A novel anti-LAG-3/TIGIT bispecific antibody exhibits potent anti-tumor efficacy in mouse models as monotherapy or in combination with PD-1 antibody.

We report the generation of a novel anti-LAG-3/TIGIT bispecific IgG4 antibody, ZGGS15, and evaluated its anti-tumor efficacy in mouse models as monotherapy or in combination with a PD-1 antibody. ZGGS15 exhibited strong affinities for human LAG-3 and TIGIT, with KDs of 3.05 nM and 2.65 nM, respectively. ZGGS15 has EC50s of 0.69 nM and 1.87 nM for binding to human LAG-3 and TIGIT on CHO-K1 cells, respectively. ZGGS15 competitively inhibited the binding of LAG-3 to MHC-II (IC50 = 0.77 nM) and the binding of TIGIT to CD155 (IC50 = 0.24 nM). ZGGS15 does not induce ADCC, CDC, or obvious cytokine production. In vivo results showed that ZGGS15 had better anti-tumor inhibition than single anti-LAG-3 or anti-TIGIT agents and demonstrated a synergistic effect when combined with nivolumab, with a significantly higher tumor growth inhibition of 95.80% (p = 0.001). The tumor volume inhibition rate for ZGGS15 at 2 mg/kg was 69.70%, and for ZGGS15 at 5 mg/kg plus nivolumab at 1 mg/kg, it was 94.03% (p < 0.001). Our data reveal that ZGGS15 exhibits potent anti-tumor efficacy without eliciting ADCC or CDC or causing cytokine production, therefore having a safe profile.

Dual molecule targeting HDAC6 leads to intratumoral CD4+ cytotoxic lymphocytes recruitment through MHC-II upregulation on lung cancer cells.

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.

Oral PD-L1 inhibitor GS-4224 selectively engages PD-L1 high cells and elicits pharmacodynamic responses in patients with advanced solid tumors.

BACKGROUND: Checkpoint inhibitors targeting the programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) pathway are effective therapies in a range of immunogenic cancer types. Blocking this pathway with an oral therapy could benefit patients through greater convenience, particularly in combination regimens, and allow flexible management of immune-mediated toxicities. METHODS: PD-L1 binding activity was assessed in engineered dimerization and primary cell target occupancy assays. Preclinical antitumor activity was evaluated in ex vivo and in vivo human PD-L1-expressing tumor models. Human safety, tolerability, pharmacokinetics, and biomarker activity were evaluated in an open-label, multicenter, sequential dose-escalation study in patients with advanced solid tumors. Biomarkers evaluated included target occupancy, flow cytometric immunophenotyping, plasma cytokine measurements, and T-cell receptor sequencing. RESULTS: GS-4224 binding caused dimerization of PD-L1, blocking its interaction with PD-1 and leading to reversal of T-cell inhibition and increased tumor killing in vitro and in vivo. The potency of GS-4224 was dependent on the density of cell surface PD-L1, with binding being most potent on PD-L1-high cells. In a phase 1 dose-escalation study in patients with advanced solid tumors, treatment was well tolerated at doses of 400-1,500 mg once daily. Administration of GS-4224 was associated with a dose-dependent increase in plasma GS-4224 exposure and reduction in free PD-L1 on peripheral blood T cells, an increase in Ki67 among the PD-1-positive T-cell subsets, and elevated plasma cytokines and chemokines. CONCLUSIONS: GS-4224 is a novel, orally bioavailable small molecule inhibitor of PD-L1. GS-4224 showed evidence of expected on-target biomarker activity, including engagement of PD-L1 and induction of immune-related pharmacodynamic responses consistent with PD-L1 blockade. TRIAL REGISTRATION NUMBER: NCT04049617.

Role of co­inhibitory molecules in the treatment of psoriasis (Review).

Psoriasis is a common chronic inflammatory skin disease characterized by abnormal activation and infiltration of T-cells and excessive proliferation of keratinocytes (KCs). Its pathogenesis is complex and frequently accompanied by the imbalance of T-cell subpopulations, contributing to its development and further exacerbation. Therefore, the immune system, especially T-cells, is mainly involved in the pathogenesis of psoriasis. While T-cell activation not only requires the first recognition of T-cell receptor and major histocompatibility complex peptide, co-stimulatory and co-inhibitory pathways are reported to promote or dampen T-cell responses through a variety of mechanisms. In recent years, immuno-related agents have been applied in the treatment of numerous clinical diseases, including psoriasis, and are starting to show promising and potential therapy prospects in autoimmune skin diseases. The present review outlined the role of co-inhibitory molecules in the pathogenesis of psoriasis and their application in the treatment of psoriasis.

[Effect of LAG3 deficiency on natural killer cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis].

OBJECTIVE: To investigate the effect of LAG-3 deficiency (LAG3-/-) on natural killer (NK) cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis. METHODS: C57BL/6 mice, each weighing (20 ± 2) g, were divided into the LAG3-/- and wild type (WT) groups, and each mouse in both groups was inoculated with 3 000 E. multilocularis protoscoleces via the hepatic portal vein. Mouse liver and spleen specimens were collected 12 weeks post-infection, sectioned and stained with sirius red, and the hepatic lesions and fibrosis were observed. Mouse hepatic and splenic lymphocytes were isolated, and flow cytometry was performed to detect the proportions of hepatic and splenic NK cells, the expression of CD44, CD25 and CD69 molecules on NK cell surface, and the secretion of interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin (IL)-4, IL-10 and IL-17A. RESULTS: Sirius red staining showed widening of inflammatory cell bands and hyperplasia of fibrotic connective tissues around mouse hepatic lesions, as well as increased deposition of collagen fibers in the LAG3-/-group relative to the WT group. Flow cytometry revealed lower proportions of mouse hepatic (6.29% ± 1.06% vs. 11.91% ± 1.85%, P < 0.000 1) and splenic NK cells (4.44% ± 1.22% vs. 5.85% ± 1.10%, P > 0.05) in the LAG3-/- group than in the WT group, and the mean fluorescence intensity of CD44 was higher on the surface of mouse hepatic NK cells in the LAG3-/- group than in the WT group (t = -3.234, P < 0.01), while no significant differences were found in the mean fluorescence intensity of CD25 or CD69 on the surface of mouse hepaticNK cells between the LAG3-/- and WT groups (both P values > 0.05). There were significant differences between the LAG3-/- and WT groups in terms of the percentages of IFN-γ (t = -0.723, P > 0.05), TNF-α (t = -0.659, P > 0.05), IL-4 (t = -0.263, P > 0.05), IL-10 (t = -0.455, P > 0.05) or IL-17A secreted by mouse hepatic NK cells (t = 0.091, P > 0.05), and the percentage of IFN-γ secreted by mouse splenic NK cells was higher in the LAG3-/- group than in the WT group (58.40% ± 1.64% vs. 50.40% ± 4.13%; t = -4.042, P < 0.01); however, there were no significant differences between the two groups in terms of the proportions of TNF-α (t = -1.902, P > 0.05), IL-4 (t = -1.333, P > 0.05), IL-10 (t = -1.356, P > 0.05) or IL-17A secreted by mouse splenic NK cells (t = 0.529, P > 0.05). CONCLUSIONS: During the course of E. multilocularis infections, LAG3-/- promotes high-level secretion of IFN-γ by splenic NK cells, which may participate in the reversal the immune function of NK cells, resulting in aggravation of hepatic fibrosis.

Targeting PARG induces tumor cell growth inhibition and antitumor immune response by reducing phosphorylated STAT3 in ovarian cancer.

BACKGROUND: Ovarian cancer is the most lethal gynecological malignancy, with limited treatment options after failure of standard therapies. Despite the potential of poly(ADP-ribose) polymerase inhibitors in treating DNA damage response (DDR)-deficient ovarian cancer, the development of resistance and immunosuppression limit their efficacy, necessitating alternative therapeutic strategies. Inhibitors of poly(ADP-ribose) glycohydrolase (PARG) represent a novel class of inhibitors that are currently being assessed in preclinical and clinical studies for cancer treatment. METHODS: By using a PARG small-molecule inhibitor, COH34, and a cell-penetrating antibody targeting the PARG's catalytic domain, we investigated the effects of PARG inhibition on signal transducer and activator of transcription 3 (STAT3) in OVCAR8, PEO1, and Brca1-null ID8 ovarian cancer cell lines, as well as in immune cells. We examined PARG inhibition-induced effects on STAT3 phosphorylation, nuclear localization, target gene expression, and antitumor immune responses in vitro, in patient-derived tumor organoids, and in an immunocompetent Brca1-null ID8 ovarian mouse tumor model that mirrors DDR-deficient human high-grade serous ovarian cancer. We also tested the effects of overexpressing a constitutively activated STAT3 mutant on COH34-induced tumor cell growth inhibition. RESULTS: Our findings show that PARG inhibition downregulates STAT3 activity through dephosphorylation in ovarian cancer cells. Importantly, overexpression of a constitutively activated STAT3 mutant in tumor cells attenuates PARG inhibitor-induced growth inhibition. Additionally, PARG inhibition reduces STAT3 phosphorylation in immune cells, leading to the activation of antitumor immune responses, shown in immune cells cocultured with ovarian cancer patient tumor-derived organoids and in immune-competent mice-bearing mouse ovarian tumors. CONCLUSIONS: We have identified a novel antitumor mechanism underlying PARG inhibition beyond its primary antitumor effects through blocking DDR in ovarian cancer. Furthermore, targeting PARG activates antitumor immune responses, thereby potentially increasing response rates to immunotherapy in patients with ovarian cancer.

Lymphocyte-Activation Gene 3 Facilitates Pathological Tau Neuron-to-Neuron Transmission.

The spread of prion-like protein aggregates is a common driver of pathogenesis in various neurodegenerative diseases, including Alzheimer's disease (AD) and related Tauopathies. Tau pathologies exhibit a clear progressive spreading pattern that correlates with disease severity. Clinical observation combined with complementary experimental studies has shown that Tau preformed fibrils (PFF) are prion-like seeds that propagate pathology by entering cells and templating misfolding and aggregation of endogenous Tau. While several cell surface receptors of Tau are known, they are not specific to the fibrillar form of Tau. Moreover, the underlying cellular mechanisms of Tau PFF spreading remain poorly understood. Here, it is shown that the lymphocyte-activation gene 3 (Lag3) is a cell surface receptor that binds to PFF but not the monomer of Tau. Deletion of Lag3 or inhibition of Lag3 in primary cortical neurons significantly reduces the internalization of Tau PFF and subsequent Tau propagation and neuron-to-neuron transmission. Propagation of Tau pathology and behavioral deficits induced by injection of Tau PFF in the hippocampus and overlying cortex are attenuated in mice lacking Lag3 selectively in neurons. These results identify neuronal Lag3 as a receptor of pathologic Tau in the brain，and for AD and related Tauopathies, a therapeutic target.

Immune checkpoint biology in health & disease: Immune checkpoint biology and autoimmunity in cancer patients.

Immune checkpoints (ICs) play a central role in maintaining immune homoeostasis. The discovery that tumours use this physiological mechanism to avoid elimination by the immune system, opened up avenues for therapeutic targeting of ICs as a novel way of treating cancer. However, this therapy a new array of autoimmune side effects, termed immune-related adverse events (irAEs). In this narrative review, we first recapitulate the physiological function of ICs that are approved targets for cancer immunotherapy (CTLA-4, PD-(L)1 and LAG-3), as the groundwork to critically discuss current knowledge on irAEs. Specifically, we summarize clinical aspects and examine a molecular classification and predisposing factors of irAEs. Finally, we discuss irAE treatment, particularly emphasizing how molecular knowledge is changing the current treatment paradigm.

Lymphocyte-activation gene 3 (LAG-3) as a promising immune checkpoint in cancer immunotherapy: From biology to the clinic.

In recent years, there have been notable advancements in the field of cancer immunotherapy, namely in the area of immune checkpoint inhibition. The Lymphocyte-activation gene 3 (LAG-3) has garnered attention as a potentially valuable focus of study in this particular field. The present study examines the biological aspects of LAG-3, its clinical consequences, and the potential therapeutic opportunities associated with its modulation. LAG-3, similar to CD4, has a regulatory role in modulating the immune system. The upregulation of this protein inside the neoplastic milieu hampers the immune system's ability to mount an effective response, hence enabling the evasion of cancer cells from immune surveillance. The LAG-3 protein interacts with ligands, inhibiting cytotoxic immune cells such as CD8+ T cells and NK cells. The potential of LAG-3 inhibitors presents intriguing prospects. Integrating these medicines with established treatments like PD-1/PD-L1 or CTLA-4 inhibitors can broaden the range of available therapy choices and address resistance issues. The advent of personalized therapy is imminent, as evidenced by the utilization of predictive biomarkers such as LAG-3 expression to inform individualized therapeutic approaches. Additionally, inhibitors of LAG-3 exhibit promise in addressing immunological depletion and resistance by revitalizing T cells and producing durable immune responses. The realization of LAG-3's promise necessitates global collaboration and equal access. Multinational trials are expected to ascertain the efficacy of the intervention in various patient groups.

Effect of LAG3 deficiency on natural killer cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis / 中国血吸虫病防治杂志

Objective To investigate the effect of LAG-3 deficiency (LAG3-/-) on natural killer (NK) cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis. Methods C57BL/6 mice, each weighing (20 ± 2) g, were divided into the LAG3-/- and wild type (WT) groups, and each mouse in both groups was inoculated with 3 000 E. multilocularis protoscoleces via the hepatic portal vein. Mouse liver and spleen specimens were collected 12 weeks post-infection, sectioned and stained with sirius red, and the hepatic lesions and fibrosis were observed. Mouse hepatic and splenic lymphocytes were isolated, and flow cytometry was performed to detect the proportions of hepatic and splenic NK cells, the expression of CD44, CD25 and CD69 molecules on NK cell surface, and the secretion of interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin (IL)-4, IL-10 and IL-17A. Results Sirius red staining showed widening of inflammatory cell bands and hyperplasia of fibrotic connective tissues around mouse hepatic lesions, as well as increased deposition of collagen fibers in the LAG3-/-group relative to the WT group. Flow cytometry revealed lower proportions of mouse hepatic (6.29% ± 1.06% vs. 11.91% ± 1.85%, P < 0.000 1) and splenic NK cells (4.44% ± 1.22% vs. 5.85% ± 1.10%, P > 0.05) in the LAG3-/- group than in the WT group, and the mean fluorescence intensity of CD44 was higher on the surface of mouse hepatic NK cells in the LAG3-/- group than in the WT group (t = −3.234, P < 0.01), while no significant differences were found in the mean fluorescence intensity of CD25 or CD69 on the surface of mouse hepaticNK cells between the LAG3-/- and WT groups (both P values > 0.05). There were significant differences between the LAG3-/- and WT groups in terms of the percentages of IFN-γ (t = −0.723, P > 0.05), TNF-α (t = −0.659, P > 0.05), IL-4 (t = −0.263, P > 0.05), IL-10 (t = −0.455, P > 0.05) or IL-17A secreted by mouse hepatic NK cells (t = 0.091, P > 0.05), and the percentage of IFN-γ secreted by mouse splenic NK cells was higher in the LAG3-/- group than in the WT group (58.40% ± 1.64% vs. 50.40% ± 4.13%; t = −4.042, P < 0.01); however, there were no significant differences between the two groups in terms of the proportions of TNF-α (t = −1.902, P > 0.05), IL-4 (t = −1.333, P > 0.05), IL-10 (t = −1.356, P > 0.05) or IL-17A secreted by mouse splenic NK cells (t = 0.529, P > 0.05). Conclusions During the course of E. multilocularis infections, LAG3-/- promotes high-level secretion of IFN-γ by splenic NK cells, which may participate in the reversal the immune function of NK cells, resulting in aggravation of hepatic fibrosis.

Elevated monocyte HLA-DR in pediatric secondary hemophagocytic lymphohistiocytosis: a retrospective study.

Introduction: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition, and its diagnosis may be challenging. In particular, some cases show close similarities to sepsis (fever, organ failure, and high ferritin), but their treatment, while urgent, differ: prompt broad-spectrum antibiotherapy for sepsis and immunosuppressive treatment for HLH. We questioned whether monocyte human leucocyte antigen (mHLA)-DR could be a diagnostic marker for secondary HLH (sHLH). Methods: We retrospectively reviewed data from patients with a sHLH diagnosis and mHLA-DR quantification. mHLA-DR data from healthy children and children with septic shock, whose HLA-DR expression is reduced, from a previously published study were also included for comparison. Results: Six patients with sHLH had mHLA-DR quantification. The median level of monocyte mHLA-DR expression in patients with sHLH [79,409 antibodies bound per cell (AB/C), interquartile range (IQR) (75,734-86,453)] was significantly higher than that in healthy children and those with septic shock (29,668 AB/C, IQR (24,335-39,199), and 7,493 AB/C, IQR (3,758-14,659), respectively). Each patient with sHLH had a mHLA-DR higher than our laboratory normal values. Four patients had a second mHLA-DR sampling 2 to 4 days after the initial analysis and treatment initiation with high-dose corticosteroids; for all patients, mHLA-DR decreased to within or close to the normal range. One patient with systemic juvenile idiopathic arthritis had repeated mHLA-DR measurements over a 200-day period during which she underwent four HLH episodes. mHLA-DR increased during relapses and normalized after treatment incrementation. Conclusion: In this small series, mHLA-DR was systematically elevated in patients with sHLH. Elevated mHLA-DR could contribute to sHLH diagnosis and help earlier distinction with septic shock.

Commentary on oncolytic viruses: past, present, and future.

Whither oncolytic viruses? From the peak of their popularity in the early 2000s, when the ONYX-015 adenovirus had just entered the clinic, and then again in 2015 when the Food and Drug Administration-approved talimogene laherparepvec (also known as OncoVEXGM-CSF), which briefly revived interest, oncolytic viruses (OVs) have mostly fallen out of favor despite the many pharmaceutical companies with OVs in development.This commentary enumerates and addresses the core conceptions, perceptions, and misconceptions that characterize the current 'trough of disillusionment' in which the field of anticancer virotherapy finds itself and suggests reasons for optimism.

A combined analysis of multi-omics data reveals the prognostic values and immunotherapy response of LAG3 in human cancers.

Lymphocyte-activation gene 3 (LAG3) is a highly anticipated immune checkpoint in the context of cancer, exerting regulatory control over immune cell proliferation and function to reinforce the advancement of cancers. However, the comprehensive functional analysis of LAG3 across various cancer types remains undisclosed; thus, this study aims to investigate the pan-cancer expression profile of LAG3. We have investigated the expression profile, prognostic significance, and genetic alterations of LAG3 in various cancers while elucidating its characteristic in immune response regulation. Our findings demonstrated that elevated LAG3 expression is significantly associated with favorable prognosis in patients with cutaneous melanoma (SKCM), and it may be a potential biomarker for SKCM. Furthermore, multiple immune algorithms have highlighted the important regulatory role of LAG3 for the tumor-infiltrating immune cells including CD8 + T cells, B cells, dendritic cells (DCs), macrophages, and natural killer (NK) cells. We also examined the distribution of LAG3 at the single-cell level and explored its functional significance. A comprehensive and systematic analysis of LAG3 would facilitate a comprehensive evaluation of LAG3 in cancer biology and provide valuable insights for cancer management.

Reduced regulatory effects of bone marrow-derived mesenchymal stem cells on activated T lymphocytes and Th1/Th2 cytokine secretion in children with aplastic anemia.

Acquired aplastic anemia (AA) is a recognized immune-mediated disorder and abnormally activated T lymphocyte-mediated bone marrow destruction is considered to be its main pathogenesis. Whether abnormal activation of T lymphocytes would also damage bone marrow-derived MSCs remains to be further studied. The aim of this study was to analyze the extent of T lymphocyte activation and the levels of Th1/Th2 cytokines of AA patients, and to explore the immunomodulatory effects of BM-MSCs on IL-2-stimulated T lymphocyte activation and cytokine production in vitro by means of transwell co-culture assay and flow cytometry measurement. The intermediate (CD25+) activated T cells were dominant in peripheral blood, while the early (CD69+) and late (HLA-DR+) activated T cells were predominant in bone marrow. Severe AA patients have an obviously higher proportion of CD3+CD8+CD69+ T cells than NSAA cases. The levels of IL-2 and IL-6 in AA patients were slightly elevated and INF-γ was mildly decreased in comparison with normal individuals. BM-MSCs derived from AA could not effectively inhibit the IL-2-induced activation of T cells with higher proportions of CD25+CD3+CD4+, CD69+CD3+CD4+ and CD25+CD3+CD8+ T cells after co-culture, and they showed a decreased ability to balance the Th1/Th2 cytokine production. Moreover, they had less robust osteogenic differentiation and more prone to adipogenic differentiation. We concluded that abnormally excessive T cell activation accompanied by abnormal cytokine secretion may impair the function of BM-MSCs in children with aplastic anemia.

A mean-field description for the expansion kinetics of activated T cell populations.

When lymphocytes encounter their cognate antigen, they become activated and undergo a limited number of cell divisions during which they differentiate into memory or effector cells or die. While the dynamics of individual cells are often heterogeneous, the expansion kinetics at the population level are highly reproducible, suggesting a mean-field description. To generate a finite division destiny, we consider two scenarios: Cells stop dividing after a certain number of iterations or their death rate increases with each cell division. The dynamics of the combined system can be mapped to a partial differential equation, and for a suitable choice of the activation rate, we obtain simple analytical solutions for the total cell number and the mean number of divisions per cell which can well describe the signal-dependent T cell expansion kinetics from in vitro experiments. Interestingly, only the division cessation mechanism yields an expression for the division destiny that does not contradict experiments. We show that the generation-dependent decrease of the division rate in individual cells leads to a time-dependent decrease at the population level which is consistent with a "time-to-die" control mechanism for the division destiny as suggested previously. We also derive mean-field equations for the total cell number which provide a basis for implementing T cell expansion kinetics into quantitative systems pharmacology models for immuno-oncology and CAR-T cell therapies.

FGL1-LAG3 axis impairs IL-10-Producing regulatory T cells associated with Systemic lupus erythematosus disease activity.

Background: Systemic Lupus Erythematosus (SLE) is a prototypic autoimmune disease, which is accompanied by liver damage. However, it remains unknown whether liver damage is associated with SLE progression. Method: ology: HepG2 and L-02 cells were stimulated with cytokines, and FGL1 mRNA and protein expression levels were determined using Real-time PCR and ELISA, respectively. Regulatory T cells (Treg) isolated from healthy individuals as well as patients with SLE and SLE and liver damage (SLE-LD) were cultured with autologous effector CD4+T cells in the presence of a functional antibody or isotype control. The expression levels of LAG3, CD25, PD-1, CXCR5, ICOS and OX40 were evaluated by flow cytometry. FGL1, IL-10, IL-17a and IL-21 levels in serum or culture supernatants were quantified by ELISA. Results: Patients with SLE-LD exhibits higher disease activity indices and anti-dsDNA antibody levels. Importantly, fibrinogen-like protein 1 (FGL1), a key factor released from the injured liver, is up-regulated in patients with SLE-LD and is associated with disease activity. FGL1 expression is induced by the inflammatory cytokine IL-6 signaling in hepatocytes. Higher expression of the FGL1 receptor lymphocyte activation gene 3 (LAG3) is detected in Treg cells from patients with SLE-LD. The FGL1-LAG3 signaling axis inhibits Treg cell proliferation and impairs the suppressive activity of Treg cells by limiting IL-10 secretion. Furthermore, FGL1-LAG3 signaling promotes the production of pathogenic IL-17a and IL-21 by CD4+T cells by reducing IL-10 level produced by Treg in patients with SLE. Conclusions: The FGL1-LAG3 signal axis is a key mechanism that subverts the suppressive function of Treg cells. This may provide a new therapeutic target for SLE and SLE-induced liver damage.

Intraperitoneal administration of a modified vaccinia virus Ankara confers single-chain interleukin-12 expression to the omentum and achieves immune-mediated efficacy against peritoneal carcinomatosis.

BACKGROUND: Peritoneal carcinomatosis is an advanced stage of cancer in which the disease has spread to the peritoneal cavity. In order to restore antitumor immunity subverted by tumor cells in this location, we evaluated intraperitoneal administrations of modified vaccinia virus Ankara (MVA) engineered to express single-chain interleukin 12 (scIL-12) to increase antitumor immune responses. METHODS: MVA encoding scIL-12 (MVA.scIL-12) was evaluated against peritoneal carcinomatosis models based on intraperitoneal engraftment of tumor cells. CD8-mediated immune responses, elucidated antitumor efficacy, and safety were evaluated following intravenous, intratumoral, or intraperitoneal administration of the viral vector. The immune response was measured by ELISpot (enzyme-linked immunosorbent spot), RNA sequencing, flow cytometry, intravital microscopy, and depletion of lymphocyte subsets with monoclonal antibodies. Safety was assessed by body-weight follow-up and blood testing. Tissue tropism on intravenous or intraperitoneal administration was assessed by bioluminescence analysis using a reporter MVA encoding luciferase. RESULTS: Intraperitoneal or locoregional administration, but not other routes of administration, resulted in a potent immune response characterized by increased levels of tumor-specific CD8+ T lymphocytes with the ability to produce both interferon-γ and tumor necrosis factor-α. The antitumor immune response was detectable not only in the peritoneal cavity but also systemically. As a result of intraperitoneal treatment, a single administration of MVA.scIL-12 encoding scIL-12 completely eradicated MC38 tumors implanted in the peritoneal cavity and also protected cured mice from subsequent subcutaneous rechallenges. Bioluminescence imaging using an MVA encoding luciferase revealed that intraperitoneal administration targets transgene to the omentum. The omentum is considered a key tissue in immune protection of the peritoneal cavity. The safety profile of intraperitoneal administration was also better than that following intravenous administration since no weight loss or hematological toxicity was observed when the vector was locally delivered into the peritoneal cavity. CONCLUSION: Intraperitoneal administration of MVA vectors encoding scIL-12 targets the omentum, which is the tissue where peritoneal carcinomatosis usually begins. MVA.scIL-12 induces a potent tumor-specific immune response that often leads to the eradication of experimental tumors disseminated to the peritoneal cavity.

Advancement of anti-LAG-3 in cancer therapy.

Immune checkpoint inhibitors have effectively transformed the treatment of many cancers, particularly those highly devastating malignancies. With their widespread popularity, the drawbacks of immune checkpoint inhibitors are also recognized, such as drug resistance and immune-related systematic side effects. Thus, it never stops investigating novel immune checkpoint inhibitors. Lymphocyte Activation Gene-3 (LAG-3) is a well-established co-inhibitory receptor that performs negative regulation on immune responses. Recently, a novel FDA-approved LAG-3 blocking agent, together with nivolumab as a new combinational immunotherapy for metastatic melanoma, brought LAG-3 back into focus. Clinical data suggests that anti-LAG-3 agents can amplify the therapeutic response of other immune checkpoint inhibitors with manageable side effects. In this review, we elucidate the intercellular and intracellular mechanisms of LAG-3, clarify the current understanding of LAG-3 in the tumor microenvironment, identify present LAG-3-associated therapeutic agents, discuss current LAG-3-involving clinical trials, and eventually address future prospects for LAG-3 inhibitors.

Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) neutralization counteracts T cell immune evasion in breast cancer.

BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT) is a key intracellular enzyme that participates in nicotinamide adenine dinucleotide (NAD) homeostasis as well as a released cytokine (eNAMPT) that is elevated in inflammatory conditions and in cancer. In patients with breast cancer, circulating eNAMPT is elevated and its plasma levels correlate with prognosis and staging. In light of this, we investigated the contribution of eNAMPT in triple negative mammary carcinoma progression by investigating the effect of its neutralization via a specific neutralizing monoclonal antibody (C269). METHODS: We used female BALB/c mice injected with 4T1 clone 5 cells and female C57BL6 injected with EO771 cells, evaluating tumoral size, spleen weight and number of metastases. We injected two times a week the anti-eNAMPT neutralizing antibody and we sacrificed the mice after 28 days. Harvested tumors were analyzed by histopathology, flow cytometry, western blot, immunohistochemistry, immunofluorescence and RNA sequencing to define tumor characteristics (isolating tumor infiltrating lymphocytes and tumoral cells) and to investigate the molecular mechanisms behind the observed phenotype. Moreover, we dissected the functional relationship between T cells and tumoral cells using three-dimensional (3D) co-cultures. RESULTS: The neutralization of eNAMPT with C269 led to decreased tumor size and reduced number of lung metastases. RNA sequencing and functional assays showed that eNAMPT controlled T-cell response via the programmed death-ligand 1/programmed cell death protein 1 (PD-L1/PD-1) axis and its neutralization led to a restoration of antitumoral immune responses. In particular, eNAMPT neutralization was able to activate CD8+IFNγ+GrzB+ T cells, reducing the immunosuppressive phenotype of T regulatory cells. CONCLUSIONS: These studies indicate for the first time eNAMPT as a novel immunotherapeutic target for triple negative breast cancer.

Characterisation of the protein expression of the emerging immunotherapy targets VISTA, LAG-3 and PRAME in primary uveal melanoma: insights from a southern French patient cohort.

Uveal melanoma (UM) is the most common intraocular tumour in adults, with dismal prognosis once metastases develop, since therapeutic options for the metastatic disease are ineffective. Over the past decade, novel cancer therapies based on immunotherapy have changed the landscape of treatment of different forms of cancer leading to many hopes of improvement in patient overall survival (OS). VISTA, LAG-3 and PRAME are novel promising targets of immunotherapy that have recently gained attention in different solid tumours, but whose relevance in UM remained to be comprehensively evaluated until now. Here, we studied the protein expression of VISTA, LAG-3 and PRAME using immunohistochemistry in representative whole tissue sections from primary UM cases in a cohort of 30 patients from a single centre (Nice University Hospital, Nice, France). The expression of each of these markers was correlated with different clinical and pathological parameters, including onset of metastases and OS. We demonstrated the protein expression of VISTA and LAG-3 in small lymphocytes infiltrating the tumour, while no expression of the proteins was detected in UM cells. For PRAME, nuclear expression was observed in UM cells, but no expression in tumour infiltrating immune cells was identified. Increased levels of VISTA expression in tumour infiltrating lymphocytes (TILs) were associated with nuclear BAP1 expression and better prognosis. Higher levels of LAG-3 in TILs were associated with higher levels of CD8-positive TILs. PRAME nuclear positivity in melanoma cells was associated with epithelioid cell dominant (>90%) UM histological subtype, higher mitotic numbers and a higher percentage of chromosome 8q gain. This study proposes VISTA as a novel relevant immune checkpoint molecule in primary UM and contributes to confirm LAG-3 and PRAME as potentially important immunotherapy targets in the treatment of UM patients, helping to expand the number of immunotherapy candidate molecules that are relevant to modulate in this aggressive cancer.