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1.
PLoS Pathog ; 19(12): e1011853, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38100526

RESUMO

Engineered T cells hold great promise to become part of an effective HIV cure strategy, but it is currently unclear how best to redirect T cells to target HIV. To gain insight, we generated engineered T cells using lentiviral vectors encoding one of three distinct HIV-specific T cell receptors (TCRs) or a previously optimized HIV-targeting chimeric antigen receptor (CAR) and compared their functional capabilities. All engineered T cells had robust, antigen-specific polyfunctional cytokine profiles when mixed with artificial antigen-presenting cells. However, only the CAR T cells could potently control HIV replication. TCR affinity enhancement did not augment HIV control but did allow TCR T cells to recognize common HIV escape variants. Interestingly, either altering Nef activity or adding additional target epitopes into the HIV genome bolstered TCR T cell anti-HIV activity, but CAR T cells remained superior in their ability to control HIV replication. To better understand why CAR T cells control HIV replication better than TCR T cells, we performed a time course to determine when HIV-specific T cells were first able to activate Caspase 3 in HIV-infected targets. We demonstrated that CAR T cells recognized and killed HIV-infected targets more rapidly than TCR T cells, which correlates with their ability to control HIV replication. These studies suggest that the speed of target recognition and killing is a key determinant of whether engineered T cell therapies will be effective against infectious diseases.


Assuntos
Infecções por HIV , HIV-1 , Receptores de Antígenos Quiméricos , Humanos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos de Linfócitos T/genética , Infecções por HIV/terapia , Replicação Viral
2.
J Immunol ; 208(1): 169-180, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34853077

RESUMO

Adoptive T cell therapy with T cells expressing affinity-enhanced TCRs has shown promising results in phase 1/2 clinical trials for solid and hematological tumors. However, depth and durability of responses to adoptive T cell therapy can suffer from an inhibitory tumor microenvironment. A common immune-suppressive agent is TGF-ß, which is secreted by tumor cells and cells recruited to the tumor. We investigated whether human T cells could be engineered to be resistant to inhibition by TGF-ß. Truncating the intracellular signaling domain from TGF-ß receptor (TGFßR) II produces a dominant-negative receptor (dnTGFßRII) that dimerizes with endogenous TGFßRI to form a receptor that can bind TGF-ß but cannot signal. We previously generated specific peptide enhanced affinity receptor TCRs recognizing the HLA-A*02-restricted peptides New York esophageal squamous cell carcinoma 1 (NY-ESO-1)157-165/l-Ag family member-1A (TCR: GSK3377794, formerly NY-ESO-1c259) and melanoma Ag gene A10254-262 (TCR: ADP-A2M10, formerly melanoma Ag gene A10c796). In this article, we show that exogenous TGF-ß inhibited in vitro proliferation and effector functions of human T cells expressing these first-generation high-affinity TCRs, whereas inhibition was reduced or abolished in the case of second-generation TCRs coexpressed with dnTGFßRII (e.g., GSK3845097). TGF-ß isoforms and a panel of TGF-ß-associated genes are overexpressed in a range of cancer indications in which NY-ESO-1 is commonly expressed, particularly in synovial sarcoma. As an example, immunohistochemistry/RNAscope identified TGF-ß-positive cells close to T cells in tumor nests and stroma, which had low frequencies of cells expressing IFN-γ in a non-small cell lung cancer setting. Coexpression of dnTGFßRII may therefore improve the efficacy of TCR-transduced T cells.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Carcinoma de Células Escamosas/terapia , Neoplasias Hematológicas/terapia , Imunoterapia Adotiva/métodos , Melanoma/terapia , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Sarcoma Sinovial/terapia , Fator de Crescimento Transformador beta/metabolismo , Antígenos de Neoplasias/imunologia , Carcinoma de Células Escamosas/imunologia , Linhagem Celular Tumoral , Engenharia Genética , Antígeno HLA-A2/metabolismo , Neoplasias Hematológicas/imunologia , Humanos , Tolerância Imunológica , Melanoma/imunologia , Proteínas de Membrana/imunologia , Proteínas de Neoplasias/imunologia , Fragmentos de Peptídeos/imunologia , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Sarcoma Sinovial/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T , Microambiente Tumoral
3.
Hepatology ; 69(5): 2061-2075, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30561769

RESUMO

Patients with hepatocellular carcinoma (HCC) have a poor prognosis and limited therapeutic options. Alpha-fetoprotein (AFP) is often expressed at high levels in HCC and is an established clinical biomarker of the disease. Expression of AFP in nonmalignant liver can occur, particularly in a subset of progenitor cells and during chronic inflammation, at levels typically lower than in HCC. This cancer-specific overexpression indicates that AFP may be a promising target for immunotherapy. We verified expression of AFP in normal and diseased tissue and generated an affinity-optimized T-cell receptor (TCR) with specificity to AFP/HLA-A*02+ tumors. Expression of AFP was investigated using database searches, by qPCR, and by immunohistochemistry (IHC) analysis of a panel of human tissue samples, including normal, diseased, and malignant liver. Using in vitro mutagenesis and screening, we generated a TCR that recognizes the HLA-A*02-restricted AFP158-166 peptide, FMNKFIYEI, with an optimum balance of potency and specificity. These properties were confirmed by an extension of the alanine scan (X-scan) and testing TCR-transduced T cells against normal and tumor cells covering a variety of tissues, cell types, and human leukocyte antigen (HLA) alleles. Conclusion: We have used a combination of physicochemical, in silico, and cell biology methods for optimizing a TCR for improved affinity and function, with properties that are expected to allow TCR-transduced T cells to differentiate between antigen levels on nonmalignant and cancer cells. T cells transduced with this TCR constitute the basis for a trial of HCC adoptive T-cell immunotherapy.


Assuntos
Carcinoma Hepatocelular/imunologia , Antígeno HLA-A2/metabolismo , Neoplasias Hepáticas/imunologia , Receptores de Antígenos de Linfócitos T/uso terapêutico , alfa-Fetoproteínas/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/terapia , Células Hep G2 , Humanos , Imunoterapia/métodos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/terapia , Receptores de Antígenos de Linfócitos T/imunologia
4.
Blood ; 122(6): 863-71, 2013 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-23770775

RESUMO

An obstacle to cancer immunotherapy has been that the affinity of T-cell receptors (TCRs) for antigens expressed in tumors is generally low. We initiated clinical testing of engineered T cells expressing an affinity-enhanced TCR against HLA-A*01-restricted MAGE-A3. Open-label protocols to test the TCRs for patients with myeloma and melanoma were initiated. The first two treated patients developed cardiogenic shock and died within a few days of T-cell infusion, events not predicted by preclinical studies of the high-affinity TCRs. Gross findings at autopsy revealed severe myocardial damage, and histopathological analysis revealed T-cell infiltration. No MAGE-A3 expression was detected in heart autopsy tissues. Robust proliferation of the engineered T cells in vivo was documented in both patients. A beating cardiomyocyte culture generated from induced pluripotent stem cells triggered T-cell killing, which was due to recognition of an unrelated peptide derived from the striated muscle-specific protein titin. These patients demonstrate that TCR-engineered T cells can have serious and not readily predictable off-target and organ-specific toxicities and highlight the need for improved methods to define the specificity of engineered TCRs.


Assuntos
Doenças Cardiovasculares/complicações , Melanoma/sangue , Mieloma Múltiplo/sangue , Proteínas Musculares/metabolismo , Miocárdio/patologia , Proteínas Quinases/metabolismo , Linfócitos T/citologia , Alelos , Motivos de Aminoácidos , Antígenos de Neoplasias/metabolismo , Técnicas de Cultura de Células , Conectina , Citocinas/metabolismo , Epitopos/metabolismo , Antígenos HLA-A/metabolismo , Humanos , Imunoterapia Adotiva , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Melanoma/terapia , Pessoa de Meia-Idade , Mieloma Múltiplo/terapia , Miocárdio/imunologia , Proteínas de Neoplasias/metabolismo , Peptídeos/metabolismo , Engenharia de Proteínas , Receptores de Antígenos de Linfócitos T/imunologia
5.
Blood ; 119(15): 3420-30, 2012 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-22318202

RESUMO

We investigated whether TCRs restricted to the more ubiquitously expressed MHC class I molecules could be used to redirect human regulatory T cells (Tregs). Using a series of HLA-A2-restricted TCRs that recognize the same peptide-MHC class I complex (pMHC) with affinities varying up to 3500 fold, we observed that TCR affinity had no effect on the ability of the introduced TCRs to confer potent Ag-specific suppressive activity. Surprisingly, we found a naturally occurring, low-affinity MHC class I-restricted TCR specific for an NY-ESO-1 epitope that was unable to redirect a functional CD4 T-effector cell response could confer potent antigen-specific suppressive activity when expressed in Tregs and severely impair the expansion of highly functional HIV-1(GAG)-specific CD8 T cells expressing a high-affinity TCR. This suppressive activity was only observed when both Ags were presented by the same cell, and no suppression was observed when the target Ags were put in distinct cells. These studies underscore the clinical utility of using MHC class I-restricted TCRs to endow Tregs with specificity to control autoimmune disease and highlight the conditions in which this approach would have most therapeutic benefit.


Assuntos
Especificidade do Receptor de Antígeno de Linfócitos T/fisiologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/fisiologia , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/fisiologia , Células Cultivadas , Genes Reporter , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Células K562 , Ativação Linfocitária/genética , Ativação Linfocitária/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Ligação Proteica , Receptores de Antígenos de Linfócitos T/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Reguladores/metabolismo , Transfecção , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia
6.
Mol Cancer Ther ; 6(7): 2081-91, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17620437

RESUMO

Tumor-associated human telomerase reverse transcriptase (hTERT) is expressed in >85% of human tumors but not in most normal cells. As a result, this antigen has received considerable attention from those interested in cancer immunotherapy. Specifically, there has been strong interest in MHC class I-associated peptides derived from hTERT because these are expressed on the cell surface and thus may enable the targeting of tumor cells. Much of this interest has focused on peptide 540-548, ILAKFLHWL, which was predicted to exhibit the strongest binding to the common HLA A*0201 presenting molecule. The hTERT(540-548) peptide is currently being assessed in therapeutic vaccination trials; however, there is controversy surrounding whether it is naturally processed and presented on the surface of neoplastic cells. Here, we generate two highly sensitive reagents to assess the presentation of hTERT(540-548) on tumor cells: (a) a CD8(+) CTL clone, and (b) a recombinant T-cell receptor (TCR) that binds with picomolar affinity and a half-life exceeding 14 h. This TCR enables the identification of individual HLA A2-hTERT(540-548) complexes on the cell surface. The use of both this TCR and the highly antigen-sensitive CTL clone shows that the hTERT(540-548) peptide cannot be detected on the surface of tumor cells, indicating that this peptide is not a naturally presented epitope. We propose that, in future, rigorous methods must be applied for the validation of peptide epitopes used for clinical applications.


Assuntos
Antígenos HLA-A/imunologia , Fragmentos de Peptídeos/imunologia , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Telomerase/imunologia , Sequência de Aminoácidos , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/imunologia , Linhagem Celular Tumoral , Separação Celular , Células Clonais , Ensaio de Imunoadsorção Enzimática , Epitopos , Antígeno HLA-A2 , Humanos , Interferon gama/farmacologia , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Inibidores de Proteassoma , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/isolamento & purificação , Linfócitos T Citotóxicos/efeitos dos fármacos , Transfecção
7.
Nat Med ; 21(8): 914-921, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26193344

RESUMO

Despite recent therapeutic advances, multiple myeloma (MM) remains largely incurable. Here we report results of a phase I/II trial to evaluate the safety and activity of autologous T cells engineered to express an affinity-enhanced T cell receptor (TCR) recognizing a naturally processed peptide shared by the cancer-testis antigens NY-ESO-1 and LAGE-1. Twenty patients with antigen-positive MM received an average 2.4 × 10(9) engineered T cells 2 d after autologous stem cell transplant. Infusions were well tolerated without clinically apparent cytokine-release syndrome, despite high IL-6 levels. Engineered T cells expanded, persisted, trafficked to marrow and exhibited a cytotoxic phenotype. Persistence of engineered T cells in blood was inversely associated with NY-ESO-1 levels in the marrow. Disease progression was associated with loss of T cell persistence or antigen escape, in accordance with the expected mechanism of action of the transferred T cells. Encouraging clinical responses were observed in 16 of 20 patients (80%) with advanced disease, with a median progression-free survival of 19.1 months. NY-ESO-1-LAGE-1 TCR-engineered T cells were safe, trafficked to marrow and showed extended persistence that correlated with clinical activity against antigen-positive myeloma.


Assuntos
Antígenos de Neoplasias/imunologia , Proteínas de Membrana/imunologia , Mieloma Múltiplo/terapia , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/imunologia , Idoso , Antígenos de Neoplasias/genética , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Feminino , Engenharia Genética , Humanos , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/mortalidade , Sindecana-1/análise
8.
Sci Transl Med ; 5(197): 197ra103, 2013 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-23926201

RESUMO

MAGE A3, which belongs to the family of cancer-testis antigens, is an attractive target for adoptive therapy given its reactivation in various tumors and limited expression in normal tissues. We developed an affinity-enhanced T cell receptor (TCR) directed to a human leukocyte antigen (HLA)-A*01-restricted MAGE A3 antigen (EVDPIGHLY) for use in adoptive therapy. Extensive preclinical investigations revealed no off-target antigen recognition concerns; nonetheless, administration to patients of T cells expressing the affinity-enhanced MAGE A3 TCR resulted in a serious adverse event (SAE) and fatal toxicity against cardiac tissue. We present a description of the preclinical in vitro functional analysis of the MAGE A3 TCR, which failed to reveal any evidence of off-target activity, and a full analysis of the post-SAE in vitro investigations, which reveal cross-recognition of an off-target peptide. Using an amino acid scanning approach, a peptide from the muscle protein Titin (ESDPIVAQY) was identified as an alternative target for the MAGE A3 TCR and the most likely cause of in vivo toxicity. These results demonstrate that affinity-enhanced TCRs have considerable effector functions in vivo and highlight the potential safety concerns for TCR-engineered T cells. Strategies such as peptide scanning and the use of more complex cell cultures are recommended in preclinical studies to mitigate the risk of off-target toxicity in future clinical investigations.


Assuntos
Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Conectina/química , Reações Cruzadas/imunologia , Antígeno HLA-A1/imunologia , Proteínas de Neoplasias/imunologia , Peptídeos/imunologia , Linfócitos T/metabolismo , Sequência de Aminoácidos , Antígenos de Neoplasias/química , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Conectina/imunologia , Reações Cruzadas/efeitos dos fármacos , Células HEK293 , Humanos , Ativação Linfocitária/efeitos dos fármacos , Dados de Sequência Molecular , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Proteínas de Neoplasias/química , Peptídeos/química , Engenharia de Proteínas , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/efeitos dos fármacos
9.
Mol Biotechnol ; 45(2): 140-9, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20143183

RESUMO

Previously, we have described the use of phage display to generate high affinity disulfide bond-linked T cell receptors (TCRs). The affinities of the mutant TCRs were analysed after refolding of separately expressed alpha and beta chains from Escherichia coli inclusion bodies. This approach is only suitable for the analysis of small numbers of TCR variants. An attractive alternative would be soluble expression within the bacterial periplasm, but the generic production of TCRs within the E. coli periplasm has so far not proved successful. Here we show that functional, soluble TCR can be produced within the cytoplasm of trxB gor mutant E. coli strains, with maximum yields of 3.4 mg/l. We also investigated the effect of coexpressing the folding modulators Skp and DsbC finding that the TCR expression levels were largely unaffected by these chaperones. Importantly, we demonstrated that the amount of protein purified from 50 ml starter cultures was sufficient to show functionality of the TCR by specific antigen binding in both ELISA and surface plasmon resonance (SPR) assays. This TCR production method has the potential to allow rapid and medium throughput analysis of affinity-matured TCRs selected from TCR phage display libraries.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Glutationa Redutase/genética , Receptores de Antígenos de Linfócitos T/biossíntese , Tiorredoxina Dissulfeto Redutase/genética , Sequência de Aminoácidos , Sequência de Bases , Cromatografia de Afinidade , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dissulfetos/metabolismo , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Glutationa Redutase/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Ressonância de Plasmônio de Superfície , Tiorredoxina Dissulfeto Redutase/metabolismo
10.
J Immunol ; 180(9): 6116-31, 2008 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-18424733

RESUMO

Single and dual amino acid substitution variants were generated in the TCR CDRs of three TCRs that recognize tumor-associated Ags. Substitutions that enhance the reactivity of TCR gene-modified T cells to the cognate Ag complex were identified using a rapid RNA-based transfection system. The screening of a panel of variants of the 1G4 TCR, that recognizes a peptide corresponding to amino acid residues 157-165 of the human cancer testis Ag NY-ESO-1 (SLLMWITQC) in the context of the HLA-A*02 class I allele, resulted in the identification of single and dual CDR3alpha and CDR2beta amino acid substitutions that dramatically enhanced the specific recognition of NY-ESO-1(+)/HLA-A*02(+) tumor cell lines by TCR gene-modified CD4(+) T cells. Within this group of improved TCRs, a dual substitution in the 1G4 TCR CDR3alpha chain was identified that enhanced Ag-specific reactivity in gene-modified CD4(+) and CD8(+) T cells. Separate experiments on two distinct TCRs that recognize the MART-1 27-35 (AAGIGILTV) peptide/HLA-A*02 Ag complex characterized single amino acid substitutions in both TCRs that enhanced CD4(+) T cell Ag-specific reactivity. These results indicate that simple TCR substitution variants that enhance T cell function can be identified by rapid transfection and assay techniques, providing the means for generating potent Ag complex-specific TCR genes for use in the study of T cell interactions and in T cell adoptive immunotherapy.


Assuntos
Substituição de Aminoácidos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Regiões Determinantes de Complementaridade/genética , Antígenos HLA-A/imunologia , Receptores de Antígenos de Linfócitos T/genética , Substituição de Aminoácidos/imunologia , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Regiões Determinantes de Complementaridade/imunologia , Antígenos HLA-A/genética , Antígeno HLA-A2 , Humanos , Antígeno MART-1 , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T/imunologia
11.
J Immunol ; 179(9): 5845-54, 2007 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17947658

RESUMO

We examined the activity of human T cells engineered to express variants of a single TCR (1G4) specific for the cancer/testis Ag NY-ESO-1, generated by bacteriophage display with a wide range of affinities (from 4 microM to 26 pM). CD8(+) T cells expressing intermediate- and high-affinity 1G4 TCR variants bound NY-ESO-1/HLA-A2 tetramers with high avidity and Ag specificity, but increased affinity was associated with a loss of target cell specificity of the TCR gene-modified cells. T cells expressing the highest affinity TCR (K(D) value of 26 pM) completely lost Ag specificity. The TCRs with affinities in the midrange, K(D) 5 and 85 nM, showed specificity only when CD8 was absent or blocked, while the variant TCRs with affinities in the intermediate range-with K(D) values of 450 nM and 4 microM-demonstrated Ag-specific recognition. Although the biological activity of these two relatively low-affinity TCRs was comparable to wild-type reactivity in CD8(+) T cells, introduction of these TCR dramatically increased the reactivity of CD4(+) T cells to tumor cell lines.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Neoplasias/imunologia , Neoplasias/patologia , Biblioteca de Peptídeos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Sequência de Aminoácidos , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Sobrevivência Celular , Células Cultivadas , Reações Cruzadas/imunologia , Antígenos de Histocompatibilidade/imunologia , Humanos , Ativação Linfocitária/imunologia , Dados de Sequência Molecular , Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos T/química , Sensibilidade e Especificidade
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