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1.
Mol Cancer Ther ; 16(6): 1102-1113, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28292939

RESUMO

Altered expression of the Fas ligand (FasL)/Fas ratio exhibits a direct impact on the prognosis of cancer patients, and its impairment in cancer cells may lead to apoptosis resistance. Thus, the development of effective therapies targeting the FasL/Fas system may play an important role in the fight against cancer. In this study, we evaluated whether a fusion protein (hcc49scFv-FasL) comprising of the cytotoxicity domain of the FasL fused to a humanized antibody (CC49) against tumor-associated glycoprotein 72, which is expressed on oral squamous cell carcinoma (OSCC), can selectively kill OSCC cells with different FasL/Fas ratios. In clinical samples, the significantly low FasL and high Fas transcripts were observed in tumors compared with normal tissues. A lower FasL/Fas ratio was correlated with a worse prognosis of OSCC patients and higher proliferative and invasive abilities of OSCC cells. The hcc49scFv-FasL showed a selective cytotoxic effect on OSCC cells (Cal-27 and SAS) but not on normal oral keratinocytes cells (HOK) through apoptosis induction. Moreover, SAS cells harboring a lower FasL/Fas ratio than Cal-27 were more sensitive to the cytotoxic effect of hcc49scFv-FasL. Unlike wild-type FasL, hcc49scFv-FasL was not cleaved by matrix metalloproteinases and did not induce nonapoptotic signaling in SAS cells. In vivo, we found that hcc49scFv-FasL drastically reduced the formation of lymph node metastasis and decreased primary tumor growth in SAS orthotopic and subcutaneous xenograft tumor models. Collectively, our data indicate that a tumor-targeting antibody fused to the FasL can be a powerful tool for OSCC treatment, especially in populations with a low FasL/Fas ratio. Mol Cancer Ther; 16(6); 1102-13. ©2017 AACR.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Citotoxicidade Celular Dependente de Anticorpos , Carcinoma de Células Escamosas/metabolismo , Proteína Ligante Fas/metabolismo , Glicoproteínas/antagonistas & inibidores , Neoplasias Bucais/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Receptor fas/metabolismo , Animais , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos de Neoplasias/imunologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Proteína Ligante Fas/genética , Glicoproteínas/imunologia , Humanos , Camundongos , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/imunologia , Neoplasias Bucais/patologia , Prognóstico , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
2.
JAMA Dermatol ; 153(5): 413-420, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28199478

RESUMO

Importance: In a phase 1 trial, single-dose O6-benzylguanine with topical carmustine for patients with early stage (stage IA through stage IIA) cutaneous T-cell lymphoma, mycosis fungoides (MF) type, resulted in clinical responses proportional to inhibition of O6-alkylguanine-DNA alkyltransferase activity, but a maximum tolerated dose (MTD) was not reached. Objective: To determine whether dose escalation of carmustine in combination with dual-dose O6-benzylguanine to prolong alkyltransferase inhibition could reach an MTD. Design, Setting, and Participants: A single-arm, phase 1-2 clinical trial conducted at a university teaching hospital enrolled 17 adults with stage IA through stage IIA cutaneous T-cell lymphoma, MF type, to evaluate treatment using topical carmustine plus 2 subsequent daily doses of intravenous O6-benzylguanine, administered every 2 weeks for up to 24 weeks (12 cycles). All patients who received treatment were included in an intent-to-treat analysis of the response rate. The study was conducted from February 17, 2010, to April 8, 2014. Data analysis was performed from May 1, 2014, to December 1, 2015. Interventions: Topical carmustine and intravenous O6-benzylguanine. Main Outcomes and Measures: Clinical disease response was assessed by the Severity-Weighted Assessment Tool (score range, 0-400; higher score indicates worse disease). Safety data were acquired by review of adverse events at study visits. Results: Of the 17 patients enrolled, 12 (71%) were men; mean (SD) age was 45.2 (14.6) years. There were 7 complete responses and 8 partial responses to combination carmustine and O6-benzylguanine treatment. The overall clinical response rate was 88%, with a mean (SD) duration of complete response of 14.43 (6.6) months. The MTD was 20 mg of carmustine applied once in combination with 2 daily doses of 120 mg/m2 of O6-benzylguanine. Most adverse events (112 [67%]) were grade I. Of 15 patients with dermatitis, 5 individuals (33%) demonstrated grade II dermatitis that was unresponsive to topical corticosteroid therapy. The dermatitis was characterized by high levels of macrophage activation, and clearance was associated with vitamin D3 administration. Conclusions and Relevance: Compared with single-dose O6-benzylguanine and carmustine, dual-dose O6-benzylguanine resulted in higher overall response rates and reduced total carmustine doses but was associated with more cutaneous adverse events. The MTD for dual-dose O6-benzylguanine plus carmustine was also ascertained. Trial Registration: clinicaltrials.gov Identifier: NCT00961220.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfoma Cutâneo de Células T/tratamento farmacológico , Micose Fungoide/tratamento farmacológico , O(6)-Metilguanina-DNA Metiltransferase/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Administração Cutânea , Administração Intravenosa , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Carmustina/administração & dosagem , Feminino , Guanina/administração & dosagem , Guanina/análogos & derivados , Hospitais Universitários , Humanos , Linfoma Cutâneo de Células T/patologia , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Micose Fungoide/patologia , Estadiamento de Neoplasias , Neoplasias Cutâneas/patologia , Resultado do Tratamento
3.
J Biomed Sci ; 20: 16, 2013 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-23497165

RESUMO

BACKGROUND: Targeted therapy of human cancers is an attractive approach and has been investigated with limited success. We have developed novel cytotoxic agents for targeted therapy of human cancers based on the extracellular cytotoxicity domain of CD178 (FasL) and the specificity offered by single chain antibodies (scFv) against dominant human tumor Ag TAG-72 (cc49scFv) and TAL6 (L6scFv). RESULTS: The cc49scFv-FasLext is highly effective in in vitro killing of human TAG-72+ Jurkat-Ras tumor cells with a 30,000 fold greater cytotoxicity as compared to soluble FasL (sFasL). On the other hand, L6scFv-FasLext only increased cytotoxicity 500-fold as compared with sFasL against TAL6+ HeLa cells in in vitro assays. The high specificity and strong cytotoxicity of cc49scFv-FasLext made it feasible to cure IP-implanted Jurkat-Ras tumors in SCID mice. CONCLUSION: Our study demonstrated that scFv-FasLext with a strong cytotoxicity against sensitive human tumor targets may be useful as effective chemotherapeutic agents.


Assuntos
Proteína Ligante Fas/genética , Neoplasias/tratamento farmacológico , Anticorpos de Cadeia Única/genética , Animais , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Proteína Ligante Fas/farmacologia , Células HeLa , Humanos , Fragmentos de Imunoglobulinas/imunologia , Células Jurkat , Camundongos , Camundongos SCID , Neoplasias/patologia , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico , Anticorpos de Cadeia Única/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
4.
J Invest Dermatol ; 133(1): 249-57, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22951729

RESUMO

Highly regulated expression of the negative costimulatory molecule cytotoxic T-lymphocyte antigen-4 (CTLA-4) on T cells modulates T-cell activation and proliferation. CTLA-4 is preferentially expressed in Th2 T cells, whose differentiation depends on the transcriptional regulator GATA3. Sézary syndrome (SS) is a T-cell malignancy characterized by Th2 cytokine skewing, impaired T-cell responses, and overexpression of GATA3 and CTLA-4. GATA3 is regulated by phosphorylation and ubiquitination. In SS cells, we detected increased polyubiquitinated proteins and activated GATA3. We hypothesized that proteasome dysfunction in SS T cells may lead to GATA3 and CTLA-4 overexpression. To test this hypothesis, we blocked proteasome function with bortezomib in normal T cells, and observed sustained GATA3 and CTLA-4 upregulation. The increased CTLA-4 was functionally inhibitory in a mixed lymphocyte reaction (MLR). GATA3 directly transactivated the CTLA-4 promoter, and knockdown of GATA3 messenger RNA and protein inhibited CTLA-4 induction mediated by bortezomib. Finally, knockdown of GATA3 in patient's malignant T cells suppressed CTLA-4 expression. Here we demonstrate a new T-cell regulatory pathway that directly links decreased proteasome degradation of GATA3, CTLA-4 upregulation, and inhibition of T-cell responses. We also demonstrate the requirement of the GATA3/CTLA-4 regulatory pathway in fresh neoplastic CD4+ T cells. Targeting of this pathway may be beneficial in SS and other CTLA-4-overexpressing T-cell neoplasms.


Assuntos
Antígeno CTLA-4/metabolismo , Fator de Transcrição GATA3/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Síndrome de Sézary/metabolismo , Neoplasias Cutâneas/metabolismo , Linfócitos T/metabolismo , Idoso , Antineoplásicos/farmacologia , Ácidos Borônicos/farmacologia , Bortezomib , Antígeno CTLA-4/genética , Células Cultivadas , Feminino , Fator de Transcrição GATA3/genética , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Teste de Cultura Mista de Linfócitos , Masculino , Pessoa de Meia-Idade , Poliubiquitina , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/genética , Pirazinas/farmacologia , Síndrome de Sézary/genética , Neoplasias Cutâneas/genética
5.
Arch Dermatol ; 148(5): 613-20, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22250189

RESUMO

OBJECTIVES: To evaluate the toxic effects and maximum tolerated dose of topical carmustine [1,3-bis(2-chloroethyl)-1-nitrosourea] following intravenous O6-benzylguanine in the treatment of cutaneous T-cell lymphoma (CTCL), and to determine pharmacodynamics of O6-alkylguanine DNA alkyltransferase activity in treated CTCL lesions. DESIGN: Open-label, dose-escalation, phase I trial. SETTING: Dermatology outpatient clinic and clinical research unit at a university teaching hospital. PATIENTS: A total of 21 adult patients (11 male, 10 female)with early-stage (IA-IIA) refractory CTCL, mycosis fungoides type, treated with topical carmustine following intravenous O6-benzylguanine. INTERVENTION: Treatment once every 2 weeks with 120 mg/m(2) intravenous O6-benzylguanine followed 1 hour later by whole-body, low-dose topical carmustine starting at 10 mg, with 10-mg incremental dose-escalation in 3 patient cohorts. Cutaneous T-cell lymphoma lesional skin biopsy specimens were taken at baseline and 6 hours, 24 hours, and 1 week after the first O6-benzylguanine infusion for analysis of O6-alkylguanine-DNA alkyltransferase activity. MAIN OUTCOME MEASURES: Clinical response measured by physical examination and severity-weighted assessment tool measurements, safety data acquired by review of adverse events at study visits, and O6-alkylguanine-DNA alkyltransferase activity in treated lesion skin biopsy specimens. RESULTS: A minimal toxic effect was observed through the 40-mg carmustine dose level with 76% of adverse events being grade 1 based on the National Cancer Institute Common Terminology Criteria for Adverse Events. Mean baseline O6-alkylguanine-DNA alkyltransferase activity in CTCL lesions was 3 times greater than in normal controls and was diminished by a median of 100% at 6 and 24 hours following O6-benzylguanine with recovery at 1 week. Clinical disease reduction correlated positively with O6-alkylguanine-DNA alkyltransferase activity at 168 hours (P=.02) and inversely with area under the curve of O6-alkylguanine-DNA alkyltransferase over 1 week (P=.01). Twelve partial responses and 4 complete responses were observed (overall response, 76% [95% CI, 0.55-0.89]). Five patients discontinued therapy owing to adverse events with a possible, probable, or definite relationship to the study drug. CONCLUSION: O6-benzylguanine significantly depletes O6-alkylguanine-DNA alkyltransferase in CTCL lesions and in combination with topical carmustine is well tolerated and shows meaningful clinical responses in CTCL at markedly reduced total carmustine treatment doses.


Assuntos
Carmustina/administração & dosagem , Guanina/análogos & derivados , Micose Fungoide/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Administração Tópica , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Alquilantes/administração & dosagem , Biomarcadores Tumorais/metabolismo , Biópsia , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Inibidores Enzimáticos/administração & dosagem , Feminino , Seguimentos , Guanina/administração & dosagem , Humanos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Micose Fungoide/enzimologia , Micose Fungoide/patologia , O(6)-Metilguanina-DNA Metiltransferase/antagonistas & inibidores , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/patologia , Resultado do Tratamento
8.
BMC Biochem ; 12: 27, 2011 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-21615933

RESUMO

BACKGROUND: Elevated numbers of regulatory T cells (T(regs)) have been implicated in certain cancers. Depletion of T(regs) has been shown to increase anti-tumor immunity. T(regs) also play a critical role in the suppression of autoimmune responses. The study of T(regs) has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32), also known as Glycoprotein A Repetitions Predominant (GARP), has been postulated as a novel surface marker of activated T(regs). However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of T(regs) expressing LRRC32. RESULTS: Using naturally-occurring freshly isolated T(regs), we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ T(regs) are distinct from LRRC32- T(regs) with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32+ T(regs) are more potent suppressors than LRRC32- T(regs). CONCLUSIONS: A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent T(reg) populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of T(regs) and the refinement of immunotherapeutic strategies aimed at targeting these cells.


Assuntos
Regulação da Expressão Gênica , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Sinais Direcionadores de Proteínas , Linfócitos T Reguladores/metabolismo , Animais , Células HEK293 , Humanos , Espaço Intracelular/metabolismo , Selectina L/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Processamento de Proteína Pós-Traducional , Transporte Proteico , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/citologia
9.
J Biol Chem ; 278(9): 7553-7, 2003 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-12477718

RESUMO

The Jo2 anti-mouse CD95 monoclonal antibody induces lethality in mice characterized by hepatocyte death and liver hemorrhage. Mice bearing a defect in Fas expression or in the Fas-mediated apoptotic pathway are resistant to Jo2. Here we show that FcgammaRII knockout mice or mice with monoclonal antibody-blocked FcgammaRII are also resistant to Jo2. The critical FcgammaRII(+) cells are radioresistant and could not be reconstituted with splenic cells. Death of sinusoidal lining cells and destruction of sinusoids were observed, consistent with the characteristic liver hemorrhage and the selective FcgammaRII expression in sinusoidal lining cells but not hepatocytes. Hemorrhage developed coincident with hepatocyte death and the sharp rise of serum alanine aminotransferase and alanine aminotransferase. Invariably, moribund mice showed severe liver hemorrhage and destruction of sinusoids. The data demonstrate a novel mechanism by which the destruction of liver sinusoids, induced by the Jo2-mediated co-engagement of Fas and FcgammaRII, leads to severe hemorrhage and lethal fulminant hepatitis.


Assuntos
Antígenos CD/metabolismo , Falência Hepática/metabolismo , Receptores de IgG/metabolismo , Receptor fas/metabolismo , Alanina Transaminase/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Antígenos CD/química , Apoptose , Morte Celular , Relação Dose-Resposta a Droga , Hepatócitos/metabolismo , Hepatócitos/patologia , Lipopolissacarídeos/metabolismo , Fígado/metabolismo , Fígado/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de IgG/química
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