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1.
J Immunother Cancer ; 12(7)2024 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-38964788

RESUMO

BACKGROUND: OX40 has been widely studied as a target for immunotherapy with agonist antibodies taken forward into clinical trials for cancer where they are yet to show substantial efficacy. Here, we investigated potential mechanisms of action of anti-mouse (m) OX40 and anti-human (h) OX40 antibodies, including a clinically relevant monoclonal antibody (mAb) (GSK3174998) and evaluated how isotype can alter those mechanisms with the aim to develop improved antibodies for use in rational combination treatments for cancer. METHODS: Anti-mOX40 and anti-hOX40 mAbs were evaluated in a number of in vivo models, including an OT-I adoptive transfer immunization model in hOX40 knock-in (KI) mice and syngeneic tumor models. The impact of FcγR engagement was evaluated in hOX40 KI mice deficient for Fc gamma receptors (FcγR). Additionally, combination studies using anti-mouse programmed cell death protein-1 (mPD-1) were assessed. In vitro experiments using peripheral blood mononuclear cells (PBMCs) examining possible anti-hOX40 mAb mechanisms of action were also performed. RESULTS: Isotype variants of the clinically relevant mAb GSK3174998 showed immunomodulatory effects that differed in mechanism; mIgG1 mediated direct T-cell agonism while mIgG2a acted indirectly, likely through depletion of regulatory T cells (Tregs) via activating FcγRs. In both the OT-I and EG.7-OVA models, hIgG1 was the most effective human isotype, capable of acting both directly and through Treg depletion. The anti-hOX40 hIgG1 synergized with anti-mPD-1 to improve therapeutic outcomes in the EG.7-OVA model. Finally, in vitro assays with human peripheral blood mononuclear cells (hPBMCs), anti-hOX40 hIgG1 also showed the potential for T-cell stimulation and Treg depletion. CONCLUSIONS: These findings underline the importance of understanding the role of isotype in the mechanism of action of therapeutic mAbs. As an hIgG1, the anti-hOX40 mAb can elicit multiple mechanisms of action that could aid or hinder therapeutic outcomes, dependent on the microenvironment. This should be considered when designing potential combinatorial partners and their FcγR requirements to achieve maximal benefit and improvement of patient outcomes.


Assuntos
Receptores OX40 , Animais , Humanos , Camundongos , Receptores OX40/agonistas , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Linhagem Celular Tumoral , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Modelos Animais de Doenças
3.
J Immunother Cancer ; 11(11)2023 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-38007239

RESUMO

BACKGROUND: Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent and durable effects in B-cell malignancies. However, antigen loss or downregulation is a frequent cause of resistance. Here, we report development of a novel CAR T-cell therapy product to target CD79b, a pan B-cell antigen, widely expressed in most B-cell lymphomas. METHODS: We generated a novel anti-CD79b monoclonal antibody by hybridoma method. The specificity of the antibody was determined by testing against isogenic cell lines with human CD79b knock-in or knock-out. A single-chain variable fragment derived from the monoclonal antibody was used to make a panel of CD79b-targeting CAR molecules containing various hinge, transmembrane, and co-stimulatory domains. These were lentivirally transduced into primary T cells and tested for antitumor activity in in vitro and in vivo B-cell lymphoma models. RESULTS: We found that the novel anti-CD79b monoclonal antibody was highly specific and bound only to human CD79b and no other cell surface protein. In testing the various CD79b-targeting CAR molecules, superior antitumor efficacy in vitro and in vivo was found for a CAR consisting CD8α hinge and transmembrane domains, an OX40 co-stimulatory domain, and a CD3ζ signaling domain. This CD79b CAR specifically recognized human CD79b-expressing lymphoma cell lines but not CD79b knock-out cell lines. CD79b CAR T cells, generated from T cells from either healthy donors or patients with lymphoma, proliferated, produced cytokines, degranulated, and exhibited robust cytotoxic activity in vitro against CD19+ and CD19- lymphoma cell lines and patient-derived lymphoma tumors relapsing after prior CD19 CAR T-cell therapy. Furthermore, CD79b CAR T cells were highly efficient at eradicating pre-established lymphoma tumors in vivo in three aggressive lymphoma xenograft models, including two cell line-derived xenografts and one patient-derived xenograft. Notably, these CAR T cells did not demonstrate any significant tonic signaling activity or markers of exhaustion. CONCLUSION: Our results indicated that this novel CD79b CAR T-cell therapy product has robust antitumor activity against B-cell lymphomas. These results supported initiation of a phase 1 clinical trial to evaluate this product in patients with relapsed or refractory B-cell lymphomas.


Assuntos
Linfoma de Células B , Receptores de Antígenos Quiméricos , Humanos , Recidiva Local de Neoplasia/tratamento farmacológico , Linfoma de Células B/tratamento farmacológico , Linfócitos T , Anticorpos Monoclonais/metabolismo
4.
Nat Med ; 28(10): 2133-2144, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36175679

RESUMO

Trogocytosis is an active process that transfers surface material from targeted to effector cells. Using multiple in vivo tumor models and clinical data, we report that chimeric antigen receptor (CAR) activation in natural killer (NK) cells promoted transfer of the CAR cognate antigen from tumor to NK cells, resulting in (1) lower tumor antigen density, thus impairing the ability of CAR-NK cells to engage with their target, and (2) induced self-recognition and continuous CAR-mediated engagement, resulting in fratricide of trogocytic antigen-expressing NK cells (NKTROG+) and NK cell hyporesponsiveness. This phenomenon could be offset by a dual-CAR system incorporating both an activating CAR against the cognate tumor antigen and an NK self-recognizing inhibitory CAR that transferred a 'don't kill me' signal to NK cells upon engagement with their TROG+ siblings. This system prevented trogocytic antigen-mediated fratricide, while sparing activating CAR signaling against the tumor antigen, and resulted in enhanced CAR-NK cell activity.


Assuntos
Receptores de Antígenos Quiméricos , Antígenos de Neoplasias , Linhagem Celular Tumoral , Imunoterapia Adotiva/métodos , Células Matadoras Naturais , Receptores de Antígenos Quiméricos/metabolismo , Trogocitose , Evasão Tumoral
5.
Biomolecules ; 12(9)2022 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-36139163

RESUMO

Coenzyme A (CoA) is an essential co-factor at the intersection of diverse metabolic pathways. Cellular CoA biosynthesis is regulated at the first committed step-phosphorylation of pantothenic acid-catalyzed by pantothenate kinases (PANK1,2,3 in humans, PANK3 being the most highly expressed). Despite the critical importance of CoA in metabolism, the differential roles of PANK isoforms remain poorly understood. Our investigations of PANK proteins as potential precision oncology collateral lethality targets (PANK1 is co-deleted as part of the PTEN locus in some highly aggressive cancers) were severely hindered by a dearth of commercial antibodies that can reliably detect endogenous PANK3 protein. While we successfully validated commercial antibodies for PANK1 and PANK2 using CRISPR knockout cell lines, we found no commercial antibody that could detect endogenous PANK3. We therefore set out to generate a mouse monoclonal antibody against human PANK3 protein. We demonstrate that a clone (Clone MDA-299-62A) can reliably detect endogenous PANK3 protein in cancer cell lines, with band-specificity confirmed by CRISPR PANK3 knockout and knockdown cell lines. Sub-cellular fractionation shows that PANK3 is overwhelmingly cytosolic and expressed broadly across cancer cell lines. PANK3 monoclonal antibody MDA-299-62A should prove a valuable tool for researchers investigating this understudied family of metabolic enzymes in health and disease.


Assuntos
Neoplasias , Ácido Pantotênico , Animais , Anticorpos Monoclonais , Coenzima A , Humanos , Camundongos , Medicina de Precisão , Isoformas de Proteínas
6.
Front Immunol ; 13: 794684, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35720386

RESUMO

Immunotherapies such as checkpoint blockade therapies are known to enhance anti-melanoma CD8+ T cell immunity, but only a fraction of patients treated with these therapies achieve durable immune response and disease control. It may be that CD8+ T cells need help from other immune cells to generate effective and long-lasting anti-tumor immunity or that CD8+ T cells alone are insufficient for complete tumor regression and cure. Melanoma contains significant numbers of B cells; however, the role of B cells in anti-melanoma immunity is controversial. In this study, B16 melanoma mouse models were used to determine the role of B cells in anti-melanoma immunity. C57BL/6 mice, B cell knockout (KO) C57BL/6 mice, anti-CD19, and anti-CXCL13 antibody-treated C57BL/6 mice were used to determine treatment efficacy and generation of tumor-specific CD8+ T cells in response to PD-L1 blockade alone or combination with TLR-7/8 activation. Whole transcriptome analysis was performed on the tumors from B cell depleted and WT mice, untreated or treated with anti-PD-L1. Both CD40-positive and CD40-negative B cells were isolated from tumors of TLR-7/8 agonist-treated wild-type mice and adoptively transferred into tumor-bearing B cell KO mice, which were treated with anti-PD-L1 and TLR-7/8 agonist. Therapeutic efficacy was determined in the presence of activated or inactivated B cells. Microarray analysis was performed on TLR-7/8-treated tumors to look for the B cell signatures. We found B cells were required to enhance the therapeutic efficacy of monotherapy with anti-PD-L1 antibody and combination therapy with anti-PD-L1 antibody plus TLR-7/8 agonist. However, B cells were not essential for anti-CTLA-4 antibody activity. Interestingly, CD40-positive but not CD40-negative B cells contributed to anti-melanoma immunity. In addition, melanoma patients' TCGA data showed that the presence of B cell chemokine CXCL13 and B cells together with CD8+ T cells in tumors were strongly associated with improved overall survival. Our transcriptome data suggest that the absence of B cells enhances immune checkpoints expression in the tumors microenvironment. These results revealed the importance of B cells in the generation of effective anti-melanoma immunity in response to PD-1-PD-L1 blockade immunotherapy. Our findings may facilitate the design of more effective anti-melanoma immunotherapy.


Assuntos
Linfócitos T CD8-Positivos , Melanoma Experimental , Animais , Anticorpos/uso terapêutico , Humanos , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos C57BL , Receptor 7 Toll-Like , Microambiente Tumoral
7.
J Biol Chem ; 298(4): 101821, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35283189

RESUMO

Antibodies that target immune checkpoint proteins such as programmed cell death protein 1, programmed death ligand 1, and cytotoxic T-lymphocyte-associated antigen 4 in human cancers have achieved impressive clinical success; however, a significant proportion of patients fail to respond to these treatments. Galectin-9 (Gal-9), a ß-galactoside-binding protein, has been shown to induce T-cell death and facilitate immunosuppression in the tumor microenvironment by binding to immunomodulatory receptors such as T-cell immunoglobulin and mucin domain-containing molecule 3 and the innate immune receptor dectin-1, suggesting that it may have potential as a target for cancer immunotherapy. Here, we report the development of two novel Gal-9-neutralizing antibodies that specifically react with the N-carbohydrate-recognition domain of human Gal-9 with high affinity. We also show using cell-based functional assays that these antibodies efficiently protected human T cells from Gal-9-induced cell death. Notably, in a T-cell/tumor cell coculture assay of cytotoxicity, these antibodies significantly promoted T cell-mediated killing of tumor cells. Taken together, our findings demonstrate potent inhibition of human Gal-9 by neutralizing antibodies, which may open new avenues for cancer immunotherapy.


Assuntos
Anticorpos Neutralizantes , Morte Celular , Galectinas , Linfócitos T , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/farmacologia , Morte Celular/efeitos dos fármacos , Galectinas/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/terapia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Microambiente Tumoral
8.
Methods Mol Biol ; 2435: 73-93, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34993940

RESUMO

Since its inception in 1975, the hybridoma technology revolutionized science and medicine, facilitating discoveries in almost any field from the laboratory to the clinic. Many technological advancements have been developed since then, to create these "magical bullets." Phage and yeast display libraries expressing the variable heavy and light domains of antibodies, single B-cell cloning from immunized animals of different species including humans or in silico approaches, all have rendered a myriad of newly developed antibodies or improved design of existing ones. However, still the majority of these antibodies or their recombinant versions are from hybridoma origin, a preferred methodology that trespass species barriers, due to the preservation of the natural functions of immune cells in producing the humoral response: antigen specific immunoglobulins. Remarkably, this methodology can be reproduced in small laboratories without the need of sophisticate equipment. In this chapter, we will describe the most recent methods utilized by our Monoclonal Antibodies Core Facility at the University of Texas-M.D. Anderson Cancer Center. During the last 10 years, the methods, techniques, and expertise implemented in our core had generated more than 350 antibodies for various applications.


Assuntos
Anticorpos Monoclonais , Linfócitos B , Animais , Anticorpos Monoclonais/genética , Antígenos , Hibridomas , Tecnologia
9.
Leuk Lymphoma ; 63(1): 19-30, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34486917

RESUMO

Plasmacytoid dendritic cells (pDCs) serve as immunoregulatory antigen-presenting cells that play a role in various inflammatory, viral, and malignant conditions. Malignant proliferation of pDCs is implicated in the pathogenesis of certain hematologic cancers, specifically blastic plasmacytoid dendritic cell neoplasm (BPDCN) and acute myelogenous leukemia with clonal expansion of pDC (pDC-AML). In recent years, BPDCN and pDC-AML have been successfully treated with targeted therapy of pDC-specific surface marker, CD123. However, relapsed and refractory BPDCN remains an elusive cancer, with limited therapeutic options. CD303 is another specific surface marker of human pDCs, centrally involved in antigen presentation and immune tolerance. Monoclonal antibodies directed against CD303 have been studied in preclinical models and have achieved disease control in patients with cutaneous lupus erythematosus. We performed a comprehensive review of benign and malignant disorders in which CD303 have been studied, as there may be a potential future CD303-directed therapy for many of these conditions.


Assuntos
Neoplasias Hematológicas , Lectinas Tipo C , Leucemia Mieloide Aguda , Glicoproteínas de Membrana , Receptores Imunológicos , Apresentação de Antígeno , Células Dendríticas , Neoplasias Hematológicas/tratamento farmacológico , Humanos , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico
12.
Clin Cancer Res ; 25(21): 6417-6428, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31332047

RESUMO

PURPOSE: Recent studies demonstrate the role of the tumor microenvironment in tumor progression. However, strategies used to overcome the malignant phenotypes of cancer cells modulated by the microenvironment have not been thoroughly explored. In this study, we evaluated the therapeutic efficacy of a newly developed mAb targeting microfibril-associated protein 5 (MFAP5), which is secreted predominately by cancer-associated fibroblast (CAF), in ovarian and pancreatic cancer models.Experimental Design: MAbs were developed using human MFAP5 recombinant protein as an antigen in mice, and antibodies from hybridoma clones were evaluated for their specificity to human and murine MFAP5. An Octet RED384 system was used to determine the kinetics of binding affinity and the specificity of the antibody clones, which were followed by epitope mapping and functional characterization by in vitro assays. The therapeutic efficacy of a lead anti-MFAP5 antibody clone 130A in tumor suppression was evaluated by ovarian tumor- and pancreatic tumor-bearing mouse models. RESULTS: Three hybridoma clones, which produced antibodies with high affinity and specificity to MFAP5, were selected for functional studies. Antibody clone 130A, which recognizes a common epitope shared between human and murine MFAP5 protein, was further selected for in vivo studies. Results showed that clone 130A downregulated MFAP5-induced collagen production in CAFs, suppressed intratumoral microvessel leakiness, and enhanced paclitaxel bioavailability in both ovarian and pancreatic cancer mouse models. CONCLUSIONS: These data suggest that MFAP5 blockade using an immunologic approach inhibits fibrosis, induces tumor vessel normalization, and enhances chemosensitivity in ovarian and pancreatic cancer, and can be used as a novel therapeutic agent.


Assuntos
Proteínas Contráteis/genética , Fibrose/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intercelular/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Fibroblastos Associados a Câncer/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proteínas Contráteis/antagonistas & inibidores , Progressão da Doença , Feminino , Fibrose/genética , Fibrose/imunologia , Fibrose/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoterapia/métodos , Camundongos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos
13.
J Clin Invest ; 128(11): 4821-4831, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30277474

RESUMO

Plasmacytoid dendritic cells (pDCs) play a key role in antiviral responses by producing type-1 IFNs. However, recent studies showed that pDCs induce immune suppression and promote tumor growth in human ovarian cancer and myeloma. The molecular mechanisms underlying pDC acquisition of these properties are unknown. Here we show that human pDCs activated by CpG inhibited growth and induced apoptosis in myeloma cells via secreted IFN-α, but direct contact with myeloma cells converted pDCs into tumor-promoting cells by suppressing pDC IFN-α production. E-cadherin, expressed on both myeloma cells and pDCs, mediated these effects via a homophilic interaction - activation of E-cadherin signaling upregulated and activated TNFAIP3 to interact with TLR9, resulting in TLR9 ubiquitination and degradation, and inhibition of IFN-α production in pDCs. These findings were supported by an in vivo study in which pDC depletion induced tumor regression and better survival in the Vk*MYC myeloma mouse model. Furthermore, IFNAR1 expression level positively correlated to overall survival of patients with multiple myeloma (MM), and the IFN-α level in patient bone marrow was significantly lower than that in marrow of healthy individuals. This study reveals a novel mechanism underlying how MM tumors educate pDCs in their microenvironment and provides new targets for improving the treatment of MM.


Assuntos
Antígenos CD/imunologia , Caderinas/imunologia , Células Dendríticas/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Tolerância Imunológica , Mieloma Múltiplo/imunologia , Proteínas de Neoplasias/imunologia , Animais , Antígenos CD/genética , Medula Óssea/imunologia , Medula Óssea/patologia , Caderinas/genética , Células Dendríticas/patologia , Feminino , Humanos , Interferon-alfa/genética , Interferon-alfa/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Proteínas de Neoplasias/genética , Oligodesoxirribonucleotídeos/farmacologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/imunologia
14.
Clin Cancer Res ; 24(5): 1114-1123, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29180606

RESUMO

Purpose: mAbs such as anti-CD20 rituximab are proven therapies in B-cell malignancies, yet many patients develop resistance. Novel therapies against alternative targets are needed to circumvent resistance mechanisms. We sought to generate mAbs against human B-cell-activating factor receptor (BAFF-R/TNFRSF13C), which has not yet been targeted successfully for cancer therapy.Experimental Design: Novel mAbs were generated against BAFF-R, expressed as a natively folded cell surface immunogen on mouse fibroblast cells. Chimeric BAFF-R mAbs were developed and assessed for in vitro and in vivo monotherapy cytotoxicity. The chimeric mAbs were tested against human B-cell tumor lines, primary patient samples, and drug-resistant tumors.Results: Chimeric antibodies bound with high affinity to multiple human malignant B-cell lines and induced potent antibody-dependent cellular cytotoxicity (ADCC) against multiple subtypes of human lymphoma and leukemia, including primary tumors from patients who had relapsed after anti-CD20 therapy. Chimeric antibodies also induced ADCC against ibrutinib-resistant and rituximab-insensitive CD20-deficient variant lymphomas, respectively. Importantly, they demonstrated remarkable in vivo growth inhibition of drug-resistant tumor models in immunodeficient mice.Conclusions: Our method generated novel anti-BAFF-R antibody therapeutics with remarkable single-agent antitumor effects. We propose that these antibodies represent an effective new strategy for targeting and treating drug-resistant B-cell malignancies and warrant further development. Clin Cancer Res; 24(5); 1114-23. ©2017 AACR.


Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos Imunológicos/farmacologia , Receptor do Fator Ativador de Células B/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linfoma de Células B/tratamento farmacológico , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antineoplásicos Imunológicos/uso terapêutico , Receptor do Fator Ativador de Células B/genética , Receptor do Fator Ativador de Células B/imunologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/imunologia , Humanos , Hibridomas , Concentração Inibidora 50 , Linfoma de Células B/sangue , Linfoma de Células B/patologia , Camundongos , Camundongos Endogâmicos BALB C , Dobramento de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Cancer Res ; 77(14): 3894-3907, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28566332

RESUMO

Oncolytic viruses selectively lyse tumor cells, disrupt immunosuppression within the tumor, and reactivate antitumor immunity, but they have yet to live up to their therapeutic potential. Immune checkpoint modulation has been efficacious in a variety of cancer with an immunogenic microenvironment, but is associated with toxicity due to nonspecific T-cell activation. Therefore, combining these two strategies would likely result in both effective and specific cancer therapy. To test the hypothesis, we first constructed oncolytic adenovirus Delta-24-RGDOX expressing the immune costimulator OX40 ligand (OX40L). Like its predecessor Delta-24-RGD, Delta-24-RGDOX induced immunogenic cell death and recruit lymphocytes to the tumor site. Compared with Delta-24-RGD, Delta-24-RGDOX exhibited superior tumor-specific activation of lymphocytes and proliferation of CD8+ T cells specific to tumor-associated antigens, resulting in cancer-specific immunity. Delta-24-RGDOX mediated more potent antiglioma activity in immunocompetent C57BL/6 but not immunodeficient athymic mice, leading to specific immune memory against the tumor. To further overcome the immune suppression mediated by programmed death-ligand 1 (PD-L1) expression on cancer cells accompanied with virotherapy, intratumoral injection of Delta-24-RGDOX and an anti-PD-L1 antibody showed synergistic inhibition of gliomas and significantly increased survival in mice. Our data demonstrate that combining an oncolytic virus with tumor-targeting immune checkpoint modulators elicits potent in situ autologous cancer vaccination, resulting in an efficacious, tumor-specific, and long-lasting therapeutic effect. Cancer Res; 77(14); 3894-907. ©2017 AACR.


Assuntos
Vacinas Anticâncer/farmacologia , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/fisiologia , Células A549 , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Glioma/imunologia , Glioma/terapia , Glioma/virologia , Células HEK293 , Humanos , Imunomodulação , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/terapia , Neoplasias Pulmonares/virologia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Melanoma Experimental/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Neoplasias/imunologia , Neoplasias/virologia , Ligante OX40/biossíntese , Ligante OX40/genética , Ligante OX40/imunologia , Vírus Oncolíticos/genética , Vírus Oncolíticos/imunologia
16.
Clin Cancer Res ; 21(13): 3041-51, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25779950

RESUMO

PURPOSE: The IL11 receptor (IL11R) is an established molecular target in primary tumors of bone, such as osteosarcoma, and in secondary bone metastases from solid tumors, such as prostate cancer. However, its potential role in management of hematopoietic malignancies has not yet been determined. Here, we evaluated the IL11R as a candidate therapeutic target in human leukemia and lymphoma. EXPERIMENTAL DESIGN AND RESULTS: First, we show that the IL11R protein is expressed in a variety of human leukemia- and lymphoma-derived cell lines and in a large panel of bone marrow samples from leukemia and lymphoma patients, whereas expression is absent from nonmalignant control bone marrow. Moreover, a targeted peptidomimetic prototype (termed BMTP-11), specifically bound to leukemia and lymphoma cell membranes, induced ligand-receptor internalization mediated by the IL11R, and resulted in a specific dose-dependent cell death induction in these cells. Finally, a pilot drug lead-optimization program yielded a new myristoylated BMTP-11 analogue with an apparent improved antileukemia cell profile. CONCLUSIONS: These results indicate (i) that the IL11R is a suitable cell surface target for ligand-directed applications in human leukemia and lymphoma and (ii) that BMTP-11 and its derivatives have translational potential against this group of malignant diseases.


Assuntos
Antineoplásicos/farmacologia , Leucemia/tratamento farmacológico , Linfoma/tratamento farmacológico , Peptídeos/farmacologia , Receptores de Interleucina-11/antagonistas & inibidores , Sequência de Aminoácidos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Concentração Inibidora 50 , Ligantes , Dados de Sequência Molecular
17.
Mol Cancer Ther ; 14(4): 941-51, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25646014

RESUMO

Anterior gradient 2 (AGR2) promotes cancer growth, metastasis, and resistance to therapy via unknown mechanisms. We investigated the effects of extracellular AGR2 signaling through the orphan glycosylphosphatidylinositol-linked receptor C4.4A in pancreatic ductal adenocarcinoma (PDAC). Proliferation, migration, invasion, and apoptosis were measured using colorimetric, Boyden chamber, and FACS analyses. We developed blocking mAbs against AGR2 and C4.4A and tested their effects, along with siRNAs, on cancer cell functions and on orthotopic tumors in nude mice. Extracellular AGR2 stimulated proliferation, migration, invasion, and chemoresistance of PDAC cell lines. AGR2 interacted with C4.4A in cell lysates and mixtures of recombinant proteins. Knockdown of C4.4A reduced migration and resistance to gemcitabine. PDAC tissues, but not adjacent healthy pancreatic tissues, expressed high levels of AGR2 and C4.4A. AGR2 signaling through C4.4A required laminins 1 or 5 and integrin ß1. Administration of antibodies against AGR2 and C4.4A reduced growth and metastasis and caused regression of aggressive xenograft tumors, leading to increased survival of mice. These data support a model in which AGR2 binds and signals via C4.4A in an autocrine loop and promotes the growth of pancreas tumors in mice. Blocking mAbs against AGR2 and C4.4A may have therapeutic potential against PDAC.


Assuntos
Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Moléculas de Adesão Celular/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Espaço Extracelular/metabolismo , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Expressão Gênica , Humanos , Cadeias beta de Integrinas/metabolismo , Laminina/metabolismo , Camundongos , Mucoproteínas , Proteínas Oncogênicas , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Ligação Proteica , Proteínas/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Calinina , Neoplasias Pancreáticas
18.
J Immunol ; 193(2): 627-34, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24928999

RESUMO

Accumulating evidence suggests elements within tumors induce exhaustion of effector T cells and infiltration of immunosuppressive regulatory T cells (Tregs), thus preventing the development of durable antitumor immunity. Therefore, the discovery of agents that simultaneously block Treg suppressive function and reinvigorate effector function of lymphocytes is key to the development of effective cancer immunotherapy. Previous studies have shown that TLR ligands (TLRLs) could modulate the function of these T cell targets; however, those studies relied on cell-free or accessory cell-based assay systems that do not accurately reflect in vivo responses. In contrast, we used a human PBMC-based proliferation assay system to simultaneously monitor the effect of TLRLs on T cells (CD4(+), CD8(+), Tregs), B cells, and NK cells, which gave different and even conflicting results. We found that the TLR7/8L:CL097 could simultaneously activate CD8(+) T cells, B cells, and NK cells plus block Treg suppression of T cells and B cells. The TLRLs TLR1/2L:Pam3CSK4, TLR5L:flagellin, TLR4L:LPS, and TLR8/7L:CL075 also blocked Treg suppression of CD4(+) or CD8(+) T cell proliferation, but not B cell proliferation. Besides CL097, TLR2L:PGN, CL075, and TLR9L:CpG-A, CpG-B, and CpG-C) were strong activators of NK cells. Importantly, we found that Pam3CSK4 could: 1) activate CD4(+) T cell proliferation, 2) inhibit the expansion of IL-10(+) naturally occurring FOXP3(+) Tregs and induction of IL-10(+) CD4(+) Tregs (IL-10-producing type 1 Treg), and 3) block naturally occurring FOXP3(+) Tregs suppressive function. Our results suggest these agents could serve as adjuvants to enhance the efficacy of current immunotherapeutic strategies in cancer patients.


Assuntos
Leucócitos Mononucleares/imunologia , Ativação Linfocitária/imunologia , Linfócitos T Reguladores/imunologia , Receptores Toll-Like/imunologia , Adulto , Análise de Variância , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Flagelina/farmacologia , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Humanos , Imidazóis/farmacologia , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Lipopeptídeos/farmacologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Quinolinas/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Tiazóis/farmacologia , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
19.
Int J Cancer ; 135(12): 2834-46, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-24771328

RESUMO

Immunotherapeutic strategies are promising approaches for the treatment of follicular lymphoma (FL). However, their efficacy may be limited by immunosuppressive elements in the immune system and tumor microenvironment. Therefore, strategies to reverse the effects of the immunosuppressive elements are needed. We observed that regulatory T cells (Tregs) were increased in the peripheral blood at diagnosis and persisted in high numbers after induction of clinical remission with a cyclophosphamide and doxorubicin-containing chemotherapy regimen in FL patients. High levels of peripheral blood Tregs prior to therapy were associated with decreased progression-free survival in FL patients treated with either chemotherapy or combination immunotherapy that targeted CD20 and PD-1 with monoclonal antibodies rituximab and pidilizumab, respectively. Intratumoral and peripheral blood Tregs potently suppressed autologous antitumor effector T cells in FL. However, the effects of FL Tregs could be reversed by triggering Toll-like receptors (TLR) with TLR ligands Pam3 CSK4 (TLR 1/2), flagellin (TLR 5), and CpG-B (TLR 9), and/or OX40. The TLR ligands synergized with each other as well as OX40 signaling to inhibit Tregs. Furthermore, they restored the function of FL tumor-specific effector T cells. Our results suggest that a state of tolerance exists in FL patients at diagnosis and after induction of clinical remission, and agents that activate TLRs 1/2, 5, and 9, and OX40 may serve as adjuvants to enhance the efficacy of antitumor immunotherapeutic strategies and preventive vaccines against infectious diseases in these patients.


Assuntos
Regulação Neoplásica da Expressão Gênica , Linfoma Folicular/tratamento farmacológico , Linfoma Folicular/metabolismo , Receptores OX40/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Receptores Toll-Like/metabolismo , Adulto , Idoso , Antígenos CD20/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Separação Celular , Ciclofosfamida/farmacologia , Intervalo Livre de Doença , Doxorrubicina/farmacologia , Feminino , Citometria de Fluxo , Humanos , Imunossupressores/farmacologia , Imunoterapia/métodos , Interleucina-10/metabolismo , Ligantes , Masculino , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/metabolismo , Indução de Remissão , Linfócitos T Reguladores/citologia , Resultado do Tratamento , Adulto Jovem
20.
Biol Proced Online ; 16(1): 3, 2014 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-24495546

RESUMO

BACKGROUND: Although genetically engineered cells have been used to generate monoclonal antibodies (mAbs) against numerous proteins, no study has used them to generate mAbs against glycosylphosphatidylinositol (GPI)-anchored proteins. The GPI-linked protein Rae-1, an NKG2D ligand member, is responsible for interacting with immune surveillance cells. However, very few high-quality mAbs against Rae-1 are available for use in multiple analyses, including Western blotting, immunohistochemistry, and flow cytometry. The lack of high-quality mAbs limits the in-depth analysis of Rae-1 fate, such as shedding and internalization, in murine models. Moreover, currently available screening approaches for identifying high-quality mAbs are excessively time-consuming and costly. RESULTS: We used Rae-1-overexpressing CT26 tumor cells to generate 60 hybridomas that secreted mAbs against Rae-1. We also developed a streamlined screening strategy for selecting the best anti-Rae-1 mAb for use in flow cytometry assay, enzyme-linked immunosorbent assay, Western blotting, and immunostaining. CONCLUSIONS: Our cell line-based immunization approach can yield mAbs against GPI-anchored proteins, and our streamlined screening strategy can be used to select the ideal hybridoma for producing such mAbs.

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