Costimulatory CD86 Research Articles

CD28 Costimulation Augments CAR Signaling in NK Cells via the LCK/CD3ζ/ZAP70 Signaling Axis.

Multiple factors in the design of a chimeric antigen receptor (CAR) influence CAR T-cell activity, with costimulatory signals being a key component. Yet, the impact of costimulatory domains on the downstream signaling and subsequent functionality of CAR-engineered natural killer (NK) cells remains largely unexplored. Here, we evaluated the impact of various costimulatory domains on CAR-NK cell activity, using a CD70-targeting CAR. We found that CD28, a costimulatory molecule not inherently present in mature NK cells, significantly enhanced the antitumor efficacy and long-term cytotoxicity of CAR-NK cells both in vitro and in multiple xenograft models of hematologic and solid tumors. Mechanistically, we showed that CD28 linked to CD3ζ creates a platform that recruits critical kinases, such as lymphocyte-specific protein tyrosine kinase (LCK) and zeta-chain-associated protein kinase 70 (ZAP70), initiating a signaling cascade that enhances CAR-NK cell function. Our study provides insights into how CD28 costimulation enhances CAR-NK cell function and supports its incorporation in NK-based CARs for cancer immunotherapy. Significance: We demonstrated that incorporation of the T-cell-centric costimulatory molecule CD28, which is normally absent in mature natural killer (NK) cells, into the chimeric antigen receptor (CAR) construct recruits key kinases including lymphocyte-specific protein tyrosine kinase and zeta-chain-associated protein kinase 70 and results in enhanced CAR-NK cell persistence and sustained antitumor cytotoxicity.

SAIL66, a next generation CLDN6-targeting T-cell engager, demonstrates potent antitumor efficacy through dual binding to CD3/CD137

BackgroundOvarian cancer remains a formidable challenge in oncology, necessitating innovative therapeutic approaches. Claudin-6 (CLDN6), a member of the tight junction molecule CLDN family, exhibits negligible expression in healthy tissues but...

Tucaresol: A Clinical Stage Oral Candidate Drug with Two Distinct Antiviral Mechanisms

Globally, approximately 39 million people are living with Human Immunodeficiency Virus, HIV, arising from approximately 86 million infections since this epidemic began in 1981. However, the number of HIV infections is unevenly distributed with two thirds of global infections confined to Sub-Saharan Africa. Due to viral drug resistance, the most effective treatment requires a triple drug combination thereby adding to the complexity and cost of therapy. As such, many people living with HIV or at risk of infection do not have access to prevention or treatment of this potentially fatal disease. There is no cure for HIV [1]. Tucaresol is an orally active clinical stage drug which functions as a host targeted antiviral agent by protection or reconstitution of CD4+ T helper immune cells. We report herein that Tucaresol also displays in-vitro activity against HIV in infected human peripheral blood mononuclear cells. Although this in-vitro antiviral activity is not potent, the excellent safety profile and bioavailability of Tucaresol, along with its low Molecular Weight, support attainment of relevant drug concentrations in man to achieve significant in-vivo activity. This is demonstrated by previously reported stabilization of viremia in a prior proof of concept phase 1b/2a HIV clinical trial [2]. It is possible that the significant in-vivo activity of Tucaresol arises from synergy between co-stimulation of CD4+ T helper cells and the direct activity against virally infected cells. A pan in-vitro viral screen of Tucaresol further revealed a weak, direct antiviral activity against human herpes virus 6B, human papillomavirus 11, measles virus and hepatitis B virus.

CD137 (4-1BB) and T-Lymphocyte Exhaustion.

CD137 (4-1BB) costimulation results in the potent activation of antitumor T lymphocytes and elicits antitumor efficacy that is synergistic with anti-PD(L)1 checkpoint inhibitors, especially when using bispecific constructs. Emerging experimental evidence indicates that 4-1BB ligation prevents and may revert T-cell exhaustion. See related article by Jeon et al., p. 4155.

The transcriptome of CD14 + CD163 - HLA-DR low monocytes predicts mortality in Idiopathic Pulmonary Fibrosis.

The association between immune-cell-specific transcriptomic profiles and Idiopathic Pulmonary Fibrosis (IPF) mortality is unknown. To determine immune-cell-specific transcriptomic profiles associated with IPF mortality. We profiled peripheral blood mononuclear cells (PBMC) in 18 participants [University of South Florida: IPF, COVID-19, post-COVID-19 Interstitial Lung Disease (Post-COVID-19 ILD), controls] by single-cell RNA sequencing (scRNA-seq) and identified 16 immune-cell-specific transcriptomic profiles. The Scoring Algorithm of Molecular Subphenotypes (SAMS) was used to calculate Up-scores based on these 16 gene profiles. Their association with outcomes was investigated in peripheral blood, Bronchoalveolar Lavage (BAL) and lung tissue of N=416 IPF patients from six cohorts. Findings were validated in an independent IPF, PBMC scRNA-seq dataset (N=38). Cox-regression models demonstrated that 230 genes from CD14 + CD163 - HLA-DR low circulating monocytes predicted IPF mortality [Pittsburgh (p=0.02), Chicago (p=0.003)]. PBMC proportions of CD14 + CD163 - HLA-DR low monocytes were higher in progressive versus stable IPF (Yale, 0.13±0.05 versus 0.09±0.05, p=0.034). Receiving operating characteristic identified a 230 gene, Up-score >41.84 (Pittsburgh) predictive of mortality in Chicago (HR: 6.58, 95%CI: 2.15-20.13, p=0.001) and in pooled analysis of BAL cohorts (HR: 2.20, 95%CI: 1.44-3.37, p=0.0003). High-risk patients had decreased expression of the T-cell co-stimulatory genes CD28 , ICOS , ITK and LCK (Pittsburgh and Chicago, p<0.01). 230 gene-up-scores negatively correlated with Forced Vital Capacity (FVC) in IPF lung tissues (LGRC, rho=-0.2, p=0.02). Results were replicated using a subset of 13 genes from the 230-gene signature (pooled PBMC cohorts - HR: 5.34, 95%CI: 2.83-10.06, p<0.0001). The transcriptome of CD14 + CD163 - HLA-DR low monocytes is associated with increased IPF mortality.

225.9: Delayed immune tolerance for cardiac allografts in nonhuman primates by targeting CD154, CD2, and CD28 costimulation pathways.

225.9: Delayed immune tolerance for cardiac allografts in nonhuman primates by targeting CD154, CD2, and CD28 costimulation pathways.

TFEB activation hallmarks antigenic experience of B lymphocytes and directs germinal center fate decisions

Ligation of the B cell antigen receptor (BCR) initiates humoral immunity. However, BCR signaling without appropriate co-stimulation commits B cells to death rather than to differentiation into immune effector cells. How BCR activation depletes potentially autoreactive B cells while simultaneously primes for receiving rescue and differentiation signals from cognate T lymphocytes remains unknown. Here, we use a mass spectrometry-based proteomic approach to identify cytosolic/nuclear shuttling elements and uncover transcription factor EB (TFEB) as a central BCR-controlled rheostat that drives activation-induced apoptosis, and concurrently promotes the reception of co-stimulatory rescue signals by supporting B cell migration and antigen presentation. CD40 co-stimulation prevents TFEB-driven cell death, while enhancing and prolonging TFEB’s nuclear residency, which hallmarks antigenic experience also of memory B cells. In mice, TFEB shapes the transcriptional landscape of germinal center B cells. Within the germinal center, TFEB facilitates the dark zone entry of light-zone-residing centrocytes through regulation of chemokine receptors and, by balancing the expression of Bcl-2/BH3-only family members, integrates antigen-induced apoptosis with T cell-provided CD40 survival signals. Thus, TFEB reprograms antigen-primed germinal center B cells for cell fate decisions.

Phase I studies of davoceticept (ALPN-202), a PD-L1-dependent CD28 co-stimulator and dual PD-L1/CTLA-4 inhibitor, as monotherapy and in combination with pembrolizumab in advanced solid tumors (NEON-1 and NEON-2)

BackgroundDavoceticept (ALPN-202) is an Fc fusion of a CD80 variant immunoglobulin domain designed to mediate programmed death-ligand 1 (PD-L1)-dependent CD28 co-stimulation while inhibiting the PD-L1 and cytotoxic T-lymphocyte-associated antigen 4...

Generation of Devil Facial Tumour Cells Co-Expressing MHC With CD80, CD86 or 41BBL to Enhance Tumour Immunogenicity.

The major histocompatibility complex (MHC) molecules play an integral role in the adaptive immune response to transmissible cancers through tumour antigen presentation and recognition of allogeneic MHC molecules. The transmissible devil facial tumours 1 and 2 (DFT1 and DFT2) modulate MHC-I antigen presentation to evade host immune responses and facilitate transmission of tumours cells to new Tasmanian devil (Sarcophilus harrisii) hosts. To enhance T-cell-driven tumour immunogenicity for vaccination and immunotherapy, DFT1 and DFT2 cells were co-transfected with (i) NLRC5 for MHC-I expression or CIITA for MHC-I and MHC-II expression, and (ii) a co-stimulatory molecule, either CD80, CD86 or 41BBL. The co-transfected DFT cells presented enhanced expression of MHC-I and/or MHC-II. As few devil-specific monoclonal antibodies exist, we used recombinant CTLA4 and 41BB fused to a fluorescent protein to confirm expression of cell surface CD80, CD86 and 41BBL. The capacity for these cells to induce T-cell responses including PD1 and IFNG expression was evaluated in in vitro co-culture assays with captive devil peripheral blood mononuclear cells (PBMCs). Although PBMC viability had increased, there was no evidence of enhanced T-cell activation. This system can be used to identify additional factors required to promote activation of naïve devil T-cells in vitro.

Extended survival of 9- and 10-gene-edited pig heart xenografts with ischemia minimization and CD154 costimulation blockade-based immunosuppression

BackgroundXenotransplantation has made significant advances recently using pigs genetically engineered to remove carbohydrate antigens, either alone or with addition of various human complement, coagulation, and anti-inflammatory ‘transgenes’. Here we evaluated results associated with gene-edited (GE) pig hearts transplanted in baboons using an established costimulation-based immunosuppressive regimen and a cold-perfused graft preservation technique. MethodsEight baboons received heterotopic abdominal heart transplants from 3-GE (GalKO.β4GalNT2KO.hCD55, n=3), 9-GE (GalKO.β4GalNT2KO.GHRKO.hCD46.hCD55. TBM.EPCR.hCD47.HO-1, n=3) or 10-G (9-GE+CMAHKO, n=2) pigs using Steen’s cold continuous perfusion for ischemia minimization. Immunosuppression (IS) included induction with anti-thymocyte globulin and αCD20, ongoing αCD154, MMF, and tapered corticosteroid. ResultsAll three 3-GE grafts functioned well initially, but failed within 5 days. One 9-GE graft was lost intraoperatively due to a technical issue and another was lost at POD 13 due to antibody mediated rejection (AMR) in a baboon with a strongly positive pre-operative cross-match. One 10-GE heart failed at POD113 with combined cellular and antibody mediated rejection. One 9-GE and one 10-GE hearts had preserved graft function with normal myocardium on protocol biopsies, but exhibited slowly progressive graft hypertrophy until elective necropsy at POD393 and 243 respectively. Elevated levels of IL-6, MCP-1, C-reactive protein, and human thrombomodulin were variably associated with conditioning, the transplant procedure, and clinically significant postoperative events. ConclusionRelative to reference genetics without thrombo-regulatory and anti-inflammatory gene expression, 9- or 10-GE pig hearts exhibit promising performance in the context of a clinically applicable regimen including ischemia minimization and αCD154-based IS, justifying further evaluation in an orthotopic model.

CD28 co-stimulation: novel insights and applications in cancer immunotherapy.

Substantial progress in understanding T cell signalling, particularly with respect to T cell co-receptors such as the co-stimulatory receptor CD28, has been made in recent years. This knowledge has been instrumental in the development of innovative immunotherapies for patients with cancer, including immune checkpoint blockade antibodies, adoptive cell therapies, tumour-targeted immunostimulatory antibodies, and immunostimulatory small-molecule drugs that regulate T cell activation. Following the failed clinical trial of a CD28 superagonist antibody in 2006, targeted CD28 agonism has re-emerged as a technologically viable and clinically promising strategy for cancer immunotherapy. In this Review, we explore recentinsights into the molecular functions and regulation of CD28. We describe how CD28 is central to the success of current cancer immunotherapies and examine how new questions arising from studies of CD28 as a clinical target have enhanced our understanding of its biological role and may guide the development of future therapeutic strategies in oncology.

Cytosolic protein translation regulates cell asymmetry and function in early TCR activation of human CD8+ T lymphocytes.

CD8+ cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide-MHC-I complex by the T-cell receptor (TCR) and co-stimulation. This cognate interaction promotes the organisation of intimate cell-cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell-cell contact, promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux, providing the necessary energy through oxidative phosphorylation, and its own protein translation on 70S ribosomes. However, the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Here, we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton, mitochondria, and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 70S by puromycin. We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs.

Preclinical studies show that Co-STARs combine the advantages of chimeric antigen and T cell receptors for the treatment of tumors with low antigen densities.

Two types of engineered T cells have been successfully used to treat patients with cancer, one with an antigen recognition domain derived from antibodies [chimeric antigen receptors (CARs)] and the other derived from T cell receptors (TCRs). CARs use high-affinity antigen-binding domains and costimulatory domains to induce T cell activation but can only react against target cells with relatively high amounts of antigen. TCRs have a much lower affinity for their antigens but can react against target cells displaying only a few antigen molecules. Here, we describe a new type of receptor, called a Co-STAR (for costimulatory synthetic TCR and antigen receptor), that combines aspects of both CARs and TCRs. In Co-STARs, the antigen-recognizing components of TCRs are replaced by high-affinity antibody fragments, and costimulation is provided by two modules that drive NF-κB signaling (MyD88 and CD40). Using a TCR-mimic antibody fragment that targets a recurrent p53 neoantigen presented in a common human leukocyte antigen (HLA) allele, we demonstrate that T cells equipped with Co-STARs can kill cancer cells bearing low densities of antigen better than T cells engineered with conventional CARs and patient-derived TCRs in vitro. In mouse models, we show that Co-STARs mediate more robust T cell expansion and more durable tumor regressions than TCRs similarly modified with MyD88 and CD40 costimulation. Our data suggest that Co-STARs may have utility for other peptide-HLA antigens in cancer and other targets where antigen density may limit the efficacy of engineered T cells.

CD28 Shapes T Cell Receptor Signaling by Regulating ZAP70 Activation and Lck Dynamics.

T cell activation requires T cell receptor (TCR) engagement, which initiates a series of proximal events including tyrosine phosphorylation of the CD3 and TCRζ chains, recruitment, and activation of the protein tyrosine kinases Lck and ZAP70, followed by recruitment of adapter and signaling proteins. CD28 co-stimulation is also required to generate a functional immune response. Currently we lack a full understanding of the molecular mechanism of CD28 activation. TCR microclusters (MC) are submicron-sized molecular condensates and basic signaling units that form immediately after TCR ligation. Our results show that CD28 co-stimulation specifically accelerated recruitment of ZAP70 to the TCRζ chain in MCs and increased ZAP70 activation. This CD28-mediated acceleration of ZAP70 recruitment was driven by enhanced Lck recruitment to the MCs. A greater spatial separation between active and inactive species of Lck was also observed in the MCs as a consequence of CD28 co-stimulation. These results suggest that CD28 co-stimulation may lower the TCR activation threshold by enhancing the activated form of Lck in the TCR MCs.

Pan-cancer mapping of single CD8+ T cell profiles reveals a TCF1:CXCR6 axis regulating CD28 co-stimulation and anti-tumor immunity

CD8+ Tcells must persist and function in diverse tumor microenvironments to exert their effects. Thus, understanding common underlying expression programs could better inform the next generation of immunotherapies. We apply a generalizable matrix factorization algorithm that recovers both shared and context-specific expression programs from diverse datasets to a single-cell RNA sequencing (scRNA-seq) compendium of 33,161 CD8+ Tcells from 132 patients with seven human cancers. Our meta-single-cell analyses uncover a pan-cancer Tcell dysfunction program that predicts clinical non-response to checkpoint blockade in melanoma and highlights CXCR6 as a pan-cancer marker of chronically activated Tcells. Cxcr6 is trans-activated by AP-1 and repressed by TCF1. Using mouse models, we show that Cxcr6 deletion in CD8+ Tcells increases apoptosis of PD1+TIM3+ cells, dampens CD28 signaling, and compromises tumor growth control. Our study uncovers a TCF1:CXCR6 axis that counterbalances PD1-mediated suppression of CD8+ cell responses and is essential for effective anti-tumor immunity.

IMMU-06. CAR DESIGN SHAPES THE BRAIN TUMOR IMMUNE MICROENVIRONMENT: IMPACT ON ACTIVITY AND PERSISTENCE OF ANTI-GLIOMA ADOPTIVE T CELL IMMUNOTHERAPY

Abstract BACKGROUND B7-H3 CAR T-cells effectively eradicate brain tumors in xenograft models and have proven safe in clinical trials, yet no objective responses were observed in patients. The limited efficacy is partly attributed to the immunosuppressive tumor microenvironment (TME), a characteristic not fully recapitulated in investigations using xenograft models. In this study, we set to optimize B7-H3 CAR structure and examine the effects of CAR design on the brain TME using an immunocompetent glioma model. METHODS We generated a panel of fully murine B7-H3 CARs with variations in transmembrane, co-stimulatory, and activation domains. This enabled us to comprehensively investigate their effector functions within the intact immune system. High-dimensional flow cytometry and single-cell RNA sequencing were then used to investigate changes in the brain TME following CAR T-cell therapy. RESULTS While five out of six B7-H3 CARs with single co-stimulatory domains exhibited potent functionality in vitro, optimizing co-stimulation and signaling failed to enhance their anti-glioma efficacy in vivo. To further enhance therapeutic effectiveness and persistence, we incorporated 4-1BB and CD28 co-stimulation through transgenic expression of 4-1BBL on CD28-based CAR T-cells. Although this design significantly improved anti-glioma efficacy in vitro, it conferred no additional benefits in vivo. Analysis of the TME revealed that CAR T-cell therapy influenced the immune composition of the brain TME. Successful anti-tumor responses were dictated by the recruitment, activation, and spatial localization of unique inflammatory ‘immune hubs’ containing specific subsets of macrophages and endogenous T-cells. Interestingly, depletion of brain macrophages using CSF1R inhibitor markedly inhibited CAR T-cell anti-tumor activity. CONCLUSION Our study highlights the critical role of CAR structural design and its modulation of the TME in mediating the efficacy of CAR T-cell therapy in brain tumors. Yet, more specific targeting and/or reprogramming of the TME is warranted for the successful design of effective adoptive immunotherapies for high-grade glioma.

Assessment of immunomodulatory effects of five commonly used parabens on human THP-1 derived macrophages: Implications for ecological and human health impacts

Parabens are widely used as broad-spectrum anti-microbials and preservatives in food, cosmetics, pharmaceuticals, and personal care products. Studies suggest that the utilization of parabens has substantially increased over the past years, particularly during the global pandemic of coronavirus disease 2019 (COVID-19). Although parabens are generally recognized as safe by the U.S. FDA, some concerns have been raised regarding the potential health effects of parabens associated with immunotoxicity. Herein, we comprehensively investigated several key characteristics of immunotoxicants of five commonly used parabens (methyl-, ethyl-, propyl-, butyl-, and benzyl parabens) in human THP-1 derived macrophages, which are effector cells serving as a first line of host defense against pathogens and tumor immunosurveillance. The results indicate parabens, at concentrations found in humans and biota, significantly dampened macrophage chemotaxis and secretion of major pro-inflammatory cytokines (TNF-α and IL-6) and anti-inflammatory cytokine (IL-10), corroborating the mRNA expression profile. Furthermore, some parabens were found to markedly alter macrophage adhesion and cell surface expression of costimulatory molecules, CD80+ and CD86+, and significantly increase macrophage phagocytosis. Collectively, these findings heighten awareness of potential immunotoxicity posed by paraben exposure at biologically relevant concentrations, providing implications for human health and ecological risks associated with immune dysfunctions.

Modulation of Mouse Dendritic Cells In Vitro by Lactobacillus gasseri Postbiotic Proteins.

Different lactobacilli are probiotics for their beneficial effects that confer to the host. Recently, some of these effects were associated with released metabolic products/constituents (postbiotics). In the present study, the potential immunomodulatory capacity of the probiotic Lactobacillus gasseri OLL2809 cell-free supernatant (sup) was investigated in murine bone marrow-derived dendritic cells (DCs). Bacteria induced significantly higher expression of all examined cytokines than those induced by the stimulatory lipopolysaccharide (LPS) itself. On the contrary, sup only induced the anti-inflammatory IL-10 similarly to LPS, whereas IL-12 and IL-6 secretions were stimulated at a lower level. Moreover, sup reduced the surface expression of the analyzed co-stimulatory markers CD40, CD80, and CD86. Treatments of sup with different digestive enzymes indicated the proteinaceous nature of these immunomodulatory metabolites. Western blot and immunoadsorption analyzes revealed cross-reactivity of sup with the surface-layer proteins (SLPs) isolated from OLL2809. Therefore, we directly tested the ability of OLL2809 SLPs to stimulate specifically cytokine expression in iDCs. Interestingly, we found that all tested cytokines were induced by SLPs and in a dose-dependent manner. In conclusion, our results highlighted distinct immune properties between L. gasseri OLL2809 and its metabolites, supporting the concept that bacterial viability is not an essential prerequisite to exert immunomodulatory effects.

Phase I study of MCLA-145, a bispecific antibody targeting CD137 and PD-L1, in solid tumors, as monotherapy or in combination with pembrolizumab.

2520 Background: MCLA-145, a human common light chain bispecific antibody targeting CD137 and the PD-1/PD-L1 axis, is designed to enhance both antigen-mediated T cell activation via CD137 costimulation, and blockade of inhibitory PD-L1. Interim data from the ongoing phase 1 study (NCT03922204) are presented. Methods: Patients (pts) with PD-L1 ≥1% advanced/metastatic solid tumors received MCLA-145 IV as monotherapy Q2W/Q3W in 21/28-d cycles respectively, or in combination with pembrolizumab 200 mg Q3W in 21-d cycles. Pts enrolled in combination had cancers that either relapsed after PD-(L)1 therapies or were immunotherapy naïve. Primary objectives are safety, tolerability and dose-limiting toxicity (DLT) of MCLA-145 alone or combined with pembrolizumab, and determination of the recommended dose for expansion (RDE). Secondary endpoints include efficacy, pharmacokinetics, pharmacodynamics (PD) and immunogenicity. Results: As of a December 4, 2023 data cutoff, 72 pts with 26 cancer types were treated; 25% of pts had non-small cell lung cancer (NSCLC). 3 pts were continuing combination therapy. Monotherapy: 53 pts (median age 60 y, 49% male) were treated across 8 dose levels (47 pts 0.4-75 mg Q2W, 6 pts 40 mg Q3W). Median number of cycles was 2 (range 1-39). 6 pts had DLTs in the 25-75 mg dose range (febrile neutropenia [2 pts], hemolytic anemia, myositis, ALT/AST increase, neutrophil/platelet decrease [1 pt each]). Most common adverse events (AEs; all grades/G3-4) were fatigue (51%/4%), decreased appetite (34%/2%), dyspnea (32%/0%), anemia (30%/9%), ALT/AST increase (25%/11%), and pyrexia (25%/0%). The incidence of any G3-4 AE was lower with 40 mg Q3W than across all Q2W dose levels (33% vs 66%). Combination: 19 pts (median age 61 y, 47% male) were treated with MCLA-145 10, 25 or 40 mg plus Q3W. Median number of cycles was 5 (range 1-16). No DLTs occurred. Most common AEs (all grades/G3-4) were fatigue (58%/11%), cough (42%/0%), constipation (32%/0%) and ALT/AST increase (21%/11%). The RDE was established at 40 mg Q3W for both monotherapy and combination. Preliminary antitumor activity was observed with monotherapy (52 evaluable pts): 5 partial responses (PRs) in glioblastoma (lasting &gt;3 y), sarcoma, cervical, anal, and gastric cancer (treated for 2-11 mo); and combination (19 evaluable pts): 1 PR in Merkel cell carcinoma (treated 12+ mo), 1 complete response in PD-L1+ NSCLC (treated 6+ mo), both after prior immunotherapy. Disease control rate was 37% with monotherapy and 68% with combination. Exposure was dose-dependent with a terminal half-life of 69 h at 75 mg. Measure of peripheral blood Ki67+ CD8 T cells supports maximal PD activity at 40 mg. Conclusions: MCLA-145 given alone or in combination with pembrolizumab had a well-tolerated and manageable safety profile with encouraging clinical activity, including in pts who relapsed after PD-(L)1 therapies. Clinical trial information: NCT03922204 .

EZH2 inhibition reduces macrophage inflammatory responses in atherosclerosis

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Dutch Heart Foundation (Hartstichting) Aim Epigenetic processes are essential modulators of macrophage inflammatory responses. We postulate that interference in the epigenetic machinery of macrophages might offer novel approaches to combat atherosclerosis. Here, we investigate the repressive histone modification H3K27Me3 deposited by the polycomb repressive complex 2 (PRC2) with its catalytic component EZH2. We studied the therapeutic potential of macrophage EZH2 inhibition in the context of atherosclerosis. Methods Human monocyte-derived macrophages and murine peritoneal and bone-marrow derived macrophages were treated with the EZH2-specific inhibitor GSK126 and subsequently activated with LPS to mimic TLR4-inflammatory responses. The impact of Ezh2 inhibition on macrophage differentiation and activation compared to vehicle (DMSO) was assessed by RNA-seq, ChIP-seq, flow cytometry, western blot, and ELISA. To study its impact on atherosclerosis, female Ldlr-/- mice on a high-cholesterol diet (9weeks) were treated the last four weeks with GSK126 or control. Results Ezh2 inhibition by GSK126 in vitro lowered global H3K27Me3 levels without altering macrophage viability and differentiation, showcasing effective EZH2 inhibition. RNA-seq revealed that of more than one-third of the LPS-induced genes were significantly downregulated by EZH2 inhibition. Subsequent pathway analysis identified cytokine and interferon signaling, co-stimulation, and cell migration as the top down-regulated pathways (padj&amp;lt;0.05). Furthermore, ChIP-seq revealed considerably altered regulation of H3K27Me3 deposition. Indeed, we confirmed that gene and cytokine expression of the inflammatory mediators IL-6, IL-12, and TNF were reduced. Furthermore, membrane marker expression of co-stimulatory CD40, CD80, and CD86 were significantly decreased. In murine atherosclerosis, we observed a reduction in the Virmani plaque severity score and are currently assessing lesion size and composition. Conclusion Overall, we show that EZH2 inhibition reduces inflammatory responses in human and murine macrophages in vitro. We are currently assessing the impact of EZH2 inhibition on lesion size and composition in murine atherosclerosis. Additionally, we are performing ex vivo experiments on human endarterectomy plaques to assess the therapeutic potential of EZH2 inhibition on human atherosclerosis.