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1.
J Immunother Cancer ; 12(4)2024 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-38609101

RESUMEN

BACKGROUND: Despite the current therapeutic treatments including surgery, chemotherapy, radiotherapy and more recently immunotherapy, the mortality rate of lung cancer stays high. Regarding lung cancer, epigenetic modifications altering cell cycle, angiogenesis and programmed cancer cell death are therapeutic targets to combine with immunotherapy to improve treatment success. In a recent study, we uncovered that a molecule called QAPHA ((E)-3-(5-((2-cyanoquinolin-4-yl)(methyl)amino)-2-methoxyphenyl)-N-hydroxyacrylamide) has a dual function as both a tubulin polymerization and HDAC inhibitors. Here, we investigate the impact of this novel dual inhibitor on the immune response to lung cancer. METHODS: To elucidate the mechanism of action of QAPHA, we conducted a chemical proteomics analysis. Using an in vivo mouse model of lung cancer (TC-1 tumor cells), we assessed the effects of QAPHA on tumor regression. Tumor infiltrating immune cells were characterized by flow cytometry. RESULTS: In this study, we first showed that QAPHA effectively inhibited histone deacetylase 6, leading to upregulation of HSP90, cytochrome C and caspases, as revealed by proteomic analysis. We confirmed that QAPHA induces immunogenic cell death (ICD) by expressing calreticulin at cell surface in vitro and demonstrated its efficacy as a vaccine in vivo. Remarkably, even at a low concentration (0.5 mg/kg), QAPHA achieved complete tumor regression in approximately 60% of mice treated intratumorally, establishing a long-lasting anticancer immune response. Additionally, QAPHA treatment promoted the infiltration of M1-polarized macrophages in treated mice, indicating the induction of a pro-inflammatory environment within the tumor. Very interestingly, our findings also revealed that QAPHA upregulated major histocompatibility complex class II (MHC-II) expression on TC-1 tumor cells both in vitro and in vivo, facilitating the recruitment of cytotoxic CD4+T cells (CD4+CTL) expressing CD4+, NKG2D+, CRTAM+, and Perforin+. Finally, we showed that tumor regression strongly correlates to MHC-II expression level on tumor cell and CD4+ CTL infiltrate. CONCLUSION: Collectively, our findings shed light on the discovery of a new multitarget inhibitor able to induce ICD and MHC-II upregulation in TC-1 tumor cell. These two processes participate in enhancing a specific CD4+ cytotoxic T cell-mediated antitumor response in vivo in our model of lung cancer. This breakthrough suggests the potential of QAPHA as a promising agent for cancer treatment.


Asunto(s)
Antineoplásicos , Neoplasias Pulmonares , Animales , Ratones , Neoplasias Pulmonares/tratamiento farmacológico , Proteómica , Regulación hacia Arriba , Antígenos de Histocompatibilidad Clase II , Linfocitos T CD4-Positivos
2.
J Nanobiotechnology ; 22(1): 27, 2024 Jan 11.
Artículo en Inglés | MEDLINE | ID: mdl-38212782

RESUMEN

BACKGROUND: The pro-inflammatory ATP-gated P2X7 receptor is widely expressed by immune and non-immune cells. Nanobodies targeting P2X7, with potentiating or antagonistic effects, have been developed. Adeno-associated virus (AAV)-mediated gene transfer represents an efficient approach to achieve long-term in vivo expression of selected nanobody-based biologics. This approach (AAVnano) was used to validate the relevance of P2X7 as a target in dextran sodium sulfate (DSS)-induced colitis in mice. RESULTS: Mice received an intramuscular injection of AAV vectors coding for potentiating (14D5-dimHLE) or antagonistic (13A7-Fc) nanobody-based biologics targeting P2X7. Long-term modulation of P2X7 activity was evaluated ex vivo from blood samples. Colitis was induced with DSS in mice injected with AAV vectors coding for nanobody-based biologics. Severity of colitis, colon histopathology and expression of chemokines and cytokines were determined to evaluate the impact of P2X7 modulation. A single injection of an AAV vector coding for 13A7-Fc or 14D5-dimHLE efficiently modulated P2X7 function in vivo from day 15 up to day 120 post-injection in a dose-dependent manner. An AAV vector coding for 13A7-Fc significantly ameliorated DSS-induced colitis and significantly reduced immune cell infiltration and expression of chemokines and proinflammatory cytokines in colonic tissue. CONCLUSIONS: We have demonstrated the validity of AAVnano methodology to modulate P2X7 functions in vivo. Applying this methodological approach to a DSS-induced colitis model, we have shown that P2X7 blockade reduces inflammation and disease severity. Hence, this study confirms the importance of P2X7 as a pharmacological target and suggests the use of nanobody-based biologics as potential therapeutics in inflammatory bowel disease.


Asunto(s)
Productos Biológicos , Colitis , Ratones , Animales , Colon/metabolismo , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Citocinas/metabolismo , Quimiocinas/metabolismo , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad
3.
Mol Ther Methods Clin Dev ; 29: 238-253, 2023 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-37090479

RESUMEN

Immunotherapy has significantly improved treatment outcomes in various cancer entities. To enhance immunogenicity and efficacy, and to further broaden its applicability, co-administration of anti-tumor vaccines is considered as a promising strategy. Here, we introduce adeno-associated virus (AAV) vectors, widely used for in vivo gene therapy, as a potent cancer vaccine platform. Our AAV vector-based vaccine combines antigen display on the capsid surface with a vector-mediated antigen overexpression targeting different components of the immune system in a unique chronological order by a single intramuscular application. Thereby, both profound and long-lasting antigen-specific T and B cell immune responses were induced. Moreover, mice receiving the vaccine were protected against tumor growth, demonstrating its efficacy in two tumor models, including the low immunogenic and aggressive B16/F10-Ova melanoma model. Remarkably, this approach was even effective in conditions of a late tumor challenge, i.e., 80 days post-vaccination, between 88% (B16/F10-Ova melanoma) and 100% (EG7 thymoma) of mice remained tumor free. Thus, decorating AAV vector particles with antigens by capsid engineering represents a potent vaccine concept for applications in cancer immunotherapy. Its modular and versatile "plug-and-play" framework enables the use of tumor antigens of choice and the easy implementation of additional modifications to enhance immunogenicity further.

4.
Front Immunol ; 13: 1012534, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36341324

RESUMEN

Adenosine triphosphate (ATP) represents a danger signal that accumulates in injured tissues, in inflammatory sites, and in the tumor microenvironment. ATP promotes tumor growth but also anti-tumor immune responses notably via the P2X7 receptor. ATP can also be catabolized by CD39 and CD73 ecto-enzymes into immunosuppressive adenosine. P2X7, CD39 and CD73 have attracted much interest in cancer as targets offering the potential to unleash anti-tumor immune responses. These membrane proteins represent novel purinergic checkpoints that can be targeted by small drugs or biologics. Here, we investigated nanobody-based biologics targeting mainly P2X7, but also CD73, alone or in combination therapies. Blocking P2X7 inhibited tumor growth and improved survival of mice in cancer models that express P2X7. P2X7-potentiation by a nanobody-based biologic was not effective alone to control tumor growth but enhanced tumor control and immune responses when used in combination with oxaliplatin chemotherapy. We also evaluated a bi-specific nanobody-based biologic that targets PD-L1 and CD73. This novel nanobody-based biologic exerted a potent anti-tumor effect, promoting tumor rejection and improving survival of mice in two tumor models. Hence, this study highlights the importance of purinergic checkpoints in tumor control and open new avenues for nanobody-based biologics that may be further exploited in the treatment of cancer.


Asunto(s)
Neoplasias , Microambiente Tumoral , Ratones , Animales , Adenosina Trifosfato/metabolismo , Adenosina , Oxaliplatino
5.
Methods Mol Biol ; 2510: 99-127, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35776322

RESUMEN

Antibodies that recognize the ATP-gated P2X7 ion channel are etablished research tools. Nanobodies correspond to the antigen-binding variable immunoglobulin domain (VHH) of heavy chain antibodies that naturally occur in camelids. Nanobodies display better solubility than the variable domains (VH) of conventional antibodies. Therefore, it is much easier to construct bivalent and multivalent fusion proteins with nanobodies than with VH domains or with paired VH-VL domains. Moreover, nanobodies can bind functional crevices that are poorly accessbile to conventional VH-VL domains. This makes nanobodies particulary well suited as functional modulators. Here we provide protocols to raise antibodies and nanobodies against mouse and human P2X7 using cDNA-immunization. This approach evokes antibodies and nanobodies that recognize the P2X7 ion channel in native confirmation, some of which inhibit or potentiate gating of P2X7 by extracellular ATP. Furthermore, we developed protocols for producing P2X7-specific nanobodies and antibodies in vivo using rAAV vectors (AAVnano). This approach can be used either to durably inhibit or potentiate P2X7 function in vivo, or to deplete P2X7-expressing cells.


Asunto(s)
Anticuerpos de Dominio Único , Adenosina Trifosfato , Animales , Anticuerpos , Cadenas Pesadas de Inmunoglobulina , Ratones , Anticuerpos de Dominio Único/química
6.
Front Immunol ; 12: 704408, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34489954

RESUMEN

On murine T cells, mono-ADP ribosyltransferase ARTC2.2 catalyzes ADP-ribosylation of various surface proteins when nicotinamide adenine dinucleotide (NAD+) is released into the extracellular compartment. Covalent ADP-ribosylation of the P2X7 receptor by ARTC2.2 thereby represents an additional mechanism of activation, complementary to its triggering by extracellular ATP. P2X7 is a multifaceted receptor that may represents a potential target in inflammatory, and neurodegenerative diseases, as well as in cancer. We present herein an experimental approach using intramuscular injection of recombinant AAV vectors (rAAV) encoding nanobody-based biologics targeting ARTC2.2 or P2X7. We demonstrate the ability of these in vivo generated biologics to potently and durably block P2X7 or ARTC2.2 activities in vivo, or in contrast, to potentiate NAD+- or ATP-induced activation of P2X7. We additionally demonstrate the ability of rAAV-encoded functional heavy chain antibodies to elicit long-term depletion of T cells expressing high levels of ARTC2.2 or P2X7. Our approach of using rAAV to generate functional nanobody-based biologics in vivo appears promising to evaluate the role of ARTC2.2 and P2X7 in murine acute as well as chronic disease models.


Asunto(s)
ADP Ribosa Transferasas , Productos Biológicos/inmunología , Dependovirus , Vectores Genéticos , Depleción Linfocítica , Receptores Purinérgicos P2X7/inmunología , Anticuerpos de Dominio Único , ADP Ribosa Transferasas/antagonistas & inhibidores , ADP Ribosa Transferasas/inmunología , Animales , Ratones , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/inmunología
7.
Front Oncol ; 10: 1699, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33042812

RESUMEN

Adenosine triphosphate (ATP) represents a danger signal that accumulates in injured tissues, in inflammatory sites, and in the tumor microenvironment. Extracellular ATP is known to signal through plasma membrane receptors of the P2Y and P2X families. Among the P2X receptors, P2X7 has attracted increasing interest in the field of inflammation as well as in cancer. P2X7 is expressed by immune cells and by most malignant tumor cells where it plays a crucial yet complex role that remains to be clarified. P2X7 activity has been associated with production and release of pro-inflammatory cytokines, modulation of the activity and survival of immune cells, and the stimulation of proliferation and migratory properties of tumor cells. Hence, P2X7 plays an intricate role in the tumor microenvironment combining beneficial and detrimental effects that need to be further investigated. For this, we developed a novel methodology termed AAVnano based on the use of Adeno-associated viral vectors (AAV) encoding nanobodies targeting P2X7. We discuss here the advantages of this tool to study the different functions of P2X7 in cancer and other pathophysiological contexts.

8.
Front Immunol ; 11: 32, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32158442

RESUMEN

CD11c+ B cells have been reported to be increased in autoimmune diseases, but they are detected in the blood of healthy individuals as well. We aimed to characterize CD11c+ B cells from healthy donors by flow cytometry, microarray analysis, and in vitro functional assays. Here, we report that CD11c+ B cells are a distinct subpopulation of B cells, enriched in the memory subpopulation even if their phenotype is heterogeneous, with overexpression of genes involved in B-cell activation and differentiation as well as in antigen presentation. Upon activation, CD11c+ B cells can differentiate into antibody-secreting cells, and CD11c could be upregulated in CD11c- B cells by B-cell receptor activation. Finally, we show that patients with pemphigus, an autoimmune disease mediated by B cells, have a decreased frequency of CD11c+ B cell after treatment, relative to baseline. Our findings show that CD11c+ B cells are mainly memory B cells prone to differentiate into antibody secreting cells that accumulate with age, independently of gender.


Asunto(s)
Antígeno CD11c/metabolismo , Diferenciación Celular/inmunología , Memoria Inmunológica , Células Plasmáticas/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Adulto , Anciano , Presentación de Antígeno , Células Cultivadas , Femenino , Voluntarios Sanos , Humanos , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Pénfigo/inmunología , Fenotipo , Adulto Joven
9.
Curr Opin Pharmacol ; 47: 110-118, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-30986625

RESUMEN

Targeting the P2X7 ion channel, a danger sensor for extracellular nucleotides, improves outcomes in models of inflammation, cancer, and brain-diseases. Antibodies and nanobodies have been developed that antagonize or potentiate gating of P2X7. Their potential advantages over small-molecule drugs include high specificity, lower off-target effects, and tunable in vivo half-life. Genetic fusion of P2X7-specific biologics to binding modules may enable targeting of specific cell subsets. Besides directly modulating P2X7 function, antibodies can also initiate specific depletion of P2X7-expressing cells. Adeno-associated viral vectors (AAV) can be used to express P2X7-specific antibodies in vivo to achieve long-lasting biological effects. Furthermore, if successfully targeted to P2X7-expressing cells, AAVs may enable modulation of the function of P2X7-expressing immune cells via encoded transgenic RNA or proteins.


Asunto(s)
Productos Biológicos/farmacología , Receptores Purinérgicos P2X7/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Anticuerpos/uso terapéutico , Productos Biológicos/uso terapéutico , Dependovirus , Humanos , Inflamación/tratamiento farmacológico , NAD/metabolismo , Neoplasias/tratamiento farmacológico , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Dolor/tratamiento farmacológico , Anticuerpos de Dominio Único/uso terapéutico
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