The metabolism of cells regulates their sensitivity to NK cells depending on p53 status

Leukemic cells proliferate faster than non-transformed counterparts. This requires them to change their metabolism to adapt to their high growth. This change can stress cells and facilitate recognition by immune cells such as cytotoxic lymphocytes, which express the activating receptor Natural Killer G2-D (NKG2D). The tumor suppressor gene p53 regulates cell metabolism, but its role in the expression of metabolism-induced ligands, and subsequent recognition by cytotoxic lymphocytes, is unknown. We show here that dichloroacetate (DCA), which induces oxidative phosphorylation (OXPHOS) in tumor cells, induces the expression of such ligands, e.g. MICA/B, ULBP1 and ICAM-I, by a wtp53-dependent mechanism. Mutant or null p53 have the opposite effect. Conversely, DCA sensitizes only wtp53-expressing cells to cytotoxic lymphocytes, i.e. cytotoxic T lymphocytes and NK cells. In xenograft in vivo models, DCA slows down the growth of tumors with low proliferation. Treatment with DCA, monoclonal antibodies and NK cells also decreased tumors with high proliferation. Treatment of patients with DCA, or a biosimilar drug, could be a clinical option to increase the effectiveness of CAR T cell or allogeneic NK cell therapies

Administration intra-articulaire d'anticorps monoclonaux forme entière par copolymère à base de PEG-PLA

L’arthrose est l’une des dix maladies les plus invalidantes dans les pays développés. Elle affecte le cartilage des articulations causant d’importantes douleurs. Sa prévalence mondiale est d’environ 27 millions de personnes. Les traitements disponibles sont majoritairement symptomatiques et présentent malheureusement des effets insuffisants pour traiter l’arthrose et modifier l’évolution de la maladie. A ce jour, l'industrie pharmaceutique n'a pas réussi à fournir un médicament sûr et efficace et le récent échec du tanezumab, un anti-NGF, vient souligner ce constat. Actuellement plus de 60 anticorps monoclonaux sont sur le marché pour une valeur globale d'environ 80 milliards de dollars par an. Parmi les conditions traitées, on trouve notamment les maladies auto-immunes et certaines maladies inflammatoires mais rien pour le traitement de l’arthrose. Ainsi les anticorps monoclonaux sont un grand espoir actuellement à l’étude pour soigner l'arthrose. Ainsi, nanobody anti-ADAMTS-5 développé par Merck, actuellement en phase II, pourrait être prometteur mais d’autres ont connu des échecs comme la phase II du Lutikizumab, un anticorps bispécifique anti IL-1a/IL-1b. Et il est parfois difficile de savoir si l’échec est dû au choix de la cible thérapeutique ou dû au mode d’administration. En effet, la voie intraveineuse est le mode d'administration le plus courant des anticorps en raison de leur faible biodisponibilité, voire nulle par voie orale. De plus, la physiologie articulaire limite l'échange avec le flux sanguin. L’administration classique systémique mène donc à une faible exposition de l’articulation au principe actif. C’est pourquoi certains de ces échecs pendant le développement clinique pourraient être dus à un manque d'exposition dans l'articulation ou à des effets secondaires associés au mode d’administration systémique. L’administration intra-articulaire (IA) permet de cibler directement le tissu affecté par la pathologie. Des approches innovantes ont bien été tentées telles que l’injection IA d’un anti-TNF-a, mais en raison de la clairance rapide de l'espace synovial, l'administration IA de molécules thérapeutiques reste une approche difficile. C’est pourquoi l’objectif de ma thèse était de développer un procédé de délivrance IA contrôlée d’anticorps monoclonaux pour le traitement des pathologies ostéo-articulaires. La stratégie était de formule r un anticorps monoclonal avec des copolymères PEG-PLA. Les copolymères forment, au site d’injection, un dépôt encapsulant la molécule d’intérêt et permettant sa libération de manière contrôlée au cours du temps. Le Rituximab (RTX) est un anticorps anti-CD20 très connu et bien caractérisé, utilisé notamment dans le traitement de la polyarthrite rhumatoïde ainsi que de différents lymphomes. Il nous a ici servi d’anticorps model afin de valider notre stratégie de formulation. L’anticorps formulé a dans un premier temps été analysé structurellement et fonctionnellement pour la liaison à sa cible et sa capacité d’activation du système immunitaire. Et dans un deuxième temps, sa fonctionnalité a été évaluée in vivo dans un modèle murin de xénogreffe de lymphome B humain. Enfin, les propriétés pharmacocinétiques intéressantes et prometteuses de la formulation ont été établies chez la souris. Ces résultats ont été reproduits ensuite avec le Daratumumab, un autre anticorps ciblant la protéine CD38. Finalement l’approche intra-articulaire a été testée dans un modèle murin d'arthrose avec un anticorps anti-récepteur à l'IL-6, une nouvelle cible potentielle dans le traitement de l’arthrose, les résultats mitigés de cette étude sont discutés en détail. Les résultats présentés dans ce manuscrit démontrent ainsi la pertinence de formuler des protéines thérapeutiques telles que les anticorps avec des copolymères PEG-PLA pour obtenir des propriétés pharmacocinétiques améliorées en utilisant la voie d’administration intra-articulaire.Pain. Its worldwide prevalence is approximately 27 million people. Available treatments are mainly symptomatic and unfortunately don’t have good bioavailability in the joint and insufficient effects to treat osteoarthritis. To the date, pharmaceutical industry has failed to provide a safe and effective drug.Currently more than 60 monoclonal antibodies are on the market with a global value of approximately 80 billion dollars per year. Among conditions treated there are auto-immune and chronic inflammatory diseases. Thus, monoclonal antibodies are a great hope currently being studied to treat osteoarthritis. However, to date, no monoclonal antibodies have been validated by authorities for the treatment of osteoarthritis. An anti-ADAMTS-5 nanobody developed by Merck, currently in phase II, could be promising, but others have failed in clinical trials, such as the Lutikizumab, an anti-IL-1a/IL-1b bispecific antibody. Intravenous route is the most common mode of administration of antibodies due to their low oral bioavailability, limited membrane permeability and limited stability toward gastrointestinal protease activity. In addition, joint physiology limits the exchange with blood stream. Therefore, classic systemic administration leads to a low exposure of the drug in the joint. This is why some of the failures observed in clinical development could be due to a lack of exposure in the joint or to side effects associated with systemic administration. Intra-articular administration allows direct targeting of the therapeutic molecule to the affected tissue. However, due to the rapid clearance of the synovial space, intra-articular administration of therapeutic drugs remains a difficult approach. Hence, the objective of my thesis was to develop a formulation for the intra-articular controlled delivery of monoclonal antibodies for the treatment of osteoarticular pathologies. The formulation relied on the use of PEG-PLA copolymers. The copolymers form a depot at the site of injection that encapsulates the molecule and will allow its release in a controlled manner. Rituximab (RTX) is a well-known and well characterized anti-CD20 antibody. It is already used in the treatment of rheumatoid arthritis as well as various lymphomas. In the present work, we used it as a model anti body to validate our antibody formulation strategy. The formulated antibody was first analyzed structurally and functionally for its binding to its target and its ability to activate the immune system. In a second step, the functionality of formulated RTX was followed in vivo in a mouse model of human B lymphoma xenograft. Then, the formulation showed promising pharmacokinetics properties in mice after intra-articular administration, as high RTX concentrations in the joint were observed for several weeks. These results were reproduced with Daratumumab, another antibody targeting CD38. Finally, the intra-articular approach was tested in a mouse model of osteoarthritis with an anti-IL-6 receptor antibody, as IL-6 could be a potential target for osteoarthritis treatment. The results presented in this manuscript demonstrate the feasibility of formulating large biomolecules like antibodies with PEG-PLA copolymers. Moreover, the formulation combined with the intra-articular route of admin istration showed the increase of local exposure in the joint for several weeks associated with a decrease of the systemic exposure. Although the presence in the injured area of the active molecule has not clearly shown a superiority over the systemic approach in osteoarthritis, the improved pharmacokinetics properties should benefit to future therapeutic developments

Naturally improving the natural cytotoxicity of natural killer (NK) cells

International audienceThe innate lymphocyte lineage natural killer (NK) is now the target of multiple clinical applications, although none has received an agreement from any regulatory agency yet. Transplant of naïve NK cells has not proven efficient enough in the vast majority of clinical trials. Hence, new protocols wish to improve their medical use by producing them from stem cells and/or modifying them by genetic engineering. These techniques have given interesting results but these improvements often hide that natural killers are mainly that: natural. We discuss here different ways to take advantage of NK physiology to improve their clinical activity without the need of additional modifications except for in vitro activation and expansion and allograft in patients. Some of these tactics include combination with monoclonal antibodies (mAb), drugs that change metabolism and engraftment of specific NK subsets with particular activity. Finally, we propose to use specific NK cell subsets found in certain patients that show increase activity against a specific disease, including the use of NK cells derived from patients

Naturally improving the natural cytotoxicity of natural killer (NK) cells

International audienceThe innate lymphocyte lineage natural killer (NK) is now the target of multiple clinical applications, although none has received an agreement from any regulatory agency yet. Transplant of naïve NK cells has not proven efficient enough in the vast majority of clinical trials. Hence, new protocols wish to improve their medical use by producing them from stem cells and/or modifying them by genetic engineering. These techniques have given interesting results but these improvements often hide that natural killers are mainly that: natural. We discuss here different ways to take advantage of NK physiology to improve their clinical activity without the need of additional modifications except for in vitro activation and expansion and allograft in patients. Some of these tactics include combination with monoclonal antibodies (mAb), drugs that change metabolism and engraftment of specific NK subsets with particular activity. Finally, we propose to use specific NK cell subsets found in certain patients that show increase activity against a specific disease, including the use of NK cells derived from patients

The pericyte–glia interface at the blood–brain barrier

International audienceThe cerebrovasculature is a multicellular structure with varying rheological and permeability properties. The outer wall of the brain capillary endothelium is enclosed by pericytes and astrocyte end feet, anatomically assembled to guarantee barrier functions. We, here, focus on the pericyte modifications occurring in disease conditions, reviewing evidence supporting the interplay amongst pericytes, the endothelium, and glial cells in health and pathology. Deconstruction and reactivity of pericytes and glial cells around the capillary endothelium occur in response to traumatic brain injury, epilepsy, and neurodegenerative disorders, impacting vascular permeability and participating in neuroinflammation. As this represents a growing field of research, addressing the multicellular reorganization occurring at the outer wall of the blood-brain barrier (BBB) in response to an acute insult or a chronic disease could disclose novel disease mechanisms and therapeutic targets

The impact of targeting repetitive BamHI-W sequences on the sensitivity and precision of EBV DNA quantification.

Viral load monitoring and early Epstein-Barr virus (EBV) DNA detection are essential in routine laboratory testing, especially in preemptive management of Post-transplant Lymphoproliferative Disorder. Targeting the repetitive BamHI-W sequence was shown to increase the sensitivity of EBV DNA quantification, but the variability of BamHI-W reiterations was suggested to be a source of quantification bias. We aimed to assess the extent of variability associated with BamHI-W PCR and its impact on the sensitivity of EBV DNA quantification using the 1st WHO international standard, EBV strains and clinical samples.Repetitive BamHI-W- and LMP2 single- sequences were amplified by in-house qPCRs and BXLF-1 sequence by a commercial assay (EBV R-gene™, BioMerieux). Linearity and limits of detection of in-house methods were assessed. The impact of repeated versus single target sequences on EBV DNA quantification precision was tested on B95.8 and Raji cell lines, possessing 11 and 7 copies of the BamHI-W sequence, respectively, and on clinical samples.BamHI-W qPCR demonstrated a lower limit of detection compared to LMP2 qPCR (2.33 log10 versus 3.08 log10 IU/mL; P = 0.0002). BamHI-W qPCR underestimated the EBV DNA load on Raji strain which contained fewer BamHI-W copies than the WHO standard derived from the B95.8 EBV strain (mean bias: - 0.21 log10; 95% CI, -0.54 to 0.12). Comparison of BamHI-W qPCR versus LMP2 and BXLF-1 qPCR showed an acceptable variability between EBV DNA levels in clinical samples with the mean bias being within 0.5 log10 IU/mL EBV DNA, whereas a better quantitative concordance was observed between LMP2 and BXLF-1 assays.Targeting BamHI-W resulted to a higher sensitivity compared to LMP2 but the variable reiterations of BamHI-W segment are associated with higher quantification variability. BamHI-W can be considered for clinical and therapeutic monitoring to detect an early EBV DNA and a dynamic change in viral load

Hepatic and hippocampal cytochrome P450 enzyme overexpression during spontaneous recurrent seizures

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Intra-articular delivery of full-length antibodies through the use of an in situ forming depot

International audienceMonoclonal antibodies (mAbs) are large size molecules that have demonstrated high therapeutic potential for the treatment of cancer or autoimmune diseases. Despite some excellent results, their intravenous administration results in high plasma concentration. This triggers off-target effects and sometimes poor targeted tissue distribution. To circumvent this issue, we investigated a local controlled-delivery approach using an in situ forming depot technology. Two clinically relevant mAbs, rituximab (RTX) and daratumumab (DARA), were formulated using an injectable technology based on biodegradable PEG-PLA copolymers. The stability and controlled release features of the formulations were investigated. HPLC and mass spectrometry revealed the preservation of the protein structure. In vitro binding of formulated antibodies to their target antigens and to their cellular FcγRIIIa natural killer cell receptor was fully maintained. Furthermore, encapsulated RTX was as efficient as classical intravenous RTX treatment to inhibit the in vivo tumor growth of malignant human B cells in immunodeficient NSG mice. Finally, the intra-articular administration of the formulated mAbs yielded a sustained local release associated with a lower plasma concentration compared to the intra-articular delivery of non-encapsulated mAbs. Our results demonstrate that the utilization of this polymeric technology is a reliable alternative for the local delivery of fully functional clinically relevant mAbs

Clinical and biological factors associated with early Epstein-Barr Virus infection in HIV-Exposed Uninfected Infants in Eastern Uganda

International audienceBackground: Immune control of Epstein-Barr virus (EBV) infection is impaired in individuals with HIV. We explored maternal factors associated with EBV acquisition in HIV-exposed uninfected (HEU) infants and the relationship between EBV infection and serious adverse events (SAEs) during the first year of life.Methods: 201 HEU infants from Uganda enrolled in the ANRS 12174 trial were tested for antiviral capsid antigen (anti-VCA) antibodies at week 50. Date of infection was estimated by testing EBV DNA at weeks 1, 6, 14, 26, 38, and 50 postpartum on dried blood spots.Results: Eighty-seven (43%) infants tested positive for anti-VCA IgG at week 50. Among the 59 infants positive for EBV DNA, 25% were infected within the first 26 weeks. Almost half (12%) were infected before week 14. Shedding of EBV in breast milk was associated with EBV DNA in maternal plasma (P = .009), HIV RNA detection (P = .039), and lower CD4 count (P = .001) and correlated with plasma EBV DNA levels (P = .002). EBV infant infection at week 50 was associated with shedding of EBV in breast milk (P = .009) and young maternal age (P = .029). Occurrence of a clinical SAE, including malaria and pneumonia, was associated with higher levels of EBV DNA in infants (P = .010).Conclusions: By assessing EBV infection in HEU infants we observed that infection during the first year is determined by HIV and EBV maternal factors and that EBV DNA levels were higher among infants with clinical SAEs.Clinical Trials Registration: NCT00640263

Competitive apnea and its effect on the human brain: focus on the redox regulation of blood–brain barrier permeability and neuronal–parenchymal integrity

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