RESUMO
Effective antitumour immunity depends on the orchestration of potent T cell responses against malignancies1. Regression of human cancers has been induced by immune checkpoint inhibitors, T cell engagers or chimeric antigen receptor T cell therapies2-4. Although CD8 T cells function as key effectors of these responses, the role of CD4 T cells beyond their helper function has not been defined. Here we demonstrate that a trispecific antibody to HER2, CD3 and CD28 stimulates regression of breast cancers in a humanized mouse model through a mechanism involving CD4-dependent inhibition of tumour cell cycle progression. Although CD8 T cells directly mediated tumour lysis in vitro, CD4 T cells exerted antiproliferative effects by blocking cancer cell cycle progression at G1/S. Furthermore, when T cell subsets were adoptively transferred into a humanized breast cancer tumour mouse model, CD4 T cells alone inhibited HER2+ breast cancer growth in vivo. RNA microarray analysis revealed that CD4 T cells markedly decreased tumour cell cycle progression and proliferation, and also increased pro-inflammatory signalling pathways. Collectively, the trispecific antibody to HER2 induced T cell-dependent tumour regression through direct antitumour and indirect pro-inflammatory/immune effects driven by CD4 T cells.
Assuntos
Neoplasias da Mama , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Feminino , Humanos , Camundongos , Receptor ErbB-2/genéticaRESUMO
Common variable immunodeficiency (CVID) is an immune defect characterized by hypogammaglobulinemia and impaired development of B cells into plasma cells. As follicular helper T cells (TFH) play a central role in humoral immunity, we examined TFH cells in CVID, and investigated whether an inducible T cell co-stimulator (ICOS) agonist, vopratelimab, could modulate TFH, B cell interactions and enhance immunoglobulin production. CVID subjects had decreased TFH17 and increased TFH1 subsets; this was associated with increased transitional B cells and decreased IgG+ B and IgD-IgM-CD27+ memory B cells. ICOS expression on CVID CD4+ T cells was also decreased. However, ICOS activation of CD4+ T cells by vopratelimab significantly increased total CVID TFH, TFH2, cell numbers, as well as IL-4, IL-10 and IL-21 secretion in vitro. Vopratelimab treatment also increased plasma cells, IgG+ B cells, reduced naïve & transitional B cells and significantly increased IgG1 secretion by CVID B cells. Interestingly, vopratelimab treatment also restored IgA secretion in PBMCs from several CVID patients who had a complete lack of endogenous serum IgA. Our data demonstrate the potential of TFH modulation in restoring TFH and enhancing B cell maturation in CVID. The effects of an ICOS agonist in antibody defects warrants further investigation. This biologic may also be of therapeutic interest in other clinical settings of antibody deficiency.
Assuntos
Linfócitos B , Imunodeficiência de Variável Comum , Proteína Coestimuladora de Linfócitos T Induzíveis , Células T Auxiliares Foliculares , Humanos , Imunodeficiência de Variável Comum/imunologia , Imunodeficiência de Variável Comum/tratamento farmacológico , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Linfócitos B/imunologia , Linfócitos B/efeitos dos fármacos , Feminino , Masculino , Pessoa de Meia-Idade , Adulto , Células T Auxiliares Foliculares/imunologia , Células T Auxiliares Foliculares/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Imunoglobulina G/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Idoso , Adulto JovemRESUMO
Anti-CD38 monoclonal antibodies (mAbs) represent a breakthrough in the treatment of multiple myeloma (MM), yet some patients fail to respond or progress quickly with this therapy, highlighting the need for novel approaches. In this study we compared the preclinical efficacy of SAR442085, a next-generation anti-CD38 mAb with enhanced affinity for activating Fcγ receptors (FcγR), with first-generation anti-CD38 mAb daratumumab and isatuximab. In surface plasmon resonance and cellular binding assays, we found that SAR442085 had higher binding affinity than daratumumab and isatuximab for FcγRIIa (CD32a) and FcγRIIIa (CD16a). SAR442085 also exhibited better in vitro antibody-dependent cellular cytotoxicity (ADCC) against a panel of MM cells expressing variable CD38 receptor densities including MM patients' primary plasma cells. The enhanced ADCC of SAR442085 was confirmed using NK-92 cells bearing low and high affinity FcγRIIIa (CD16a)-158F/V variants. Using MM patients' primary bone marrow cells, we confirmed that SAR442085 had an increased ability to engage FcγRIIIa, resulting in higher natural killer (NK) cell activation and degranulation against primary plasma cells than preexisting Fc wild-type anti-CD38 mAbs. Finally, using huFcgR transgenic mice that express human Fcγ receptors under the control of their human regulatory elements, we demonstrated that SAR442085 had higher NK cell-dependent in vivo antitumor efficacy and better survival than daratumumab and isatuximab against EL4 thymoma or VK*MYC myeloma cells overexpressing human CD38. These results highlight the preclinical efficacy of SAR442085 and support the current evaluation of this next-generation anti-CD38 antibody in phase I clinical development in patients with relapsed/refractory MM.
Assuntos
ADP-Ribosil Ciclase 1/antagonistas & inibidores , Antineoplásicos Imunológicos/farmacologia , Células da Medula Óssea , Glicoproteínas de Membrana/antagonistas & inibidores , Mieloma Múltiplo , Proteínas de Neoplasias/antagonistas & inibidores , ADP-Ribosil Ciclase 1/metabolismo , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos Transgênicos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Proteínas de Neoplasias/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mechanisms of unassisted delivery of RNA therapeutics, including inhibitors of microRNAs, remain poorly understood. We observed that the hepatocellular carcinoma cell line SKHEP1 retains productive free uptake of a miR-21 inhibitor (anti-miR-21). Uptake of anti-miR-21, but not a mismatch (MM) control, induces expression of known miR-21 targets (DDAH1, ANKRD46) and leads to dose-dependent inhibition of cell growth. To elucidate mechanisms of SKHEP1 sensitivity to anti-miR-21, we conducted an unbiased shRNA screen that revealed tumor susceptibility gene 101 (TSG101), a component of the endosomal sorting complex required for transport (ESCRT-I), as an important determinant of anti-proliferative effects of anti-miR-21. RNA interference-mediated knockdown of TSG101 and another ESCRT-I protein, VPS28, improved uptake of anti-miR-21 in parental SKHEP1 cells and restored productive uptake to SKHEP1 clones with acquired resistance to anti-miR-21. Depletion of ESCRT-I in several additional cancer cell lines with inherently poor uptake resulted in improved activity of anti-miR-21. Finally, knockdown of TSG101 increased uptake of anti-miR-21 by cancer cells in vivo following systemic delivery. Collectively, these data support an important role for the ESCRT-I complex in the regulation of productive free uptake of anti-miRs and reveal potential avenues for improving oligonucleotide free uptake by cancer cells.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , MicroRNAs/antagonistas & inibidores , Neoplasias/metabolismo , Oligonucleotídeos/metabolismo , Animais , Transporte Biológico , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/antagonistas & inibidores , Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Feminino , Humanos , Camundongos SCID , MicroRNAs/metabolismo , Neoplasias/genética , Fatores de Transcrição/fisiologiaRESUMO
Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg(2+). A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors' selectivity for the mutant enzyme.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/química , Isocitrato Desidrogenase/química , Neoplasias/tratamento farmacológico , Sítio Alostérico , Cristalografia por Raios X , Metilação de DNA/genética , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/uso terapêutico , Escherichia coli , Regulação Neoplásica da Expressão Gênica , Humanos , Isocitrato Desidrogenase/antagonistas & inibidores , Isocitrato Desidrogenase/biossíntese , Isocitrato Desidrogenase/genética , Magnésio/química , Proteínas Mutantes/química , Proteínas Mutantes/genética , Neoplasias/genética , Neoplasias/patologia , Conformação ProteicaRESUMO
Postchemotherapy relapse presents a major unmet medical need in acute myeloid leukemia (AML), where treatment options are limited. CD25 is a leukemic stem cell marker and a conspicuous prognostic marker for overall/relapse-free survival in AML. Rare occurrence of genetic alterations among PIM family members imposes a substantial hurdle in formulating a compelling patient stratification strategy for the clinical development of selective PIM inhibitors in cancer. Here we show that CD25, a bona fide STAT5 regulated gene, is a mechanistically relevant predictive biomarker for sensitivity to PIM kinase inhibitors. Alone or in combination with tyrosine kinase inhibitors, PIM inhibitors can suppress STAT5 activation and significantly shorten the half-life of MYC to achieve substantial growth inhibition of high CD25-expressing AML cells. Our results highlight the importance of STAT5 and MYC in rendering cancer cells sensitive to PIM inhibitors. Because the presence of a CD25-positive subpopulation in leukemic blasts correlates with poor overall or relapse-free survival, our data suggest that a combination of PIM inhibitors with chemotherapy and tyrosine kinase inhibitors could improve long-term therapeutic outcomes in CD25-positive AML.
Assuntos
Antineoplásicos/farmacologia , Crise Blástica , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Fator de Transcrição STAT5/metabolismo , Antineoplásicos/química , Crise Blástica/tratamento farmacológico , Crise Blástica/genética , Crise Blástica/metabolismo , Crise Blástica/patologia , Feminino , Células HL-60 , Humanos , Subunidade alfa de Receptor de Interleucina-2/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-pim-1/genética , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Fator de Transcrição STAT5/genéticaRESUMO
PRP4 kinase is known for its roles in regulating pre-mRNA splicing and beyond. Therefore, a wider spectrum of PRP4 kinase substrates could be expected. The role of PRP4 kinase in cancer is also yet to be fully elucidated. Attaining specific and potent PRP4 inhibitors would greatly facilitate the study of PRP4 biological function and its validation as a credible cancer target. In this report, we verified the requirement of enzymatic activity of PRP4 in regulating cancer cell growth and identified an array of potential novel substrates through orthogonal proteomics approaches. The ensuing effort in structural biology unveiled for the first time unique features of PRP4 kinase domain and its potential mode of interaction with a low molecular weight inhibitor. These results provide new and important information for further exploration of PRP4 kinase function in cancer.
Assuntos
Proteínas de Neoplasias , Neoplasias , Inibidores de Proteínas Quinases , Ribonucleoproteína Nuclear Pequena U4-U6 , Linhagem Celular Tumoral , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/genética , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteômica/métodos , Ribonucleoproteína Nuclear Pequena U4-U6/antagonistas & inibidores , Ribonucleoproteína Nuclear Pequena U4-U6/química , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismoRESUMO
Tumor-associated macrophages (TAM) play an important role in maintaining the immunosuppressive state of the tumor microenvironment (TME). High levels of CD163+ TAMs specifically are associated with poor prognosis in many solid tumor types. Targeting TAMs may represent a key approach in development of the next generation of cancer immune therapeutics. Members of the leukocyte immunoglobulin-like receptor B (LILRB) family, including LILRB2 (ILT4), are known to transmit inhibitory signals in macrophages and other myeloid cells. Leveraging bulk and single cell RNA-sequencing datasets, as well as extensive immunophenotyping of human tumors, we found that LILRB2 is highly expressed on CD163+ CD11b+ cells in the TME and that LILRB2 expression correlates with CD163 expression across many tumor types. To target LILRB2, we have developed JTX-8064, a highly potent and selective antagonistic mAb. JTX-8064 blocks LILRB2 binding to its cognate ligands, including classical and nonclassical MHC molecules. In vitro, JTX-8064 drives the polarization of human macrophages and dendritic cells toward an immunostimulatory phenotype. As a result, human macrophages treated with a LILRB2 blocker are reprogrammed to increase the activation of autologous T cells in co-culture systems. Furthermore, JTX-8064 significantly potentiates the activity of anti-PD-1 in allogeneic mixed lymphocyte reaction. In a human tumor explant culture, pharmacodynamic activity of JTX-8064 was observed in monotherapy and in combination with anti-PD-1. Collectively, our work provides strong translational and preclinical rationale to target LILRB2 in cancer.
Assuntos
Neoplasias , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Macrófagos/metabolismo , Ativação Linfocitária , Técnicas de Cocultura , Linfócitos T , Microambiente Tumoral , Glicoproteínas de Membrana/genética , Receptores ImunológicosRESUMO
BACKGROUND: Cancer immunotherapy has revolutionized cancer treatment. However, considering the limited success of immunotherapy to only some cancer types and patient cohorts, there is an unmet need for developing new treatments that will result in higher response rates in patients with cancer. Immunoglobulin-like transcript 2 (ILT2), a LILRB family member, is an inhibitory receptor expressed on a variety of immune cells including T cells, natural killer (NK) cells and different myeloid cells. In the tumor microenvironment, binding of class I MHC (in particular HLA-G) to ILT2 on immune cells mediates a strong inhibitory effect, which manifests in inhibition of antitumor cytotoxicity of T and NK cells, and prevention of phagocytosis of the tumor cells by macrophages. METHODS: We describe here the development and characteristics of BND-22, a novel, humanized monoclonal antibody that selectively binds to ILT2 and blocks its interaction with classical MHC I and HLA-G. BND-22 was evaluated for its binding and blocking characteristics as well as its ability to increase the antitumor activity of macrophages, T cells and NK cells in various in vitro, ex vivo and in vivo systems. RESULTS: Collectively, our data suggest that BND-22 enhances activity of both innate and adaptive immune cells, thus generating robust and comprehensive antitumor immunity. In humanized mice models, blocking ILT2 with BND-22 decreased the growth of human tumors, hindered metastatic spread to the lungs, and prolonged survival of the tumor-bearing mice. In addition, BND-22 improved the antitumor immune response of approved therapies such as anti-PD-1 or anti-EGFR antibodies. CONCLUSIONS: BND-22 is a first-in-human ILT2 blocking antibody which has demonstrated efficient antitumor activity in various preclinical models as well as a favorable safety profile. Clinical evaluation of BND-22 as a monotherapy or in combination with other therapeutics is under way in patients with cancer. TRIAL REGISTRATION NUMBER: NCT04717375.
Assuntos
Antígenos HLA-G , Neoplasias , Animais , Antígenos HLA-G/metabolismo , Humanos , Imunoterapia , Células Matadoras Naturais , Camundongos , Neoplasias/tratamento farmacológico , Linfócitos T , Microambiente TumoralRESUMO
The presence of T regulatory (Treg) cells in the tumor microenvironment is associated with poor prognosis and resistance to therapies aimed at reactivating anti-tumor immune responses. Therefore, depletion of tumor-infiltrating Tregs is a potential approach to overcome resistance to immunotherapy. However, identifying Treg-specific targets to drive such selective depletion is challenging. CCR8 has recently emerged as one of these potential targets. Here, we describe GS-1811, a novel therapeutic monoclonal antibody that specifically binds to human CCR8 and is designed to selectively deplete tumor-infiltrating Tregs. We validate previous findings showing restricted expression of CCR8 on tumor Tregs, and precisely quantify CCR8 receptor densities on tumor and normal tissue T cell subsets, demonstrating a window for selective depletion of Tregs in the tumor. Importantly, we show that GS-1811 depleting activity is limited to cells expressing CCR8 at levels comparable to tumor-infiltrating Tregs. Targeting CCR8 in mouse tumor models results in robust anti-tumor efficacy, which is dependent on Treg depleting activity, and synergizes with PD-1 inhibition to promote anti-tumor responses in PD-1 resistant models. Our data support clinical development of GS-1811 to target CCR8 in cancer and drive tumor Treg depletion in order to promote anti-tumor immunity.
Assuntos
Neoplasias , Linfócitos T Reguladores , Camundongos , Animais , Humanos , Linfócitos T Reguladores/metabolismo , Receptor de Morte Celular Programada 1 , Imunoterapia/métodos , Neoplasias/terapia , Microambiente Tumoral , Fragmentos Fc das Imunoglobulinas/metabolismo , Receptores CCR8/metabolismoRESUMO
Deregulation of the PI3K signaling pathway is observed in many human cancers and occurs most frequently through loss of PTEN phosphatase tumor suppressor function or through somatic activating mutations in the Class IA PI3K, PIK3CA. Tumors harboring activated p110alpha, the protein product of PIK3CA, require p110alpha activity for growth and survival and hence are expected to be responsive to inhibitors of its lipid kinase activity. Whether PTEN-deficient cancers similarly depend on p110alpha activity to sustain activation of the PI3K pathway has been unclear. In this study, we used a single-vector lentiviral inducible shRNA system to selectively inactivate the three Class IA PI3Ks, PIK3CA, PIK3CB, and PIK3CD, to determine which PI3K isoforms are responsible for driving the abnormal proliferation of PTEN-deficient cancers. Down-regulation of PIK3CA in colorectal cancer cells harboring mutations in PIK3CA inhibited downstream PI3K signaling and cell growth. Surprisingly, PIK3CA depletion affected neither PI3K signaling nor cell growth in 3 PTEN-deficient cancer cell lines. In contrast, down-regulation of the PIK3CB isoform, which encodes p110beta, resulted in pathway inactivation and subsequent inhibition of growth in both cell-based and in vivo settings. This essential function of PIK3CB in PTEN-deficient cancer cells required its lipid kinase activity. Our findings demonstrate that although p110alpha activation is required to sustain the proliferation of established PIK3CA-mutant tumors, PTEN-deficient tumors are dependent instead on p110beta signaling. This unexpected finding demonstrates the need to tailor therapeutic approaches to the genetic basis of PI3K pathway activation to achieve optimal treatment response.
Assuntos
Neoplasias/enzimologia , PTEN Fosfo-Hidrolase/deficiência , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Classe I de Fosfatidilinositol 3-Quinases , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Regulação para Baixo , Humanos , Masculino , Camundongos , Camundongos Nus , Mutação/genética , Neoplasias/patologia , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/deficiência , Fosfoproteínas/metabolismo , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Transplante HeterólogoRESUMO
Novel therapies are needed for effective treatment of AML. In the relapsed setting, prognosis is very poor despite salvage treatment with chemotherapy. Evidence suggests that leukemic stem cells (LSCs) cause relapse. The cell surface receptor CD123 is highly expressed in blast cells and LSCs from AML patients and is a potential therapeutic target. CD123 cross-over dual-variable domain T-cell engager (CD123-CODV-TCE) is a bispecific antibody with an innovative format. One arm targets the CD3εδ subunit of T-cell co-receptors on the surface of T cells, while the other targets CD123 on malignant cells, leading to cell-specific cytotoxic activity. Here, we describe the preclinical activity of CD123-CODV-TCE. CD123-CODV-TCE effectively binds to human and cynomolgus monkey CD3 and CD123 and is a highly potent T-cell engager. It mediates T-cell activation and T-cell-directed killing of AML cells in vitro. In vivo, CD123-CODV-TCE suppresses AML tumor growth in leukemia xenograft mouse models, where it achieves an effective half-life of 3.2 days, which is a significantly longer half-life compared to other bispecific antibodies with no associated Fc fragment. The in vitro safety profile is as expected for compounds with similar modes of action. These results suggest that CD123-CODV-TCE may be a promising therapy for patients with relapsed/refractory AML.
Assuntos
Anticorpos Biespecíficos , Leucemia Mieloide Aguda , Animais , Complexo CD3 , Humanos , Subunidade alfa de Receptor de Interleucina-3 , Leucemia Mieloide Aguda/tratamento farmacológico , Macaca fascicularis , Camundongos , Linfócitos TRESUMO
Immune checkpoint blockade elicits durable anti-cancer responses in the clinic, however a large proportion of patients do not benefit from treatment. Several mechanisms of innate and acquired resistance to checkpoint blockade have been defined and include mutations of MHC I and IFNγ signaling pathways. However, such mutations occur in a low frequency of patients and additional mechanisms have yet to be elucidated. In an effort to better understand acquired resistance to checkpoint blockade, we generated a mouse tumor model exhibiting in vivo resistance to anti-PD-1 antibody treatment. MC38 tumors acquired resistance to PD-1 blockade following serial in vivo passaging. Lack of sensitivity to PD-1 blockade was not attributed to dysregulation of PD-L1 or ß2M expression, as both were expressed at similar levels in parental and resistant cells. Similarly, IFNγ signaling and antigen processing and presentation pathways were functional in both parental and resistant cell lines. Unbiased gene expression analysis was used to further characterize potential resistance mechanisms. RNA-sequencing revealed substantial differences in global gene expression, with tumors resistant to anti-PD-1 displaying a marked reduction in expression of immune-related genes relative to parental MC38 tumors. Indeed, resistant tumors exhibited reduced immune infiltration across multiple cell types, including T and NK cells. Pathway analysis revealed activation of TGFß and Notch signaling in anti-PD-1 resistant tumors, and activation of these pathways was associated with poorer survival in human cancer patients. While pharmacological inhibition of TGFß and Notch in combination with PD-1 blockade decelerated tumor growth, a local mRNA-based immunotherapy potently induced regression of resistant tumors, resulting in complete tumor remission, and resensitized tumors to treatment with anti-PD-1. Overall, this study describes a novel anti-PD-1 resistant mouse tumor model and underscores the role of two well-defined signaling pathways in response to immune checkpoint blockade. Furthermore, our data highlights the potential of intratumoral mRNA therapy in overcoming acquired resistance to PD-1 blockade.
Assuntos
Imunoterapia , Neoplasias , Animais , Apresentação de Antígeno , Modelos Animais de Doenças , Humanos , Camundongos , RNA Mensageiro/genéticaRESUMO
Local immunotherapy ideally stimulates immune responses against tumors while avoiding toxicities associated with systemic administration. Current strategies for tumor-targeted, gene-based delivery, however, are limited by adverse effects such as off-targeting or antivector immunity. We investigated the intratumoral administration of saline-formulated messenger (m)RNA encoding four cytokines that were identified as mediators of tumor regression across different tumor models: interleukin-12 (IL-12) single chain, interferon-α (IFN-α), granulocyte-macrophage colony-stimulating factor, and IL-15 sushi. Effective antitumor activity of these cytokines relied on multiple immune cell populations and was accompanied by intratumoral IFN-γ induction, systemic antigen-specific T cell expansion, increased granzyme B+ T cell infiltration, and formation of immune memory. Antitumor activity extended beyond the treated lesions and inhibited growth of distant tumors and disseminated tumors. Combining the mRNAs with immunomodulatory antibodies enhanced antitumor responses in both injected and uninjected tumors, thus improving survival and tumor regression. Consequently, clinical testing of this cytokine-encoding mRNA mixture is now underway.
Assuntos
Citocinas , Neoplasias , Citocinas/genética , Humanos , Neoplasias/genética , Neoplasias/terapia , RNA MensageiroRESUMO
Bmi-1 and Mel-18 are structural homologues that belong to the Polycomb group of transcriptional regulators and are believed to stably maintain repression of gene expression by altering the state of chromatin at specific promoters. While a number of clinical and experimental observations have implicated Bmi-1 in human tumorigenesis, the role of Mel-18 in cancer cell growth has not been investigated. We report here that short hairpin RNA-mediated knockdown of either Bmi-1 or Mel-18 in human medulloblastoma DAOY cells results in the inhibition of proliferation, loss of clonogenic survival, anchorage-independent growth, and suppression of tumor formation in nude mice. Furthermore, overexpression of both Bmi-1 and Mel-18 significantly increases the clonogenic survival of Rat1 fibroblasts. In contrast, stable downregulation of Bmi-1 or Mel-18 alone does not affect the growth of normal human WI38 fibroblasts. Proteomics-based characterization of Bmi-1 and Mel-18 protein complexes isolated from cancer cells revealed substantial similarities in their respective compositions. Finally, gene expression analysis identified a number of cancer-relevant pathways that may be controlled by Bmi-1 and Mel-18 and also showed that these Polycomb proteins regulate a set of common gene targets. Taken together, these results suggest that Bmi-1 and Mel-18 may have overlapping functions in cancer cell growth.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Meduloblastoma/patologia , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Homologia de Sequência de Aminoácidos , Animais , Morte Celular , Proliferação de Células , Sobrevivência Celular , Proteínas de Ligação a DNA/genética , Regulação para Baixo/genética , Fibroblastos/citologia , Expressão Gênica , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Meduloblastoma/genética , Camundongos , Proteínas Nucleares/genética , Complexo Repressor Polycomb 1 , Proteínas do Grupo Polycomb , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/metabolismo , Ratos , Proteínas Repressoras/genética , Transplante HeterólogoRESUMO
TGFß is a pleiotropic cytokine that may have both tumor inhibiting and tumor promoting properties, depending on tissue and cellular context. Emerging data support a role for TGFß in suppression of antitumor immunity. Here we show that SAR439459, a pan-TGFß neutralizing antibody, inhibits all active isoforms of human and murine TGFß, blocks TGFß-mediated pSMAD signaling, and TGFß-mediated suppression of T cells and NK cells. In vitro, SAR439459 synergized with anti-PD1 to enhance T cell responsiveness. In syngeneic tumor models, SAR439459 treatment impaired tumor growth, while the combination of SAR439459 with anti-PD-1 resulted in complete tumor regression and a prolonged antitumor immunity. Mechanistically, we found that TGFß inhibition with PD-1 blockade augmented intratumoral CD8+ T cell proliferation, reduced exhaustion, evoked proinflammatory cytokines, and promoted tumor-specific CD8+ T cell responses. Together, these data support the hypothesis that TGFß neutralization using SAR439459 synergizes with PD-1 blockade to promote antitumor immunity and formed the basis for the ongoing clinical investigation of SAR439459 in patients with cancer (NCT03192345).
Assuntos
Terapia de Imunossupressão , Receptor de Morte Celular Programada 1 , Animais , Anticorpos Monoclonais/farmacologia , Linhagem Celular Tumoral , Humanos , Tolerância Imunológica , CamundongosRESUMO
Isatuximab is a monoclonal antibody targeting the transmembrane receptor and ectoenzyme CD38, a protein highly expressed on hematological malignant cells, including those in multiple myeloma (MM). Upon binding to CD38-expressing MM cells, isatuximab is thought to induce tumor cell killing via fragment crystallizable (Fc)-dependent mechanisms, including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), as well as via direct Fc-independent mechanisms. Here, these mechanisms of action were investigated in MM and diffuse large B-cell lymphoma (DLBCL) cell lines, as well as in peripheral blood mononuclear cells derived from healthy donors, and in MM patient-derived samples. Our findings show that isatuximab-mediated cytotoxicity occurred primarily via ADCC and ADCP in MM cell lines and via ADCC and apoptosis in DLBCL cell lines expressing high levels of CD38. We identified the programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) pathway and MM cell-secreted transforming growth factor-beta (TGF-ß) as tumor cell-related features that could suppress CD38-mediated ADCC. Furthermore, we established that isatuximab can directly activate natural killer (NK) cells and promote NK cell-mediated cytotoxicity via crosslinking of CD38 and CD16. Finally, isatuximab-induced CDC was observed in cell lines with high CD38 receptor density (>250,000 molecules/cell) and limited expression of inhibitory complement regulatory proteins (CD46, CD55, and CD59; <50,000 molecules/cell). Taken together, our findings highlight mechanistic insights for isatuximab and provide support for a range of combination therapy approaches that could be tested for isatuximab in the future.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos Imunológicos/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/imunologia , Mieloma Múltiplo/imunologia , Apoptose/efeitos dos fármacos , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Ativação Linfocitária/efeitos dos fármacosRESUMO
VPS34 is a key regulator of endomembrane dynamics and cargo trafficking, and is essential in cultured cell lines and in mice. To better characterize the role of VPS34 in cell growth, we performed unbiased cell line profiling studies with the selective VPS34 inhibitor PIK-III and identified RKO as a VPS34-dependent cellular model. Pooled CRISPR screen in the presence of PIK-III revealed endolysosomal genes as genetic suppressors. Dissecting VPS34-dependent alterations with transcriptional profiling, we found the induction of hypoxia response and cholesterol biosynthesis as key signatures. Mechanistically, acute VPS34 inhibition enhanced lysosomal degradation of transferrin and low-density lipoprotein receptors leading to impaired iron and cholesterol uptake. Excess soluble iron, but not cholesterol, was sufficient to partially rescue the effects of VPS34 inhibition on mitochondrial respiration and cell growth, indicating that iron limitation is the primary driver of VPS34-dependency in RKO cells. Loss of RAB7A, an endolysosomal marker and top suppressor in our genetic screen, blocked transferrin receptor degradation, restored iron homeostasis and reversed the growth defect as well as metabolic alterations due to VPS34 inhibition. Altogether, our findings suggest that impaired iron mobilization via the VPS34-RAB7A axis drive VPS34-dependence in certain cancer cells.
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Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Ferro/metabolismo , Neoplasias/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células , Colesterol/biossíntese , Colesterol/genética , Classe III de Fosfatidilinositol 3-Quinases/genética , Endossomos/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Receptores de LDL/metabolismo , Transferrina/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
PURPOSE: We have previously reported that matrix metalloproteinases MMP-2, MMP-9, and the complex MMP-9/NGAL can be detected in urine of patients with a variety of cancers including prostate and bladder carcinoma. In addition, we also detected several unidentified urinary gelatinase activities with molecular weights >125 kDa. The objective of the current study was to identify these high molecular weight (HMW) species, determine their potential as predictors of disease status, and ask whether a tumor-specific pattern existed based on urinary MMP analysis. EXPERIMENTAL DESIGN: Chromatography, zymography, and mass spectrometry was used to identify HMW gelatinase species of approximately 140, 190, and >220 kDa in urine of cancer patients. To determine whether a tumor-specific pattern of appearance existed among the MMPs detected, we analyzed the urine of 189 patients with prostate or bladder cancer and controls. RESULTS: The approximately 140, >220 kDa, and approximately 190 HMW gelatinase species were identified as MMP-9/tissue inhibitor of metalloproteinase 1 complex, MMP-9 dimer, and ADAMTS-7, respectively. The frequency of detection of any MMP species was significantly higher in urine from prostate and bladder cancer groups than controls. MMP-9 dimer and MMP-9 were independent predictors for distinguishing between patients with prostate and bladder cancer (P < 0.001 for each) by multivariable analysis. CONCLUSIONS: This study is the first to identify a tumor-specific urinary MMP fingerprint that may noninvasively facilitate identification of cancer presence and type. This information may be of diagnostic and prognostic value in the detection and/or clinical monitoring of disease progression and therapeutic efficacy in patients with bladder or prostate cancer.