Systematic study of tissue factor expression in solid tumors.

BACKGROUND: Elevated tissue factor (TF) expression, although restricted in normal tissue, has been reported in multiple solid cancers, and expression has been associated with poor prognosis. This manuscript compares TF expression across various solid tumor types via immunohistochemistry in a single study, which has not been performed previously. AIMS: To increase insight in the prevalence and cellular localization of TF expression across solid cancer types, we performed a detailed and systematic analysis of TF expression in tumor tissue obtained from patients with ovarian, esophageal, bladder, cervical, endometrial, pancreatic, prostate, colon, breast, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), and glioblastoma. The spatial and temporal variation of TF expression was analyzed over time and upon disease progression in patient-matched biopsies taken at different timepoints. In addition, TF expression in patient-matched primary tumor and metastatic lesions was also analyzed. METHODS AND RESULTS: TF expression was detected via immunohistochemistry (IHC) using a validated TF-specific antibody. TF was expressed in all cancer types tested, with highest prevalence in pancreatic cancer, cervical cancer, colon cancer, glioblastoma, HNSCC, and NSCLC, and lowest in breast cancer. Staining was predominantly membranous in pancreatic, cervical, and HNSCC, and cytoplasmic in glioblastoma and bladder cancer. In general, expression was consistent between biopsies obtained from the same patient over time, although variability was observed for individual patients. NSCLC biopsies of primary tumor and matched lymph node metastases showed no clear difference in TF expression overall, although individual patient changes were observed. CONCLUSION: This study shows that TF is expressed across a broad range of solid cancer types, and expression is present upon tumor dissemination and over the course of treatment.

Mechanistic and pharmacodynamic studies of DuoBody-CD3x5T4 in preclinical tumor models.

CD3 bispecific antibodies (bsAbs) show great promise as anticancer therapeutics. Here, we show in-depth mechanistic studies of a CD3 bsAb in solid cancer, using DuoBody-CD3x5T4. Cross-linking T cells with tumor cells expressing the oncofetal antigen 5T4 was required to induce cytotoxicity. Naive and memory CD4+ and CD8+ T cells were equally effective at mediating cytotoxicity, and DuoBody-CD3x5T4 induced partial differentiation of naive T-cell subsets into memory-like cells. Tumor cell kill was associated with T-cell activation, proliferation, and production of cytokines, granzyme B, and perforin. Genetic knockout of FAS or IFNGR1 in 5T4+ tumor cells abrogated tumor cell kill. In the presence of 5T4+ tumor cells, bystander kill of 5T4- but not of 5T4-IFNGR1- tumor cells was observed. In humanized xenograft models, DuoBody-CD3x5T4 antitumor activity was associated with intratumoral and peripheral blood T-cell activation. Lastly, in dissociated patient-derived tumor samples, DuoBody-CD3x5T4 activated tumor-infiltrating lymphocytes and induced tumor-cell cytotoxicity, even when most tumor-infiltrating lymphocytes expressed PD-1. These data provide an in-depth view on the mechanism of action of a CD3 bsAb in preclinical models of solid cancer.

DuoBody-CD40x4-1BB induces dendritic-cell maturation and enhances T-cell activation through conditional CD40 and 4-1BB agonist activity.

BACKGROUND: Despite the preclinical promise of CD40 and 4-1BB as immuno-oncology targets, clinical efforts evaluating CD40 and 4-1BB agonists as monotherapy have found limited success. DuoBody-CD40×4-1BB (GEN1042/BNT312) is a novel investigational Fc-inert bispecific antibody for dual targeting and conditional stimulation of CD40 and 4-1BB to enhance priming and reactivation of tumor-specific immunity in patients with cancer. METHODS: Characterization of DuoBody-CD40×4-1BB in vitro was performed in a broad range of functional immune cell assays, including cell-based reporter assays, T-cell proliferation assays, mixed-lymphocyte reactions and tumor-infiltrating lymphocyte assays, as well as live-cell imaging. The in vivo activity of DuoBody-CD40×4-1BB was assessed in blood samples from patients with advanced solid tumors that were treated with DuoBody-CD40×4-1BB in the dose-escalation phase of the first-in-human clinical trial (NCT04083599). RESULTS: DuoBody-CD40×4-1BB exhibited conditional CD40 and 4-1BB agonist activity that was strictly dependent on crosslinking of both targets. Thereby, DuoBody-CD40×4-1BB strengthened the dendritic cell (DC)/T-cell immunological synapse, induced DC maturation, enhanced T-cell proliferation and effector functions in vitro and enhanced expansion of patient-derived tumor-infiltrating lymphocytes ex vivo. The addition of PD-1 blocking antibodies resulted in potentiation of T-cell activation and effector functions in vitro compared with either monotherapy, providing combination rationale. Furthermore, in a first-in-human clinical trial, DuoBody-CD40×4-1BB mediated clear immune modulation of peripheral antigen presenting cells and T cells in patients with advanced solid tumors. CONCLUSION: DuoBody-CD40×4-1BB is capable of enhancing antitumor immunity by modulating DC and T-cell functions and shows biological activity in patients with advanced solid tumors. These findings demonstrate that targeting of these two pathways with an Fc-inert bispecific antibody may be an efficacious approach to (re)activate tumor-specific immunity and support the clinical investigation of DuoBody-CD40×4-1BB for the treatment of cancer.

An Fc-inert PD-L1×4-1BB bispecific antibody mediates potent anti-tumor immunity in mice by combining checkpoint inhibition and conditional 4-1BB co-stimulation.

Immune checkpoint inhibitors (ICI) targeting the PD-1/PD-L1 axis have changed the treatment paradigm for advanced solid tumors; however, many patients experience treatment resistance. In preclinical models 4-1BB co-stimulation synergizes with ICI by activating cytotoxic T- and NK-cell-mediated anti-tumor immunity. Here we characterize the mechanism of action of a mouse-reactive Fc-inert PD-L1×4-1BB bispecific antibody (mbsAb-PD-L1×4-1BB) and provide proof-of-concept for enhanced anti-tumor activity. In reporter assays mbsAb-PD-L1×4-1BB exhibited conditional 4-1BB agonist activity that was dependent on simultaneous binding to PD-L1. mbsAb-PD-L1×4-1BB further blocked the PD-L1/PD-1 interaction independently of 4-1BB binding. By combining both mechanisms, mbsAb-PD-L1×4-1BB strongly enhanced T-cell proliferation, cytokine production and antigen-specific cytotoxicity using primary mouse cells in vitro. Furthermore, mbsAb-PD-L1×4-1BB exhibited potent anti-tumor activity in the CT26 and MC38 models in vivo, leading to the rejection of CT26 tumors that were unresponsive to PD-L1 blockade alone. Anti-tumor activity was associated with increased tumor-specific CD8+ T cells and reduced regulatory T cells within the tumor microenvironment and tumor-draining lymph nodes. In immunocompetent tumor-free mice, mbsAb-PD-L1×4-1BB treatment neither induced T-cell infiltration into the liver nor elevated liver enzymes in the blood. Dual targeting of PD-L1 and 4-1BB with a bispecific antibody may therefore address key limitations of first generation 4-1BB-agonistic antibodies, and may provide a novel approach to improve PD-1/PD-L1 checkpoint blockade.

Preclinical Characterization and Phase I Trial Results of a Bispecific Antibody Targeting PD-L1 and 4-1BB (GEN1046) in Patients with Advanced Refractory Solid Tumors.

Checkpoint inhibitors (CPI) have revolutionized the treatment paradigm for advanced solid tumors; however, there remains an opportunity to improve response rates and outcomes. In preclinical models, 4-1BB costimulation synergizes with CPIs targeting the programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) axis by activating cytotoxic T-cell-mediated antitumor immunity. DuoBody-PD-L1×4-1BB (GEN1046) is an investigational, first-in-class bispecific immunotherapy agent designed to act on both pathways by combining simultaneous and complementary PD-L1 blockade and conditional 4-1BB stimulation in one molecule. GEN1046 induced T-cell proliferation, cytokine production, and antigen-specific T-cell-mediated cytotoxicity superior to clinically approved PD-(L)1 antibodies in human T-cell cultures and exerted potent antitumor activity in transplantable mouse tumor models. In dose escalation of the ongoing first-in-human study in heavily pretreated patients with advanced refractory solid tumors (NCT03917381), GEN1046 demonstrated pharmacodynamic immune effects in peripheral blood consistent with its mechanism of action, manageable safety, and early clinical activity [disease control rate: 65.6% (40/61)], including patients resistant to prior PD-(L)1 immunotherapy. SIGNIFICANCE: DuoBody-PD-L1×4-1BB (GEN1046) is a first-in-class bispecific immunotherapy with a manageable safety profile and encouraging preclinical and early clinical activity. With its ability to confer clinical benefit in tumors typically less sensitive to CPIs, GEN1046 may fill a clinical gap in CPI-relapsed or refractory disease or as a combination therapy with CPIs. See related commentary by Li et al., p. 1184. This article is highlighted in the In This Issue feature, p. 1171.

Transcriptome analysis reveals the contribution of oligodendrocyte and radial glia-derived cues for maintenance of microglia identity.

Microglia are increasingly being recognized as druggable targets in neurodegenerative disorders, and good in vitro models are crucial to address cell biological questions. Major challenges are to recapitulate the complex microglial morphology and their in vivo transcriptome. We have therefore exposed primary microglia from adult rhesus macaques to a variety of different culture conditions including exposure to soluble factors as M-CSF, IL-34, and TGF-ß as well as serum replacement approaches, and compared their morphologies and transcriptomes to those of mature, homeostatic in vivo microglia. This enabled us to develop a new, partially serum-free, monoculture protocol, that yields high numbers of ramified cells. We also demonstrate that exposure of adult microglia to M-CSF or IL-34 induces similar transcriptomes, and that exposure to TGF-ß has much less pronounced effects than it does on rodent microglia. However, regardless of culture conditions, the transcriptomes of in vitro and in vivo microglia remained substantially different. Analysis of differentially expressed genes inspired us to perform 3D-spherical coculture experiments of microglia with oligodendrocytes and radial glia. In such spheres, microglia signature genes were strongly induced, even in the absence of neurons and astrocytes. These data reveal a novel role for oligodendrocyte and radial glia-derived cues in the maintenance of microglial identity, providing new anchor points to study microglia in health and disease.

Transcriptome and proteome profiling of neural stem cells from the human subventricular zone in Parkinson's disease.

It is currently accepted that the human brain has a limited neurogenic capacity and an impaired regenerative potential. We have previously shown the existence of CD271-expressing neural stem cells (NSCs) in the subventricular zone (SVZ) of Parkinson's disease (PD) patients, which proliferate and differentiate towards neurons and glial cells in vitro. To study the molecular profile of these NSCs in detail, we performed RNA sequencing and mass spectrometry on CD271+ NSCs isolated from human post-mortem SVZ and on homogenates of the SVZ. CD271+ cells were isolated through magnetic cell separation (MACS). We first compared the molecular profile of CD271+ NSCs to the SVZ homogenate from control donors and then compared CD271+ cells to CD11b+ microglia. These results confirmed their neural stem cell identity. Finally we compared controls and PD patients to establish a specific molecular profile of NSCs and the SVZ in PD. While our transcriptome analysis did not identify any differentially expressed genes in the SVZ between control and PD patients, our proteome analysis revealed several proteins that were differentially expressed in PD. Some of these proteins are involved in cytoskeletal organization and mitochondrial function. Transcriptome and proteome analyses of NSCs from PD revealed changes in the expression of genes and proteins involved in metabolism, transcriptional activity and cytoskeletal organization. Our data suggest that NSCs may transit into a primed-quiescent state, that is in an "alert" non-proliferative phase in PD. Our results not only confirm pathological hallmarks of PD (e.g. impaired mitochondrial function), but also show that the NSCs from SVZ undergo significant changes at both transcriptome and proteome level following PD.

TLR-Induced IL-12 and CCL2 Production by Myeloid Cells Is Dependent on Adenosine A3 Receptor-Mediated Signaling.

TLR-induced signaling potently activates cells of the innate immune system and is subject to regulation at different levels. Inflammatory conditions are associated with increased levels of extracellular adenosine, which can modulate TLR-induced production of cytokines through adenosine receptor-mediated signaling. There are four adenosine receptor subtypes that induce different signaling cascades. In this study, we demonstrate a pivotal contribution of adenosine A3 receptor (A3R)-mediated signaling to the TLR4-induced expression of IL-12 in different types of human myeloid APC. In dendritic cells, IL-12 and CCL2 responses as evoked by TLR2, 3, 4, 5, and 8, as well as IL-12 responses evoked by whole pathogens, were all reduced when A3R-mediated signaling was blocked. As a result, concomitant production of IFN-γ and IL-17 by T cells was significantly inhibited. We further show that selective inhibition of A3R-mediated signaling reduced TLR-induced phosphorylation of the transcription factor STAT1 at tyrosine 701. Next-generation sequencing revealed that A3R-mediated signaling controls the expression of metallothioneins, known inhibitors of STAT1 phosphorylation. Together our results reveal a novel regulatory layer of innate immune responses, with a central role for metallothioneins and autocrine/paracrine signaling via A3Rs.

VISTA expression by microglia decreases during inflammation and is differentially regulated in CNS diseases.

V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA) is a negative checkpoint regulator (NCR) involved in inhibition of T cell-mediated immunity. Expression changes of other NCRs (PD-1, PD-L1/L2, CTLA-4) during inflammation of the central nervous system (CNS) were previously demonstrated, but VISTA expression in the CNS has not yet been explored. Here, we report that in the human and mouse CNS, VISTA is most abundantly expressed by microglia, and to lower levels by endothelial cells. Upon TLR stimulation, VISTA expression was reduced in primary neonatal mouse and adult rhesus macaque microglia in vitro. In mice, microglial VISTA expression was reduced after lipopolysaccharide (LPS) injection, during experimental autoimmune encephalomyelitis (EAE), and in the accelerated aging Ercc1 Δ/- mouse model. After LPS injection, decreased VISTA expression in mouse microglia was accompanied by decreased acetylation of lysine residue 27 in histone 3 in both its promoter and enhancer region. ATAC-sequencing indicated a potential regulation of VISTA expression by Pu.1 and Mafb, two transcription factors crucial for microglia function. Finally, our data suggested that VISTA expression was decreased in microglia in multiple sclerosis lesion tissue, whereas it was increased in Alzheimer's disease patients. This study is the first to demonstrate that in the CNS, VISTA is expressed by microglia, and that VISTA is differentially expressed in CNS pathologies.

An Overview of in vitro Methods to Study Microglia.

Neuroinflammation is a common feature in neurodegenerative diseases and strategies to modulate neuroinflammatory processes are increasingly considered as therapeutic options. In such strategies, glia cells rather than neurons represent the cellular targets. Microglia, the resident macrophages of the central nervous system, are principal players in neuroinflammation and detailed cellular biological knowledge of this particular cell type is therefore of pivotal importance. The last decade has shed new light on the origin, characteristics and functions of microglia, underlining the need for specific in vitro methodology to study these cells in detail. In this review we provide a comprehensive overview of existing methodology such as cell lines, stem cell-derived microglia and primary dissociated cell cultures, as well as discuss recent developments. As there is no in vitro method available yet that recapitulates all hallmarks of adult homeostatic microglia, we also discuss the advantages and limitations of existing models across different species.

Colony-Stimulating Factor 1 Receptor (CSF1R) Regulates Microglia Density and Distribution, but Not Microglia Differentiation In Vivo.

Microglia are brain-resident macrophages with trophic and phagocytic functions. Dominant loss-of-function mutations in a key microglia regulator, colony-stimulating factor 1 receptor (CSF1R), cause adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), a progressive white matter disorder. Because it remains unclear precisely how CSF1R mutations affect microglia, we generated an allelic series of csf1r mutants in zebrafish to identify csf1r-dependent microglia changes. We found that csf1r mutations led to aberrant microglia density and distribution and regional loss of microglia. The remaining microglia still had a microglia-specific gene expression signature, indicating that they had differentiated normally. Strikingly, we also observed lower microglia numbers and widespread microglia depletion in postmortem brain tissue of ALSP patients. Both in zebrafish and in human disease, local microglia loss also presented in regions without obvious pathology. Together, this implies that CSF1R mainly regulates microglia density and that early loss of microglia may contribute to ALSP pathogenesis.

Inflammasome-induced IL-1ß secretion in microglia is characterized by delayed kinetics and is only partially dependent on inflammatory caspases.

Inflammasomes are multiprotein complexes that link pathogen recognition and cellular stress to the processing of the proinflammatory cytokine interleukin-1ß (IL-1ß). Whereas inflammasome-mediated activation is heavily studied in hematopoietic macrophages and dendritic cells, much less is known about microglia, resident tissue macrophages of the brain that originate from a distinct progenitor. To directly compare inflammasome-mediated activation in different types of macrophages, we isolated primary microglia and hematopoietic macrophages from adult, healthy rhesus macaques. We analyzed the expression profile of NOD (nucleotide-binding oligomerization domain)-like receptors, adaptor proteins, and caspases and characterized inflammasome activation and regulation in detail. We here demonstrate that primary microglia can respond to the same innate stimuli as hematopoietic macrophages. However, microglial responses are more persistent due to lack of negative regulation on pro-IL-1ß expression. In addition, we show that while caspase 1, 4, and 5 activation is pivotal for inflammasome-induced IL-1ß secretion by hematopoietic macrophages, microglial secretion of IL-1ß is only partially dependent on these inflammatory caspases. These results identify key cell type-specific differences that may aid the development of strategies to modulate innate immune responses in the brain.

Alternative methods for the use of non-human primates in biomedical research.

The experimental use of non-human primates (NHP) in Europe is tightly regulated and is only permitted when there are no alternatives available. As a result, NHP are most often used in late, pre-clinical phases of biomedical research. Although the impetus for scientists, politicians and the general public to replace, reduce and refine NHP in biomedical research is strong, the development of 3Rs technology for NHP poses specific challenges. In February 2014 a workshop on "Alternative methods for the use of NHP in biomedical research" was organized within the international exchange program of EUPRIM-Net II, a European infrastructure initiative that links biomedical primate research centers. The workshop included lectures by key scientists in the field of alternatives as well as by experts from governmental and non-governmental organizations. Furthermore, parallel sessions were organized to stimulate discussion on the challenges of advancing the use of alternative methods for NHP. Subgroups voted on four statements and together composed a list with opportunities and priorities. This report summarizes the presentations that were held, the content of the discussion sessions and concludes with recommendations on 3Rs development for NHP specifically. These include technical, conceptual as well as political topics.