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P60, a Foxp3 inhibitory peptide, can hinder the regulatory T cell (Treg) activity and impair tumor proliferation. However, low systemic stability and poor specificity have led to daily dosing to achieve therapeutic effect. Therefore, this study aims to improve P60 stability and specific delivery through its encapsulation in liposomes targeting CD25, constitutively expressed in Tregs. P60 liposomes formulated with DSPE-PEG750 or DSPE-PEG2000 were incubated with DSPE-PEG2000-Maleimide micelles conjugated to Fab' fragments of anti-CD25 to develop two targeted formulations or immunoliposomes (IL): IL-P602000 (DSPE-PEG2000 only) and IL-P60750 (combining DSPE-PEG750 and DSPE-PEG2000). P60 encapsulation efficiency was 50%-60% irrespective of PEG chain length. Treg uptake was 2.5 and 14 times higher for IL-PEG750 compared with IL-PEG2000 and non-targeted liposomes, respectively, in in-vitro assays. In fact, IL-P60750 allowed CD8+ T cells ex-vivo proliferation in presence of Treg at doses 10-20 times lower than for free P60. Antitumor response of P60 and IL-P60750 in monotherapy and combined with anti-PD-1 was evaluated in MC38 and LLCOVA tumor bearing mice. In MC38 model, IL-P60750 monotherapy induced total tumor regression in 40% of mice reaching 100% for anti-PD-1 combination. This effect was associated with a significant increase in activated CD8+ T cells in tumors. Notably, IL-P60750 also inhibited human Treg in ex-vivo assay, showing the translational capability of this formulation. In conclusion, IL-P60750 formulated with different PEG chain lengths, has demonstrated antitumor efficacy by selective inhibition of Treg activity and enhances the effect of anti-PD1. Altogether, this novel IL represents a promising nanoplatform for cancer immunotherapies.
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Despite the potential of CAR-T therapies for hematological malignancies, their efficacy in patients with relapse and refractory Acute Myeloid Leukemia has been limited. The aim of our study has been to develop and manufacture a CAR-T cell product that addresses some of the current limitations. We initially compared the phenotype of T cells from AML patients and healthy young and elderly controls. This analysis showed that T cells from AML patients displayed a predominantly effector phenotype, with increased expression of activation (CD69 and HLA-DR) and exhaustion markers (PD1 and LAG3), in contrast to the enriched memory phenotype observed in healthy donors. This differentiated and more exhausted phenotype was also observed, and corroborated by transcriptomic analyses, in CAR-T cells from AML patients engineered with an optimized CAR construct targeting CD33, resulting in a decreased in vivo antitumoral efficacy evaluated in xenograft AML models. To overcome some of these limitations we have combined CRISPR-based genome editing technologies with virus-free gene-transfer strategies using Sleeping Beauty transposons, to generate CAR-T cells depleted of HLA-I and TCR complexes (HLA-IKO/TCRKO CAR-T cells) for allogeneic approaches. Our optimized protocol allows one-step generation of edited CAR-T cells that show a similar phenotypic profile to non-edited CAR-T cells, with equivalent in vitro and in vivo antitumoral efficacy. Moreover, genomic analysis of edited CAR-T cells revealed a safe integration profile of the vector, with no preferences for specific genomic regions, with highly specific editing of the HLA-I and TCR, without significant off-target sites. Finally, the production of edited CAR-T cells at a larger scale allowed the generation and selection of enough HLA-IKO/TCRKO CAR-T cells that would be compatible with clinical applications. In summary, our results demonstrate that CAR-T cells from AML patients, although functional, present phenotypic and functional features that could compromise their antitumoral efficacy, compared to CAR-T cells from healthy donors. The combination of CRISPR technologies with transposon-based delivery strategies allows the generation of HLA-IKO/TCRKO CAR-T cells, compatible with allogeneic approaches, that would represent a promising option for AML treatment.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , Animais , Humanos , Idoso , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/metabolismo , Imunoterapia Adotiva/métodos , Modelos Animais de DoençasRESUMO
Immunotherapy resistance in non-small cell lung cancer (NSCLC) may be mediated by an immunosuppressive microenvironment, which can be shaped by the mutational landscape of the tumor. Here, we observed genetic alterations in the PTEN/PI3K/AKT/mTOR pathway and/or loss of PTEN expression in >25% of patients with NSCLC, with higher frequency in lung squamous carcinomas (LUSC). Patients with PTEN-low tumors had higher levels of PD-L1 and PD-L2 and showed worse progression-free survival when treated with immunotherapy. Development of a Pten-null LUSC mouse model revealed that tumors with PTEN loss were refractory to antiprogrammed cell death protein 1 (anti-PD-1), highly metastatic and fibrotic, and secreted TGFß/CXCL10 to promote conversion of CD4+ lymphocytes into regulatory T cells (Treg). Human and mouse PTEN-low tumors were enriched in Tregs and expressed higher levels of immunosuppressive genes. Importantly, treatment of mice bearing Pten-null tumors with TLR agonists and anti-TGFß antibody aimed to alter this immunosuppressive microenvironment and led to tumor rejection and immunologic memory in 100% of mice. These results demonstrate that lack of PTEN causes immunotherapy resistance in LUSCs by establishing an immunosuppressive tumor microenvironment that can be reversed therapeutically. SIGNIFICANCE: PTEN loss leads to the development of an immunosuppressive microenvironment in lung cancer that confers resistance to anti-PD-1 therapy, which can be overcome by targeting PTEN loss-mediated immunosuppression.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares , PTEN Fosfo-Hidrolase , Linfócitos T Reguladores , Animais , Humanos , Camundongos , Antígeno B7-H1/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Imunoterapia/métodos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Microambiente Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêuticoRESUMO
[This corrects the article DOI: 10.3389/fimmu.2022.1044025.].
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The historical lack of preclinical models reflecting the genetic heterogeneity of multiple myeloma (MM) hampers the advance of therapeutic discoveries. To circumvent this limitation, we screened mice engineered to carry eight MM lesions (NF-κB, KRAS, MYC, TP53, BCL2, cyclin D1, MMSET/NSD2 and c-MAF) combinatorially activated in B lymphocytes following T cell-driven immunization. Fifteen genetically diverse models developed bone marrow (BM) tumors fulfilling MM pathogenesis. Integrative analyses of â¼500 mice and â¼1,000 patients revealed a common MAPK-MYC genetic pathway that accelerated time to progression from precursor states across genetically heterogeneous MM. MYC-dependent time to progression conditioned immune evasion mechanisms that remodeled the BM microenvironment differently. Rapid MYC-driven progressors exhibited a high number of activated/exhausted CD8+ T cells with reduced immunosuppressive regulatory T (Treg) cells, while late MYC acquisition in slow progressors was associated with lower CD8+ T cell infiltration and more abundant Treg cells. Single-cell transcriptomics and functional assays defined a high ratio of CD8+ T cells versus Treg cells as a predictor of response to immune checkpoint blockade (ICB). In clinical series, high CD8+ T/Treg cell ratios underlie early progression in untreated smoldering MM, and correlated with early relapse in newly diagnosed patients with MM under Len/Dex therapy. In ICB-refractory MM models, increasing CD8+ T cell cytotoxicity or depleting Treg cells reversed immunotherapy resistance and yielded prolonged MM control. Our experimental models enable the correlation of MM genetic and immunological traits with preclinical therapy responses, which may inform the next-generation immunotherapy trials.
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Mieloma Múltiplo , Camundongos , Animais , Mieloma Múltiplo/terapia , Mieloma Múltiplo/tratamento farmacológico , Linfócitos T CD8-Positivos , Evasão da Resposta Imune , Linfócitos T Reguladores , Imunoterapia/efeitos adversos , Microambiente Tumoral/genéticaRESUMO
Regulatory T cells overwhelm conventional T cells in the tumor microenvironment (TME) thanks to a FOXP3-driven metabolic program that allows them to engage different metabolic pathways. Using a melanoma model of adoptive T cell therapy (ACT), we show that FOXP3 overexpression in mature CD8 T cells improved their antitumor efficacy, favoring their tumor recruitment, proliferation, and cytotoxicity. FOXP3-overexpressing (Foxp3UP) CD8 T cells exhibited features of tissue-resident memory-like and effector T cells, but not suppressor activity. Transcriptomic analysis of tumor-infiltrating Foxp3UP CD8 T cells showed positive enrichment in a wide variety of metabolic pathways, such as glycolysis, fatty acid (FA) metabolism, and oxidative phosphorylation (OXPHOS). Intratumoral Foxp3UP CD8 T cells exhibited an enhanced capacity for glucose and FA uptake as well as accumulation of intracellular lipids. Interestingly, Foxp3UP CD8 T cells compensated for the loss of mitochondrial respiration-driven ATP production by activating aerobic glycolysis. Moreover, in limiting nutrient conditions these cells engaged FA oxidation to drive OXPHOS for their energy demands. Importantly, their ability to couple glycolysis and OXPHOS allowed them to sustain proliferation under glucose restriction. Our findings demonstrate a hitherto unknown role for FOXP3 in the adaptation of CD8 T cells to TME that may enhance their efficacy in ACT.
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Linfócitos T CD8-Positivos , Fatores de Transcrição Forkhead , Imunoterapia Adotiva , Melanoma , Humanos , Linfócitos T CD8-Positivos/imunologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Glucose/metabolismo , Melanoma/terapia , Microambiente TumoralRESUMO
Immune checkpoint inhibitor (ICI)-based immunotherapy in triple negative breast cancer (TNBC) is achieving limited therapeutic results, requiring the development of more potent strategies. Combination of ICI with vaccination strategies would enhance antitumor immunity and response rates to ICI in patients having poorly infiltrated tumors. In heavily mutated tumors, neoantigens (neoAgs) resulting from tumor mutations have induced potent responses when used as vaccines. Thus, our aim was the identification of immunogenic neoAgs suitable as vaccines in TNBC patients. By using whole exome sequencing, RNAseq and HLA binding algorithms of tumor samples from a cohort of eight TNBC patients, we identified a median of 60 mutations/patient, which originated a putative median number of 98 HLA class I-restricted neoAgs. Considering a group of 27 predicted neoAgs presented by HLA-A*02:01 allele in two patients, peptide binding to HLA was experimentally confirmed in 63% of them, whereas 55% were immunogenic in vivo in HLA-A*02:01+ transgenic mice, inducing T-cells against the mutated but not the wild-type peptide sequence. Vaccination with peptide pools or DNA plasmids expressing these neoAgs induced polyepitopic T-cell responses, which recognized neoAg-expressing tumor cells. These results suggest that TNBC tumors harbor neoAgs potentially useful in therapeutic vaccines, opening the way for new combined immunotherapies.
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Neoplasias de Mama Triplo Negativas , Humanos , Camundongos , Animais , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/terapia , Imunoterapia/métodos , Antígenos de Neoplasias , Antígeno HLA-A2 , Peptídeos , Camundongos TransgênicosRESUMO
Identification of new markers associated with long-term efficacy in patients treated with CAR T cells is a current medical need, particularly in diseases such as multiple myeloma. In this study, we address the impact of CAR density on the functionality of BCMA CAR T cells. Functional and transcriptional studies demonstrate that CAR T cells with high expression of the CAR construct show an increased tonic signaling with up-regulation of exhaustion markers and increased in vitro cytotoxicity but a decrease in in vivo BM infiltration. Characterization of gene regulatory networks using scRNA-seq identified regulons associated to activation and exhaustion up-regulated in CARHigh T cells, providing mechanistic insights behind differential functionality of these cells. Last, we demonstrate that patients treated with CAR T cell products enriched in CARHigh T cells show a significantly worse clinical response in several hematological malignancies. In summary, our work demonstrates that CAR density plays an important role in CAR T activity with notable impact on clinical response.
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Current vaccines against SARS-CoV-2, based on the original Wuhan sequence, induce antibodies with different degrees of cross-recognition of new viral variants of concern. Despite potent responses generated in vaccinated and infected individuals, the Omicron (B.1.1.529) variant causes breakthrough infections, facilitating viral transmission. We previously reported a vaccine based on a cyclic peptide containing the 446-488 S1 sequence (446-488cc) of the SARS-CoV-2 spike (S) protein from Wuhan isolate. To provide the best immunity against Omicron, here we compared Omicron-specific immunity induced by a Wuhan-based 446-488cc peptide, by a Wuhan-based recombinant receptor-binding domain (RBD) vaccine and by a new 446-488cc peptide vaccine based on the Omicron sequence. Antibodies induced by Wuhan peptide 446-488cc in three murine strains not only recognized the Wuhan and Omicron 446-488 peptides similarly, but also Wuhan and Omicron RBD protein variants. By contrast, antibodies induced by the Wuhan recombinant RBD vaccine showed a much poorer cross-reactivity for the Omicron RBD despite similar recognition of Wuhan and Omicron peptide variants. Finally, although the Omicron-based 446-488cc peptide vaccine was poorly immunogenic in mice due to the loss of T cell epitopes, co-immunization with Omicron peptide 446-488cc and exogenous T cell epitopes induced strong cross-reactive antibodies that neutralized Omicron SARS-CoV-2 virus. Since mutations occurring within this sequence do not alter T cell epitopes in humans, these results indicate the robust immunogenicity of 446-488cc-based peptide vaccines that induce antibodies with a high cross-recognition capacity against Omicron, and suggest that this sequence could be included in future vaccines targeting the Omicron variant.
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COVID-19 , SARS-CoV-2 , Humanos , Animais , Camundongos , Vacinas contra COVID-19 , Epitopos de Linfócito T , COVID-19/prevenção & controle , Vacinas de Subunidades Antigênicas , AnticorposRESUMO
Identification of relevant epitopes is crucial for the development of subunit peptide vaccines inducing neutralizing and cellular immunity against SARS-CoV-2. Our aim was the characterization of epitopes in the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein to generate a peptide vaccine. Epitope mapping using a panel of 10 amino acid overlapped 15-mer peptides covering region 401-515 from RBD did not identify linear epitopes when tested with sera from infected individuals or from RBD-immunized mice. However, immunization of mice with these 15-mer peptides identified four peptides located at region 446-480 that induced antibodies recognizing the peptides and RBD/S1 proteins. Immunization with peptide 446-480 from S protein formulated with Freund's adjuvant or with CpG oligodeoxinucleotide/Alum induced polyepitopic antibody responses in BALB/c and C56BL/6J mice, recognizing RBD (titres of 3 × 104-3 × 105, depending on the adjuvant) and displaying neutralizing capacity (80-95% inhibition capacity; p < 0.05) against SARS-CoV-2. Murine CD4 and CD8T-cell epitopes were identified in region 446-480 and vaccination experiments using HLA transgenic mice suggested the presence of multiple human T-cell epitopes. Antibodies induced by peptide 446-480 showed broad recognition of S proteins and S-derived peptides belonging to SARS-CoV-2 variants of concern. Importantly, vaccination with peptide 446-480 or with a cyclic version of peptide 446-488 containing a disulphide bridge between cysteines 480 and 488, protected humanized K18-hACE2 mice from a lethal dose of SARS-CoV-2 (62.5 and 75% of protection; p < 0.01 and p < 0.001, respectively). This region could be the basis for a peptide vaccine or other vaccine platforms against Covid-19.
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Anticorpos Neutralizantes/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Imunidade Celular , Imunidade Humoral , SARS-CoV-2/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Neutralizantes/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , Vacinas contra COVID-19/normas , Reações Cruzadas/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B , Epitopos de Linfócito T/imunologia , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/imunologiaRESUMO
BACKGROUND: Adoptive immunotherapy with tumour-infiltrating lymphocytes (TIL) may benefit from the use of selective markers, such as PD-1, for tumour-specific T-cell enrichment, and the identification of predictive factors that help identify those patients capable of rendering tumour-reactive TILs. We have investigated this in ovarian cancer (OC) patients as candidates for TIL therapy implementation. METHODS: PD-1- and PD-1+ CD8 TILs were isolated from ovarian tumours and expanded cells were tested against autologous tumour cells. Baseline tumour samples were examined using flow cytometry, multiplexed immunofluorescence and Nanostring technology, for gene expression analyses, as well as a next-generation sequencing gene panel, for tumour mutational burden (TMB) calculation. RESULTS: Tumour-reactive TILs were detected in half of patients and were exclusively present in cells derived from the PD-1+ fraction. Importantly, a high TIL density in the fresh tumour, the presence of CD137+ cells within the PD-1+CD8+ TIL subset and their location in the tumour epithelium, together with a baseline T-cell-inflamed genetic signature and/or a high TMB, are features that identify patients rendering tumour-reactive TIL products. CONCLUSION: We have demonstrated that PD-1 identifies ovarian tumour-specific CD8 TILs and has uncovered predictive factors that identify OC patients who are likely to render tumour-specific cells from PD-1+ TILs.
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Biomarcadores Tumorais/genética , Linfócitos T CD8-Positivos/imunologia , Regulação Neoplásica da Expressão Gênica , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Ovarianas/patologia , Receptor de Morte Celular Programada 1/metabolismo , Biomarcadores Tumorais/metabolismo , Feminino , Seguimentos , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/imunologia , Fenótipo , Prognóstico , Estudos RetrospectivosRESUMO
Analyzing immunomodulatory elements operating during antitumor vaccination in prostate cancer patients and murine models we identified IL-10-producing DC as a subset with poorer immunogenicity and clinical efficacy. Inhibitory TAM receptors MER and AXL were upregulated on murine IL-10+ DC. Thus, we analyzed conditions inducing these molecules and the potential benefit of their blockade during vaccination. MER and AXL upregulation was more efficiently induced by a vaccine containing Imiquimod than by a poly(I:C)-containing vaccine. Interestingly, MER expression was found on monocyte-derived DC, and was dependent on IL-10. TAM blockade improved Imiquimod-induced DC activation in vitro and in vivo, resulting in increased vaccine-induced T-cell responses, which were further reinforced by concomitant IL-10 inhibition. In different tumor models, a triple therapy (including vaccination, TAM inhibition and IL-10 blockade) provided the strongest therapeutic effect, associated with enhanced T-cell immunity and enhanced CD8+ T cell tumor infiltration. Finally, MER levels in DC used for vaccination in cancer patients correlated with IL-10 expression, showing an inverse association with vaccine-induced clinical response. These results suggest that TAM receptors upregulated during vaccination may constitute an additional target in combinatorial therapeutic vaccination strategies.
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Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Imunoterapia/métodos , Melanoma Experimental/terapia , Neoplasias da Próstata/terapia , Adjuvantes Imunológicos/administração & dosagem , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/administração & dosagem , Linhagem Celular Tumoral , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Imiquimode/administração & dosagem , Imunogenicidade da Vacina/efeitos dos fármacos , Interleucina-10/metabolismo , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Masculino , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Transgênicos , Poli I-C/administração & dosagem , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Pirimidinas , Quinolinas , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , c-Mer Tirosina Quinase/antagonistas & inibidores , c-Mer Tirosina Quinase/genética , Receptor Tirosina Quinase AxlRESUMO
Cancer is characterized by genomic instability leading to deletion or amplification of oncogenes or tumor suppressors. However, most of the altered regions are devoid of known cancer drivers. Here, we identify lncRNAs frequently lost or amplified in cancer. Among them, we found amplified lncRNA associated with lung cancer-1 (ALAL-1) as frequently amplified in lung adenocarcinomas. ALAL-1 is also overexpressed in additional tumor types, such as lung squamous carcinoma. The RNA product of ALAL-1 is able to promote the proliferation and tumorigenicity of lung cancer cells. ALAL-1 is a TNFα- and NF-κB-induced cytoplasmic lncRNA that specifically interacts with SART3, regulating the subcellular localization of the protein deubiquitinase USP4 and, in turn, its function in the cell. Interestingly, ALAL-1 expression inversely correlates with the immune infiltration of lung squamous tumors, while tumors with ALAL-1 amplification show lower infiltration of several types of immune cells. We have thus unveiled a pro-oncogenic lncRNA that mediates cancer immune evasion, pointing to a new target for immune potentiation.
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Variações do Número de Cópias de DNA/genética , Evasão da Resposta Imune/genética , Neoplasias Pulmonares/genética , RNA Longo não Codificante/genética , Células A549 , Adenocarcinoma de Pulmão/genética , Antígenos de Neoplasias/genética , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , NF-kappa B/genética , Oncogenes/genética , Proteases Específicas de Ubiquitina/genéticaRESUMO
Harnessing the immune system by blocking the programmed cell death protein 1 (PD-1) pathway has been a major breakthrough in non-small-cell lung cancer treatment. Nonetheless, many patients fail to respond to PD-1 inhibition. Using three syngeneic models, we demonstrate that short-term starvation synergizes with PD-1 blockade to inhibit lung cancer progression and metastasis. This antitumor activity was linked to a reduction in circulating insulin-like growth factor 1 (IGF-1) and a downregulation of IGF-1 receptor (IGF-1R) signaling in tumor cells. A combined inhibition of IGF-1R and PD-1 synergistically reduced tumor growth in mice. This effect required CD8 cells, boosted the intratumoral CD8/Treg ratio and led to the development of tumor-specific immunity. In patients with non-small-cell lung cancer, high plasma levels of IGF-1 or high IGF-1R expression in tumors was associated with resistance to anti-PD-1-programmed death-ligand 1 immunotherapy. In conclusion, our data strongly support the clinical evaluation of IGF-1 modulators in combination with PD-1 blockade.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Humanos , Inibidores de Checkpoint Imunológico , Fator de Crescimento Insulin-Like I/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Receptor de Morte Celular Programada 1RESUMO
BACKGROUND/AIM: Cytokine-induced killer (CIK) cells are ex vivo expanded major histocompatibility complex (MHC)-unrestricted cytotoxic cells with promising effects against a variety of cancer types. Regulatory T-cells (T-reg) have been shown to reduce the effectiveness of CIK cells against tumor cells. Peptide P60 has been shown to inhibit the immunosuppressive functions of T-regs. This study aimed at examining the effect of p60 on CIK cells efficacy against renal and pancreatic cancer cells. MATERIALS AND METHODS: The effect of P60 on CIK cytotoxicity was examined using flow cytometry, WST-8-based cell viability assay and interferon γ (IFNγ) ELISA. RESULTS: P60 treatment resulted in a significant decrease in the viability of renal and pancreatic cancer cell lines co-cultured with CIK cells. No increase in IFNγ secretion from CIK cells was detected following treatment with P60. P60 caused no changes in the distribution of major effector cell populations in CIK cell cultures. CONCLUSION: P60 may potentiate CIK cell cytotoxicity against tumor cells.
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Células Matadoras Induzidas por Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Fatores de Transcrição Forkhead/antagonistas & inibidores , Neoplasias Renais/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Peptídeos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura/métodos , Células Matadoras Induzidas por Citocinas/metabolismo , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Interferon gama/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Neoplasias Renais/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Neoplasias Pancreáticas/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismoRESUMO
Immune checkpoint blockade has shown anti-cancer efficacy, but requires systemic administration of monoclonal antibodies (mAbs), often leading to adverse effects. To avoid toxicity, mAbs could be expressed locally in tumors. We developed adeno-associated virus (AAV) and Semliki Forest virus (SFV) vectors expressing anti-programmed death ligand 1 (aPDL1) mAb. When injected intratumorally in MC38 tumors, both viral vectors led to similar local mAb expression at 24 h, diminishing quickly in SFV-aPDL1-treated tumors. However, SFV-aPDL1 induced >40% complete regressions and was superior to AAV-aPDL1, as well as to aPDL1 mAb given systemically or locally. SFV-aPDL1 induced abscopal effects and was also efficacious against B16-ovalbumin (OVA). The higher SFV-aPDL1 antitumor activity could be related to local upregulation of interferon-stimulated genes because of SFV RNA replication. This was confirmed by combining local SFV-LacZ administration and systemic aPDL1 mAb, which provided higher antitumor effects than each separated agent. SFV-aPDL1 promoted tumor-specific CD8 T cells infiltration in both tumor models. In MC38, SFV-aPDL1 upregulated co-stimulatory markers (CD137/OX40) in tumor CD8 T cells, and its combination with anti-CD137 mAb showed more pronounced antitumor effects than each single agent. These results indicate that local transient expression of immunomodulatory mAbs using non-propagative RNA vectors inducing type I interferon (IFN-I) responses represents a potent and safe approach for cancer treatment.
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Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Expressão Gênica , Vetores Genéticos/genética , Neoplasias/genética , Neoplasias/imunologia , Vírus de RNA/genética , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linhagem Celular , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Humanos , Imunomodulação/efeitos dos fármacos , Imunofenotipagem , Injeções Intralesionais , Camundongos , Neoplasias/patologia , Neoplasias/terapia , Proteínas Recombinantes de Fusão/genética , Vírus da Floresta de Semliki/genética , Taxa de Sobrevida , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Carga TumoralRESUMO
Immune checkpoint inhibitors are currently tested in different combinations in patients with advanced hepatocellular carcinoma (HCC). Nivolumab, an anti-PD-1 agent, has gained approval in the second-line setting in the USA. Epigenetic drugs have immune-mediated antitumor effects that may improve the activity of immunotherapy agents. Our aim was to study the therapeutic efficacy of checkpoint inhibitors (anti-CTLA-4 and anti-PD-1 antibodies) in combination with the histone deacetylase inhibitor (HDACi) Belinostat. In a subcutaneous Hepa129 murine HCC model, we demonstrated that Belinostat improves the antitumor activity of anti-CTLA-4 but not of anti-PD-1 therapy. This effect correlated with enhanced IFN-γ production by antitumor T-cells and a decrease in regulatory T-cells. Moreover, the combination induced early upregulation of PD-L1 on tumor antigen-presenting cells and late expression of PD-1 on tumor-infiltrating effector T-cells, suggesting the suitability of PD-1 blockade. Indeed, Belinostat combined with the simultaneous blockade of CTLA-4 and PD-1 led to complete tumor rejection. These results provide a rationale for testing Belinostat in combination with checkpoint inhibitors to enhance their therapeutic activity in patients with HCC.
Assuntos
Antígeno CTLA-4/antagonistas & inibidores , Carcinoma Hepatocelular/tratamento farmacológico , Inibidores de Histona Desacetilases/administração & dosagem , Ácidos Hidroxâmicos/administração & dosagem , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Sulfonamidas/administração & dosagem , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Humanos , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C3H , Linfócitos T Reguladores/imunologiaRESUMO
Adoptive immunotherapy with ex vivo-expanded tumor-infiltrating lymphocytes (TILs) has achieved objective clinical responses in a significant number of patients with cancer. The failure of many patients to develop long-term tumor control may be, in part, due to exhaustion of transferred T cells in the presence of a hostile tumor microenvironment. In several tumor types, growth and survival of carcinoma cells appear to be sustained by a network of receptors/ligands of the ErbB family. We speculated that if transferred T cells could benefit from EGFR ligands produced by the tumor, they might proliferate better and exert their anti-tumor activities more efficiently. We found that CD8+ T cells transduced with a retrovirus to express EGFR responded to EGFR ligands activating the EGFR signaling pathway. These EGFR-expressing effector T cells proliferated better and produced more IFN-γ and TNF-α in the presence of EGFR ligands produced by tumor cells in vitro. EGFR-expressing CD8 T cells from OT-1 mice were more efficient killing B16-OVA cells than control OT-1 CD8 T cells. Importantly, EGFR-expressing OT-1 T cells injected into B16-OVA tumor bearing mice were recruited into the tumor, expressed lower levels of the exhaustion markers PD1, TIGIT, and LAG3, and were more efficient in delaying tumor growth. Our results suggest that genetic modification of CD8+ T cells to express EGFR might be considered in immunotherapeutic strategies based on adoptive transfer of anti-tumor T cells against cancers expressing EGFR ligands.
Assuntos
Transferência Adotiva , Linfócitos T CD8-Positivos , Receptores ErbB , Vetores Genéticos , Neoplasias , Retroviridae , Transdução Genética , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/transplante , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/imunologia , Feminino , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapiaRESUMO
Disruption of the programmed cell death protein 1 (PD-1) pathway with immune checkpoint inhibitors represents a major breakthrough in the treatment of non-small cell lung cancer. We hypothesized that combined inhibition of C5a/C5aR1 and PD-1 signaling may have a synergistic antitumor effect. The RMP1-14 antibody was used to block PD-1, and an L-aptamer was used to inhibit signaling of complement C5a with its receptors. Using syngeneic models of lung cancer, we demonstrate that the combination of C5a and PD-1 blockade markedly reduces tumor growth and metastasis and leads to prolonged survival. This effect is accompanied by a negative association between the frequency of CD8 T cells and myeloid-derived suppressor cells within tumors, which may result in a more complete reversal of CD8 T-cell exhaustion. Our study provides support for the clinical evaluation of anti-PD-1 and anti-C5a drugs as a novel combination therapeutic strategy for lung cancer.Significance: Using a variety of preclinical models of lung cancer, we demonstrate that the blockade of C5a results in a substantial improvement in the efficacy of anti-PD-1 antibodies against lung cancer growth and metastasis. This study provides the preclinical rationale for the combined blockade of PD-1/PD-L1 and C5a to restore antitumor immune responses, inhibit tumor cell growth, and improve outcomes of patients with lung cancer. Cancer Discov; 7(7); 694-703. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 653.