RESUMO
Lipid nanoparticle (LNP)-encapsulated mRNA has been used for in vivo production of several secreted protein classes, such as IgG, and has enabled the development of personalized vaccines in oncology. Establishing the feasibility of delivering complex multispecific modalities that require higher-order structures important for their function could help expand the use of mRNA/LNP biologic formulations. Here, we evaluated whether in vivo administration of mRNA/LNP formulations of SIRPα-Fc-CD40L and TIGIT-Fc-LIGHT could achieve oligomerization and extend exposure, on-target activity, and antitumor responses comparable with that of the corresponding recombinant fusion proteins. Intravenous infusion of the formulated LNP-encapsulated mRNAs led to rapid and sustained production of functional hexameric proteins in vivo, which increased the overall exposure relative to the recombinant protein controls by â¼28 to 140 fold over 96 hours. High concentrations of the mRNA-encoded proteins were also observed in secondary lymphoid organs and within implanted tumors, with protein concentrations in tumors up to 134-fold greater than with the recombinant protein controls 24 hours after treatment. In addition, SIRPα-Fc-CD40L and TIGIT-Fc-LIGHT mRNAs induced a greater increase in antigen-specific CD8+ T cells in the tumors. These mRNA/LNP formulations were well tolerated and led to a rapid increase in serum and intratumoral IL2, delayed tumor growth, extended survival, and outperformed the activities of benchmark mAb controls. Furthermore, the mRNA/LNPs demonstrated improved efficacy in combination with anti-PD-L1 relative to the recombinant fusion proteins. These data support the delivery of complex oligomeric biologics as mRNA/LNP formulations, where high therapeutic expression and exposure could translate into improved patient outcomes. SIGNIFICANCE: Lipid nanoparticle-encapsulated mRNA can efficiently encode complex fusion proteins encompassing immune checkpoint blockers and costimulators that functionally oligomerize in vivo with extended pharmacokinetics and durable exposure to induce potent antitumor immunity.
Assuntos
Nanopartículas , RNA Mensageiro , Proteínas Recombinantes de Fusão , Animais , Camundongos , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Nanopartículas/química , Humanos , Feminino , Camundongos Endogâmicos C57BL , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Lipídeos/química , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Linhagem Celular TumoralRESUMO
Although immunotherapy with PD-(L)1 blockade is routine for lung cancer, little is known about acquired resistance. Among 1,201 patients with non-small cell lung cancer (NSCLC) treated with PD-(L)1 blockade, acquired resistance is common, occurring in >60% of initial responders. Acquired resistance shows differential expression of inflammation and interferon (IFN) signaling. Relapsed tumors can be separated by upregulated or stable expression of IFNγ response genes. Upregulation of IFNγ response genes is associated with putative routes of resistance characterized by signatures of persistent IFN signaling, immune dysfunction, and mutations in antigen presentation genes which can be recapitulated in multiple murine models of acquired resistance to PD-(L)1 blockade after in vitro IFNγ treatment. Acquired resistance to PD-(L)1 blockade in NSCLC is associated with an ongoing, but altered IFN response. The persistently inflamed, rather than excluded or deserted, tumor microenvironment of acquired resistance may inform therapeutic strategies to effectively reprogram and reverse acquired resistance.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Animais , Camundongos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Transdução de Sinais , Imunoterapia , Apresentação de Antígeno , Antígeno B7-H1/metabolismo , Microambiente TumoralRESUMO
The extracellular domain of tumor necrosis factor receptors (TNFR) generally require assembly into a homotrimeric quaternary structure as a prerequisite for initiation of signaling via the cytoplasmic domains. TNF receptor homotrimers are natively activated by similarly homo-trimerized TNF ligands, but can also be activated by synthetic agonists including engineered antibodies and Fc-ligand fusion proteins. A large body of literature from pre-clinical models supports the hypothesis that synthetic agonists targeting a diverse range of TNF receptors (including 4-1BB, CD40, OX40, GITR, DR5, TNFRSF25, HVEM, LTßR, CD27, and CD30) could amplify immune responses to provide clinical benefit in patients with infectious diseases or cancer. Unfortunately, however, the pre-clinical attributes of synthetic TNF receptor agonists have not translated well in human clinical studies, and have instead raised fundamental questions regarding the intrinsic biology of TNF receptors. Clinical observations of bell-shaped dose response curves have led some to hypothesize that TNF receptor overstimulation is possible and can lead to anergy and/or activation induced cell death of target cells. Safety issues including liver toxicity and cytokine release syndrome have also been observed in humans, raising questions as to whether those toxicities are driven by overstimulation of the targeted TNF receptor, a non-TNF receptor related attribute of the synthetic agonist, or both. Together, these clinical findings have limited the development of many TNF receptor agonists, and may have prevented generation of clinical data which reflects the full potential of TNF receptor agonism. A number of recent studies have provided structural insights into how different TNF receptor agonists bind and cluster TNF receptors, and these insights aid in deconvoluting the intrinsic biology of TNF receptors with the mechanistic underpinnings of synthetic TNF receptor agonist therapeutics.
Assuntos
Neoplasias , Receptores do Fator de Necrose Tumoral , Humanos , Ligantes , Receptores do Fator de Necrose Tumoral/metabolismo , Antígenos CD40 , Transdução de SinaisRESUMO
Despite over a decade of clinical trials combining inhibition of emerging checkpoints with a PD-1/L1 inhibitor backbone, meaningful survival benefits have not been shown in PD-1/L1 inhibitor resistant or refractory solid tumours, particularly tumours dominated by a myelosuppressive microenvironment. Achieving durable anti-tumour immunity will therefore likely require combination of adaptive and innate immune stimulation, myeloid repolarisation, enhanced APC activation and antigen processing/presentation, lifting of the CD47/SIRPα (Cluster of Differentiation 47/signal regulatory protein alpha) 'do not eat me' signal, provision of an apoptotic 'pro-eat me' or 'find me' signal, and blockade of immune checkpoints. The importance of effectively targeting mLILRB2 and SIRPAyeloid cells to achieve improved response rates has recently been emphasised, given myeloid cells are abundant in the tumour microenvironment of most solid tumours. TNFSF14, or LIGHT, is a tumour necrosis superfamily ligand with a broad range of adaptive and innate immune activities, including (1) myeloid cell activation through Lymphotoxin Beta Receptor (LTßR), (2) T/NK (T cell and natural killer cell) induced anti-tumour immune activity through Herpes virus entry mediator (HVEM), (3) potentiation of proinflammatory cytokine/chemokine secretion through LTßR on tumour stromal cells, (4) direct induction of tumour cell apoptosis in vitro, and (5) the reorganisation of lymphatic tissue architecture, including within the tumour microenvironment (TME), by promoting high endothelial venule (HEV) formation and induction of tertiary lymphoid structures. LTBR (Lymphotoxin beta receptor) and HVEM rank highly amongst a range of costimulatory receptors in solid tumours, which raises interest in considering how LIGHT-mediated costimulation may be distinct from a growing list of immunotherapy targets which have failed to provide survival benefit as monotherapy or in combination with PD-1 inhibitors, particularly in the checkpoint acquired resistant setting.
Assuntos
Receptor beta de Linfotoxina , Neoplasias , Humanos , Receptor de Morte Celular Programada 1 , Células Mieloides , Citocinas , Neoplasias/tratamento farmacológico , Imunoterapia , Microambiente TumoralRESUMO
Vγ9Vδ2+ T cell-targeted immunotherapy is of interest to harness its MHC-independent cytotoxic potential against a variety of cancers. Recent studies have identified heterodimeric butyrophilin (BTN) 2A1 and BTN3A1 as the molecular entity providing "signal 1" to the Vγ9Vδ2 TCR, but "signal 2" costimulatory requirements remain unclear. Using a tumor cell-free assay, we demonstrated that a BTN2A1/3A1 heterodimeric fusion protein activated human Vγ9Vδ2+ T cells, but only in the presence of costimulatory signal via CD28 or NK group 2 member D. Nonetheless, addition of a bispecific γδ T cell engager BTN2A1/3A1-Fc-CD19scFv alone enhanced granzyme B-mediated killing of human CD19+ lymphoma cells when cocultured with Vγ9Vδ2+ T cells, suggesting expression of costimulatory ligand(s) on tumor cells is sufficient to satisfy the "signal 2" requirement. These results highlight the parallels of signal 1 and signal 2 requirements in αß and γδ T cell activation and demonstrate the utility of heterodimeric BTNs to promote targeted activation of γδ T cells.
Assuntos
Antígenos CD28 , Receptores de Antígenos de Linfócitos T gama-delta , Antígenos CD/metabolismo , Butirofilinas/metabolismo , Granzimas , Humanos , Ligantes , Ativação Linfocitária , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Receptores de Antígenos de Linfócitos T gama-delta/metabolismoRESUMO
Coinhibition of TIGIT (T cell immunoreceptor with Ig and ITIM domains) and PD-1/PD-L1 (PD-1/L1) may improve response rates compared with monotherapy PD-1/L1 blockade in checkpoint naive non-small cell lung cancer with PD-L1 expression >50%. TIGIT mAbs with an effector-competent Fc can induce myeloid cell activation, and some have demonstrated effector T cell depletion, which carries a clinical liability of unknown significance. TIGIT Ab blockade translates to antitumor activity by enabling PVR signaling through CD226 (DNAM-1), which can be directly inhibited by PD-1. Furthermore, DNAM-1 is downregulated on tumor-infiltrating lymphocytes (TILs) in advanced and checkpoint inhibition-resistant cancers. Therefore, broadening clinical responses from TIGIT blockade into PD-L1low or checkpoint inhibition-resistant tumors, may be induced by immune costimulation that operates independently from PD-1/L1 inhibition. TNFSF14 (LIGHT) was identified through genomic screens, in vitro functional analysis, and immune profiling of TILs as a TNF ligand that could provide broad immune activation. Accordingly, murine and human bifunctional fusion proteins were engineered linking the extracellular domain of TIGIT to the extracellular domain of LIGHT, yielding TIGIT-Fc-LIGHT. TIGIT competitively inhibited binding to all PVR ligands. LIGHT directly activated myeloid cells through interactions with LTßR (lymphotoxin ß receptor), without the requirement for a competent Fc domain to engage Fcγ receptors. LIGHT costimulated CD8+ T and NK cells through HVEM (herpes virus entry mediator A). Importantly, HVEM was more widely expressed than DNAM-1 on T memory stem cells and TILs across a range of tumor types. Taken together, the mechanisms of TIGIT-Fc-LIGHT promoted strong antitumor activity in preclinical tumor models of primary and acquired resistance to PD-1 blockade, suggesting that immune costimulation mediated by LIGHT may broaden the clinical utility of TIGIT blockade.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Animais , Antígeno B7-H1/genética , Humanos , Camundongos , Células Mieloides/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Receptores Imunológicos , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genéticaRESUMO
Disrupting the binding of CD47 to SIRPα has emerged as a promising immunotherapeutic strategy for advanced cancers by potentiating antibody-dependent cellular phagocytosis (ADCP) of targeted antibodies. Preclinically, CD47/SIRPα blockade induces antitumor activity by increasing the phagocytosis of tumor cells by macrophages and enhancing the cross-presentation of tumor antigens to CD8+ T cells by dendritic cells; both of these processes are potentiated by CD40 signaling. Here we generated a novel, two-sided fusion protein incorporating the extracellular domains of SIRPα and CD40L, adjoined by a central Fc domain, termed SIRPα-Fc-CD40L. SIRPα-Fc-CD40L bound CD47 and CD40 with high affinity and activated CD40 signaling in the absence of Fc receptor cross-linking. No evidence of hemolysis, hemagglutination, or thrombocytopenia was observed in vitro or in cynomolgus macaques. Murine SIRPα-Fc-CD40L outperformed CD47 blocking and CD40 agonist antibodies in murine CT26 tumor models and synergized with immune checkpoint blockade of PD-1 and CTLA4. SIRPα-Fc-CD40L activated a type I interferon response in macrophages and potentiated the activity of ADCP-competent targeted antibodies both in vitro and in vivo These data illustrated that whereas CD47/SIRPα inhibition could potentiate tumor cell phagocytosis, CD40-mediated activation of a type I interferon response provided a bridge between macrophage- and T-cell-mediated immunity that significantly enhanced durable tumor control and rejection.
Assuntos
Antígenos CD40/metabolismo , Antígeno CD47/antagonistas & inibidores , Linfócitos T CD8-Positivos/imunologia , Interferon Tipo I/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Proteínas Recombinantes de Fusão/farmacologia , Imunidade Adaptativa , Animais , Ligante de CD40/genética , Ligante de CD40/imunologia , Antígeno CD47/imunologia , Antígeno CD47/metabolismo , Linhagem Celular Tumoral , Humanos , Imunidade Inata , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Interferon Tipo I/metabolismo , Macaca fascicularis , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/metabolismo , Neoplasias/patologia , Distribuição Aleatória , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Simultaneous blockade of immune checkpoint molecules and co-stimulation of the TNF receptor superfamily (TNFRSF) is predicted to improve overall survival in human cancer. TNFRSF co-stimulation depends upon coordinated antigen recognition through the T cell receptor followed by homotrimerization of the TNFRSF, and is most effective when these functions occur simultaneously. To address this mechanism, we developed a two-sided human fusion protein incorporating the extracellular domains (ECD) of PD-1 and OX40L, adjoined by a central Fc domain, termed PD1-Fc-OX40L. The PD-1 end of the fusion protein binds PD-L1 and PD-L2 with affinities of 2.08 and 1.76 nM, respectively, and the OX40L end binds OX40 with an affinity of 246 pM. High binding affinity on both sides of the construct translated to potent stimulation of OX40 signaling and PD1:PD-L1/L2 blockade, in multiple in vitro assays, including improved potency as compared to pembrolizumab, nivolumab, tavolixizumab and combinations of those antibodies. Furthermore, when activated human T cells were co-cultured with PD-L1 positive human tumor cells, PD1-Fc-OX40L was observed to concentrate to the immune synapse, which enhanced proliferation of T cells and production of IL-2, IFNγ and TNFα, and led to efficient killing of tumor cells. The therapeutic activity of PD1-Fc-OX40L in established murine tumors was significantly superior to either PD1 blocking, OX40 agonist, or combination antibody therapy; and required CD4+ T cells for maximum response. Importantly, all agonist functions of PD1-Fc-OX40L are independent of Fc receptor cross-linking. Collectively, these data demonstrate a highly potent fusion protein that is part of a platform, capable of providing checkpoint blockade and TNFRSF costimulation in a single molecule, which uniquely localizes TNFRSF costimulation to checkpoint ligand positive tumor cells.
Assuntos
Ligante de CD40/metabolismo , Fragmentos Fc das Imunoglobulinas , Imunomodulação , Neoplasias/metabolismo , Neoplasias/terapia , Receptor de Morte Celular Programada 1/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Antígeno B7-H1/metabolismo , Biomarcadores Tumorais , Ligante de CD40/química , Linhagem Celular , Citotoxicidade Imunológica , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Ativação Linfocitária/imunologia , Camundongos , Neoplasias/imunologia , Neoplasias/mortalidade , Receptor de Morte Celular Programada 1/química , Ligação Proteica , Receptores OX40/metabolismo , Proteínas Recombinantes de Fusão/química , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Increased tryptophan (Trp) catabolism in the tumor microenvironment (TME) can mediate immune suppression by upregulation of interferon (IFN)-γ-inducible indoleamine 2,3-dioxygenase (IDO1) and/or ectopic expression of the predominantly liver-restricted enzyme tryptophan 2,3-dioxygenase (TDO). Whether these effects are due to Trp depletion in the TME or mediated by the accumulation of the IDO1 and/or TDO (hereafter referred to as IDO1/TDO) product kynurenine (Kyn) remains controversial. Here we show that administration of a pharmacologically optimized enzyme (PEGylated kynureninase; hereafter referred to as PEG-KYNase) that degrades Kyn into immunologically inert, nontoxic and readily cleared metabolites inhibits tumor growth. Enzyme treatment was associated with a marked increase in the tumor infiltration and proliferation of polyfunctional CD8+ lymphocytes. We show that PEG-KYNase administration had substantial therapeutic effects when combined with approved checkpoint inhibitors or with a cancer vaccine for the treatment of large B16-F10 melanoma, 4T1 breast carcinoma or CT26 colon carcinoma tumors. PEG-KYNase mediated prolonged depletion of Kyn in the TME and reversed the modulatory effects of IDO1/TDO upregulation in the TME.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Hidrolases/uso terapêutico , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Cinurenina/metabolismo , Neoplasias/tratamento farmacológico , Animais , Vacinas Anticâncer/uso terapêutico , Linhagem Celular Tumoral , Humanos , Neoplasias/enzimologia , Neoplasias/imunologia , Neoplasias/metabolismo , Microambiente TumoralRESUMO
Stimulation of several TNF receptor family proteins has been shown to dampen inflammatory disease in murine models through augmenting the number and/or activity of regulatory T cells (Tregs). We recently found that one molecule, 4-1BB, used binding to Galectin-9 to exert its immunosuppressive effects and drive expansion of CD8+Foxp3- Tregs. We now show that ligation of another TNFR family molecule, DR3, which has previously been found to strongly expand CD4+Foxp3+ Tregs and suppress inflammation, also requires Galectin-9. We found that the extracellular region of DR3 directly binds to Galectin-9, and that Galectin-9 associates with DR3 in Tregs. From studies in vitro with Galectin-9-/- CD4+ T cells and Tregs, we found that stimulatory activity induced by ligating DR3 was in part dependent on Galectin-9. In vivo, in a model of experimental autoimmune encephalomyelitis, we show that an agonist of DR3 suppressed disease, correlating with expansion of CD4+Foxp3+ Tregs, and this protective effect was lost in Galectin-9-/- mice. Similar results were seen in an allergic lung inflammation model. Thus, we demonstrate a novel function of Galectin-9 in facilitating activity of DR3 related to Treg-mediated suppression.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Galectinas/metabolismo , Inflamação/imunologia , Esclerose Múltipla/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Fatores de Transcrição Forkhead/metabolismo , Galectinas/genética , Humanos , Tolerância Imunológica , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Membro 25 de Receptores de Fatores de Necrose Tumoral/metabolismo , Transdução de SinaisRESUMO
T-cell costimulation typically occurs in a defined microenvironment that is not recapitulated by agonistic antibody therapy. To deliver such stimulation under more favorable conditions, we investigated whether an allogeneic cell-based vaccine that secreted Fc-OX40L, Fc-ICOSL, or Fc-4-1BBL would activate and expand T cells comparably with systemically administered agonist antibodies. Among these costimulators, locally secreted Fc-OX40L provided superior priming of antigen-specific CD8(+) T cells, compared with combinations with OX40 antibodies or vaccine alone. Vaccine-expressed Fc-OX40L also stimulated IFNγ, TNFα, granzyme B, and IL2 by antigen-specific CD8(+) T cells similarly to OX40 antibodies, without off-target consequences such as proinflammatory cytokine induction. Vaccine-secreted Fc-OX40L increased CD127(+)KLRG-1(-) memory precursor cells during the contraction phase, resulting in improved proliferation upon secondary antigen challenge, as compared with OX40 antibody. A cell-based vaccine cosecreting gp96-Ig and Fc-OX40L led to even more pronounced tumor control, complete tumor rejection, and increased tumor antigen-specific T-cell proliferation, including in tumor-infiltrating lymphocytes, as compared with combinations of gp96-Ig vaccine and OX40 antibodies, in mice with established melanoma or colorectal carcinoma. These data suggest that local modulation of the vaccine microenvironment has unexpected advantages over systemic costimulation with agonistic antibodies, which may simplify the clinical translation of such combination immunotherapies into humans. Cancer Immunol Res; 4(9); 766-78. ©2016 AACR.
Assuntos
Vacinas Anticâncer/imunologia , Imunidade , Memória Imunológica , Neoplasias/imunologia , Linfócitos T/imunologia , Ligante 4-1BB/antagonistas & inibidores , Ligante 4-1BB/imunologia , Transferência Adotiva , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Ligante de CD40/antagonistas & inibidores , Ligante de CD40/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Melanoma Experimental , Glicoproteínas de Membrana , Camundongos , Neoplasias/mortalidade , Neoplasias/patologia , Neoplasias/terapia , Linfócitos T/metabolismoRESUMO
Epilepsy or seizure disorder is among the least understood chronic medical conditions affecting over 65 million people worldwide. Here, we show that disruption of the polycystic kidney disease 2-like 1 (Pkd2l1 or Pkdl), encoding polycystin-L (PCL), a non-selective cation channel, increases neuronal excitability and the susceptibility to pentylenetetrazol-induced seizure in mice. PCL interacts with ß2-adrenergic receptor (ß2AR) and co-localizes with ß2AR on the primary cilia of neurons in the brain. Pkdl deficiency leads to the loss of ß2AR on neuronal cilia, which is accompanied with a remarkable reduction in cAMP levels in the central nervous system (CNS). The reduction of cAMP levels is associated with a reduction in the activation of cAMP response element-binding protein, but not the activation of Ca(2+)/calmodulin-dependent protein kinase II, Akt or mitogen-activated protein kinases. Our data, thus, indicate for the first time that a ciliary protein complex is required for the control of neuronal excitability in the CNS.
Assuntos
Canais de Cálcio/genética , Córtex Cerebral/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Epilepsia/genética , Hipocampo/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores de Superfície Celular/genética , Tálamo/metabolismo , Animais , Canais de Cálcio/deficiência , Córtex Cerebral/patologia , Cílios/metabolismo , Cílios/patologia , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Epilepsia/induzido quimicamente , Epilepsia/metabolismo , Epilepsia/patologia , Potenciais Pós-Sinápticos Excitadores , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Hipocampo/patologia , Humanos , Transporte de Íons , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Pentilenotetrazol , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Superfície Celular/deficiência , Transdução de Sinais , Tálamo/patologiaRESUMO
The classical model for identification and clinical development of anticancer agents was based on small molecules, which were often quite toxic. Early studies in small groups of patients would seek to identify a maximum tolerated dose and major dose-limiting toxicities. Tumor response (shrinkage) would be assessed after a minimum number of doses in phase II testing. The decision to take the drug into the randomized phase III clinical setting was usually based on the proportion and duration of objective tumor responses, along with overall survival compared with historical controls. Immune-oncologics that are designed to fight cancer by direct CD8(+) T-cell priming and activation or by blocking a negative regulatory molecule have a number of sharp distinctions from cytotoxic drugs. These include cytoreductive effects that may be very different in timing of onset from traditional chemotherapy and the potential for inducing long-term durable remissions even in heavily pretreated patients with metastatic disease. In this paper we review the different classes of immune-oncologic drugs in clinical development with particular attention to the biostatistical challenges associated with evaluating efficacy in clinical trials. Confronting these issues upfront is particularly important given the rapidly expanding number of clinical trials with both monotherapy and combination trials in immunooncology.
Assuntos
Antineoplásicos/uso terapêutico , Fatores Imunológicos/imunologia , Fatores Imunológicos/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Ensaios Clínicos como Assunto , Humanos , Projetos de PesquisaRESUMO
Antitumor T cells either avoid or are inhibited in hypoxic and extracellular adenosine-rich tumor microenvironments (TMEs) by A2A adenosine receptors. This may limit further advances in cancer immunotherapy. There is a need for readily available and safe treatments that weaken the hypoxia-A2-adenosinergic immunosuppression in the TME. Recently, we reported that respiratory hyperoxia decreases intratumoral hypoxia and concentrations of extracellular adenosine. We show that it also reverses the hypoxia-adenosinergic immunosuppression in the TME. This, in turn, stimulates (i) enhanced intratumoral infiltration and reduced inhibition of endogenously developed or adoptively transfered tumor-reactive CD8 T cells, (ii) increased proinflammatory cytokines and decreased immunosuppressive molecules, such as transforming growth factor-ß (TGF-ß), (iii) weakened immunosuppression by regulatory T cells, and (iv) improved lung tumor regression and long-term survival in mice. Respiratory hyperoxia also promoted the regression of spontaneous metastasis from orthotopically grown breast tumors. These effects are entirely T cell- and natural killer cell-dependent, thereby justifying the testing of supplemental oxygen as an immunological coadjuvant to combine with existing immunotherapies for cancer.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Oxigênio/uso terapêutico , Adenosina/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Hiperóxia/complicações , Hiperóxia/patologia , Hipóxia/complicações , Hipóxia/imunologia , Hipóxia/patologia , Terapia de Imunossupressão , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Neoplasias/patologia , Oxigênio/farmacologia , Indução de Remissão , Respiração/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
UNLABELLED: Intratumoral hypoxia and hypoxia inducible factor-1α (HIF-1-α)-dependent CD39/CD73 ectoenzymes may govern the accumulation of tumor-protecting extracellular adenosine and signaling through A2A adenosine receptors (A2AR) in tumor microenvironments (TME). Here, we explored the conceptually novel motivation to use supplemental oxygen as a treatment to inhibit the hypoxia/HIF-1α-CD39/CD73-driven accumulation of extracellular adenosine in the TME in order to weaken the tumor protection. We report that hyperoxic breathing (60 % O2) decreased the TME hypoxia, as well as levels of HIF-1α and downstream target proteins of HIF-1α in the TME according to proteomic studies in mice. Importantly, oxygenation also downregulated the expression of adenosine-generating ectoenzymes and significantly lowered levels of tumor-protecting extracellular adenosine in the TME. Using supplemental oxygen as a tool in studies of the TME, we also identified FHL-1 as a potentially useful marker for the conversion of hypoxic into normoxic TME. Hyperoxic breathing resulted in the upregulation of antigen-presenting MHC class I molecules on tumor cells and in the better recognition and increased susceptibility to killing by tumor-reactive cytotoxic T cells. Therapeutic breathing of 60 % oxygen resulted in the significant inhibition of growth of established B16.F10 melanoma tumors and prolonged survival of mice. Taken together, the data presented here provide proof-of principle for the therapeutic potential of systemic oxygenation to convert the hypoxic, adenosine-rich and tumor-protecting TME into a normoxic and extracellular adenosine-poor TME that, in turn, may facilitate tumor regression. We propose to explore the combination of supplemental oxygen with existing immunotherapies of cancer. KEY MESSAGES: Oxygenation decreases levels of tumor protecting hypoxia. Oxygenation decreases levels of tumor protecting extracellular adenosine. Oxygenation decreases expression of HIF-1alpha dependent tumor-protecting proteins. Oxygenation increases MHC class I expression and enables tumor regression.
Assuntos
Adenosina/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/complicações , Hipóxia/terapia , Neoplasias/complicações , Neoplasias/terapia , Oxigênio/uso terapêutico , Animais , Hipóxia Celular , Linhagem Celular Tumoral , Feminino , Hipóxia/metabolismo , Camundongos Endogâmicos C57BL , Neoplasias/metabolismo , Microambiente TumoralRESUMO
Over the past decade, our laboratory has developed a secreted heat shock protein (HSP), chaperone gp96, cell-based vaccine that generates effective anti-tumor and anti-infectious immunity in vivo. Gp96-peptide complexes were identified as an extremely efficient stimulator of MHC I-mediated antigen cross-presentation, generating CD8 cytotoxic T-lymphocyte responses detectable in blood, spleen, gut and reproductive tract to femto-molar concentrations of antigen. These studies provided the first evidence that cell-based gp96-Ig-secreting vaccines may serve as a potent modality to induce both systemic and mucosal immunity. This approach takes advantage of the combined adjuvant and antigen delivery capacity of gp96 for the generation of cytotoxic immunity against a wide range of antigens in both anti-vial and anti-cancer vaccination. Here, we review the vaccine design that utilizes the unique property/ability of endoplasmic HSP gp96 to bind antigenic peptides and deliver them to antigen-presenting cells.
Assuntos
Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Glicoproteínas de Membrana/imunologia , Vacinas Sintéticas/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Controle de Doenças Transmissíveis/métodos , Controle de Doenças Transmissíveis/tendências , Doenças Transmissíveis/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Imunoterapia , Glicoproteínas de Membrana/metabolismo , Mucosa/imunologia , Mucosa/metabolismo , Neoplasias/imunologia , Neoplasias/mortalidade , Neoplasias/terapia , Vacinas Sintéticas/administração & dosagemRESUMO
TNFRSF25 is an understudied broad-acting T cell costimulator with high homology to TNFR1, however, the overall role of this receptor in T cell immunobiology is unclear. Ligation of TNFRSF25 by its monogamous ligand, TNFSF15 (TL1A), leads to recruitment of TNFR-associated factor 2 and TNFR-associated death domain in primary T cells with downstream activation of both NFκB as well as the PI3K/Akt axis. These signaling pathways are dependent upon coordinated engagement of the T cell receptor and interleukin-2 receptor and leads to the constitutive proliferation of CD4+FoxP3+ regulatory T cells (Treg) as a result of tonic exposure to self-antigen. Concurrent activation of CD4+ or CD8+ conventional T cell clones is dependent upon the availability of cognate foreign antigen. Here, we provide a review of both the literature and our work on this receptor and propose that the overall function of TL1A signaling to TNFRSF25 in T cells is to provide simultaneous costimulation of foreign-antigen-specific effector T cells and pre-existing Treg in order to focus the clonality of effector immunity to pathogen-derived antigens and reduce the risk of bystander inflammation toward self- or endogenous microbial antigens.
Assuntos
Membro 25 de Receptores de Fatores de Necrose Tumoral/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Imunidade Adaptativa/fisiologia , Animais , Humanos , Membro 25 de Receptores de Fatores de Necrose Tumoral/química , Membro 25 de Receptores de Fatores de Necrose Tumoral/genética , Transdução de Sinais , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/química , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genéticaRESUMO
TNF superfamily member 15 (TL1A) is the ligand for TNFR superfamily (TNFRSF)25. We previously reported that TNFRSF25 stimulation with an agonist Ab, 4C12, expands pre-existing CD4(+)Foxp3(+) regulatory T cells (Tregs) in vivo. To determine how the physiological ligand differs from the Ab, we generated a soluble mouse TL1A-Ig fusion protein that forms a dimer of TL1A trimers in solution with an apparent molecular mass of 516 kDa. In vitro, TL1A-Ig mediated rapid proliferation of Foxp3(+) Tregs and a population of CD4(+)Foxp3(-) conventional T cells. TL1A-Ig also blocked de novo biogenesis of inducible Tregs and it attenuated the suppressive function of Tregs. TNFRSF25 stimulation by TL1A-Ig in vivo induced expansion of Tregs such that they increased to 30-35% of all CD4(+) T cells in the peripheral blood within 5 d of treatment. Treg proliferation in vivo was dependent on TCR engagement with MHC class II. Elevated Treg levels can be maintained for at least 20 d with daily injections of TL1A-Ig. TL1A-Ig-expanded Tregs expressed high levels of activation/memory markers KLRG1 and CD103 and were highly suppressive ex vivo. TL1A-Ig-mediated Treg expansion in vivo was protective against allergic lung inflammation, a mouse model for asthma, by reversing the ratio of conventional T cells to Tregs in the lung and blocking eosinophil exudation into the bronchoalveolar fluid. Thus, TL1A-Ig fusion proteins are highly active and tightly controllable agents to stimulate Treg proliferation in vivo, and they are uniquely able to maintain high levels of expanded Tregs by repeated administration.
Assuntos
Proteínas Recombinantes de Fusão/genética , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/isolamento & purificação , Animais , Células CHO , Linhagem Celular Tumoral , Clonagem Molecular , Cricetinae , Citometria de Fluxo , Genes Reporter , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Insercional , Células NIH 3T3 , Plasmídeos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Hipersensibilidade Respiratória/genética , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/metabolismo , Transfecção , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/fisiologiaRESUMO
The mechanisms by which B lymphocytes inhibit anti-tumor immunity remain poorly understood. Murine EMT-6 mammary tumors grow readily in immune competent mice (BALB/c), but poorly in B-cell-deficient µ(-/-) BALB/c mice (BCDM). T regulatory cell (Treg) expansion and function were impaired in BCDM compared with BALB/c. In this study, we compared tumor growth, Treg cell proliferation, tumor lymphocyte infiltration and cytolytic T cell activity in BALB/c, BCDM and BCDM partially reconstituted with B cells by adoptive transfer (BCDM+B). Partial reconstitution of BCDM with adoptively transferred B cells restored EMT-6 tumor growth, which was independent of IL-10 secretion by B cells. Instead, high frequencies of intratumoral B cells were associated with increased recruitment and proliferation of Treg cells within the tumor microenvironment. The B-cell-dependent accumulation of Treg within the tumor microenvironment was associated with reduced tumor infiltration by CD49+ NK and CD8+ T cells and reduced cytotoxic T cell activity against EMT-6 targets. Our studies indicate that tumor-dependent immunosuppression of T-cell-mediated anti-tumor immunity is coordinated within the tumor microenvironment by B-cell-dependent cross talk with Treg cells, which does not require production of IL-10 by B cells.
Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Interleucina-10/metabolismo , Neoplasias Mamárias Experimentais/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Microambiente Tumoral/imunologia , Transferência Adotiva , Animais , Proliferação de Células , Citometria de Fluxo , Interleucina-10/imunologia , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptor Cross-Talk/imunologiaRESUMO
TNFR superfamily (TNFRSF)4 (OX40, CD134) and TNFRSF25 are costimulatory receptors that influence CD4(+) and CD8(+) T cell responses to cognate Ag. Independently, these receptors have been described to stimulate overlapping functions, including enhanced proliferation and activation for both regulatory T cells (CD4(+)Foxp3(+); Tregs) and conventional T cells (CD4(+)Foxp3(-) or CD8(+)Foxp3(-); Tconvs). To determine the relative functionality of TNFRSF4 and TNFRSF25 in T cell immunity, the activity of TNFRSF4 and TNFRS25 agonistic Abs was compared in the context of both traditional protein/adjuvant (OVA/aluminum hydroxide) and CD8(+)-specific heat shock protein-based (gp96-Ig) vaccine approaches. These studies demonstrate that both TNFRSF4 and TNFRSF25 independently and additively costimulate vaccine-induced CD8(+) T cell proliferation following both primary and secondary Ag challenge. In contrast, the activities of TNFRSF4 and TNFRSF25 were observed to be divergent in the costimulation of CD4(+) T cell immunity. TNFRSF4 agonists were potent costimulators of OVA/aluminum hydroxide-induced CD4(+) Tconv proliferation, but they only weakly costimulated Treg proliferation and IgG2a production, whereas TNFRSF25 agonists were strong costimulators of Treg proliferation, producers of IgG1, IgG2a, and IgG2b, and weak costimulators of CD4(+) Tconv proliferation. Interestingly, Ag-specific cellular and humoral responses were uncoupled upon secondary immunization, which was dramatically affected by the presence of TNFRSF4 or TNFRSF25 costimulation. These studies highlight the overlapping but nonredundant activities of TNFRSF4 and TNFRSF25 in T cell immunity, which may guide the application of receptor agonistic agents as vaccine adjuvants for infectious disease and tumor immunity.