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1.
Cytotherapy ; 23(9): 793-798, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34215503

RESUMO

BACKGROUND AIMS: Human myeloperoxidase has been shown to be overexpressed in many types of leukemia, such as chronic myeloid leukemia, acute myeloid leukemia and myelodysplastic syndrome. The authors identified two myeloperoxidase-derived HLA-A2-restricted peptides, MY4 and MY8, as novel leukemia-associated antigens. METHODS: Ex vivo-elicited MY4- and MY8-specific cytotoxic T lymphocytes were generated, and tested for leukemia cell lysis in vitro and in NOD/SCID AML xenograft model. RESULTS: These MY4- and MY8-specific cytotoxic T lymphocytes killed leukemic blasts while sparing healthy donor bone marrow cells. In addition, co-injection of MY4- and MY8-specific cytotoxic T lymphocytes into nonobese diabetic/severe combined immunodeficiency mice with acute myeloid leukemia drastically reduced tumor burden in vivo. The authors also found that MY4- and MY8-specific T cells could be detected in the peripheral blood mononuclear cells of allogeneic stem cell transplant recipients. CONCLUSIONS: These antigen-specific T cells were significantly increased in blood samples from patients compared with healthy donors, suggesting that both MY4 and MY8 are immunogenic and that MY4- and MY8-specific cytotoxic T lymphocytes may play a role in reducing leukemia in vivo. Thus, the discovery of MY4 and MY8 as novel leukemia-associated antigens paves the way for targeting these antigens in immunotherapy against myeloid leukemia.


Assuntos
Antígeno HLA-A2 , Leucemia Mieloide Aguda , Animais , Humanos , Leucemia Mieloide Aguda/terapia , Leucócitos Mononucleares , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Peptídeos , Peroxidase , Linfócitos T Citotóxicos
2.
Leukemia ; 34(6): 1626-1636, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31908357

RESUMO

Immunotherapy targeting leukemia-associated antigens has shown promising results. Because of the heterogeneity of leukemia, vaccines with a single peptide have elicited only a limited immune response. Targeting several peptides together elicited peptide-specific cytotoxic T lymphocytes (CTLs) in leukemia patients, and this was associated with clinical responses. Thus, the discovery of novel antigens is essential. In the current study, we investigated cyclin E as a novel target for immunotherapy. Cyclin E1 and cyclin E2 were found to be highly expressed in hematologic malignancies, according to reverse transcription polymerase chain reaction and western blot analysis. We identified two HLA-A*0201 binding nonameric peptides, CCNE1M from cyclin E1 and CCNE2L from cyclin E2, which both elicited the peptide-specific CTLs. The peptide-specific CTLs specifically kill leukemia cells. Furthermore, CCNE1M and CCNE2L CTLs were increased in leukemia patients who underwent allogeneic hematopoietic stem cell transplantation, and this was associated with desired clinical outcomes. Our findings suggest that cyclin E1 and cyclin E2 are potential targets for immunotherapy in leukemia.


Assuntos
Antígenos de Neoplasias/metabolismo , Vacinas Anticâncer/imunologia , Ciclina E/imunologia , Ciclinas/imunologia , Antígeno HLA-A2/imunologia , Leucemia/imunologia , Proteínas Oncogênicas/imunologia , Adulto , Idoso , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/farmacologia , Epitopos de Linfócito T/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linfócitos T Citotóxicos/imunologia , Adulto Jovem
4.
Br J Haematol ; 185(4): 679-690, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30828801

RESUMO

Acute myeloid leukaemia (AML) is a lethal haematological malignancy characterized by an immunosuppressive milieu in the tumour microenvironment (TME) that fosters disease growth and therapeutic resistance. Hypomethylating agents (HMAs) demonstrate clinical efficacy in AML patients and exert immunomodulatory activities. In the present study, we show that guadecitabine augments both antigen processing and presentation, resulting in increased AML susceptibility to T cell-mediated killing. Exposure to HMA results in the activation of the endogenous retroviral pathway with concomitant downstream amplification of critical mediators of inflammation. In an immunocompetent murine leukaemia model, guadecitabine negatively regulates inhibitory accessory cells in the TME by decreasing PD-1 (also termed PDCD1) expressing T cells and reducing AML-mediated expansion of myeloid-derived suppressor cells. Therapy with guadecitabine results in enhanced leukaemia-specific immunity, as manifested by increased CD4 and CD8 cells targeting syngeneic leukaemia cells. We have previously reported that vaccination with AML/dendritic cell fusions elicits the expansion of leukaemia-specific T cells and protects against disease relapse. In the present study, we demonstrate that vaccination in conjunction with HMA therapy results in enhanced anti-leukaemia immunity and survival. The combination of a novel personalized dendritic cell/AML fusion vaccine and an HMA has therapeutic potential, and a clinical trial investigating this combination is planned.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Azacitidina/análogos & derivados , Vacinas Anticâncer/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Microambiente Tumoral/imunologia , Animais , Antineoplásicos Imunológicos/imunologia , Azacitidina/imunologia , Azacitidina/farmacologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Células Dendríticas/imunologia , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Humanos , Imunidade Celular/efeitos dos fármacos , Leucemia Mieloide Aguda/imunologia , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Receptor de Morte Celular Programada 1/metabolismo , Retroviridae/imunologia , Ativação Viral/imunologia
5.
Biol Blood Marrow Transplant ; 25(7): 1347-1354, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30826465

RESUMO

Although bortezomib and rituximab have synergistic activity in patients with lymphoma and both can attenuate graft-versus-host disease (GVHD), the drugs have not been used together in patients undergoing allogeneic stem cell transplantation (alloSCT). In this phase I/II trial, we assessed the safety and activity of bortezomib added to the rituximab (R) plus BEAM (carmustine, etoposide, cytarabine, melphalan) regimen in patients with relapsed lymphoma undergoing alloSCT. Primary GVHD prophylaxis consisted of tacrolimus and methotrexate. Bortezomib (1 to 1.3 mg/m2 per dose) was administered i.v. on days -13, -6, -1, and +2. We performed inverse probability weighting analysis to compare GVHD and survival results with an historical control group that received R-BEAM without bortezomib. Thirty-nine patients were assessable for toxic effects and response. The median age was 54 years. The most common diagnosis was diffuse large B cell lymphoma (41%). Twenty-two patients (56%) and 17 patients (44%) received their transplants from matched related and matched unrelated donors, respectively. The maximum tolerated bortezomib dose was 1 mg/m2. The weighted cumulative incidences of grades II to IV and III or IV acute GVHD were 50% and 34%, respectively; these incidences and survival rates were not significantly different from those of the control group. Median survival was not reached in patients age ≤ 50 years and with a long follow-up time of 60.7 months. The R-BEAM regimen has a survival benefit in lymphoma patients age ≤ 50 years undergoing alloSCT. The addition of bortezomib has no impact on survival or incidence of GVHD.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Bortezomib/administração & dosagem , Neoplasias Hematológicas , Linfoma Difuso de Grandes Células B , Rituximab/administração & dosagem , Transplante de Células-Tronco , Adulto , Fatores Etários , Idoso , Aloenxertos , Carmustina/administração & dosagem , Citarabina/administração & dosagem , Intervalo Livre de Doença , Feminino , Neoplasias Hematológicas/mortalidade , Neoplasias Hematológicas/patologia , Neoplasias Hematológicas/terapia , Humanos , Linfoma Difuso de Grandes Células B/mortalidade , Linfoma Difuso de Grandes Células B/patologia , Linfoma Difuso de Grandes Células B/terapia , Masculino , Melfalan/administração & dosagem , Pessoa de Meia-Idade , Podofilotoxina/administração & dosagem , Taxa de Sobrevida
6.
Clin Cancer Res ; 25(8): 2610-2620, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30647079

RESUMO

PURPOSE: Inefficient homing of adoptively transferred cytotoxic T lymphocytes (CTLs) to tumors is a major limitation to the efficacy of adoptive cellular therapy (ACT) for cancer. However, through fucosylation, a process whereby fucosyltransferases (FT) add fucose groups to cell surface glycoproteins, this challenge may be overcome. Endogenously fucosylated CTLs and ex vivo fucosylated cord blood stem cells and regulatory T cells were shown to preferentially home to inflamed tissues and marrow. Here, we show a novel approach to enhance CTL homing to leukemic marrow and tumor tissue. EXPERIMENTAL DESIGN: Using the enzyme FT-VII, we fucosylated CTLs that target the HLA-A2-restricted leukemia antigens CG1 and PR1, the HER2-derived breast cancer antigen E75, and the melanoma antigen gp-100. We performed in vitro homing assays to study the effects of fucosylation on CTL homing and target killing. We used in vivo mouse models to demonstrate the effects of ex vivo fucosylation on CTL antitumor activities against leukemia, breast cancer, and melanoma. RESULTS: Our data show that fucosylation increases in vitro homing and cytotoxicity of antigen-specific CTLs. Furthermore, fucosylation enhances in vivo CTL homing to leukemic bone marrow, breast cancer, and melanoma tissue in NOD/SCID gamma (NSG) and immunocompetent mice, ultimately boosting the antitumor activity of the antigen-specific CTLs. Importantly, our work demonstrates that fucosylation does not interfere with CTL specificity. CONCLUSIONS: Together, our data establish ex vivo CTL fucosylation as a novel approach to improving the efficacy of ACT, which may be of great value for the future of ACT for cancer.


Assuntos
Citotoxicidade Imunológica , Epitopos de Linfócito T/imunologia , Imunoterapia Adotiva , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Animais , Biomarcadores , Linhagem Celular Tumoral , Quimiotaxia de Leucócito/imunologia , Regulação da Expressão Gênica , Glicosilação , Humanos , Imunofenotipagem , Imunoterapia Adotiva/métodos , Ativação Linfocitária , Camundongos , Peptídeos/imunologia , Migração Transendotelial e Transepitelial
7.
Nat Commun ; 10(1): 254, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30651550

RESUMO

Although B cell response is frequently found in cancer, there is little evidence that it alters tumor development or progression. The process through which tumor-associated antigens trigger humoral response is not well delineated. We investigate the repertoire of antigens associated with humoral immune response in pancreatic ductal adenocarcinoma (PDAC) using in-depth proteomic profiling of immunoglobulin-bound proteins from PDAC patient plasmas and identify tumor antigens that induce antibody response together with exosome hallmark proteins. Additional profiling of PDAC cell-derived exosomes reveals significant overlap in their protein content with immunoglobulin-bound proteins in PDAC plasmas, and significant autoantibody reactivity is observed between PDAC cell-derived exosomes and patient plasmas compared to healthy controls. Importantly, PDAC-derived exosomes induce a dose-dependent inhibition of PDAC serum-mediated complement-dependent cytotoxicity towards cancer cells. In summary, we provide evidence that exosomes display a large repertoire of tumor antigens that induce autoantibodies and exert a decoy function against complement-mediated cytotoxicity.


Assuntos
Formação de Anticorpos/imunologia , Antígenos de Neoplasias/imunologia , Linfócitos B/imunologia , Carcinoma Ductal Pancreático/imunologia , Proteínas do Sistema Complemento/imunologia , Exossomos/imunologia , Neoplasias Pancreáticas/imunologia , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/metabolismo , Autoanticorpos/imunologia , Carcinoma Ductal Pancreático/sangue , Linhagem Celular Tumoral , Estudos de Coortes , Conjuntos de Dados como Assunto , Exossomos/metabolismo , Exossomos/ultraestrutura , Feminino , Perfilação da Expressão Gênica , Voluntários Saudáveis , Humanos , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Neoplasias Pancreáticas/sangue , Proteômica/métodos , Análise de Sequência de RNA
8.
Blood Adv ; 2(16): 2052-2062, 2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30115642

RESUMO

T-cell responses to minor histocompatibility antigens (mHAs) mediate both antitumor immunity (graft-versus-leukemia [GVL]) and graft-versus-host disease (GVHD) in allogeneic stem cell transplant. Identifying mHAs with high allele frequency, tight binding affinity to common HLA molecules, and narrow tissue restriction could enhance immunotherapy against leukemia. Genotyping and HLA allele data from 101 HLA-matched donor-recipient pairs (DRPs) were computationally analyzed to predict both class I and class II mHAs likely to induce either GVL or GVHD. Roughly twice as many mHAs were predicted in HLA-matched unrelated donor (MUD) stem cell transplantation (SCT) compared with HLA-matched related transplants, an expected result given greater genetic disparity in MUD SCT. Computational analysis predicted 14 of 18 previously identified mHAs, with 2 minor antigen mismatches not being contained in the patient cohort, 1 missed mHA resulting from a noncanonical translation of the peptide antigen, and 1 case of poor binding prediction. A predicted peptide epitope derived from GRK4, a protein expressed in acute myeloid leukemia and testis, was confirmed by targeted differential ion mobility spectrometry-tandem mass spectrometry. T cells specific to UNC-GRK4-V were identified by tetramer analysis both in DRPs where a minor antigen mismatch was predicted and in DRPs where the donor contained the allele encoding UNC-GRK4-V, suggesting that this antigen could be both an mHA and a cancer-testis antigen. Computational analysis of genomic and transcriptomic data can reliably predict leukemia-associated mHA and can be used to guide targeted mHA discovery.


Assuntos
Simulação por Computador , Transplante de Células-Tronco Hematopoéticas , Leucemia Mielogênica Crônica BCR-ABL Positiva , Leucemia Mieloide Aguda , Antígenos de Histocompatibilidade Menor/imunologia , Modelos Imunológicos , Síndromes Mielodisplásicas , Aloenxertos , Feminino , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Efeito Enxerto vs Leucemia/imunologia , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/terapia , Masculino , Síndromes Mielodisplásicas/imunologia , Síndromes Mielodisplásicas/patologia , Síndromes Mielodisplásicas/terapia , Doadores não Relacionados
9.
J Immunol ; 201(5): 1389-1399, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30021768

RESUMO

Proteinase 3 (P3), a serine protease expressed by myeloid cells, localized within azurophil granules, and also expressed on the cellular membrane of polymorphonuclear neutrophils (PMN), is the target of autoimmunity in granulomatosis with polyangiitis. PR1, an HLA-A2 restricted nonameric peptide derived from P3, has been targeted effectively in myeloid leukemia. We previously showed (Molldrem et al. 2003. JClinInvest 111: 639-647) that overexpression of P3 in chronic myeloid leukemia induces apoptosis of high-affinity PR1-specific T cells, leading to deletional tolerance and leukemia outgrowth. In this study, we investigated the effect of membrane P3 (mP3)-expressing PMN and acute myeloid leukemia (AML) blasts on the proliferation of CD4 and CD8 T cells in vitro. We demonstrate that mP3-expressing PMN significantly inhibits autologous healthy donor T cell proliferation but does not affect cytokine production in activated T cells and that this effect requires cell proximity and was abrogated by P3 blockade. This inhibition required P3 enzyme activity. However, suppression was not reversed by either the addition of catalase or the inhibition of arginase I. In addition to P3 blockade, anti-low density lipoprotein receptor-related protein 1 (LRP1) Ab also restored T cells' capacity to proliferate. Last, we show dose-dependent inhibition of T cell proliferation by mP3-expressing AML blasts. Together, our findings demonstrate a novel mechanism whereby PMN- and AML-associated mP3 inhibits T cell proliferation via direct LRP1 and mP3 interaction, and we identify P3 as a novel target to modulate immunity in myeloid leukemia and autoimmune disease.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células , Leucemia Mieloide Aguda/imunologia , Mieloblastina/imunologia , Proteínas de Neoplasias/imunologia , Neutrófilos/imunologia , Adulto , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Feminino , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Neutrófilos/patologia
10.
Clin Cancer Res ; 24(14): 3386-3396, 2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-29661776

RESUMO

Purpose: PR1 is a human leukocyte antigen (HLA)-A2 nonameric peptide derived from neutrophil elastase (NE) and proteinase 3 (P3). We have previously shown that PR1 is cross-presented by solid tumors, leukemia, and antigen-presenting cells, including B cells. We have also shown that cross-presentation of PR1 by solid tumors renders them susceptible to killing by PR1-targeting immunotherapies. As multiple myeloma is derived from B cells, we investigated whether multiple myeloma is also capable of PR1 cross-presentation and subsequently capable of being targeted by using PR1 immunotherapies.Experimental Design: We tested whether multiple myeloma is capable of cross-presenting PR1 and subsequently becomes susceptible to PR1-targeting immunotherapies, using multiple myeloma cell lines, a xenograft mouse model, and primary multiple myeloma patient samples.Results: Here we show that multiple myeloma cells lack endogenous NE and P3, are able to take up exogenous NE and P3, and cross-present PR1 on HLA-A2. Cross-presentation by multiple myeloma utilizes the conventional antigen processing machinery, including the proteasome and Golgi, and is not affected by immunomodulating drugs (IMiD). Following PR1 cross-presentation, we are able to target multiple myeloma with PR1-CTL and anti-PR1/HLA-A2 antibody both in vitro and in vivoConclusions: Collectively, our data demonstrate that PR1 is a novel tumor-associated antigen target in multiple myeloma and that multiple myeloma is susceptible to immunotherapies that target cross-presented antigens. Clin Cancer Res; 24(14); 3386-96. ©2018 AACR.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Antígeno HLA-A2/imunologia , Mieloma Múltiplo/imunologia , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/imunologia , Animais , Apresentação do Antígeno/efeitos dos fármacos , Apresentação do Antígeno/imunologia , Células Apresentadoras de Antígenos/imunologia , Transporte Biológico , Linhagem Celular Tumoral , Ativação do Complemento , Apresentação Cruzada/efeitos dos fármacos , Apresentação Cruzada/imunologia , Citotoxicidade Imunológica , Modelos Animais de Doenças , Antígeno HLA-A2/química , Antígeno HLA-A2/metabolismo , Humanos , Fatores Imunológicos/farmacologia , Imunomodulação/efeitos dos fármacos , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Oncotarget ; 9(4): 4280-4281, 2018 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-29435102
12.
Front Immunol ; 9: 3153, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30713535

RESUMO

Despite substantial advances in the treatment of acute myeloid leukemia (AML), only 30% of patients survive more than 5 years. Therefore, new therapeutics are much needed. Here, we present a novel therapeutic strategy targeting PR1, an HLA-A2 restricted myeloid leukemia antigen. Previously, we have developed and characterized a novel T-cell receptor-like monoclonal antibody (8F4) that targets PR1/HLA-A2 and eliminates AML xenografts by antibody-dependent cellular cytotoxicity (ADCC). To improve the potency of 8F4, we adopted a strategy to link T-cell cytotoxicity with a bi-specific T-cell-engaging antibody that binds PR1/HLA-A2 on leukemia and CD3 on neighboring T-cells. The 8F4 bi-specific antibody maintained high affinity and specific binding to PR1/HLA-A2 comparable to parent 8F4 antibody, shown by flow cytometry and Bio-Layer Interferometry. In addition, 8F4 bi-specific antibody activated donor T-cells in the presence of HLA-A2+ primary AML blasts and cell lines in a dose dependent manner. Importantly, activated T-cells lysed HLA-A2+ primary AML blasts and cell lines after addition of 8F4 bi-specific antibody. In conclusion, our studies demonstrate the therapeutic potential of a novel bi-specific antibody targeting the PR1/HLA-A2 leukemia-associated antigen, justifying further clinical development of this strategy.


Assuntos
Anticorpos Biespecíficos/imunologia , Antígenos de Neoplasias/imunologia , Antígeno HLA-A2/imunologia , Leucemia Mieloide Aguda/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Biespecíficos/farmacologia , Especificidade de Anticorpos/imunologia , Antígenos de Neoplasias/metabolismo , Células CHO , Linhagem Celular , Cricetulus , Citotoxicidade Imunológica , Antígeno HLA-A2/metabolismo , Humanos , Imunoterapia Adotiva , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/terapia , Ativação Linfocitária , Ligação Proteica , Linfócitos T/metabolismo
13.
Cancer Res ; 77(19): 5374-5383, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28819024

RESUMO

Early-phase clinical trials evaluating CD8+ T cell-eliciting, HER2-derived peptide vaccines administered to HER2+ breast cancer patients in the adjuvant setting suggest synergy between the vaccines and trastuzumab, the mAb targeting the HER2 protein. Among 60 patients enrolled in clinical trials evaluating the E75 + GM-CSF and GP2 + GM-CSF vaccines, there have been no recurrences in patients vaccinated after receiving trastuzumab as part of standard therapy in the per treatment analyses conducted after a median follow-up of greater than 34 months. Here, we describe a mechanism by which this synergy may occur. Flow cytometry showed that trastuzumab facilitated uptake of HER2 by dendritic cells (DC), which was mediated by the Fc receptor and was specific to trastuzumab. In vitro, increased HER2 uptake by DC increased cross-presentation of E75, the immunodominant epitope derived from the HER2 protein, an observation confirmed in two in vivo mouse models. This increased E75 cross-presentation, mediated by trastuzumab treatment, enabled more efficient expansion of E75-specific cytotoxic T cells (E75-CTL). These results demonstrate a mechanism by which trastuzumab links innate and adaptive immunity by facilitating activation of antigen-specific T cells. On the basis of these data, we conclude that HER2-positive breast cancer patients that have been treated with trastuzumab may experience a more robust antitumor immune response by restimulation of T cells with the E75 peptide vaccine, thereby accounting for the improved disease-free survival observed with combination therapy. Cancer Res; 77(19); 5374-83. ©2017 AACR.


Assuntos
Neoplasias da Mama/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Neoplasias Ovarianas/imunologia , Receptor ErbB-2/metabolismo , Linfócitos T Citotóxicos/imunologia , Trastuzumab/uso terapêutico , Animais , Apoptose , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Vacinas Anticâncer/uso terapêutico , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Receptor ErbB-2/imunologia , Células Tumorais Cultivadas , Vacinas de Subunidades/uso terapêutico
14.
J Biol Chem ; 292(24): 10295-10305, 2017 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-28468826

RESUMO

Neutrophil elastase (NE) can be rapidly taken up by tumor cells that lack endogenous NE expression, including breast cancer, which results in cross-presentation of PR1, an NE-derived HLA-A2-restricted peptide that is an immunotherapy target in hematological and solid tumor malignancies. The mechanism of NE uptake, however, remains unknown. Using the mass spectrometry-based approach, we identify neuropilin-1 (NRP1) as a NE receptor that mediates uptake and PR1 cross-presentation in breast cancer cells. We demonstrated that soluble NE is a specific, high-affinity ligand for NRP1 with a calculated Kd of 38.7 nm Furthermore, we showed that NRP1 binds to the RRXR motif in NE. Notably, NRP1 knockdown with interfering RNA or CRISPR-cas9 system and blocking using anti-NRP1 antibody decreased NE uptake and, subsequently, susceptibility to lysis by PR1-specific cytotoxic T cells. Expression of NRP1 in NRP1-deficient cells was sufficient to induce NE uptake. Altogether, because NRP1 is broadly expressed in tumors, our findings suggest a role for this receptor in immunotherapy strategies that target cross-presented antigens.


Assuntos
Absorção Fisiológica , Neoplasias da Mama/metabolismo , Apresentação Cruzada , Elastase de Leucócito/metabolismo , Proteínas de Neoplasias/metabolismo , Neuropilina-1/metabolismo , Motivos de Aminoácidos , Anticorpos Bloqueadores/metabolismo , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Feminino , Humanos , Cinética , Elastase de Leucócito/química , Elastase de Leucócito/imunologia , Ligantes , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Neuropilina-1/antagonistas & inibidores , Neuropilina-1/química , Neuropilina-1/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo
15.
Cancer Res ; 77(12): 3144-3150, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28428279

RESUMO

Human prostate cancer often metastasizes to bone, but the biological basis for such site-specific tropism remains largely unresolved. Recent work led us to hypothesize that this tropism may reflect pathogenic interactions between RAGE, a cell surface receptor expressed on malignant cells in advanced prostate cancer, and proteinase 3 (PR3), a serine protease present in inflammatory neutrophils and hematopoietic cells within the bone marrow microenvironment. In this study, we establish that RAGE-PR3 interaction mediates homing of prostate cancer cells to the bone marrow. PR3 bound to RAGE on the surface of prostate cancer cells in vitro, inducing tumor cell motility through a nonproteolytic signal transduction cascade involving activation and phosphorylation of ERK1/2 and JNK1. In preclinical models of experimental metastasis, ectopic expression of RAGE on human prostate cancer cells was sufficient to promote bone marrow homing within a short timeframe. Our findings demonstrate how RAGE-PR3 interactions between human prostate cancer cells and the bone marrow microenvironment mediate bone metastasis during prostate cancer progression, with potential implications for prognosis and therapeutic intervention. Cancer Res; 77(12); 3144-50. ©2017 AACR.


Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mieloblastina/metabolismo , Invasividade Neoplásica/patologia , Neoplasias da Próstata/patologia , Microambiente Tumoral/fisiologia , Animais , Medula Óssea/metabolismo , Medula Óssea/patologia , Neoplasias Ósseas/secundário , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Xenoenxertos , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias da Próstata/metabolismo
16.
Cancer Immunol Res ; 5(4): 319-329, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28254787

RESUMO

Immunotherapies targeting immune checkpoints have proven efficacious in reducing the burden of lung cancer in patients; however, the antigenic targets of these reinvigorated T cells remain poorly defined. Lung cancer tumors contain tumor-associated macrophages (TAM) and neutrophils, which release the serine proteases neutrophil elastase (NE) and proteinase 3 (P3) into the tumor microenvironment. NE and P3 shape the antitumor adaptive immune response in breast cancer and melanoma. In this report, we demonstrate that lung cancer cells cross-presented the tumor-associated antigen PR1, derived from NE and P3. Additionally, NE and P3 enhanced the expression of human leukocyte antigen (HLA) class I molecules on lung cancer cells and induced unique, endogenous peptides in the immunopeptidome, as detected with mass spectrometry sequencing. Lung cancer patient tissues with high intratumoral TAMs were enriched for MHC class I genes and T-cell markers, and patients with high TAM and cytotoxic T lymphocyte (CTL) infiltration had improved overall survival. We confirmed the immunogenicity of unique, endogenous peptides with cytotoxicity assays against lung cancer cell lines, using CTLs from healthy donors that had been expanded against select peptides. Finally, CTLs specific for serine proteases-induced endogenous peptides were detected in lung cancer patients using peptide/HLA-A2 tetramers and were elevated in tumor-infiltrating lymphocytes. Thus, serine proteases in the tumor microenvironment of lung cancers promote the presentation of HLA class I immunogenic peptides that are expressed by lung cancer cells, thereby increasing the antigen repertoire that can be targeted in lung cancer. Cancer Immunol Res; 5(4); 319-29. ©2017 AACR.


Assuntos
Apresentação do Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Imunomodulação , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Serina Proteases/metabolismo , Sequência de Aminoácidos , Biomarcadores , Linhagem Celular Tumoral , Citocinas/metabolismo , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunofenotipagem , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Neoplasias Pulmonares/patologia , Ativação Linfocitária , Peptídeos/química , Peptídeos/imunologia , Peptídeos/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
17.
Front Immunol ; 8: 1975, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29422892

RESUMO

Cathepsin G (CG) is a myeloid azurophil granule protease that is highly expressed by acute myeloid leukemia (AML) blasts and leukemia stem cells. We previously identified CG1 (FLLPTGAEA), a human leukocyte antigen-A2-restricted nonameric peptide derived from CG, as an immunogenic target in AML. In this report, we aimed to assess the level of CG expression in acute lymphoid leukemia (ALL) and its potential as an immunotherapeutic target in ALL. Using RT-PCR and western blots, we identified CG mRNA and protein, respectively, in B-ALL patient samples and cell lines. We also examined CG expression in a large cohort of 130 patients with ALL via reverse-phase protein array (RPPA). Our data show that CG is widely expressed by ALL and is a poor prognosticator. In addition to endogenous expression, we also provide evidence that CG can be taken up by ALL cells. Finally, we demonstrate that patient ALL can be lysed by CG1-specific cytotoxic T lymphocytes in vitro. Together, these data show high expression of CG by ALL and implicate CG as a target for immunotherapy in ALL.

19.
Cytotherapy ; 18(8): 995-1001, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27378343

RESUMO

BACKGROUND AIMS: PR1 is an HLA-A2 restricted leukemia-associated antigen derived from neutrophil elastase and proteinase 3, both of which are normally stored in the azurophil granules of myeloid cells but overexpressed in myeloid leukemic cells. PR1-specific cytotoxic lymphocytes (PR1-CTLs) have activity against primary myeloid leukemia in vitro and in vivo and thus could have great potential in the setting of adoptive cellular therapy (ACT). Adult peripheral blood-derived PR1-CTLs are infrequent but preferentially lyse myeloid leukemia cells. We sought to examine PR1-CTLs in umbilical cord blood (UCB) because UCB units provide a rapidly available cell source and a lower risk of graft-versus-host disease, even in the setting of mismatched human leukocyte antigen (HLA) loci. METHODS: We first determined the frequency of PR1-CTLs in HLA-A2(+) UCB units and then successfully expanded them ex vivo using repeated stimulation with PR1 peptide-pulsed antigen-presenting cells (APCs). After expansion, we assessed the PR1-CTL phenotype (naive, effector, memory) and function against PR1-expressing target cells. RESULTS: PR1-CTLs are detected at an average frequency of 0.14% within the CD8(+) population of fresh UCB units, which is 45 times higher than in healthy adult peripheral blood. UCB PR1-CTLs are phenotypically naive, consistent with the UCB CD8(+) population as a whole. In addition, the cells can be expanded by stimulation with PR1 peptide-pulsed APCs. Expansion results in an increased frequency of PR1-CTLs, up to 4.56%, with an average 20-fold increase in total number. After expansion, UCB PR1-CTLs express markers consistent with effector memory T cells. Expanded UCB PR1-CTLs are functional in vitro as they are able to produce cytokines and lyse PR1-expressing leukemia cell lines. CONCLUSIONS: This study is the first report to show that T cells specific for a leukemia-associated antigen are found at a significantly higher frequency in UCB than adult blood. Our results also demonstrate specific cytotoxicity of expanded UCB-derived PR1-CTLs against PR1-expressing targets. Together, our data suggest that UCB PR1-CTLs could be useful to prevent or treat leukemia relapse in myeloid leukemia patients.


Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Sangue Fetal/citologia , Antígeno HLA-A2/imunologia , Imunoterapia Adotiva , Leucemia Mieloide/terapia , Mieloblastina/imunologia , Linfócitos T Citotóxicos/imunologia , Adulto , Células Cultivadas , Sangue Fetal/imunologia , Antígeno HLA-A2/química , Antígeno HLA-A2/metabolismo , Humanos , Células K562 , Leucemia Mieloide/imunologia , Contagem de Linfócitos , Mieloblastina/química , Mieloblastina/metabolismo , Linfócitos T Citotóxicos/metabolismo , Células U937
20.
PLoS One ; 11(7): e0160004, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27467256

RESUMO

Skin toxicity is the most common toxicity caused by Epidermal Growth Factor Receptor (EGFR) inhibitors, and has been associated with clinical efficacy. As EGFR inhibitors enhance the expression of antigen presenting molecules in affected skin keratinocytes, they may concurrently facilitate neo-antigen presentation in lung cancer tumor cells contributing to anti-tumor immunity. Here, we investigated the modulatory effect of the EGFR inhibitor, erlotinib on antigen presenting molecules and PD-L1, prominent immune checkpoint protein, of skin keratinocytes and lung cancer cell lines to delineate the link between EGFR signaling pathway inhibition and potential anti-tumor immunity. Erlotinib up-regulated MHC-I and MHC-II proteins on IFNγ treated keratinocytes but abrogated IFNγ-induced expression of PD-L1, suggesting the potential role of infiltrating autoreactive T cells in the damage of keratinocytes in affected skin. Interestingly, the surface expression of MHC-I, MHC-II, and PD-L1 was up-regulated in response to IFNγ more often in lung cancer cell lines sensitive to erlotinib, but only expression of PD-L1 was inhibited by erlotinib. Further, erlotinib significantly increased T cell mediated cytotoxicity on lung cancer cells. Lastly, the analysis of gene expression dataset of 186 lung cancer cell lines from Cancer Cell Line Encyclopedia demonstrated that overexpression of PD-L1 was associated with sensitivity to erlotinib and higher expression of genes related to antigen presenting pathways and IFNγ signaling pathway. Our findings suggest that the EGFR inhibitors can facilitate anti-tumor adaptive immune responses by breaking tolerance especially in EGFR driven lung cancer that are associated with overexpression of PD-L1 and genes related to antigen presentation and inflammation.


Assuntos
Receptores ErbB/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Antineoplásicos , Antígeno B7-H1/imunologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Cloridrato de Erlotinib/uso terapêutico , Humanos , Neoplasias Pulmonares/imunologia
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