RESUMO
BACKGROUND: Antibody-drug conjugates are an exceptional and useful therapeutic tool for multiple diseases, particularly for cancer treatment. We previously showed that the fusion of the serine protease granzyme B (GrB), the effector molecule or T and B cells, to a binding domain allows the controlled and effective delivery of the cytotoxic payload into the target cell. The production of these constructs induced the formation of high molecular aggregates with a potential impact on the efficacy and safety of the protein. METHODS: Our laboratory designed a new Fn14 targeted fusion construct designated GrB(C210A)-Fc-IT4 which contains a modified GrB payload for improved protein production and preserved biological activity. We assessed the construct's enzymatic activity, as well as in vitro cytotoxicity and internalization into target cells. We also assessed pharmacokinetics, efficacy and toxicology parameters in vivo. RESULTS: GrB(C210A)-Fc-IT4 protein exhibited high affinity and selective cytotoxicity within the nanomolar range when tested against a panel of Fn14-positive human cancer cell lines. The construct rapidly internalized into target cells, activating the caspase cascade and causing mitochondrial membrane depolarization. Pharmacokinetic studies in mice revealed that GrB(C210A)-Fc-IT4 displayed a bi-exponential clearance from plasma with a fast initial clearance (t1/2α=0.36 hour) followed by a prolonged terminal-phase plasma half-life (t1/2ß=35 hours). Mice bearing MDA-MB-231 orthotopic tumor xenografts treated with vehicle or GrB(C210A)-Fc-IT4 construct (QODx5) demonstrated tumor regression and long-term (>80 days) suppression of tumor growth. Treatment of mice bearing established, subcutaneous A549 lung tumors showed impressive, long-term tumor suppression compared with a control group treated with vehicle alone. Administration of GrB(C210A)-Fc-IT4 (100 mg/kg total dose) was well-tolerated by mice and resulted in significant reduction of tumor burden in a lung cancer patient-derived xenograft model. Toxicity studies revealed no statistically significant changes in aspartate transferase, alanine transferase or lactate dehydrogenase in treated mice. Histopathological analysis of tissues from treated mice did not demonstrate any specific drug-related changes. CONCLUSION: GrB(C210A)-Fc-IT4 demonstrated excellent, specific cytotoxicity in vitro and impressive in vivo efficacy with no significant toxicity in normal murine models. These studies show GrB(C210A)-Fc-IT4 is an excellent candidate for further preclinical development.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Granzimas/metabolismo , Receptor de TWEAK/metabolismo , Animais , Feminino , Humanos , Camundongos , Camundongos NusRESUMO
A commonly employed method to determine the function of a particular cell population and to assess its contribution to the overall system in vivo is to selectively deplete that population and observe the effects. Using monoclonal antibodies to deliver toxins to target cells can achieve this with a high degree of efficiency. Here, we describe an in vivo model combining the use of immunotoxins and multidrug resistant (MDR) gene deficient mice so that only MDR deficient cells expressing the target molecule would be depleted while target molecule expressing, but MDR sufficient, cells are spared. This allows targeted depletion at a higher degree of specificity than has been previously achieved. We have applied this technique to study trogocytosis, the intercellular transfer of cell surface molecules, but this principle could also be adapted using technology already available for use in other fields of study.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Citotoxicidade Imunológica/efeitos dos fármacos , Genes MDR/fisiologia , Imunotoxinas/toxicidade , Depleção Linfocítica/métodos , Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/deficiência , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Feminino , Sobrevivência de Enxerto/efeitos dos fármacos , Transplante de Coração , Antígenos de Histocompatibilidade Classe II/imunologia , Imunoconjugados/toxicidade , Fragmentos Fab das Imunoglobulinas/toxicidade , Transplante de Rim , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Proteínas Inativadoras de Ribossomos Tipo 1/toxicidade , Baço/efeitos dos fármacos , Baço/imunologia , Baço/patologia , Tolerância ao Transplante/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 objective of this study was to develop and explore a novel CD133-targeting immunotoxin (IT) for use in combination with the endosomal escape method photochemical internalization (PCI). scFvCD133/rGelonin was recombinantly constructed by fusing a gene (scFvCD133) encoding the scFv that targets both non-glycosylated and glycosylated forms of both human and murine CD133/prominin-1 to a gene encoding the ribosome-inactivating protein (RIP) gelonin (rGelonin). RIP-activity was assessed in a cell-free translation assay. Selective binding and intracellular accumulation of scFvCD133/rGelonin was evaluated by flow cytometry and fluorescence microscopy. PCI of scFvCD133/rGelonin was explored in CD133high and CD133low cell lines and a CD133neg cell line, where cytotoxicity was evaluated by the MTT assay. scFvCD133/rGelonin exhibited superior binding to and a higher accumulation in CD133high cells compared to CD133low cells. No cytotoxic responses were detected in either CD133high or CD133low cells after 72 h incubation with <100 nM scFvCD133/rGelonin. Despite a severe loss in RIP-activity of scFvCD133/rGelonin compared to free rGelonin, PCI of scFvCD133/rGelonin induced log-fold reduction of viability compared to PCI of rGelonin. Strikingly, PCI of scFvCD133/rGelonin exceeded the cytotoxicity of PCI of rGelonin also in CD133low cells. In conclusion, PCI promotes strong cytotoxic activity of the per se non-toxic scFvCD133/rGelonin in both CD133high and CD133low cancer cells.
RESUMO
Interactions between stromal cells and tumor cells pay a major role in cancer growth and progression. This is reflected in the composition of anticancer drugs which includes compounds directed towards the immune system and tumor-vasculature in addition to drugs aimed at the cancer cells themselves. Drug-based treatment regimens are currently designed to include compounds targeting the tumor stroma in addition to the cancer cells. Treatment limiting adverse effects remains, however, one of the major challenges for drug-based therapy and novel tolerable treatment modalities with diverse high efficacy on both tumor cells and stroma is therefore of high interest. It was hypothesized that the vascular targeted fusion toxin VEGF121/rGel in combination with the intracellular drug delivery technology photochemical internalization (PCI) stimulate direct cancer parenchymal cell death in addition to inhibition of tumor perfusion, and that an immune mediated response is relevant for treatment outcome. The aim of the present study was therefore to elucidate the anticancer mechanisms of VEGF121/rGel-PCI. In contrast to VEGF121/rGel monotherapy, VEGF121/rGel-PCI was found to mediate its effect through VEGFR1 and VEGFR2, and a targeted treatment effect was shown on two VEGFR1 expressing cancer cell lines. A cancer parenchymal treatment effect was further indicated on H&E stains of CT26-CL25 and 4â¯T1 tumors. VEGF121/rGel-PCI was shown, by dynamic contrast enhanced MRI, to induce a sustained inhibition of tumor perfusion in both tumor models. A 50% complete remission (CR) of CT26.CL25 colon carcinoma allografts was found in immunocompetent mice while no CR was detected in CT26.CL25 bearing athymic mice. In conclusion, the present report indicate VEGF121/rGel -PCI as a treatment modality with multimodal tumor targeted efficacy that should be further developed towards clinical utilization.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Sistemas de Liberação de Medicamentos , Neoplasias/tratamento farmacológico , Proteínas Inativadoras de Ribossomos Tipo 1/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Animais , Linhagem Celular Tumoral , Feminino , Luz , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
RESUMO
Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism, and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk-associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by the two alleles of a long ncRNA.
Assuntos
Glutaminase/genética , Neoplasias/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Alelos , Processamento Alternativo , Metabolismo Energético , Células HCT116 , Humanos , Neoplasias/genética , Precursores de RNA/química , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismoRESUMO
HER2-targeted therapy has been shown to have limited efficacy in ovarian cancer despite frequent overexpression of this receptor. Photochemical internalization (PCI) is a modality for cytosolic drug delivery, currently undergoing clinical evaluation. In the present project we studied the application of PCI in combination with the HER2-targeted recombinant fusion toxin, MH3-B1/rGel, for the treatment of ovarian cancer. The SKOV-3 cell line, resistant to trastuzumab- and MH3-B1/rGel- monotherapy, was shown to respond strongly to PCI of MH3-B1/rGel to a similar extent as observed for the treatment-sensitive SK-BR-3 breast cancer cells. Extensive hydrolytic degradation of MH3-B1/rGel in acidic endocytic vesicles was indicated as the mechanism of MH3-B1/rGel resistance in SKOV-3 cells. This was shown by the positive Pearson's correlation coefficient between Alexa488-labeled MH3-B1/rGel and Lysotracker in SKOV-3 cells in contrast to the negative Pearson's correlation coefficient in SK-BR-3 cells. The application of PCI to induce the release of MH3-B1/rGel was also demonstrated to be effective on SKOV-3 xenografts. Application of PCI with MH3-B1/rGel was further found highly effective in the HER2 expressing HOC-7 and NuTu-19 ovarian cancer cell lines. The presented results warrant future development of PCI in combination with MH3-B1/rGel as a novel therapeutic approach in preclinical models of ovarian cancer.
Assuntos
Antineoplásicos/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Imunotoxinas/farmacologia , Terapia de Alvo Molecular/métodos , Neoplasias Ovarianas/metabolismo , Receptor ErbB-2/metabolismo , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Western Blotting , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Neoplasias Ovarianas/patologia , Fármacos Fotossensibilizantes/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Cadeia Única/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The cytokine TWEAK and its receptor, Fn14, have emerged as potentially valuable targets for cancer therapy. Granzyme B (GrB)-containing Fn14-targeted constructs were generated containing either the Fn14 ligand TWEAK (GrB-TWEAK) or an anti-Fn14 humanized single-chain antibody (GrB-Fc-IT4) as the targeting moieties. Both constructs showed high affinity and selective cytotoxicity against a panel of Fn14-expressing human tumor cells including triple-negative breast cancer (TNBC) lines. Cellular expression of the GrB inhibitor PI-9 in target cells had no impact on the cytotoxic effect of either construct. Cellular expression of MDR1 showed no cross-resistance to the fusion constructs. GrB-TWEAK and GrB-Fc-IT4 activated intracellular caspase cascades and cytochrome c-related proapoptotic pathways consistent with the known intracellular functions of GrB in target cells. Treatment of mice bearing established HT-29 xenografts with GrB-TWEAK showed significant tumor growth inhibition compared with vehicle alone (P < 0.05). Both GrB-TWEAK and GrB-Fc-IT4 displayed significant tumor growth inhibition when administered to mice bearing orthotopic MDA-MB-231 (TNBC) tumor xenografts. The Cancer Genome Atlas analysis revealed that Fn14 mRNA expression was significantly higher in TNBC and in HER2-positive disease (P < 0.0001) compared with hormone receptor-positive breast cancer, and in basal-like 2 tumors (P = 0.01) compared with other TNBC molecular subtypes. IHC analysis of a 101 patient TNBC tumor microarray showed that 55 of 101 (54%) of tumors stained positive for Fn14, suggesting that this may be an excellent potential target for precision therapeutic approaches. Targeting Fn14 using fully human, GrB-containing fusion constructs may form the basis for a new class of novel, potent, and highly effective constructs for targeted therapeutic applications.
Assuntos
Receptores do Fator de Necrose Tumoral/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Granzimas/genética , Granzimas/farmacologia , Células HEK293 , Células HT29 , Humanos , Células Jurkat , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Terapia de Alvo Molecular , Receptores do Fator de Necrose Tumoral/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacologia , Receptor de TWEAK , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
HER2 is overexpressed in 20-30% of breast tumors and is associated with aggressiveness and increased risk of recurrence and death. The HER2 protein is internalized as a part of its activity, and may therefore be utilized as a target for the specific intracellular delivery of drugs. Photochemical internalization (PCI) is a novel technology now undergoing clinical evaluation for its ability to improve the release into the cytosol of drugs entrapped in the endo/lysosomal compartment. PCI employs an amphiphilic photosensitizer which localizes in the membranes of endo/lysosomes. Subsequent light exposure (visible light) causes destabilization of the endo/lysosomal membranes. PCI has been proven highly effective for improving the cytosolic delivery of targeted toxins based on type I ribosome inactivating protein toxins such as gelonin. We examined the impact of the level of target antigen expression on PCI efficacy. Four human breast cancer cell lines (MDA-MB-231, BT-20, Zr-75-1 and SK-BR-3) covering a wide range of HER2 expression were included in the present study. PCI of the HER2-targeted fusion toxin MH3-B1/rGel was found to be highly effective in all four cell lines. The increase in PCI-mediated efficacy was not directly correlated with the cellular levels of HER2 as assessed by western blots, the overall uptake of MH3-B1/rGel as measured by flow cytometry, the amount of MH3-B1/rGel localized to endo/lysosomes assessed by confocal microscopy or the cell sensitivity to the photochemical treatment itself (photosensitizer and light without MH3-B1/rGel). However, correcting the PCI efficacy for the baseline cellular sensitivity to rGel revealed a linear correlation (R(2)=0.80) with HER2 expression. The present report therefore concludes the cellular sensitivity to the toxin as an important parameter for PCI efficacy and also indicates PCI of a HER2-targeted fusion toxin as an attractive treatment alternative for breast cancer patients with both HER2-low and -high expression.
Assuntos
Anticorpos/administração & dosagem , Imunotoxinas/administração & dosagem , Luz , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Inativadoras de Ribossomos Tipo 1/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Processos Fotoquímicos , Fármacos Fotossensibilizantes/administração & dosagem , Porfirinas/administração & dosagem , Receptor ErbB-2/imunologiaRESUMO
Vascular targeting for cancer is increasingly recognized as a therapeutic strategy although the lack of objective responses and the development of resistance are major limitations for clinically-available drugs. Endothelial targeted toxins exert increased toxicity compared to antiangiogenic drugs and may therefore overcome these limitations. The specificity and toxicity of targeted toxins may be increased by utilization of a drug delivery system which provides selective release of the targeted toxins in the target cells. Photochemical internalization (PCI) is a non-invasive modality which causes translocation into the cytosol of agents that are trapped in endosomes. This study describes the first use of PCI in combination with a recombinant fusion toxin targeting tumor vasculature. Endothelial cells bearing VEGFR2 treated with VEGF121/rGel showed dramatic enhancement of toxicity after PCI utilizing the photosensitizer TPCS2a (Amphinex®). We compared the PCI of VEGF121/rGel to that of bleomycin which is currently under clinical evaluation. The VEGFR2 specificity of VEGF121/rGel was shown to be preserved by the PCI treatment. PCI of VEGF121/rGel was further shown to induce vascular collapse and edema in the invasive areas of CT26.CL25 colon carcinoma tumors as shown by CD31 IHC. Antitumor effects, as assessed by tumor growth delay were found for PCI of VEGF121/rGel and PCI of bleomycin with cure rates of 40% and 33% respectively. PCI of VEGF121/rGel was, however, better tolerated compared to PCI of bleomycin. Thus, PCI of vascular targeted toxins provides higher specificity and increased tolerability compared to PCI of bleomycin and may represent an interesting clinical future for the PCI technology.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Proteínas Recombinantes de Fusão/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/uso terapêutico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular , Linhagem Celular Tumoral , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Fármacos Fotossensibilizantes/administração & dosagem , Fármacos Fotossensibilizantes/uso terapêutico , Porfirinas/administração & dosagem , Porfirinas/uso terapêutico , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Suínos , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
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
The TNF-like weak inducer of apoptosis (TWEAK; TNFSF12) receptor Fn14 (TNFRSF12A) is expressed at low levels in normal tissues but frequently highly expressed in a wide range of tumor types such as lung, melanoma, and breast, and therefore it is a potentially unique therapeutic target for these diverse tumor types. We have generated a recombinant protein containing a humanized, dimeric single-chain anti-fibroblast growth factor-inducible 14-kDa protein (Fn14) antibody fused to recombinant gelonin toxin as a potential therapeutic agent (designated hSGZ). The hSGZ immunotoxin is a highly potent and selective agent that kills Fn14-positive (Fn14(+)) tumor cells in vitro. Treatment of cells expressing the MDR protein MDR1 (ABCB1B) showed no cross-resistance to hSGZ. Induced overexpression of Fn14 levels in MCF7 cells through HER2 (ERBB2) signaling translated to an improved therapeutic index of hSGZ treatment. In combination with trastuzumab, hSGZ showed an additive or synergistic cytotoxic effect on HER2(+)/Fn14(+) breast cancer cell lines. Also, hSGZ treatment inhibited Erb3/Akt signaling in HER2-overexpressing breast cancer cells. Pharmacokinetic studies in mice revealed that hSGZ exhibited a biexponential clearance from plasma with a rapid initial clearance (t1/2α = 1.26 hours) followed by a seven-fold longer plasma half-life (t1/2ß = 7.29 hours). At 24, 48, and 72 hours after injection, uptake of the hSGZ into tumors was 5.1, 4.8, and 4.7%ID/g, with a tumor-to-muscle ratio of 5.6, 6.2, and 9.0, respectively. Therapeutic efficacy studies showed significant tumor inhibition effects using an MDA-MB-231/Luc breast cancer xenograft model. Our findings show that hSGZ is an effective anticancer agent and a potential candidate for clinical studies.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Imunotoxinas/farmacologia , Receptores do Fator de Necrose Tumoral/imunologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Anticorpos Biespecíficos/farmacocinética , Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacocinética , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Feminino , Meia-Vida , Humanos , Imunotoxinas/farmacocinética , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 1/farmacocinética , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Receptor de TWEAK , Trastuzumab , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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
Fibroblast growth factor-inducible protein 14 (Fn14), the cell surface receptor for tumor necrosis factor-like weak inducer of apoptosis (TWEAK), is overexpressed in various human solid tumor types and can be a negative prognostic indicator. We detected Fn14 expression in â¼60% of the melanoma cell lines we tested, including both B-Raf WT and B-Raf(V600E) lines. Tumor tissue microarray analysis indicated that Fn14 expression was low in normal skin, but elevated in 173/190 (92%) of primary melanoma specimens and in 86/150 (58%) of melanoma metastases tested. We generated both a chemical conjugate composed of the recombinant gelonin (rGel) toxin and the anti-Fn14 antibody ITEM-4 (designated ITEM4-rGel) and a humanized, dimeric single-chain antibody of ITEM-4 fused to rGel (designated hSGZ). Both ITEM4-rGel and hSGZ were highly cytotoxic to a panel of different melanoma cell lines. Mechanistic studies showed that both immunotoxins induced melanoma cell necrosis. In addition, these immunotoxins could upregulate the cellular expression of Fn14 and trigger cell-signaling events similar to the Fn14 ligand TWEAK. Finally, treatment of mice bearing human melanoma MDA-MB-435 xenografts with either ITEM4-rGel or hSGZ showed significant tumor growth inhibition compared with controls. We conclude that Fn14 is a therapeutic target in melanoma and the hSGZ construct appears to warrant further development as a therapeutic agent against Fn14-positive melanoma.
Assuntos
Fatores Imunológicos/farmacocinética , Imunotoxinas/farmacocinética , Melanoma/tratamento farmacológico , Receptores do Fator de Necrose Tumoral/imunologia , Neoplasias Cutâneas/tratamento farmacológico , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Desenho de Fármacos , Feminino , Humanos , Fatores Imunológicos/genética , Fatores Imunológicos/imunologia , Imunotoxinas/genética , Imunotoxinas/imunologia , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Ligação Proteica/imunologia , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Neoplasias Cutâneas/imunologia , Receptor de TWEAK , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We conducted a phase 1 study of an anti-CD33 immunotoxin, humanized monoclonal antibody M195 conjugated to recombinant gelonin (HUM-195/rGEL), in patients with relapsed, refractory myeloid leukemias. Twenty-eight patients received the construct intravenously at four dose levels (12, 18, 28 and 40 mg/m(2) per course) in a "3+3" study design. The dose-limiting toxicity was infusion-related allergic reaction including hypoxia and hypotension. The 28 mg/m(2) total dose was considered the maximally tolerated dose. Four patients developed a reduction in peripheral blood blasts of at least 50%. Three patients treated with the 10, 12 and 28 mg/m(2) doses showed a 38-50% reduction in bone marrow blasts. There was normalization of platelets in one patient treated with 40 mg/m(2). Pharmacokinetic analysis demonstrated that the highest blood levels achieved were 200-300 ng/mL which cleared with a half-life of â¼20 hours. Antigenicity was low with one patient at the 12 mg/m(2) dose and one patient at the 18 mg/m(2) dose (2/23, <10%) developing antibodies to the recombinant gelonin component after 28 days. We concluded that HUM-195/rGel can be safely administered in a multi-dose cycle to patients with advanced myeloid malignancies and warrants further investigation.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Imunotoxinas/uso terapêutico , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/patologia , Proteínas Inativadoras de Ribossomos Tipo 1/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunotoxinas/imunologia , Imunotoxinas/farmacologia , Leucemia Mieloide/imunologia , Masculino , Pessoa de Meia-Idade , Proteínas Inativadoras de Ribossomos Tipo 1/imunologia , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Resultado do TratamentoRESUMO
As a part of an ongoing assessment of its mechanism of action, we evaluated the in vivo pharmacokinetics, tissue distribution, toxicity and antitumor efficacy of VEGF(121)/rGel, a novel fusion protein. Pharmacokinetic studies showed that VEGF(121)/rGel cleared from the circulation in a biphasic manner with calculated half-lives of 0.3 and 6h for the alpha and beta phases, respectively. Pharmacokinetic evaluation of (64)Cu-DOTA-VEGF(121)/rGel showed relatively high blood retention 30 min after injection (26.6 ± 1.73% ID/g), dropping to 11.8 ± 2.83% and 0.82 ± 0.11% ID/g at 60 and 240 min post injection, respectively. Tissue uptake studies showed that kidneys, liver and tumor had the highest drug concentrations 48 h after administration. The maximum tolerated dose (MTD), based on a QOD×5 i.v. administration schedule, was found to be 18 mg/kg with an LD(50) of 25mg/kg. Treatment of BALB/c mice with VEGF(121)/rGel at doses up to the MTD caused no alterations in hematologic parameters. However, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) parameters increased in a dose-related manner. The no-observable-adverse-effect-level (NOAEL) was determined to be 20% of the MTD (3.6 mg/kg). VEGF(121)/rGel treatment of mice bearing orthotopically-placed MDA-MB-231 breast tumors caused increased vascular permeability of tumor tissue by 53% compared to saline-treated controls. Immunohistochemical analysis showed significant tumor hypoxia and necrosis as a consequence of vascular damage. In summary, VEGF(121)/rGel appears to be an effective therapeutic agent causing focused damage to tumor vasculature with minimal toxic effects to normal organs. This agent appears to be an excellent candidate for further clinical development.
Assuntos
Fator A de Crescimento do Endotélio Vascular/farmacocinética , Alanina Transaminase/metabolismo , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Aspartato Aminotransferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Distribuição Tecidual , Fator A de Crescimento do Endotélio Vascular/farmacologia , Fator A de Crescimento do Endotélio Vascular/uso terapêuticoRESUMO
Mantle cell lymphoma (MCL) is an incurable, aggressive histo-type of B-cell non-Hodgkin lymphoma associated with both high relapsed rates and relatively short survival. Because MCL over-expresses receptors for B lymphocyte stimulator (BLyS) and displays constitutively active NF-κB, agents targeting these pathways may be of therapeutic relevance in this disease. To investigate the potential clinical use of the rGel/BLyS fusion toxin in combination with bortezomib, we evaluated this fusion toxin for its ability to inhibit MCL growth in severe combined immunodeficiency (SCID) xenograft model. Compared with PBS-treated mice, mice treated with this fusion toxin prolonged both median (84 days vs. 125 days) and overall survival (0% vs. 40%) (p=0.0027). Compared with bortezomib alone-treated mice, mice treated with rGel/BLyS plus bortezomib showed significantly increased median (91 days vs. 158 days) and overall survival (0% vs. 20%) (p=0.0127). Histopathologic analysis of peritoneal intestinal mesentery from MCL-SCID mice showed no demonstrable microscopic lymphomatous involvement at 225 days after treatment with rGel/BLyS. Combination treatment resulted in a synergistic growth inhibition, down-regulation of NF-κB DNA-binding activity, inhibition of cyclin D1, Bcl-x(L), p-Akt, Akt, p-mTOR, and p-Bad, up-regulation of Bax, and induction of cellular apoptosis. Our findings demonstrate that rGel/BLyS is an effective therapeutic agent for both primary and salvage treatment of aggressive MCL expressing constitutively active NF-κB and BLyS receptors and may be an excellent candidate for clinical development.
Assuntos
Antineoplásicos/farmacologia , Fator Ativador de Células B/genética , Linfoma de Célula do Manto/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 1/genética , Toxinas Biológicas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Humanos , Linfoma de Célula do Manto/patologia , Camundongos , Camundongos SCID , NF-kappa B/metabolismo , Transplante de Neoplasias , Inibidores de Proteassoma , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirazinas/farmacologia , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais , Toxinas Biológicas/genética , Transplante HeterólogoRESUMO
Noninvasive imaging techniques have been considered important strategies in the clinic to monitor tumor early response to therapy. In the present study, we applied RGD peptides conjugated to iron oxide nanoparticles (IONP-RGD) as contrast agents in magnetic resonance imaging (MRI) to noninvasively monitor the response of a vascular disrupting agent VEGF(121)/rGel in an orthotopic glioblastoma model. RGD peptides were firstly coupled to IONPs coated with a crosslinked PEGylated amphiphilic triblock copolymer. In vitro binding assays confirmed that cellular uptake of particles was mainly dependent on the interaction between RGD and integrin α(v)ß(3) of human umbilical vein endothelial cells (HUVEC). The tumor targeting of IONP-RGD was observed in an orthotopic U87 glioblastoma model. Finally, noninvasive monitoring of the tumor response to VEGF(121)/rGel therapy at early stages of treatment was successfully accomplished using IONP-RGD as a contrast agent for MRI, a superior method over common anatomical approaches which are based on tumor size measurements. This preclinical study can accelerate anticancer drug development and promote clinical translation of nanoprobes.