InVivoMAb mouse IgG2a isotype control, unknown specificity

The macrophage checkpoint receptor SIRPα signals against phagocytosis by binding CD47 expressed on all cells - including macrophages. Here, inhibiting cis interactions between SIRPα and CD47 on the same macrophage increases eating approximately the same as inhibiting trans interactions. Antibody blockade of CD47, as pursued in clinical trials against cancer, is applied separately to human-derived macrophages and to red blood cell (RBC) targets for phagocytosis, and both scenarios produce surprisingly similar increases in RBC engulfment. Blockade of both macrophages and targets results in hyper-phagocytosis, and knockdown of macrophage-CD47 likewise increases eating of 'foreign' cells and particles, decreases SIRPα's baseline inhibitory signaling, and linearly increases binding of soluble-CD47 in trans, consistent with cis-trans competition. Many cell types express both SIRPα and CD47, including mouse melanoma B16 cells, and CRISPR-mediated deletions modulate B16 phagocytosis consistent with cis-trans competition. Additionally, soluble SIRPα binding to human-CD47 displayed on Chinese hamster ovary (CHO) cells is suppressed by SIRPα co-display, and atomistic computations confirm SIRPα bends and binds CD47 in cis. Safety and efficacy profiles for CD47-SIRPα blockade might therefore reflect a disruption of both cis and trans interactions. © 2020. Published by The Company of Biologists Ltd.

Cancer onset and progression involves the accumulation of multiple oncogenic hits, which are thought to dominate or bypass the physiologic regulatory mechanisms in tissue development and homeostasis. We demonstrate in T-cell acute lymphoblastic leukemia (T-ALL) that, irrespective of the complex oncogenic abnormalities underlying tumor progression, experimentally induced, persistent T-cell receptor (TCR) signaling has antileukemic properties and enforces a molecular program resembling thymic negative selection, a major developmental event in normal T-cell development. Using mouse models of T-ALL, we show that induction of TCR signaling by high-affinity self-peptide/MHC or treatment with monoclonal antibodies to the CD3ε chain (anti-CD3) causes massive leukemic cell death. Importantly, anti-CD3 treatment hampered leukemogenesis in mice transplanted with either mouse- or patient-derived T-ALLs. These data provide a strong rationale for targeted therapy based on anti-CD3 treatment of patients with TCR-expressing T-ALL and demonstrate that endogenous developmental checkpoint pathways are amenable to therapeutic intervention in cancer cells. T-ALLs are aggressive malignant lymphoid proliferations of T-cell precursors characterized by high relapse rates and poor prognosis, calling for the search for novel therapeutic options. Here, we report that the lineage-specific TCR/CD3 developmental checkpoint controlling cell death in normal T-cell progenitors remains switchable to induce massive tumor cell apoptosis in T-ALL and is amenable to preclinical therapeutic intervention. Cancer Discov; 6(9); 972-85. ©2016 AACR.See related commentary by Lemonnier and Mak, p. 946This article is highlighted in the In This Issue feature, p. 932. ©2016 American Association for Cancer Research.

Triple-negative breast cancer (TNBC) has poorer outcomes than other breast cancers (BC), including HER2+ BC. Cathepsin D (CathD) is a poor prognosis marker overproduced by BC cells, hypersecreted in the tumour microenvironment with tumour-promoting activity. Here, we characterized the immunomodulatory activity of the anti-CathD antibody F1 and its improved Fab-aglycosylated version (F1M1) in immunocompetent mouse models of TNBC (C57BL/6 mice harbouring E0771 cell grafts) and HER2-amplified BC (BALB/c mice harbouring TUBO cell grafts).

Antibodies targeting TGF-β and PD-L1 initially showed promise as second-generation PD-L1 agents. However, consecutive trial failures have limited their clinical success. Our study reveals that the efficacy of the TGF-β×PD-L1 bispecific antibody (BsAb) is compromised by insufficient activation of innate immune responses. To address this, we combine STING agonists with the BsAb, significantly enhancing tumor suppression beyond that achieved with standard STING agonist plus anti-PD-L1 combinations in preclinical models. Unexpectedly, even STING agonist monotherapy is improved by TGF-β blockade, suggesting that TGF-β suppresses STING-driven immune activation. We find that this synergy is mediated by the CXCL16-CXCR6 axis, where STING activation and TGF-β blockade promote CXCL16 expression in macrophages and dendritic cells, recruiting and sustaining cytotoxic CXCR6+ T cells. Additionally, PD-L1 blockade further enhances their antitumor activity. To optimize this strategy, we develop Y101S, an antibody-drug conjugate targeting TGF-β, PD-L1, and STING, which demonstrates superior tumor control and immune modulation in preclinical models. These findings highlight the therapeutic potential of this triple-targeting approach.

Psoriasis and atopic dermatitis (AD) are two prevalent inflammatory skin disorders, each characterized by distinct adaptive immune responses. However, recent evidence suggests that these diseases may share overlapping immune mechanisms, especially concerning keratinocyte function. The specific cytokines that coordinate these inflammatory pathways remain largely undefined.

The human respiratory syncytial virus (hRSV) is a pathogen of global concern, causing significant morbidity and mortality, mainly in preterm infants. To date, all licenced monoclonal antibodies (mAbs) developed against hRSV have targeted its surface fusion or pre-fusion protein (F-hRSV).

Natural Killer (NK) cells play a critical role in regulating tumor growth, but our understanding of the mechanisms underlying their anti-tumor activity remains limited. We identified the histone methyltransferase EHMT2 as a key suppressor of NK cell-mediated cytotoxicity. EHMT2 inhibition in cancer cells enhanced NK cell-mediated elimination of diverse cancers, including uveal melanoma, breast cancer, and pancreatic cancer. EHMT2 loss increased AZGP1 and decreased TGF-β1 levels, resulting in the autocrine elevation of NKG2D ligands MICB and ULBP3, chemokines in cancer cells, and the paracrine stimulation of NK cell function. In a syngeneic pancreatic cancer model, EHMT2 inhibition suppressed tumors in an NK cell-dependent manner, as NK cell depletion restored tumor growth. This effect persisted and remained dependent on NK cells in Rag2 knockout mice (lacking T and B cells), but not in NSG mice (lacking T-, B- and NK-cells). Furthermore, EHMT2 and TGF-β1 inhibitors suppressed tumors in immunocompetent, but not in immunodeficient mice. These findings establish EHMT2 as a suppressor of NK cell-mediated anti-tumor immunity and a promising therapeutic target.

The incorporation of the current immunotherapy, GD2-targeting monoclonal antibodies, into the standard of care has moderately improved clinical outcomes in children with high-risk neuroblastoma (HR-NB); however, overall survival remains low. More than 50% of patients with HR-NB are refractory to or eventually develop resistance to anti-GD2 treatment. HR-NBs are generally known to have a low tumor mutational burden, are immunologically cold and possess an immunosuppressive tumor microenvironment. Understanding the mechanisms of immune evasion may provide novel targets for improving the efficacy of immunotherapies for these immunologically cold HR-NBs. Here, utilizing immunocompetent mouse models of immunologically cold HR-NB, we revealed a novel function of IGF2BP1 in promoting the immune escape of neuroblastoma tumors. We demonstrate that neuroblastoma cell-specific knockdown of IGF2BP1 favorably alters the tumor microenvironment of HR-NBs, turning these "immunologically cold" tumors into an immunogenic type, thereby priming them for anti-GD2 therapy-induced immune responses. Downregulation of IGF2BP1 in NB cells decreased the number of immunosuppressive T-regulatory and dysfunctional/exhausted CD8+ T cells and promoted the accumulation of effector MHCII +  macrophages at the tumor site. Importantly, knockdown of IGF2BP1 along with anti-GD2 immunotherapy induced a synergistic immunogenic effect and achieved a potent antitumor response in an HR-NB mouse model, with increased accumulation of effector CD8+ T cells and CD86+  macrophages but decreased MDSC numbers in the tumor microenvironment. Thus, disrupting NB cancer cell IGF2BP1-mediated immunosuppression is a potential approach for improving the efficacy of anti-GD2 immunotherapy towards HR-NBs.

Chaperone-mediated autophagy (CMA) is a selective autophagic process essential for maintaining cellular quality and responding to stress. Dysregulation of the CMA pathway is increasingly recognized in various cancers, yet the mechanisms behind CMA hyperactivation in cancer cells remain unclear. Here, we show that CMA is upregulated in patient-derived glioblastoma stem cells (GSCs), indicated by a significant increase in the lysosomal abundance of the CMA receptor, lysosome-associated membrane protein 2 A (LAMP2A). This increase results from MST4-mediated phosphorylation of LAMP2A, enhancing its stability and promoting homotrimer formation while inhibiting degradation by Cathepsin A. CMA supports GSC proliferation and self-renewal by activating mTORC1 through the selective degradation of its negative regulators, TSC1 and TSC2. Additionally, CMA is involved in epigenetic silencing of the cGAS-STING pathway, promoting tumor immune escape via lysosomal degradation of the DNA demethylase TET3. Inhibition of CMA synergizes with immune checkpoint therapy in glioblastoma models, highlighting a potential therapeutic target.

Resistance to artemisinin-based combination therapies (ACTs) is steadily increasing in malaria-endemic countries, and new medicines to treat this disease are urgently needed. Drug discovery efforts are hindered by species differences in drug metabolism as new chemical entities must survive metabolism by diverse enzymes across multiple species, enabling cures in preclinical disease models before progression to the clinic. Here, we show how the use of a mouse line extensively genetically humanized for enzymes of the cytochrome P450 superfamily and their transcriptional regulators, the "8HUM" line, can circumvent this issue and improve the translational accuracy of data generated. Engraftment of human erythrocytes into 8HUM/Rag2-/-, an immunocompromised version of the 8HUM line lacking mature T and B cells, was insufficient to permit infection with Plasmodium falciparum, and depletion of natural killer cells by antibody treatment did not alter this outcome. However, infection of 8HUM with Plasmodium berghei permitted assessment of drug efficacy against this Plasmodium species. Approved antimalarials were generally more metabolically stable in 8HUM than in wild-type mice. Major species differences between humans and mice in routes of metabolic elimination for quinine derivatives were removed with 8HUM. Therefore, the 8HUM P. berghei model described here will be of value early in the critical path for antimalarial drug discovery, improving alignment of drug metabolism with the clinical situation while bypassing mouse-specific issues of metabolism to facilitate proof-of-concept in vivo demonstration of efficacy, a key requirement for validation of new drug targets and chemical series.

Previous studies have suggested that activation of the IL-33/ST2 axis as well as elevated expression of the full-length IL-33 precursor acting in an ST2-independent fashion both contribute to pulmonary fibrosis. The protective effect of genetic ST2 deficiency on pulmonary fibrosis is known to be partial, with unclear mechanisms preventing a more complete protection. Here, we report that ST2 deficiency failed to fully protect the lungs from excess collagen accumulation after the profibrotic bleomycin injury and simultaneously facilitated elevations in pulmonary levels of a previously suggested profibrotic mediator, IL-9, as well as a known activator of IL-9 expression, TSLP. Pulmonary CD4+ T cells were the main producers of IL-9. Neutralizing antibody-mediated in vivo blockade of TSLP potently attenuated pulmonary levels of both IL-9 and collagen in the bleomycin injury model in wild-type and particularly ST2-deficient mice. All these observations were markedly pronounced in mice with single deficiency of ST2 and the overall pattern of findings was also preserved in mice with dual deficiency of ST2 and IL-33. It was concluded that the antifibrotic effect of ST2 deficiency is hindered by the simultaneous activation of the TSLP-IL-9 axis in experimental bleomycin-induced pulmonary fibrosis. These findings inform further development of antifibrotic therapies.

Cancer immuno-surveillance and response to therapy are affected by environmental factors, including nutrition. However, the direct effects of individual nutrients remain poorly understood. Here we investigate the impact of dietary ligands of Aryl hydrocarbon receptor (AhR), a transcription factor activated by tryptophan catabolites generated through food digestion and microbiota metabolism. By analyzing pre-clinical tumor models in mice fed on a diet naturally poor in AhR ligands or the same diet supplemented with Indole-3-carbinol, we show that diet-derived AhR ligands are required for the optimal efficacy of anti-PD1 therapy. Using conditional knockout mice, we evidence an essential role for AhR in CD8 T cells, but not NK cells or myeloid cells. Mechanistically, AhR promotes anti-PD1-mediated reinvigoration of progenitor exhausted CD8 T cells and licences the functional response of effector CD8 T cells. Our work allows a better understanding of the role of nutrients in anti-tumor immune responses and has implications for the rational design of dietary interventions for improving the efficacy of checkpoint blockade therapy.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with limited effective treatment options. Emerging evidence links enriched environment (EE)-induced eustress to PDAC inhibition. However, the underlying mechanisms remain unclear. In this study, we explored the role of gut microbiota in PDAC-suppressive effects of EE. We demonstrated that depletion of gut microbiota with antibiotics abolished EE-induced tumor suppression, while fecal microbiota transplantation (FMT) from EE mice significantly inhibited tumor growth in both subcutaneous and orthotopic PDAC models housed in standard environment. 16S rRNA sequencing revealed that EE enhanced gut microbiota diversity and selectively enriched probiotic Lactobacillus, particularly L. reuteri. Treatment with L. reuteri significantly suppressed PDAC tumor growth and increased natural killer (NK) cell infiltration into the tumor microenvironment. Depletion of NK cells alleviated the anti-tumor effects of L. reuteri, underscoring the essential role of NK cell-mediated immunity in anti-tumor response. Clinical analysis of PDAC patients showed that higher fecal Lactobacillus abundance correlated with improved progression-free and overall survival, further supporting the therapeutic potential of L. reuteri in PDAC. Overall, this study identifies gut microbiota as a systemic regulator of PDAC under psychological stress. Supplementation of psychobiotic Lactobacillus may offer a novel therapeutic strategy for PDAC.

Metabolic reprogramming, particularly upregulated de novo pyrimidine biosynthesis, drives cancer progression and immune evasion. Dihydroorotate dehydrogenase (DHODH), a key enzyme in this pathway, is a promising therapeutic target, but its inhibitors often face resistance in immune-refractory melanoma, linked to low basal stimulator of interferon genes (STING) expression.

The molecular and functional changes in endothelial cells during disease progression such as cancer have been noted but the mechanism of their activation is still under-studied. Previously we discovered that tumor-derived Oncostatin M induced tumor-associated vascular phenotypes, and the activated endothelial cells in turn promoted tumor progression and metastasis of clear-cell renal cell carcinoma (ccRCC). However, the mechanism of Oncostatin M action remains unknown. Here, we reveal that Oncostatin M signaling triggers specific epigenetic reprogramming of endothelial cells through upregulation of lysine acetyltransferase 6B, leading to increased histone 3 lysine 14 acetylation (H3K14ac) in vitro and in vivo. H3K14ac-modified chromatins upregulate specific gene sets associated with hypoxic response, hyper-angiogenesis, inflammation, and mesenchymal transition. Targeting H3K14ac in endothelial cells by interfering with acetyltransferase 6B function or neutralizing Oncostatin M ameliorates the premalignant hyperplastic phenotypes in the autochthonous ccRCC mouse model and diminishes tumor growth and metastasis in the ccRCC xenograft model.

Prevotella melaninogenica is enriched in the lungs of people with cystic fibrosis (pwCF), yet its functional impact on respiratory tract homeostasis remains incompletely understood. Prior studies identified immune modulatory effects following lung exposure to Prevotella, but the relevance of these findings for CF infections is unknown.

Chemokines and their receptors play a pivotal role in shaping the tumor microenvironment (TME) and modulating immune responses by orchestrating immune cell recruitment, spatial positioning, and facilitating cell-cell interactions. However, the exact mechanisms underlying chemokine signaling across different cell populations within the TME remain poorly understood. In this study, we utilized multiple-omics approaches to explore the relationship between CCR1+ macrophages, CD8+ exhausted T (Tex) cells, and immune checkpoint blockade (ICB) therapy response, as well as the role of chemokine signaling in the formation of CCR1+ macrophage and CD8+ Tex cell niches. We found that CCR1+ macrophages were closely associated with ICB outcomes in melanoma. Additionally, combination therapy with a CCR1 antagonist and anti-PD-1 monoclonal antibody significantly reduced tumor burden in melanoma mouse models, which was attributed to the substantial depletion of CD8+ Tex cells. Further, CCR1+ macrophages were found to co-localize with CD8+ Tex cells in human melanoma tissue, and the CCR1+ macrophage-CD8+ Tex cell niche was correlated with ICB treatment response in mice. Importantly, the CCR1-CCL3 axis was identified as a critical mediator in the formation of this niche. Overall, our study underscores the spatial relationship between CCR1+ macrophages and CD8+ Tex cells in ICB therapy, providing a promising strategy to overcome ICB resistance in melanoma.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint inflammation that leads to tissue damage and disability. RA affects approximately 0.5-1% of the global population and is driven by a complex interplay of genetic susceptibility, environmental factors, and immune dysregulation. While biologic and targeted synthetic DMARDs improved RA treatment, they have limitations in efficacy, safety, and accessibility. B-cell-targeting therapies, such as anti-CD20, have shown effectiveness, but only with broad immunosuppression, which can increase infection risk and compromise humoral immunity. Therefore, there is an unmet need for more selective therapeutic strategies that modulate pathogenic immune pathways while preserving protective immune functions. It has been suggested that targeting the BAFF pathway may offer a more favorable therapeutic approach compared to targeting CD20.

Immunotherapy combinations can improve patient outcomes, yet the interactions within the tumor microenvironment (TME) that drive therapeutic synergy are poorly understood. Tumor establishment drives monocyte recruitment and differentiation into tumor-associated macrophages (TAMs), which have essential roles in coordinating immune responses and are thus attractive targets for therapeutic modulation. In a murine model of combination anti-programmed cell death protein 1 (PD-1) and its ligand (anti-PD-L1) checkpoint blockade, tumor control was associated with increased infiltration of CD8+ T cells and M1-like repolarization of TAMs. Live-cell imaging of the tumor microenvironment revealed close contacts between tumor-infiltrating CD8+ T cells and TAMs, in which the extent of the contact interfaces increased with combination immunotherapy. Treatment with anti-PD-L1 was able to increase macrophage expression of pro-inflammatory factors and phagocytic activity, suggesting a role for TAMs in reactivating CD8+ T cells in the TME. However, co-treatment with anti-PD-1 was ultimately necessary for tumor control, indicating the need for combination targeting of the TME.

Influenza A viruses remain a global health threat, yet no universal antibody therapy exists. Clinical programs have centered on neutralizing mAbs, only to be thwarted by strain specificity and rapid viral escape. We instead engineered three non-neutralizing IgG2a mAbs that target distinct, overlapping epitopes within the conserved N terminus of the M2 ectodomain (M2e). Combined at low dose, this "triple M2e-mAb" confers robust prophylactic and therapeutic protection in mice challenged with diverse human and zoonotic IAV strains, including highly pathogenic variants. Therapeutic efficacy depends on Fc-mediated effector activity via FcγRI, FcγRIII, and FcγRIV, rather than in vitro neutralization. Serial passaging in triple M2e-mAb-treated immunocompetent and immunodeficient hosts failed to generate viral escape mutants. Our findings redefine the influenza-specific antibody therapeutic design and support Fc-optimized, non-neutralizing M2e-mAbs as a broadly effective, mutation-resistant, off-the-shelve therapy with direct relevance to human pandemic preparedness.