RESUMO
The trematode Schistosoma mansoni Sm14 antigen was expressed in the yeast Pichia pastoris and secreted into the culture medium at yields of approximately 250 mg L-1. Sm14 belongs to a family of fatty-acid binding proteins and appears to play an important role in uptake, transport, and compartmentalization of lipids in S. mansoni and it is a potential vaccine candidate in both humans and domesticated animals. The Sm14 gene was codon-optimized for expression in P. pastoris, and placed under transcription of the strong methanol inducible AOX1 promoter. Mut+ transformants were selected and used in fed-batch cultivation using a 2.5L fermenter equipped with an on-line methanol control system in order to maintain constant methanol levels during induction. Optimal conditions for the expression of Sm14 by P. pastoris were found to be: dissolved oxygen at 40%, temperature of 25 °C, pH 5.0, and a constant methanol concentration of 1 gL-1. Our results show that a correctly processed Sm14 was secreted into the culture medium at levels of approximately 250 mg L-1. Sm14 from clarified culture medium was purified using a two-step procedure: anion-exchange chromatography followed by hydrophobic interaction chromatography, resulting in >95% purity with a final yield of 40% from the starting cell culture medium. This product has been tested in preliminary clinical trials and shown to elicit an antibody response with no adverse reactions.
Assuntos
Antígenos de Helmintos , Proteínas de Transporte de Ácido Graxo , Expressão Gênica , Proteínas de Helminto , Pichia/metabolismo , Schistosoma mansoni/genética , Vacinas , Animais , Antígenos de Helmintos/biossíntese , Antígenos de Helmintos/genética , Antígenos de Helmintos/imunologia , Antígenos de Helmintos/isolamento & purificação , Proteínas de Transporte de Ácido Graxo/biossíntese , Proteínas de Transporte de Ácido Graxo/genética , Proteínas de Transporte de Ácido Graxo/imunologia , Proteínas de Transporte de Ácido Graxo/isolamento & purificação , Proteínas de Helminto/biossíntese , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Proteínas de Helminto/isolamento & purificação , Humanos , Pichia/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Schistosoma mansoni/imunologia , Vacinas/biossíntese , Vacinas/genética , Vacinas/imunologia , Vacinas/isolamento & purificaçãoRESUMO
The development of cancer immunotherapy has long been a challenge. Here, we report that prophylactic vaccination with a highly attenuated Trypanosoma cruzi strain expressing NY-ESO-1 (CL-14-NY-ESO-1) induces both effector memory and effector CD8(+) T lymphocytes that efficiently prevent tumor development. However, the therapeutic effect of such a vaccine is limited. We also demonstrate that blockade of Cytotoxic T Lymphocyte Antigen 4 (CTLA-4) during vaccination enhances the frequency of NY-ESO-1-specific effector CD8(+) T cells producing IFN-γ and promotes lymphocyte migration to the tumor infiltrate. As a result, therapy with CL-14-NY-ESO-1 together with anti-CTLA-4 is highly effective in controlling the development of an established melanoma.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/imunologia , Vacinas Anticâncer/imunologia , Imunoterapia/métodos , Melanoma Experimental/terapia , Proteínas de Membrana/imunologia , Animais , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/genética , Linfócitos T CD8-Positivos/parasitologia , Antígeno CTLA-4/antagonistas & inibidores , Feminino , Humanos , Melanoma Experimental/imunologia , Melanoma Experimental/parasitologia , Proteínas de Membrana/administração & dosagem , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologiaRESUMO
PURPOSE: The molecular specificity of monoclonal antibodies (mAbs) directed against tumor antigens has proven effective for targeted therapy of human cancers, as shown by a growing list of successful antibody-based drug products. We describe a novel, nonlinear compartmental model using PET-derived data to determine the "best-fit" parameters and model-derived quantities for optimizing biodistribution of intravenously injected (124)I-labeled antitumor antibodies. METHODS: As an example of this paradigm, quantitative image and kinetic analyses of anti-A33 humanized mAb (also known as "A33") were performed in 11 colorectal cancer patients. Serial whole-body PET scans of (124)I-labeled A33 and blood samples were acquired and the resulting tissue time-activity data for each patient were fit to a nonlinear compartmental model using the SAAM II computer code. RESULTS: Excellent agreement was observed between fitted and measured parameters of tumor uptake, "off-target" uptake in bowel mucosa, blood clearance, tumor antigen levels, and percent antigen occupancy. CONCLUSION: This approach should be generally applicable to antibody-antigen systems in human tumors for which the masses of antigen-expressing tumor and of normal tissues can be estimated and for which antibody kinetics can be measured with PET. Ultimately, based on each patient's resulting "best-fit" nonlinear model, a patient-specific optimum mAb dose (in micromoles, for example) may be derived.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias Colorretais/diagnóstico por imagem , Neoplasias Colorretais/tratamento farmacológico , Modelos Biológicos , Terapia de Alvo Molecular , Tomografia por Emissão de Pósitrons , Medicina de Precisão , Animais , Anticorpos Monoclonais/metabolismo , Neoplasias Colorretais/patologia , Humanos , Radioisótopos do Iodo , Cinética , CamundongosRESUMO
Recombinant poxviruses (vaccinia and fowlpox) expressing tumor-associated antigens are currently being evaluated in clinical trials as cancer vaccines to induce tumor-specific immune responses that will improve clinical outcome. To test whether a diversified prime and boost regimen targeting NY-ESO-1 will result in clinical benefit, we conducted two parallel phase II clinical trials of recombinant vaccinia-NY-ESO-1 (rV-NY-ESO-1), followed by booster vaccinations with recombinant fowlpox-NY-ESO-1 (rF-NY-ESO-1) in 25 melanoma and 22 epithelial ovarian cancer (EOC) patients with advanced disease who were at high risk for recurrence/progression. Integrated NY-ESO-1-specific antibody and CD4(+) and CD8(+) T cells were induced in a high proportion of melanoma and EOC patients. In melanoma patients, objective response rate [complete and partial response (CR+PR)] was 14%, mixed response was 5%, and disease stabilization was 52%, amounting to a clinical benefit rate (CBR) of 72% in melanoma patients. The median PFS in the melanoma patients was 9 mo (range, 0-84 mo) and the median OS was 48 mo (range, 3-106 mo). In EOC patients, the median PFS was 21 mo (95% CI, 16-29 mo), and median OS was 48 mo (CI, not estimable). CD8(+) T cells derived from vaccinated patients were shown to lyse NY-ESO-1-expressing tumor targets. These data provide preliminary evidence of clinically meaningful benefit for diversified prime and boost recombinant pox-viral-based vaccines in melanoma and ovarian cancer and support further evaluation of this approach in these patient populations.
Assuntos
Antígenos de Neoplasias/imunologia , Vetores Genéticos/genética , Melanoma/imunologia , Proteínas de Membrana/imunologia , Neoplasias Ovarianas/imunologia , Vacinação , Vacinas Sintéticas/imunologia , Vaccinia virus/genética , Formação de Anticorpos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Vírus da Varíola das Aves Domésticas/genética , Humanos , Melanoma/patologia , Neoplasias Ovarianas/patologia , Resultado do TratamentoRESUMO
The prognosis of head and neck squamous cell carcinoma (HNSCC) patients remains poor. The identification of high-risk subgroups is needed for the development of custom-tailored therapies. The expression of cancer-testis antigens (CTAs) has been linked to a worse prognosis in other cancer types; however, their prognostic value in HNSCC is unclear because only few patients have been examined and data on CTA protein expression are sparse. A tissue microarray consisting of tumor samples from 453 HNSCC patients was evaluated for the expression of CTA proteins using immunohistochemistry. Frequency of expression and the subcellular expression pattern (nuclear, cytoplasmic, or both) was recorded. Protein expression of melanoma antigen (MAGE)-A family CTA, MAGE-C family CTA and NY-ESO-1 was found in approximately 30, 7 and 4% of tumors, respectively. The subcellular expression pattern in particular had a marked impact on the patients' prognosis. Median overall survival (OS) of patients with (i) simultaneous cytoplasmic and nuclear expression compared to (ii) either cytoplasmic or nuclear expression and (iii) negative patients was 23.0 versus 109.0 versus 102.5 months, for pan-MAGE (p < 0.0001), 46.6 versus 50.0 versus 109.0 for MAGE-A3/A4 (p = 0.0074) and 13.3 versus 50.0 versus 100.2 months for NY-ESO-1 (p = 0.0019). By multivariate analysis, these factors were confirmed as independent markers for poor survival. HNSCC patients showing protein expression of MAGE-A family members or NY-ESO-1 represent a subgroup with an extraordinarily poor survival. The development of immunotherapeutic strategies targeting these CTA may, therefore, be a promising approach to improve the outcome of HNSCC patients.
Assuntos
Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/imunologia , Biomarcadores Tumorais/biossíntese , Carcinoma de Células Escamosas/mortalidade , Neoplasias de Cabeça e Pescoço/mortalidade , Antígenos Específicos de Melanoma/biossíntese , Proteínas de Membrana/biossíntese , Proteínas de Neoplasias/imunologia , Adulto , Idoso , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/radioterapia , Citoplasma/imunologia , Feminino , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/imunologia , Prognóstico , Risco , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
Cancer vaccines have yet to yield clinical benefit, despite the measurable induction of humoral and cellular immune responses. As immunosuppression by CD4(+) CD25(+) regulatory T (Treg) cells has been linked to the failure of cancer immunotherapy, blocking suppression is therefore critical for successful clinical strategies. Here, we addressed whether a lyophilized preparation of Streptococcus pyogenes (OK-432), which stimulates Toll-like receptors, could overcome Treg-cell suppression of CD4(+) T-cell responses in vitro and in vivo. OK-432 significantly enhanced in vitro proliferation of CD4(+) effector T cells by blocking Treg-cell suppression and this blocking effect depended on IL-12 derived from antigen-presenting cells. Direct administration of OK-432 into tumor-associated exudate fluids resulted in a reduction of the frequency and suppressive function of CD4(+) CD25(+) Foxp3(+) Treg cells. Furthermore, when OK-432 was used as an adjuvant of vaccination with HER2 and NY-ESO-1 for esophageal cancer patients, NY-ESO-1-specific CD4(+) T-cell precursors were activated, and NY-ESO-1-specific CD4(+) T cells were detected within the effector/memory T-cell population. CD4(+) T-cell clones from these patients had high-affinity TCRs and recognized naturally processed NY-ESO-1 protein presented by dendritic cells. OK-432 therefore inhibits Treg-cell function and contributes to the activation of high-avidity tumor antigen-specific naive T-cell precursors.
Assuntos
Terapia de Imunossupressão , Streptococcus pyogenes/imunologia , Linfócitos T Reguladores/imunologia , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/imunologia , Antígenos CD4/metabolismo , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Exsudatos e Transudatos/imunologia , Humanos , Interleucina-12/farmacologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Proteínas de Membrana/administração & dosagem , Proteínas de Membrana/imunologia , Neoplasias/imunologia , Picibanil/administração & dosagem , Picibanil/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismoRESUMO
Tumor Ag-specific CD4(+) T cells play important functions in tumor immunosurveillance, and in certain cases they can directly recognize HLA class II-expressing tumor cells. However, the underlying mechanism of intracellular Ag presentation to CD4(+) T cells by tumor cells has not yet been well characterized. We analyzed two naturally occurring human CD4(+) T cell lines specific for different peptides from cytosolic tumor Ag NY-ESO-1. Whereas both lines had the same HLA restriction and a similar ability to recognize exogenous NY-ESO-1 protein, only one CD4(+) T cell line recognized NY-ESO-1(+) HLA class II-expressing melanoma cells. Modulation of Ag processing in melanoma cells using specific molecular inhibitors and small interfering RNA revealed a previously undescribed peptide-selective Ag-presentation pathway by HLA class II(+) melanoma cells. The presentation required both proteasome and endosomal protease-dependent processing mechanisms, as well as cytosolic heat shock protein 90-mediated chaperoning. Such tumor-specific pathway of endogenous HLA class II Ag presentation is expected to play an important role in immunosurveillance or immunosuppression mediated by various subsets of CD4(+) T cells at the tumor local site. Furthermore, targeted activation of tumor-recognizing CD4(+) T cells by vaccination or adoptive transfer could be a suitable strategy for enhancing the efficacy of tumor immunotherapy.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Proteínas de Choque Térmico HSP90/imunologia , Peptídeos/imunologia , Apresentação de Antígeno , Antígenos de Neoplasias/genética , Linfócitos T CD4-Positivos/citologia , Linhagem Celular Tumoral , Clonagem Molecular , Citocinas/biossíntese , Citocinas/imunologia , Citosol/imunologia , Citosol/metabolismo , Endossomos/genética , Endossomos/imunologia , Epitopos de Linfócito T/genética , Feminino , Proteínas de Choque Térmico HSP90/genética , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Ativação Linfocitária/imunologia , Melanoma/imunologia , Melanoma/patologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Peptídeos/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/imunologia , RNA Interferente Pequeno/genéticaRESUMO
One of the main challenges in cancer research is the development of vaccines that induce effective and long-lived protective immunity against tumors. Significant progress has been made in identifying members of the cancer testis antigen family as potential vaccine candidates. However, an ideal form for antigen delivery that induces robust and sustainable antigen-specific T-cell responses, and in particular of CD8(+) T lymphocytes, remains to be developed. Here we report the use of a recombinant nonpathogenic clone of Trypanosoma cruzi as a vaccine vector to induce vigorous and long-term T cell-mediated immunity. The rationale for using the highly attenuated T. cruzi clone was (i) the ability of the parasite to persist in host tissues and therefore to induce a long-term antigen-specific immune response; (ii) the existence of intrinsic parasite agonists for Toll-like receptors and consequent induction of highly polarized T helper cell type 1 responses; and (iii) the parasite replication in the host cell cytoplasm, leading to direct antigen presentation through the endogenous pathway and consequent induction of antigen-specific CD8(+) T cells. Importantly, we found that parasites expressing a cancer testis antigen (NY-ESO-1) were able to elicit human antigen-specific T-cell responses in vitro and solid protection against melanoma in a mouse model. Furthermore, in a therapeutic protocol, the parasites expressing NY-ESO-1 delayed the rate of tumor development in mice. We conclude that the T. cruzi vector is highly efficient in inducing T cell-mediated immunity and protection against cancer cells. More broadly, this strategy could be used to elicit a long-term T cell-mediated immunity and used for prophylaxis or therapy of chronic infectious diseases.
Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Proteínas de Membrana/imunologia , Trypanosoma cruzi/imunologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Western Blotting , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/parasitologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/parasitologia , Vacinas Anticâncer/administração & dosagem , Linhagem Celular Tumoral , Células Cultivadas , Doença de Chagas/imunologia , Doença de Chagas/parasitologia , Doença de Chagas/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos/administração & dosagem , Vetores Genéticos/imunologia , Humanos , Imunização/métodos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Neoplasias Experimentais/prevenção & controle , Transfecção/métodos , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismoRESUMO
Ipilimumab, a monoclonal antibody against cytotoxic T lymphocyte antigen 4 (CTLA-4), has been shown to improve survival in patients with advanced metastatic melanoma. It also enhances immunity to NY-ESO-1, a cancer/testis antigen expressed in a subset of patients with melanoma. To characterize the association between immune response and clinical outcome, we first analyzed NY-ESO-1 serum antibody by ELISA in 144 ipilimumab-treated patients with melanoma and found 22 of 140 (16%) seropositive at baseline and 31 of 144 (22%) seropositive following treatment. These NY-ESO-1-seropositive patients had a greater likelihood of experiencing clinical benefit 24 wk after ipilimumab treatment than NY-ESO-1-seronegative patients (P = 0.02, relative risk = 1.8, two-tailed Fisher test). To understand why some patients with NY-ESO-1 antibody failed to experience clinical benefit, we analyzed NY-ESO-1-specific CD4(+) and CD8(+) T-cell responses by intracellular multicytokine staining in 20 NY-ESO-1-seropositive patients and found a surprising dissociation between NY-ESO-1 antibody and CD8 responses in some patients. NY-ESO-1-seropositive patients with associated CD8(+) T cells experienced more frequent clinical benefit (10 of 13; 77%) than those with undetectable CD8(+) T-cell response (one of seven; 14%; P = 0.02; relative risk = 5.4, two-tailed Fisher test), as well as a significant survival advantage (P = 0.01; hazard ratio = 0.2, time-dependent Cox model). Together, our data suggest that integrated NY-ESO-1 immune responses may have predictive value for ipilimumab treatment and argue for prospective studies in patients with established NY-ESO-1 immunity. The current findings provide a strong rationale for the clinical use of modulators of immunosuppression with concurrent approaches to favor tumor antigen-specific immune responses, such as vaccines or adoptive transfer, in patients with cancer.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/imunologia , Melanoma/imunologia , Proteínas de Membrana/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Ipilimumab , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Análise de SobrevidaRESUMO
Specific targets of cellular immunity in human premalignancy are largely unknown. Monoclonal gammopathy of undetermined significance (MGUS) represents a precursor lesion to myeloma (MM). We show that antigenic targets of spontaneous immunity in MGUS differ from MM. MGUS patients frequently mount a humoral and cellular immune response against SOX2, a gene critical for self-renewal in embryonal stem cells. Intranuclear expression of SOX2 marks the clonogenic CD138(-) compartment in MGUS. SOX2 expression is also detected in a proportion of CD138(+) cells in MM patients. However, these patients lack anti-SOX2 immunity. Cellular immunity to SOX2 inhibits the clonogenic growth of MGUS cells in vitro. Detection of anti-SOX2 T cells predicts favorable clinical outcome in patients with asymptomatic plasmaproliferative disorders. Harnessing immunity to antigens expressed by tumor progenitor cells may be critical for prevention and therapy of human cancer.
Assuntos
Células-Tronco Embrionárias/imunologia , Proteínas HMGB/imunologia , Paraproteinemias/imunologia , Paraproteinemias/metabolismo , Fatores de Transcrição/imunologia , Biomarcadores , Proliferação de Células , Células Cultivadas , Progressão da Doença , Proteínas HMGB/metabolismo , Saúde , Humanos , Imunoglobulina G/imunologia , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Paraproteinemias/patologia , Paraproteinemias/terapia , Fatores de Transcrição SOXB1 , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Resultado do TratamentoRESUMO
Based on their inability to express argininosuccinate synthetase (ASS), some cancer entities feature the characteristic of L-arginine (Arg) auxotrophy. This inability to intrinsically generate Arg makes them applicable for arginine deiminase (ADI) treatment, an Arg-depleting drug. Arg is also used for the synthesis of endothelial nitric oxide (NO), which mainly confers vasodilatation but is also considered to have a major influence on tumor vascularization. The purpose of this study was to define changes in tumor vasculature in an ADI-treated melanoma xenograft mouse model using the blood pool agent AngioSense 750 and fluorescence molecular tomography (FMT). We used an ASS-negative melanoma xenograft mouse model and subjected it to weekly ADI treatment. Changes in tumor size were measured, and alterations in tumor vasculature were depicted by FMT and CD31 immunohistochemistry (IHC). On ADI treatment and effective antitumor therapy, we observed a drop in NO plasma levels and visualized changes in tumor vascularization with FMT and IHC. ADI treatment in melanoma xenografts has a tumor-reducing effect, which can be noninvasively imaged by quantifying tumor vascularization with FMT and IHC.
Assuntos
Hidrolases/farmacologia , Melanoma/irrigação sanguínea , Melanoma/tratamento farmacológico , Imagem Óptica/métodos , Tomografia/métodos , Animais , Argininossuccinato Sintase/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Camundongos SCID , Imagem Molecular/métodos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Óxido Nítrico/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
During cancer progression, malignant cells may evade immunosurveillance. However, evidence for immunological escape in humans is scarce. We report here the clinical course of a melanoma patient whose initial tumor was positive for the antigens NY-ESO-1, MAGE-C1, and Melan-A. Upon immunization with a recombinant vaccinia/fowlpox NY-ESO-1 construct, the patient experienced a mixed clinical response and spreading of the NY-ESO-1 epitopes in the CD4+ T cell compartment. After NY-ESO-1 protein + CpG immunization, the patient's anti-NY-ESO-1 IgG response increased. Over the following years, progressing lesions were resected and found to be NY-ESO-1-negative while being positive for MAGE-C1, Melan-A, and MHC-I. The fatal, inoperable brain metastasis was analyzed after his death and also proved to be NY-ESO-1-negative, while being positive for MAGE-C1 and Melan-A, as well as MHC-I. We propose that cancer control and cancer escape in this patient were governed by NY-ESO-1-specific immunological pressure. Our findings provide evidence for the existence of immunoediting and immunoescape in this cancer patient.
Assuntos
Antígenos de Neoplasias/imunologia , Melanoma/imunologia , Proteínas de Membrana/imunologia , Humanos , Imuno-Histoquímica , Masculino , Melanoma/patologia , Melanoma/secundário , Pessoa de Meia-IdadeRESUMO
We investigated whether antibodies against intracellular tumor-associated antigens support tumor-specific immunity when administered together with a treatment that destroys the tumor. We propose that released antigens form immune complexes with the antibodies, which are then efficiently taken up by dendritic cells. We cloned the first human monoclonal antibodies against the Cancer/Testis (CT) antigen, NY-ESO-1. We tested whether the monoclonal anti-NY-ESO-1 antibody (12D7) facilitates cross-presentation of a NY-ESO-1-derived epitope by dendritic cells to human CD8+ T cells, and whether this results in the maturation of dendritic cells in vitro. We investigated the efficacy of 12D7 in combination with chemotherapy using BALB/c mice bearing syngeneic CT26 tumors that express intracellular NY-ESO-1. Human dendritic cells that were incubated with NY-ESO-1:12D7 immune complexes efficiently stimulated NY-ESO-1(157-165)/HLA-A2-specific human CD8+ T cells to produce interferon-γ, whereas NY-ESO-1 alone did not. Furthermore, the incubation of dendritic cells with NY-ESO-1:12D7 immune complexes resulted in the maturation of dendritic cells. Treatment of BALB/c mice that bear CT26/NY-ESO-1 tumors with 5-fluorouracil (5-FU) plus 12D7 was significantly more effective than chemotherapy alone. We propose systemic injection of monoclonal antibodies (mAbs) against tumor-associated antigens plus a treatment that promotes the local release of those antigens resulting in immune complex formation as a novel therapeutic modality for cancer.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Antígenos de Neoplasias/imunologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas de Membrana/imunologia , Neoplasias/tratamento farmacológico , Animais , Anticorpos Monoclonais/farmacologia , Diferenciação Celular/efeitos dos fármacos , Clonagem Molecular , Apresentação Cruzada/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Mapeamento de Epitopos , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/imunologia , Neoplasias/patologia , Resultado do TratamentoRESUMO
Melan-A is a cancer testis antigen commonly found in melanoma, and has been shown to stimulate the body's immune response against cancerous cells. We have developed and executed a process utilizing current good manufacturing practices (cGMP) to produce the 6 times-His tagged protein in C41DE3 Escherichia coli for use in Phase I clinical trials. Approximately 11 g of purified Melan-A were produced from a 20 L fed-batch fermentation. Purification was achieved through a three column process utilizing immobilized metal affinity, anion exchange, and cation exchange chromatography with a buffer system optimized for low-solubility, high LPS binding capacity proteins. The host cell proteins, residual DNA, and endotoxin concentration were well below limits for a prescribed dose with a final purity level of 91%.
Assuntos
Vacinas Anticâncer , Histidina/metabolismo , Antígeno MART-1/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Pesquisa Biomédica , Química Farmacêutica , Cromatografia por Troca Iônica , Fermentação , Histidina/química , Histidina/genética , Antígeno MART-1/química , Antígeno MART-1/genética , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Reprodutibilidade dos TestesRESUMO
Immunization of cancer patients with vaccines containing full-length tumor Ags aims to elicit specific Abs and both CD4(+) and CD8(+) T cells. Vaccination with protein Ags, however, often elicits only CD4(+) T cell responses without inducing Ag-specific CD8(+) T cells, as exogenous protein is primarily presented to CD4(+) T cells. Recent data revealed that Ab-mediated targeting of protein Ags to cell surface receptors on dendritic cells could enhance the induction of both CD4(+) and CD8(+) T cells. We investigated in this study if these observations were applicable to NY-ESO-1, a cancer-testis Ag widely used in clinical cancer vaccine trials. We generated two novel targeting proteins consisting of the full-length NY-ESO-1 fused to the C terminus of two human mAbs against the human mannose receptor and DEC-205, both internalizing molecules expressed on APC. These targeting proteins were evaluated for their ability to activate NY-ESO-1-specific human CD4(+) and CD8(+) T cells in vitro. Both targeted NY-ESO-1 proteins rapidly bound to their respective targets on APC. Whereas nontargeted and Ab-targeted NY-ESO-1 proteins similarly activated CD4(+) T cells, cross-presentation to CD8(+) T cells was only efficiently induced by targeted NY-ESO-1. In addition, both mannose receptor and DEC-205 targeting elicited specific CD4(+) and CD8(+) T cells from PBLs of cancer patients. Receptor-specific delivery of NY-ESO-1 to APC appears to be a promising vaccination strategy to efficiently generate integrated and broad Ag-specific immune responses against NY-ESO-1 in cancer patients.
Assuntos
Antígenos CD/metabolismo , Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo , Proteínas de Membrana/imunologia , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Células Clonais , Apresentação Cruzada/genética , Apresentação Cruzada/imunologia , Testes Imunológicos de Citotoxicidade , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Receptor de Manose , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Ligação Proteica/imunologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Autoantibodies, a hallmark of both autoimmunity and cancer, represent an easily accessible surrogate for measuring adaptive immune responses to cancer. Sera can now be assayed for reactivity against thousands of proteins using microarrays, but there is no agreed-upon standard to analyze results. We developed a set of tailored quality control and normalization procedures based on ELISA validation to allow patient comparisons and determination of individual cutoffs for specificity and sensitivity. Sera from 60 patients with pancreatic cancer, 51 patients with ovarian cancer, and 53 age-matched healthy donors were used to assess the binding of IgG antibodies against a panel of >8000 human antigens using protein microarrays and fluorescence detection. The resulting data interpretation led to the definition and ranking of proteins with preferred recognition by the sera from cancer patients in comparison with healthy donors, both by frequency and strength of signal. We found that 202 proteins were preferentially immunogenic in ovarian cancer sera compared to 29 in pancreatic cancer, with few overlaps. Correlates of autoantibody signatures with known tumor expression of corresponding antigens, functional pathways, clinical stage, and outcome were examined. Serological analysis of arrays displaying the complete human proteome (seromics) represents a new era in cancer immunology, opening the way to defining the repertoire of the humoral immune response to cancer.
Assuntos
Neoplasias Ovarianas/sangue , Neoplasias Pancreáticas/sangue , Análise Serial de Proteínas/métodos , Proteoma/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Especificidade de Anticorpos , Autoanticorpos/imunologia , Biomarcadores Tumorais/imunologia , Doadores de Sangue , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Genes Neoplásicos , Humanos , Pessoa de Meia-Idade , Proteínas de Neoplasias/sangue , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/imunologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/imunologia , Reprodutibilidade dos TestesRESUMO
Early detection of colorectal cancer (CRC) is currently based on fecal occult blood testing (FOBT) and colonoscopy, both which can significantly reduce CRC-related mortality. However, FOBT has low-sensitivity and specificity, whereas colonoscopy is labor- and cost-intensive. Therefore, the discovery of novel biomarkers that can be used for improved CRC screening, diagnosis, staging and as targets for novel therapies is of utmost importance. To identify novel CRC biomarkers we utilized representational difference analysis (RDA) and characterized a colon cancer associated transcript (CCAT1), demonstrating consistently strong expression in adenocarcinoma of the colon, while being largely undetectable in normal human tissues (p < 000.1). CCAT1 levels in CRC are on average 235-fold higher than those found in normal mucosa. Importantly, CCAT1 is strongly expressed in tissues representing the early phase of tumorigenesis: in adenomatous polyps and in tumor-proximal colonic epithelium, as well as in later stages of the disease (liver metastasis, for example). In CRC-associated lymph nodes, CCAT1 overexpression is detectable in all H&E positive, and 40.0% of H&E and immunohistochemistry negative lymph nodes, suggesting very high sensitivity. CCAT1 is also overexpressed in 40.0% of peripheral blood samples of patients with CRC but not in healthy controls. CCAT1 is therefore a highly specific and readily detectable marker for CRC and tumor-associated tissues.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias do Colo/genética , Precursores de RNA/genética , RNA Neoplásico/biossíntese , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Pólipos Adenomatosos/diagnóstico , Pólipos Adenomatosos/genética , Adolescente , Sequência de Aminoácidos , Sequência de Bases , Biomarcadores Tumorais/análise , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/patologia , Detecção Precoce de Câncer/métodos , Células HCT116 , Células HT29 , Humanos , Mucosa Intestinal/metabolismo , Linfonodos/metabolismo , Dados de Sequência Molecular , Mucosa/metabolismo , Metástase Neoplásica , Precursores de RNA/análise , RNA Neoplásico/genética , Sensibilidade e Especificidade , Regulação para CimaRESUMO
NY-ESO-1 is a prototypic cancer/testis antigen. In a recent phase I clinical trial, we vaccinated 13 patients bearing NY-ESO-1-expressing tumors with a complex of cholesterol-bearing hydrophobized pullulan (CHP) and NY-ESO-1 protein (CHP-NY-ESO-1) and showed efficient induction of NY-ESO-1 antibody, and CD4 and CD8 T cell responses using peripheral blood from the patients. In our study, we analyzed heteroclitic serological responses in those patients after vaccination. Serological response against 11 tumor antigens including MAGE-A1, MAGE-A3, MAGE-A4, CT7/MAGEC1, CT10/MAGEC2, CT45, CT46/HORMAD1, SOX2, SSX2, XAGE1B and p53 was examined by enzyme-linked immunosorbent assay (ELISA) using sera from ten vaccinated patients. Expression of tumor antigens was determined by reverse transcription-polymerase chain reaction or immunohistochemistry. Eight of nine patients who showed antibody responses against NY-ESO-1 also showed an antibody response against at least 1 of these 11 tumor antigens after vaccination. In one patient, seven tumor antigens were recognized. Specificity analysis of the antibody response by ELISA using control recombinant proteins and synthetic peptides and by Western blot showed that the response was not against His6-tag and/or bacterial products included in a preparation of CHP-NY-ESO-1 used for vaccination. Thus, heteroclitic serological responses appear to be indicative of the overall immune response against the tumor, and their analysis could be useful for immune monitoring in cancer vaccine.
Assuntos
Anticorpos Antineoplásicos/sangue , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Neoplasias Esofágicas/imunologia , Proteínas de Membrana/imunologia , Neoplasias da Próstata/imunologia , Animais , Anticorpos Antineoplásicos/imunologia , Antígenos de Neoplasias/metabolismo , Linhagem Celular Tumoral , Neoplasias Esofágicas/sangue , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias da Próstata/sangueRESUMO
Epidermal Growth Factor Receptor (EGFR) is involved in stimulating the growth of many human tumors, but the success of therapeutic agents has been limited in part by interference from the EGFR on normal tissues. Previously, we reported an antibody (mab806) against a truncated form of EGFR found commonly in gliomas. Remarkably, it also recognizes full-length EGFR on tumor cells but not on normal cells. However, the mechanism for this activity was unclear. Crystallographic structures for Fab:EGFR(287-302) complexes of mAb806 (and a second, related antibody, mAb175) show that this peptide epitope adopts conformations similar to those found in the wtEGFR. However, in both conformations observed for wtEGFR, tethered and untethered, antibody binding would be prohibited by significant steric clashes with the CR1 domain. Thus, these antibodies must recognize a cryptic epitope in EGFR. Structurally, it appeared that breaking the disulfide bond preceding the epitope might allow the CR1 domain to open up sufficiently for antibody binding. The EGFR(C271A/C283A) mutant not only binds mAb806, but binds with 1:1 stoichiometry, which is significantly greater than wtEGFR binding. Although mAb806 and mAb175 decrease tumor growth in xenografts displaying mutant, overexpressed, or autocrine stimulated EGFR, neither antibody inhibits the in vitro growth of cells expressing wtEGFR. In contrast, mAb806 completely inhibits the ligand-associated stimulation of cells expressing EGFR(C271A/C283A). Clearly, the binding of mAb806 and mAb175 to the wtEGFR requires the epitope to be exposed either during receptor activation, mutation, or overexpression. This mechanism suggests the possibility of generating antibodies to target other wild-type receptors on tumor cells.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/imunologia , Receptores ErbB/imunologia , Proteínas de Neoplasias/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antineoplásicos/uso terapêutico , Complexo Antígeno-Anticorpo/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Cristalografia por Raios X , Epitopos , Humanos , Camundongos , Camundongos Nus , Conformação Proteica , Desnaturação Proteica/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Vaccination with cancer/testis Ag MAGE-A3 in the form of recombinant protein often induces specific humoral and cellular immune responses. Although Ag-specific CD4+ T cells following vaccination are detectable by cytokine production after a single in vitro stimulation, their detection before vaccination is difficult because of low frequency. In this study, we have applied a sensitive method using CD154 (CD40L) staining to detect MAGE-A3-specific CD4+ T cells. MAGE-A3-specific T cell responses were analyzed in four healthy donors, two lung cancer patients with spontaneous serum Abs to MAGE-A3, and two baseline seronegative lung cancer patients throughout vaccination with MAGE-A3 protein. MAGE-A3-specific CD4+ T cells were detected in all individuals tested, at low frequency in healthy donors and seronegative cancer patients and higher frequency in patients seropositive for MAGE-A3. Polyclonal expansion of CD154-expressing CD4+ T cells after cell sorting generated a large number of MAGE-A3-specific CD4+ T cell lines from all individuals tested, enabling full characterization of peptide specificity, HLA-restriction, and avidity. Application of this method to cancer patients vaccinated with MAGE-A3 protein with or without adjuvant revealed that protein vaccination induced oligoclonal activation of MAGE-A3-specific CD4+ T cells. It appeared that MAGE-A3 protein vaccination in the presence of adjuvant selectively expanded high avidity CD4+ T cells, whereas high avidity T cells disappeared after multiple vaccinations with MAGE-A3 protein alone.