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Anticalin proteins are a novel class of clinical-stage biopharmaceuticals with high potential in various disease areas. Anticalin proteins, derived from extracellular human lipocalins are single-chain proteins, with a highly stable structure that can be engineered to bind with high specificity and potency to targets of therapeutic relevance. The small size and stable structure support their development as inhalable biologics in the field of respiratory diseases as already demonstrated for PRS-060/AZD1402, an Anticalin protein currently undergoing clinical development for the treatment of asthma. Anticalin proteins provide formatting flexibility which allows fusion with the same or other Anticalin proteins, or with other biologics to generate multivalent, multiparatopic or multispecific fusion proteins. The fusion of Anticalin proteins to antibodies allows the generation of potent therapeutic proteins with new modes of action, such as antibody-Anticalin bispecific proteins with tumor-localized activity. Cinrebafusp alfa and PRS-344/S095012 antibody-Anticalin bispecific proteins were designed to reduce potential systemic toxicity by localizing the activity to the tumor, and are currently in clinical development in immuno-oncology. Furthermore, the ease in generating bi- and multispecifics as well as the small and stable structure prompted the investigation of Anticalin proteins for the CAR T space, opening additional potential treatment options based on Anticalin protein therapies.
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Produtos Biológicos , Neoplasias , Produtos Biológicos/uso terapêutico , Desenvolvimento de Medicamentos , Humanos , Lipocalinas/uso terapêutico , Neoplasias/tratamento farmacológico , ProteínasRESUMO
PURPOSE: While patients responding to checkpoint blockade often achieve remarkable clinical responses, there is still significant unmet need due to resistant or refractory tumors. A combination of checkpoint blockade with further T-cell stimulation mediated by 4-1BB agonism may increase response rates and durability of response. A bispecific molecule that blocks the programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) axis and localizes 4-1BB costimulation to a PD-L1-positive (PD-L1+) tumor microenvironment (TME) or tumor draining lymph nodes could maximize antitumor immunity and increase the therapeutic window beyond what has been reported for anti-4-1BB mAbs. EXPERIMENTAL DESIGN: We generated and characterized the PD-L1/4-1BB bispecific molecule PRS-344/S095012 for target binding and functional activity in multiple relevant in vitro assays. Transgenic mice expressing human 4-1BB were transplanted with human PD-L1-expressing murine MC38 cells to assess in vivo antitumoral activity. RESULTS: PRS-344/S095012 bound to its targets with high affinity and efficiently blocked the PD-1/PD-L1 pathway, and PRS-344/S095012-mediated 4-1BB costimulation was strictly PD-L1 dependent. We demonstrated a synergistic effect of both pathways on T-cell stimulation with the bispecific PRS-344/S095012 being more potent than the combination of mAbs. PRS-344/S095012 augmented CD4-positive (CD4+) and CD8-positive (CD8+) T-cell effector functions and enhanced antigen-specific T-cell stimulation. Finally, PRS-344/S095012 demonstrated strong antitumoral efficacy in an anti-PD-L1-resistant mouse model in which soluble 4-1BB was detected as an early marker for 4-1BB agonist activity. CONCLUSIONS: The PD-L1/4-1BB bispecific PRS-344/S095012 efficiently combines checkpoint blockade with a tumor-localized 4-1BB-mediated stimulation burst to antigen-specific T cells, more potent than the combination of mAbs, supporting the advancement of PRS-344/S095012 toward clinical development. See related commentary by Shu et al., p. 3182.
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Antígeno B7-H1 , Neoplasias , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/imunologia , Humanos , Fatores Imunológicos/uso terapêutico , Imunoterapia , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/imunologia , Microambiente TumoralRESUMO
Extracellular vesicles (EVs) are sub-micron-sized membranous spheres secreted by cells. EVs play a functional role as intercellular communicators and are associated with a number of diseases. Research into EVs is an area of growing interest due their many potential uses as therapeutic agents, as diagnostic and theranostic biomarkers, and as regulators of cellular biology. Flow cytometry is a popular method for enumerating and phenotyping EVs, even though the majority of EVs are below the detection sensitivity of most commercially available flow cytometers. Here, we present optimized protocols for EV labeling that increase the signal-to-noise ratio of EVs by removing residual antibody. Protocols for alignment of high-resolution jet-in-air flow cytometers are also provided. Published 2020. U.S. Government. Basic Protocol 1: Bulk EV staining with CFSE protein binding dye Basic Protocol 2: Antigen-specific staining of EV markers with fluorochrome-conjugated antibodies Basic Protocol 3: Astrios EQ instrument setup and sample acquisition Basic Protocol 4: Counting particles and EVs on Astrios EQ with spike-in reference beads.
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Vesículas Extracelulares , Citometria de Fluxo , Nanotecnologia , Vírus/isolamento & purificação , Animais , Linhagem Celular , Citometria de Fluxo/métodos , Corantes Fluorescentes , Camundongos , Nanotecnologia/métodos , Coloração e RotulagemRESUMO
Extracellular vesicles (EVs) are small, heterogeneous and difficult to measure. Flow cytometry (FC) is a key technology for the measurement of individual particles, but its application to the analysis of EVs and other submicron particles has presented many challenges and has produced a number of controversial results, in part due to limitations of instrument detection, lack of robust methods and ambiguities in how data should be interpreted. These complications are exacerbated by the field's lack of a robust reporting framework, and many EV-FC manuscripts include incomplete descriptions of methods and results, contain artefacts stemming from an insufficient instrument sensitivity and inappropriate experimental design and lack appropriate calibration and standardization. To address these issues, a working group (WG) of EV-FC researchers from ISEV, ISAC and ISTH, worked together as an EV-FC WG and developed a consensus framework for the minimum information that should be provided regarding EV-FC. This framework incorporates the existing Minimum Information for Studies of EVs (MISEV) guidelines and Minimum Information about a FC experiment (MIFlowCyt) standard in an EV-FC-specific reporting framework (MIFlowCyt-EV) that supports reporting of critical information related to sample staining, EV detection and measurement and experimental design in manuscripts that report EV-FC data. MIFlowCyt-EV provides a structure for sharing EV-FC results, but it does not prescribe specific protocols, as there will continue to be rapid evolution of instruments and methods for the foreseeable future. MIFlowCyt-EV accommodates this evolution, while providing information needed to evaluate and compare different approaches. Because MIFlowCyt-EV will ensure consistency in the manner of reporting of EV-FC studies, over time we expect that adoption of MIFlowCyt-EV as a standard for reporting EV- FC studies will improve the ability to quantitatively compare results from different laboratories and to support the development of new instruments and assays for improved measurement of EVs.
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The benefits of anti-cancer agents extend beyond direct tumor killing. One aspect of cell death is the potential to release antigens that initiate adaptive immune responses. Here, a diffusion enhanced formulation, INT230-6, containing potent anti-cancer cytotoxic agents, was administered intratumorally into large (approx. 300mm3) subcutaneous murine Colon26 tumors. Treatment resulted in regression from baseline in 100% of the tumors and complete response in up to 90%. CD8+ or CD8+/CD4+ T cell double-depletion at treatment onset prevented complete responses, indicating a critical role of T cells in promoting complete tumor regression. Mice with complete response were protected from subcutaneous and intravenous re-challenge of Colon26 cells in a CD4+/CD8+ dependent manner. Thus, immunological T cell memory was induced by INT230-6. Colon26 tumors express the endogenous retroviral protein gp70 containing the CD8+ T-cell AH-1 epitope. AH-1-specific CD8+ T cells were detected in peripheral blood of tumor-bearing mice and their frequency increased 14 days after treatment onset. AH-1-specific CD8+ T cells were also significantly enriched in tumors of untreated mice. These cells had an activated phenotype and highly expressed Programmed cell-death protein-1 (PD-1) but did not lead to tumor regression. CD8+ T cell tumor infiltrate also increased 11 days after treatment. INT230-6 synergized with checkpoint blockade, inducing a complete remission of the primary tumors and shrinking of untreated contralateral tumors, which demonstrates not only a local but also systemic immunological effect of the combined therapy. Similar T-cell dependent inhibition of tumor growth was also found in an orthotopic 4T1 breast cancer model.
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Biological nanoparticles, including viruses and extracellular vesicles (EVs), are of interest to many fields of medicine as biomarkers and mediators of or treatments for disease. However, exosomes and small viruses fall below the detection limits of conventional flow cytometers due to the overlap of particle-associated scattered light signals with the detection of background instrument noise from diffusely scattered light. To identify, sort, and study distinct subsets of EVs and other nanoparticles, as individual particles, we developed nanoscale Fluorescence Analysis and Cytometric Sorting (nanoFACS) methods to maximise information and material that can be obtained with high speed, high resolution flow cytometers. This nanoFACS method requires analysis of the instrument background noise (herein defined as the "reference noise"). With these methods, we demonstrate detection of tumour cell-derived EVs with specific tumour antigens using both fluorescence and scattered light parameters. We further validated the performance of nanoFACS by sorting two distinct HIV strains to >95% purity and confirmed the viability (infectivity) and molecular specificity (specific cell tropism) of biological nanomaterials sorted with nanoFACS. This nanoFACS method provides a unique way to analyse and sort functional EV- and viral-subsets with preservation of vesicular structure, surface protein specificity and RNA cargo activity.
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The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the establishment of reproducible, scalable, and high-throughput methods for the production and purification of clinical grade EV. Methods including ultracentrifugation (U/C), ultrafiltration, immunoprecipitation, and size-exclusion chromatography (SEC) have been employed to isolate EV, each facing limitations such as efficiency, particle purity, lengthy processing time, and/or sample volume. We developed a cGMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving bioreactor culture, tangential flow filtration (TFF), and preparative SEC. We applied this purification method for the isolation of engineered EV carrying multiple complexes of a novel human immunostimulatory cytokine-fusion protein, heterodimeric IL-15 (hetIL-15)/lactadherin. HEK293 cells stably expressing the fusion cytokine were cultured in a hollow-fibre bioreactor. Conditioned medium was collected and EV were isolated comparing three procedures: U/C, SEC, or TFF + SEC. SEC demonstrated comparable particle recovery, size distribution, and hetIL-15 density as U/C purification. Relative to U/C, SEC preparations achieved a 100-fold reduction in ferritin concentration, a major protein-complex contaminant. Comparative proteomics suggested that SEC additionally decreased the abundance of cytoplasmic proteins not associated with EV. Combination of TFF and SEC allowed for bulk processing of large starting volumes, and resulted in bioactive EV, without significant loss in particle yield or changes in size, morphology, and hetIL-15/lactadherin density. Taken together, the combination of bioreactor culture with TFF + SEC comprises a scalable, efficient method for the production of highly purified, bioactive EV carrying hetIL-15/lactadherin, which may be useful in targeted cancer immunotherapy approaches.
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Extracellular vesicles (EVs), including exosomes and microvesicles, are 30-800 nm vesicles that are released by most cell types, as biological packages for intercellular communication. Their importance in cancer and inflammation makes EVs and their cargo promising biomarkers of disease and cell-free therapeutic agents. Emerging high-resolution cytometric methods have created a pressing need for efficient fluorescent labeling procedures to visualize and detect EVs. Suitable labels must be bright enough for one EV to be detected without the generation of label-associated artifacts. To identify a strategy that robustly labels individual EVs, we used nanoFACS, a high-resolution flow cytometric method that utilizes light scattering and fluorescence parameters along with sample enumeration, to evaluate various labels. Specifically, we compared lipid-, protein-, and RNA-based staining methods and developed a robust EV staining strategy, with the amine-reactive fluorescent label, 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester, and size exclusion chromatography to remove unconjugated label. By combining nanoFACS measurements of light scattering and fluorescence, we evaluated the sensitivity and specificity of EV labeling assays in a manner that has not been described for other EV detection methods. Efficient characterization of EVs by nanoFACS paves the way towards further study of EVs and their roles in health and disease.
Assuntos
Vesículas Extracelulares/metabolismo , Citometria de Fluxo , Coloração e Rotulagem , Aminas , Animais , Linhagem Celular , Micropartículas Derivadas de Células/metabolismo , Cromatografia em Gel , Citometria de Fluxo/métodos , Corantes Fluorescentes , Camundongos , Micelas , Coloração e Rotulagem/métodosRESUMO
To analyze EVs with conventional flow cytometers, most researchers will find it necessary to bind EVs to beads that are large enough to be individually resolved on the flow cytometer available in their lab or facility. Although high-resolution flow cytometers are available and are being used for EV analysis, the use of these instruments for studying EVs requires careful use and validation by experienced small-particle flow cytometrists, beyond the scope of this chapter. Shown here is a method for using streptavidin-coated beads to capture biotinylated antibodies, and stain the bead-bound EVs with directly conjugated antibodies. We find that this method is a useful tool not only on its own, without further high resolution flow cytometric analysis, but also as a means for optimizing staining methods and testing new labels for later use in high resolution, single EV flow cytometric studies. The end of the chapter includes sphere-packing calculations to quantify aspects of EV- and bead-surface geometry, as a reference for use as readers of this chapter optimize their own flow cytometry assays with EVs.
Assuntos
Vesículas Extracelulares/metabolismo , Citometria de Fluxo , Biomarcadores , Linhagem Celular , Exossomos , Vesículas Extracelulares/química , Citometria de Fluxo/métodos , Humanos , Tamanho da PartículaRESUMO
Extracellular vesicles (EV), including exosomes and microvesicles, are nano-sized intercellular communication vehicles that participate in a multitude of physiological processes. Due to their biological properties, they are also promising candidates for the systemic delivery of therapeutic compounds, such as cytokines, chemotherapeutic drugs, siRNAs and viral vectors. However, low EV production yield and rapid clearance of administered EV by liver macrophages limit their potential use as therapeutic vehicles. We have used a hollow-fiber bioreactor for the efficient production of bioactive EV bearing the heterodimeric cytokine complex Interleukin-15:Interleukin-15 receptor alpha. Bioreactor culture yielded â¼40-fold more EV per mL conditioned medium, as compared to conventional cell culture. Biophysical analysis and comparative proteomics suggested a more diverse population of EV in the bioreactor preparations, while serum protein contaminants were detectable only in conventional culture EV preparations. We also identified the Scavenger Receptor Class A family (SR-A) as a novel monocyte/macrophage uptake receptor for EV. In vivo blockade of SR-A with dextran sulfate dramatically decreased EV liver clearance in mice, while enhancing tumor accumulation. These findings facilitate development of EV therapeutic methods.
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Técnicas de Cultura Celular por Lotes/instrumentação , Reatores Biológicos , Fracionamento Celular/instrumentação , Vesículas Extracelulares/fisiologia , Vesículas Extracelulares/ultraestrutura , Macrófagos/metabolismo , Neoplasias Experimentais/ultraestrutura , Técnicas de Cultura Celular por Lotes/métodos , Fracionamento Celular/métodos , Células Cultivadas , Desenho de Equipamento , Células HEK293 , Humanos , Engenharia Tecidual/instrumentaçãoRESUMO
Hypoxia is a common feature in solid tumors that has been implicated in immune evasion. Previous studies from our group have shown that hypoxia upregulates the co-stimulatory receptor CD137 on activated T lymphocytes and on vascular endothelial cells. In this study, we show that exposure of mouse and human tumor cell lines to hypoxic conditions (1% O2) promotes CD137 transcription. However, the resulting mRNA is predominantly an alternatively spliced form that encodes for a soluble variant, lacking the transmembrane domain. Accordingly, soluble CD137 (sCD137) is detectable by ELISA in the supernatant of hypoxia-exposed cell lines and in the serum of tumor-bearing mice. sCD137, as secreted by tumor cells, is able to bind to CD137-Ligand (CD137L). Our studies on primed T lymphocytes in co-culture with stable transfectants for CD137L demonstrate that tumor-secreted sCD137 prevents co-stimulation of T lymphocytes. Such an effect results from preventing the interaction of CD137L with the transmembrane forms of CD137 expressed on T lymphocytes undergoing activation. Indeed, silencing CD137 with shRNA renders more immunogenic tumor-cell variants upon inoculation to immunocompetent mice but which readily grafted on immunodeficient or CD8+ T-cell-depleted mice. These mechanisms are interpreted as a molecular strategy deployed by tumors to repress lymphocyte co-stimulation via CD137/CD137L.
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UNLABELLED: Weak and ineffective antitumor cytotoxic T lymphocyte (CTL) responses can be rescued by immunomodulatory mAbs targeting PD-1 or CD137. Using Batf3(-/-) mice, which are defective for cross-presentation of cell-associated antigens, we show that BATF3-dependent dendritic cells (DC) are essential for the response to therapy with anti-CD137 or anti-PD-1 mAbs. Batf3(-/-) mice failed to prime an endogenous CTL-mediated immune response toward tumor-associated antigens, including neoantigens. As a result, the immunomodulatory mAbs could not amplify any therapeutically functional immune response in these mice. Moreover, administration of systemic sFLT3L and local poly-ICLC enhanced DC-mediated cross-priming and synergized with anti-CD137- and anti-PD-1-mediated immunostimulation in tumor therapy against B16-ovalbumin-derived melanomas, whereas this function was lost in Batf3(-/-) mice. These experiments show that cross-priming of tumor antigens by FLT3L- and BATF3-dependent DCs is crucial to the efficacy of immunostimulatory mAbs and represents a very attractive point of intervention to enhance their clinical antitumor effects. SIGNIFICANCE: Immunotherapy with immunostimulatory mAbs is currently achieving durable clinical responses in different types of cancer. We show that cross-priming of tumor antigens by BATF3-dependent DCs is a key limiting factor that can be exploited to enhance the antitumor efficacy of anti-PD-1 and anti-CD137 immunostimulatory mAbs.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Fatores de Transcrição de Zíper de Leucina Básica/genética , Células Dendríticas/imunologia , Melanoma Experimental/terapia , Proteínas Repressoras/genética , Animais , Anticorpos Monoclonais/uso terapêutico , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linhagem Celular Tumoral , Células Dendríticas/citologia , Humanos , Imunoterapia/métodos , Ativação Linfocitária/imunologia , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/imunologia , Proteínas Repressoras/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
Cancer immunotherapy is undergoing significant progress due to recent clinical successes by refined adoptive T-cell transfer and immunostimulatory monoclonal Ab (mAbs). B16F10-derived OVA-expressing mouse melanomas resist curative immunotherapy with either adoptive transfer of activated anti-OVA OT1 CTLs or agonist anti-CD137 (4-1BB) mAb. However, when acting in synergistic combination, these treatments consistently achieve tumor eradication. Tumor-infiltrating lymphocytes that accomplish tumor rejection exhibit enhanced effector functions in both transferred OT-1 and endogenous cytotoxic T lymphocytes (CTLs). This is consistent with higher levels of expression of eomesodermin in transferred and endogenous CTLs and with intravital live-cell two-photon microscopy evidence for more efficacious CTL-mediated tumor cell killing. Anti-CD137 mAb treatment resulted in prolonged intratumor persistence of the OT1 CTL-effector cells and improved function with focused and confined interaction kinetics of OT-1 CTL with target cells and increased apoptosis induction lasting up to six days postadoptive transfer. The synergy of adoptive T-cell therapy and agonist anti-CD137 mAb thus results from in vivo enhancement and sustainment of effector functions.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Imunoterapia Adotiva/métodos , Melanoma Experimental/terapia , Linfócitos T Citotóxicos/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Proteínas Aviárias/genética , Linhagem Celular Tumoral , Terapia Combinada , Linfócitos do Interstício Tumoral/imunologia , Masculino , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/genética , Proteínas com Domínio T/metabolismo , Microambiente Tumoral/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/deficiência , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genéticaRESUMO
A current pressing need in cancer immunology is the development of preclinical model systems that are immunocompetent for the study of human tumors. Here, we report the development of a humanized murine model that can be used to analyze the pharmacodynamics and antitumor properties of immunostimulatory monoclonal antibodies (mAb) in settings where the receptors targeted by the mAbs are expressed. Human lymphocytes transferred into immunodeficient mice underwent activation and redistribution to murine organs, where they exhibited cell-surface expression of hCD137 and hPD-1. Systemic lymphocyte infiltrations resulted in a lethal CD4(+) T cell-mediated disease (xenograft-versus-host disease), which was aggravated when murine subjects were administered clinical-grade anti-hCD137 (urelumab) and anti-hPD-1 (nivolumab). In mice engrafted with human colorectal HT-29 carcinoma cells and allogeneic human peripheral blood mononuclear cells (PBMC), or with a patient-derived gastric carcinoma and PBMCs from the same patient, we found that coadministration of urelumab and nivolumab was sufficient to significantly slow tumor growth. Correlated with this result were increased numbers of activated human T lymphocytes producing IFNγ and decreased numbers of human regulatory T lymphocytes in the tumor xenografts, possibly explaining the efficacy of the therapeutic regimen. Our results offer a proof of concept for the use of humanized mouse models for surrogate efficacy and histology investigations of immune checkpoint drugs and their combinations.
Assuntos
Neoplasias Colorretais/imunologia , Proteínas de Ligação a DNA/genética , Subunidade gama Comum de Receptores de Interleucina/genética , Receptor de Morte Celular Programada 1/biossíntese , Linfócitos T/efeitos dos fármacos , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/biossíntese , Animais , Anticorpos Monoclonais/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA/imunologia , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Células HT29 , Humanos , Subunidade gama Comum de Receptores de Interleucina/imunologia , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/imunologia , Camundongos , Nivolumabe , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T/patologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/antagonistas & inibidores , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
Immunostimulatory monoclonal antibodies can be given in combinations, hence modulating the activity of 2 or more receptors of the immune system. Some of these combinations have been shown to synergize at the elicitation of therapeutically relevant immune responses in transgenic mice developing spontaneous, oncogene-driven tumors, including multifocal hepatocellular carcinomas expressing ovalbumin as a surrogate tumor-associated antigen.
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The immune system poses obstacles to viral vectors, even in the first administration to preimmunized hosts. We have observed that the livers of B cell-deficient mice were more effectively transduced by a helper-dependent adenovirus serotype-5 (HDA) vector than those of WT mice. This effect was T-cell independent as shown in athymic mice. Passive transfer of the serum from adenovirus-naïve WT to Rag1KO mice resulted in a reduction in gene transfer that was traced to IgM purified from serum of adenovirus-naïve mice. To ascribe the gene transfer inhibition activity to either adenoviral antigen-specific or antigen-unspecific functions of IgM, we used a monoclonal IgM antibody of unrelated specificity. Both the polyclonal and the irrelevant monoclonal IgM inhibited gene transfer by the HDA vector to either cultured hepatocellular carcinoma cells or to the liver of mice in vivo. Adsorption of polyclonal or monoclonal IgMs to viral capsids was revealed by ELISAs on adenovirus-coated plates. These observations indicate the existence of an inborn IgM mechanism deployed against a prevalent virus to reduce early post-infection viremia. In conclusion, innate IgM binding to adenovirus serotype-5 capsids restrains gene-transfer and offers a mechanism to be targeted for optimization of vector dosage in gene therapy with HDA vectors.
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Anticorpos Monoclonais/química , Técnicas de Transferência de Genes , Vetores Genéticos/imunologia , Imunidade Inata , Imunoglobulina M/química , Adenoviridae/genética , Adenoviridae/imunologia , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Capsídeo/química , Hepatócitos/imunologia , Hepatócitos/patologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Soros Imunes/administração & dosagem , Fígado/citologia , Fígado/imunologia , Depleção Linfocítica , Camundongos , Camundongos Knockout , Camundongos Nus , Ligação Proteica , Linfócitos T/citologia , Linfócitos T/imunologia , Células Tumorais CultivadasRESUMO
Immunotherapies often permit combinations to increase efficacy. Two approaches are currently leading our field: adoptive therapy with T cells transfected with chimeric antigen receptors and monoclonal antibodies blocking the PD-1/PD-L1 (B7-H1) axis. In this issue of Clinical Cancer Research, preclinical evidence for a synergistic combination of such approaches is reported.
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Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Neoplasias/genética , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , HumanosRESUMO
PURPOSE: Immunostimulatory monoclonal antibodies (ISmAb) that unleash antitumor immune responses are showing efficacy in cancer clinical trials. Anti-B7-H1 (PD-L1) monoclonal antibodies (mAb) block a critical inhibitory pathway in T cells, whereas anti-CD137 and OX40 mAbs provide T-cell costimulation. A combination of these ISmAbs (anti-CD137 + anti-OX40 + anti-B7-H1) was tested using a transgenic mouse model of multifocal and rapidly progressing hepatocellular carcinoma, in which c-myc drives transformation and cytosolic ovalbumin (OVA) is expressed in tumor cells as a model antigen. EXPERIMENTAL DESIGN: Flow-cytometry and immunohistochemistry were used to quantify tumor-infiltrating lymphocytes (TIL) elicited by treatment and assess their activation status and cytolytic potential. Tolerance induction and its prevention/reversal by treatment with the combination of ISmAbs were revealed by in vivo killing assays. RESULTS: The triple combination of ISmAbs extended survival of mice bearing hepatocellular carcinomas in a CD8-dependent fashion and synergized with adoptive T-cell therapy using activated OVA-specific TCR-transgenic OT-1 and OT-2 lymphocytes. Mice undergoing therapy showed clear increases in tumor infiltration by activated and blastic CD8(+) and CD4(+) T lymphocytes containing perforin/granzyme B and expressing the ISmAb-targeted receptors on their surface. The triple combination of ISmAbs did not result in enhanced OVA-specific cytotoxic T lymphocyte (CTL) activity but other antigens expressed by cell lines derived from such hepatocellular carcinomas were recognized by endogenous TILs. Adoptively transferred OVA-specific OT-1 lymphocytes into tumor-bearing mice were rendered tolerant, unless given the triple mAb therapy. CONCLUSION: Extension of survival and dense T-cell infiltrates emphasize the translational potential of combinational immunotherapy strategies for hepatocellular carcinoma. Clin Cancer Res; 19(22); 6151-62. ©2013 AACR.
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Anticorpos Monoclonais/uso terapêutico , Carcinoma Hepatocelular/imunologia , Imunoterapia/métodos , Neoplasias Hepáticas/imunologia , Transferência Adotiva , Animais , Anticorpos Monoclonais/imunologia , Antígeno B7-H1/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/mortalidade , Linhagem Celular Tumoral , Citotoxicidade Imunológica/imunologia , Modelos Animais de Doenças , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/mortalidade , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Linfócitos do Interstício Tumoral/imunologia , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligante OX40 , Ovalbumina/biossíntese , Ovalbumina/imunologia , Sobrevida , Linfócitos T Citotóxicos/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Fatores de Necrose Tumoral/imunologiaRESUMO
Agonist anti-CD137 (4-1BB) mAbs enhance CD8-mediated antitumor immunity. Agonist anti-human CD137 mAbs binding to four distinct epitopes on the CD137 glycoprotein costimulated T cell activation irrespective of the engaged epitope or its interference with CD137L binding. CD137 perturbation with all these agonist mAbs resulted in Ag and Ab internalization toward an endosomal vesicular compartment. Internalization was observed in activated T lymphocytes from humans and mice, not only in culture but also in Ab-injected living animals. These in vivo experiments were carried out upon systemic i.v. injections with anti-CD137 mAbs and showed CD137 internalization in tumor-infiltrating lymphocytes and in activated human T cells transferred to immunodeficient mice. Efficient CD137 internalization required K63 polyubiquitination and endocytosed CD137-containing vesicles recruited TNFR-associated factor (TRAF) 2 and were decorated with K63 polyubiquitins. CD137 stimulation activates NF-κB through a K63-linked polyubiquitination-dependent route, and CD137-associated TRAF2 becomes K63 polyubiquitinated. Consistent with a role for TRAF2 in CD137 signaling, transgenic mice functionally deficient in TRAF2 showed delayed immunotherapeutic activity of anti-CD137 mAbs. As a whole, these findings advance our knowledge of the mechanisms of action of anti-CD137 immunostimulatory mAbs such as those currently undergoing clinical trials in cancer patients.
Assuntos
Anticorpos Monoclonais/imunologia , Ativação Linfocitária/imunologia , Neoplasias Experimentais/imunologia , Linfócitos T/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Western Blotting , Linhagem Celular , Endocitose/efeitos dos fármacos , Endocitose/imunologia , Endossomos/efeitos dos fármacos , Endossomos/imunologia , Endossomos/metabolismo , Feminino , Humanos , Imunoprecipitação , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NF-kappa B/imunologia , NF-kappa B/metabolismo , Neoplasias Experimentais/terapia , Poliubiquitina/imunologia , Poliubiquitina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Fator 2 Associado a Receptor de TNF/imunologia , Fator 2 Associado a Receptor de TNF/metabolismo , Transfecção , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
Therapy for cancer can be achieved by artificially stimulating antitumor T and natural killer (NK) lymphocytes with agonist monoclonal antibodies (mAb). T and NK cells express several members of the TNF receptor (TNFR) family specialized in delivering a costimulatory signal on their surface. Engagement of these receptors is typically associated with proliferation, elevated effector functions, resistance to apoptosis, and differentiation into memory cells. These receptors lack any intrinsic enzymatic activity and their signal transduction relies on associations with TNFR-associated factor (TRAF) adaptor proteins. Stimulation of CD137 (4-1BB), CD134 (OX40), and glucocorticoid-induced TNFR (GITR; CD357) promotes impressive tumor-rejecting immunity in a variety of murine tumor models. The mechanisms of action depend on a complex interplay of CTL, T-helper cells, regulatory T cells, dendritic cells, and vascular endothelium in tumors. Agonist mAbs specific for CD137 have shown signs of objective clinical activity in patients with metastatic melanoma, whereas anti-OX40 and anti-GITR mAbs have entered clinical trials. Preclinical evidence suggests that engaging TNFR members would be particularly active with conventional cancer therapies and additional immunotherapeutic approaches. Indeed, T-cell responses elicited to tumor antigens by means of immunogenic tumor cell death are amplified by these immunostimulatory agonist mAbs. Furthermore, anti-CD137 mAbs have been shown to enhance NK-mediated cytotoxicity elicited by rituximab and trastuzumab. Combinations with other immunomodulatory mAb that block T-cell checkpoint blockade receptors such as CTLA-4 and PD-1 are also promising.