered with GrB-loaded CPRPs at a dosage of 2.88â¯nmol GrB equiv./kg showed complete tumor growth inhibition with little body weight loss throughout the treatment period, resulting in significantly improved survival rate over the non-targeted and non-treated controls. These cell-selective penetrating and reduction-responsive polymersomes provide a targeted protein therapy for cancers.
Assuntos
Sistemas de Liberação de Medicamentos , Granzimas/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Células A549 , Animais , Humanos , Camundongos Nus , Polímeros/administração & dosagemRESUMO
Therapeutic human papillomaviruse (HPV) vaccines have the potential to inhibit the tumor growth by targeting HPV E6 and E7 oncoproteins. Among different vaccine strategies, DNA and protein-based approaches are the most effective candidates for stimulation of the immune responses against HPV infections. Our study was designed to assess the efficacy of small heat shock proteins B1 (Hsp27) and B6 (Hsp20) as an adjuvant accompanied by HPV16 E7 and hPP10-E7 antigens in tumor mouse model. A major key for successful DNA and protein transfer into cells is the development of delivery systems with high efficiency and low cytotoxicity. Herein, we used hPP10 and MPG cell penetrating peptides (CPPs) for protein and DNA delivery in vivo, respectively. Our data indicated that the combination of Hsp27 with the recombinant hPP10-E7 protein in homologous protein/protein (hPP10-E7 + Hsp27) and heterologous DNA/protein (pcDNA-E7 + MPG/ hPP10-E7 + Hsp27) significantly enhanced the E7-specific T cell responses. Indeed, these regimens induced high levels of IgG2a, IFN-γ and IL-2 directed toward Th1 responses and also Granzyme B secretion as compared to other immunization strategies, and also displayed complete protection more than 60 days after treatment. These data suggest that the use of Hsp27 as an adjuvant and MPG and hPP10 as a gene and protein carrier would represent promising applications for improvement of HPV therapeutic vaccines. © 2018 IUBMB Life, 70(10):1002-1011, 2018.
Assuntos
Proteínas de Choque Térmico HSP20/genética , Proteínas de Choque Térmico/administração & dosagem , Proteínas de Neoplasias/administração & dosagem , Vacinas contra Papillomavirus/administração & dosagem , Neoplasias do Colo do Útero/genética , Adjuvantes Imunológicos/administração & dosagem , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/administração & dosagem , Proteínas de Ligação a DNA/administração & dosagem , Feminino , Granzimas/administração & dosagem , Proteínas de Choque Térmico/genética , Humanos , Chaperonas Moleculares , Proteínas de Neoplasias/genética , Proteínas E7 de Papillomavirus/administração & dosagem , Vacinas contra Papillomavirus/genética , Vacinas contra Papillomavirus/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/prevenção & controle , Neoplasias do Colo do Útero/virologiaRESUMO
Peripheral pain signaling reflects a balance of pronociceptive and antinociceptive influences; the contribution by the gastrointestinal microbiota to this balance has received little attention. Disorders, such as inflammatory bowel disease and irritable bowel syndrome, are associated with exaggerated visceral nociceptive actions that may involve altered microbial signaling, particularly given the evidence for bacterial dysbiosis. Thus, we tested whether a community of commensal gastrointestinal bacteria derived from a healthy human donor (microbial ecosystem therapeutics; MET-1) can affect the excitability of male mouse DRG neurons. MET-1 reduced the excitability of DRG neurons by significantly increasing rheobase, decreasing responses to capsaicin (2 µm) and reducing action potential discharge from colonic afferent nerves. The increase in rheobase was accompanied by an increase in the amplitude of voltage-gated K+ currents. A mixture of bacterial protease inhibitors abrogated the effect of MET-1 effects on DRG neuron rheobase. A serine protease inhibitor but not inhibitors of cysteine proteases, acid proteases, metalloproteases, or aminopeptidases abolished the effects of MET-1. The serine protease cathepsin G recapitulated the effects of MET-1 on DRG neurons. Inhibition of protease-activated receptor-4 (PAR-4), but not PAR-2, blocked the effects of MET-1. Furthermore, Faecalibacterium prausnitzii recapitulated the effects of MET-1 on excitability of DRG neurons. We conclude that serine proteases derived from commensal bacteria can directly impact the excitability of DRG neurons, through PAR-4 activation. The ability of microbiota-neuronal interactions to modulate afferent signaling suggests that therapies that induce or correct microbial dysbiosis may impact visceral pain.SIGNIFICANCE STATEMENT Commercially available probiotics have the potential to modify visceral pain. Here we show that secretory products from gastrointestinal microbiota derived from a human donor signal to DRG neurons. Their secretory products contain serine proteases that suppress excitability via activation of protease-activated receptor-4. Moreover, from this community of commensal microbes, Faecalibacterium prausnitzii strain 16-6-I 40 fastidious anaerobe agar had the greatest effect. Our study suggests that therapies that induce or correct microbial dysbiosis may affect the excitability of primary afferent neurons, many of which are nociceptive. Furthermore, identification of the bacterial strains capable of suppressing sensory neuron excitability, and their mechanisms of action, may allow therapeutic relief for patients with gastrointestinal diseases associated with pain.
Assuntos
Gânglios Espinais/enzimologia , Microbioma Gastrointestinal/fisiologia , Granzimas/administração & dosagem , Neurônios/enzimologia , Simbiose/fisiologia , Animais , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/microbiologia , Peptídeo Hidrolases/administração & dosagem , Simbiose/efeitos dos fármacosRESUMO
Protein drugs as one of the most potent biotherapeutics have a tremendous potential in cancer therapy. Their application is, nevertheless, restricted by absence of efficacious, biocompatible, and cancer-targeting nanosystems. In this paper, we report that 2-[3-[5-amino-1-carboxypentyl]-ureido]-pentanedioic acid (Acupa)-decorated pH-responsive chimaeric polymersomes (Acupa-CPs) efficiently deliver therapeutic proteins into prostate cancer cells. Acupa-CPs had a unimodal distribution with average sizes ranging from 157-175 nm depending on amounts of Acupa. They displayed highly efficient loading of both model proteins, bovine serum albumin (BSA) and cytochrome C (CC), affording high protein loading contents of 9.1-24.5 wt.%. The in vitro release results showed that protein release was markedly accelerated at mildly acidic pH due to the hydrolysis of acetal bonds in the vesicular membrane. CLSM and MTT studies demonstrated that CC-loaded Acupa10-CPs mediated efficient delivery of protein drugs into PSMA positive LNCaP cells leading to pronounced antitumor effect, in contrast to their non-targeting counterparts and free CC. Remarkably, granzyme B (GrB)-loaded Acupa10-CPs caused effective apoptosis of LNCaP cells with a low half-maximal inhibitory concentration (IC50) of 1.6 nM. Flow cytometry and CLSM studies using MitoCapture™ revealed obvious depletion of mitochondria membrane potential in LNCaP cells treated with GrB-loaded Acupa10-CPs. The preliminary in vivo experiments showed that Acupa-CPs had a long circulation time with an elimination phase half-life of 3.3h in nude mice. PSMA-targeted, pH-responsive, and chimaeric polymersomes have appeared as efficient protein nanocarriers for targeted prostate cancer therapy.
Assuntos
Antígenos de Superfície/metabolismo , Antineoplásicos/administração & dosagem , Citocromos c/administração & dosagem , Portadores de Fármacos , Glutamato Carboxipeptidase II/metabolismo , Granzimas/administração & dosagem , Neoplasias Pancreáticas/tratamento farmacológico , Polímeros/química , Animais , Antígenos de Superfície/química , Antineoplásicos/química , Antineoplásicos/farmacocinética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Química Farmacêutica , Citocromos c/química , Citocromos c/farmacocinética , Relação Dose-Resposta a Droga , Glutamato Carboxipeptidase II/química , Granzimas/química , Granzimas/farmacocinética , Meia-Vida , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Nus , Nanopartículas , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Polietilenoglicóis/química , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Solubilidade , Succinatos/químicaRESUMO
Growing interest in natural killer (NK) cell-based therapy for treating human cancer has made it imperative to develop new tools to measure early events in cell death. We recently demonstrated that protease-cleavable luciferase biosensors detect granzyme B and pro-apoptotic caspase activation within minutes of target cell recognition by murine cytotoxic lymphocytes. Here we report successful adaptation of the biosensor technology to assess perforin-dependent and -independent induction of death pathways in tumor cells recognized by human NK cell lines and primary cells. Cell-cell signaling via both Fc receptors and NK-activating receptors led to measurable luciferase signal within 15 minutes. In addition to the previously described aspartase-cleavable biosensors, we report development of granzyme A and granzyme K biosensors, for which no other functional reporters are available. The strength of signaling for granzyme biosensors was dependent on perforin expression in IL-2-activated NK effectors. Perforin-independent induction of apoptotic caspases was mediated by death receptor ligation and was detectable after 45 minutes of conjugation. Evidence of both FasL and TRAIL-mediated signaling was seen after engagement of Jurkat cells by perforin-deficient human cytotoxic lymphocytes. Although K562 cells have been reported to be insensitive to TRAIL, robust activation of pro-apoptotic caspases by NK cell-derived TRAIL was detectable in K562 cells. These studies highlight the sensitivity of protease-cleaved luciferase biosensors to measure previously undetectable events in live cells in real time. Further development of caspase and granzyme biosensors will allow interrogation of additional features of granzyme activity in live cells including localization, timing, and specificity.
Assuntos
Apoptose/fisiologia , Técnicas Biossensoriais , Granzimas/imunologia , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Caspases/imunologia , Linhagem Celular Tumoral , Ativação Enzimática/fisiologia , Citometria de Fluxo , Granzimas/administração & dosagem , Humanos , Immunoblotting , Células Jurkat , Células K562 , Proteínas Recombinantes , TransfecçãoRESUMO
In spite of their high potency and specificity, few protein drugs have advanced to the clinical settings due to lack of safe and efficient delivery vehicles. Here, novel anisamide-decorated pH-sensitive degradable chimaeric polymersomes (Anis-CPs) were designed, prepared, and investigated for efficient and targeted delivery of apoptotic protein, granzyme B (GrB), to lung cancer cells. Anis-CPs were readily prepared with varying Anis surface densities from anisamide end-capped poly(ethylene glycol)-b-poly(2,4,6- trimethoxybenzylidene-1,1,1-tris(hydroxymethyl)ethane methacrylate)-b-poly(acrylic acid) (Anis-PEG-PTTMA-PAA) and PEG-PTTMA-PAA copolymers. Using cytochrome C (CC) as a model protein, Anis-CPs displayed high protein loading efficiencies (40.5-100%) and loading contents (up to 16.8 wt %). CC-loaded Anis-CPs had narrow distribution (PDI 0.04-0.13) and small sizes ranging from 152 to 171 nm, which increased with increasing CC contents. Notably, the release of proteins from Anis-CPs was accelerated under mildly acidic conditions, due to the hydrolysis of acetal bonds in PTTMA. MTT assays showed that GrB-loaded Anis-CPs (GrB-Anis-CPs) displayed high targetability to sigma receptor overexpressing cancer cells such as H460 and PC-3 cells. For example, GrB-Anis-CPs exhibited increasing antitumor efficacy to H460 cells with increasing Anis contents from 0 to 80%. The antitumor activity of GrB-Anis-CPs was significantly reduced upon pretreating H460 cells with haloperidol (a competitive antagonist). Notably, the half-maximal inhibitory concentrations (IC50) of GrB-Anis70-CPs were determined to be 6.25 and 5.94 nM for H460 and PC-3 cells, respectively, which were 2-3 orders of magnitude lower than that of chemotherapeutic drugs, such as paclitaxel. Flow cytometry studies demonstrated that GrB-Anis70-CPs induced widespread apoptosis of H460 cells. The confocal laser scanning microscopy (CLSM) experiments using FITC-labeled CC-loaded Anis-CPs confirmed fast internalization and intracellular protein release into H460 cells. GrB-Anis-CPs with high potency and specificity are particularly interesting for targeted therapy of lung cancers.
Assuntos
Benzamidas/química , Plásticos Biodegradáveis/química , Portadores de Fármacos/química , Células Epiteliais/efeitos dos fármacos , Granzimas/administração & dosagem , Plásticos Biodegradáveis/síntese química , Linhagem Celular Tumoral , Portadores de Fármacos/síntese química , Granzimas/farmacologia , Humanos , Metacrilatos/química , Polietilenoglicóis/químicaRESUMO
CMML (chronic myelomonocytic leukemia) belongs to the group of myeloid neoplasms known as myelodysplastic and myeloproliferative diseases. In some patients with a history of CMML, the disease transforms to acute myelomonocytic leukemia (AMML). There are no specific treatment options for patients suffering from CMML except for supportive care and DNA methyltransferase inhibitors in patients with advanced disease. New treatment strategies are urgently required, so we have investigated the use of immunotherapeutic directed cytolytic fusion proteins (CFPs), which are chimeric proteins comprising a selective domain and a toxic component (preferably of human origin to avoid immunogenicity). The human serine protease granzyme B is a prominent candidate for tumor immunotherapy because it is expressed in cytotoxic T lymphocytes and natural killer cells. Here, we report the use of CD64 as a novel target for specific CMML and AMML therapy, and correlate CD64 expression with typical surface markers representing these diseases. We demonstrate that CD64-specific human CFPs kill CMML and AMML cells ex vivo, and that the mutant granzyme B protein R201K is more cytotoxic than the wild-type enzyme in the presence of the granzyme B inhibitor PI9. Besides, the human CFP based on the granzyme B mutant was also able to kill AMML or CMML probes resistant to Pseudomonas exotoxin A.
Assuntos
Granzimas/administração & dosagem , Leucemia Mielomonocítica Aguda/tratamento farmacológico , Leucemia Mielomonocítica Crônica/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Estabilidade de Medicamentos , Feminino , Células HL-60 , Humanos , Imunotoxinas/administração & dosagem , Imunotoxinas/sangue , Imunotoxinas/imunologia , Imunotoxinas/farmacocinética , Leucemia Mielomonocítica Aguda/imunologia , Leucemia Mielomonocítica Crônica/imunologia , Masculino , Pessoa de Meia-Idade , Receptores de IgG/biossíntese , Receptores de IgG/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacocinética , Anticorpos de Cadeia Única/administração & dosagem , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacocinéticaRESUMO
The serine protease granzyme B (GrB) induces apoptosis through both caspase-dependent and -independent multiple-cascade mechanisms. VEGF121 binds to both VEGF receptor (VEGFR)-1 and VEGFR-2 receptors. We engineered a unique GrB/VEGF121 fusion protein and characterized its properties in vitro and in vivo. Endothelial and tumor cell lines showed varying levels of sensitivity to GrB/VEGF121 that correlated closely to total VEGFR-2 expression. GrB/VEGF121 localized efficiently into VEGFR-2-expressing cells, whereas the internalization into VEGFR-1-expressing cells was significantly reduced. Treatment of VEGFR-2(+) cells caused mitochondrial depolarization in 48% of cells by 48 hours. Exposure to GrB/VEGF121 induced apoptosis in VEGFR-2(+), but not in VEGFR-1(+), cells and rapid caspase activation was observed that could not be inhibited by treatment with a pan-caspase inhibitor. In vivo, GrB/VEGF121 localized in perivascular tumor areas adjacent to microvessels and in other areas in the tumor less well vascularized, whereas free GrB did not specifically localize to tumor tissue. Administration (intravenous) of GrB/VEGF121 to mice at doses up to 40 mg/kg showed no toxicity. Treatment of mice bearing established PC-3 tumor xenografts with GrB/VEGF121 showed significant antitumor effect versus treatment with GrB or saline. Treatment with GrB/VEGF121 at 27 mg/kg resulted in the regression of four of five tumors in this group. Tumors showed a two-fold lower Ki-67-labeling index compared with controls. Our results show that targeted delivery of GrB to tumor vascular endothelial cells or to tumor cells activates apoptotic cascades and this completely human construct may have significant therapeutic potential.
Assuntos
Granzimas/genética , Neoplasias/tratamento farmacológico , Proteínas Recombinantes de Fusão/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Administração Intravenosa , Animais , Apoptose/efeitos dos fármacos , Caspases/genética , Caspases/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Granzimas/administração & dosagem , Humanos , Camundongos , Neoplasias/genética , Neoplasias/patologia , Proteínas Recombinantes de Fusão/administração & dosagem , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/administração & dosagem , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/administração & dosagemRESUMO
Immunotoxins containing bacterial or plant toxins have shown promise in cancer-targeted therapy, but their long-term clinical use may be hampered by vascular leak syndrome and immunogenicity of the toxin. We incorporated human granzyme B (GrB) as an effector and generated completely human chimeric fusion proteins containing the humanized anti-Her2/neu single-chain antibody 4D5 (designated GrB/4D5). Introduction of a pH-sensitive fusogenic peptide (designated GrB/4D5/26) resulted in comparatively greater specific cytotoxicity although both constructs showed similar affinity to Her2/neu-positive tumor cells. Compared with GrB/4D5, GrB/4D5/26 showed enhanced and long-lasting cellular uptake and improved delivery of GrB to the cytosol of target cells. Treatment with nanomolar concentrations of GrB/4D5/26 resulted in specific cytotoxicity, induction of apoptosis, and efficient downregulation of PI3K/Akt and Ras/ERK pathways. The endogenous presence of the GrB proteinase inhibitor 9 did not impact the response of cells to the fusion construct. Surprisingly, tumor cells resistant to lapatinib or Herceptin, and cells expressing MDR-1 resistant to chemotherapeutic agents showed no cross-resistance to the GrB-based fusion proteins. Administration (intravenous, tail vein) of GrB/4D5/26 to mice bearing BT474 M1 breast tumors resulted in significant tumor suppression. In addition, tumor tissue excised from GrB/4D5/26-treated mice showed excellent delivery of GrB to tumors and a dramatic induction of apoptosis compared with saline treatment. This study clearly showed that the completely human, functionalized GrB construct can effectively target Her2/neu-expressing cells and displays impressive in vitro and in vivo activity. This construct should be evaluated further for clinical use.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Granzimas/administração & dosagem , Receptor ErbB-2/administração & dosagem , Anticorpos de Cadeia Única/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Camundongos , Terapia de Alvo Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Serina Proteases/administração & dosagem , Serina Proteases/genética , Serina Proteases/imunologia , Transdução de Sinais/efeitos dos fármacos , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologiaRESUMO
PURPOSE: Luteinizing hormone receptor (LHR) is found in abundance on human ovarian, breast, endometrial and prostate carcinomas but at only low levels on non-gonadal tissues. To selectively kill LHR-expressing tumors, granzyme B (GrB) was linked to a protein in which both chains of human chorionic gonadotropin were yoked together (YCG). METHODS: GrB-YCG was expressed and secreted from insect Sf9 cells. Its GrB enzymatic activity and binding affinity for hLHR were then characterized. The differential cytotoxicity of GrB-YCG versus GrB alone was tested in a panel of LHR-expressing tumor cells by SRB assay, and the mechanisms involved in the cell death were investigated by confocal fluorescence microscopy, flow cytometry, and western blot analysis. RESULTS: GrB-YCG was successfully expressed and secreted from Sf9 insect cells and purified from cell culture supernatants. The serine protease activity of GrB-YCG was equivalent to that of human recombinant GrB. An in vitro hormone binding assay revealed that the GrB-YCG molecule also retained the ability to bind to the LHR receptor with an affinity similar to that of native hCG. Upon cell binding, GrB-YCG was rapidly internalized into LHR-expressing human ovarian cancer cells and produced selective and potent tumor cell killing by inducing apoptosis through activation of caspase-3. CONCLUSIONS: These results validate LHR as a therapeutic target and indicate that delivery of the human pro-apoptotic enzyme GrB to tumor cells by yoked hCG has substantial selectivity and therapeutic potential for human tumors that express high levels of LHR such as ovarian carcinomas.