Activating Inducible T-cell Costimulator Yields Antitumor Activity Alone and in Combination with Anti-PD-1 Checkpoint Blockade.

In recent years, there has been considerable interest in mAb-based induction of costimulatory receptor signaling as an approach to combat cancer. However, promising nonclinical data have yet to translate to a meaningful clinical benefit. Inducible T-cell costimulator (ICOS) is a costimulatory receptor important for immune responses. Using a novel clinical-stage anti-ICOS immunoglobulin G4 mAb (feladilimab), which induces but does not deplete ICOS+ T cells and their rodent analogs, we provide an end-to-end evaluation of the antitumor potential of antibody-mediated ICOS costimulation alone and in combination with programmed cell death protein 1 (PD-1) blockade. We demonstrate, consistently, that ICOS is expressed in a range of cancers, and its induction can stimulate growth of antitumor reactive T cells. Furthermore, feladilimab, alone and with a PD-1 inhibitor, induced antitumor activity in mouse and humanized tumor models. In addition to nonclinical evaluation, we present three patient case studies from a first-time-in-human, phase I, open-label, dose-escalation and dose-expansion clinical trial (INDUCE-1; ClinicalTrials.gov: NCT02723955), evaluating feladilimab alone and in combination with pembrolizumab in patients with advanced solid tumors. Preliminary data showing clinical benefit in patients with cancer treated with feladilimab alone or in combination with pembrolizumab was reported previously; with example cases described here. Additional work is needed to further validate the translation to the clinic, which includes identifying select patient populations that will benefit from this therapeutic approach, and randomized data with survival endpoints to illustrate its potential, similar to that shown with CTLA-4 and PD-1 blocking antibodies. Significance: Stimulation of the T-cell activation marker ICOS with the anti-ICOS agonist mAb feladilimab, alone and in combination with PD-1 inhibition, induces antitumor activity across nonclinical models as well as select patients with advanced solid tumors.

Tumor-immune profiling of murine syngeneic tumor models as a framework to guide mechanistic studies and predict therapy response in distinct tumor microenvironments.

Mouse syngeneic tumor models are widely used tools to demonstrate activity of novel anti-cancer immunotherapies. Despite their widespread use, a comprehensive view of their tumor-immune compositions and their relevance to human tumors has only begun to emerge. We propose each model possesses a unique tumor-immune infiltrate profile that can be probed with immunotherapies to inform on anti-tumor mechanisms and treatment strategies in human tumors with similar profiles. In support of this endeavor, we characterized the tumor microenvironment of four commonly used models and demonstrate they encompass a range of immunogenicities, from highly immune infiltrated RENCA tumors to poorly infiltrated B16F10 tumors. Tumor cell lines for each model exhibit different intrinsic factors in vitro that likely influence immune infiltration upon subcutaneous implantation. Similarly, solid tumors in vivo for each model are unique, each enriched in distinct features ranging from pathogen response elements to antigen presentation machinery. As RENCA tumors progress in size, all major T cell populations diminish while myeloid-derived suppressor cells become more enriched, possibly driving immune suppression and tumor progression. In CT26 tumors, CD8 T cells paradoxically increase in density yet are restrained as tumor volume increases. Finally, immunotherapy treatment across these different tumor-immune landscapes segregate into responders and non-responders based on features partially dependent on pre-existing immune infiltrates. Overall, these studies provide an important resource to enhance our translation of syngeneic models to human tumors. Future mechanistic studies paired with this resource will help identify responsive patient populations and improve strategies where immunotherapies are predicted to be ineffective.

CRP is a novel ligand for the oxidized LDL receptor LOX-1.

C-reactive protein (CRP) is a risk factor for cardiovascular events and functions to amplify vascular inflammation through promoting endothelial dysfunction. Lectin-like oxidized low-density lipoprotein (oxLDL) receptor-1 (LOX-1) is the primary endothelial receptor for oxLDL, and both its expression and function are associated with vascular inflammation. As a scavenger receptor, LOX-1 is capable of binding to a variety of structurally unrelated ligands. Evidence is provided that demonstrates that CRP can act as a novel ligand for LOX-1. The direct interaction between these two proteins was demonstrated with purified protein in both ELISA and AlphaScreen assays. This interaction could be disrupted with known LOX-1 ligands, such as oxLDL and carrageenan. Moreover, the CRP interaction with cell surface-expressed LOX-1 was confirmed in cell-based immunofluorescent-binding studies. Mutagenesis studies demonstrated that the arginine residues forming the basic spine structure on the LOX-1 ligand-binding interface were dispensable for CRP binding, suggesting a novel ligand-binding mechanism for LOX-1, distinct from that used for oxLDL binding. The treatment of human endothelial cells with CRP led to the activation of proinflammatory genes including IL-8, ICAM-1, and VCAM-1. The inductions of these genes by CRP were LOX-1 dependent, as demonstrated by their attenuation in cells transfected with LOX-1 small-interfering RNA. Our study identifies and characterizes the direct interaction between LOX-1 and CRP and suggests that this interaction may mediate CRP-induced endothelial dysfunction.

Peripheral nerve regeneration is delayed in neuropilin 2-deficient mice.

Peripheral nerve transection or crush induces expression of class 3 semaphorins by epineurial and perineurial cells at the injury site and of the neuropilins neuropilin-1 and neuropilin-2 by Schwann and perineurial cells in the nerve segment distal to the injury. Neuropilin-dependent class 3 semaphorin signaling guides axons during neural development, but the significance of this signaling system for regeneration of adult peripheral nerves is not known. To test the hypothesis that neuropilin-2 facilitates peripheral-nerve axonal regeneration, we crushed sciatic nerves of adult neuropilin-2-deficient and littermate control mice. Axonal regeneration through the crush site and into the distal nerve segment, repression by the regenerating axons of Schwann cell p75 neurotrophin receptor expression, remyelination of the regenerating axons, and recovery of normal gait were all significantly slower in the neuropilin-2-deficient mice than in the control mice. Thus, neuropilin-2 facilitates peripheral-nerve axonal regeneration.

Bone morphogenetic proteins 4, 6, and 7 are up-regulated in mouse spinal cord during experimental autoimmune encephalomyelitis.

Although spontaneous remyelination occurs in multiple sclerosis (MS), the extent of myelin repair is often inadequate to restore normal function. Oligodendrocyte precursors remaining in nonremyelinating MS plaques may be restricted by an inhibitory signal. Bone morphogenetic proteins (BMPs) have been implicated as repressors of oligodendrocyte development and inducers of astrogliogenesis. We hypothesized that BMPs are up-regulated in MS lesions and play a role in demyelination and astrogliosis. We examined expression of BMPs in an animal model of MS, chronic experimental autoimmune encephalomyelitis (EAE) induced by the myelin oligodendrocyte glycoprotein (MOG) peptide in C57BL/6 mice. By 14 days postimmunization, compared to those of control mice, the lumbar spinal cords of MOG-peptide EAE mice demonstrated prominent astrogliosis, infiltration of inflammatory cells, and disrupted expression of myelin proteins. Quantitative RT-PCR showed that expression of BMP4, BMP6, and BMP7 mRNA increased 2- to 4-fold in the lumbar spinal cords of animals with symptomatic EAE versus in vehicle-treated and untreated controls on days 14, 21, and 42 postimmunization. BMP2 mRNA expression was not altered. BMP4 mRNA was much more abundant in the spinal cords of all animals than was mRNA encoding BMP2, BMP6, and BMP7. Immunoblot analysis confirmed the increased expression of BMP4 in the EAE animals. Immunohistochemistry revealed increased BMP4 immunoreactivity in areas of inflammation in MOG-peptide EAE animals. BMP4 labeling was mostly limited to macrophages but was sometimes associated with astrocytes and oligodendrocytes. These results indicate that members of the BMP family are differentially expressed in adult spinal cord and are up-regulated during EAE. (c) 2007 Wiley-Liss, Inc.

GluR2-free alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors intensify demyelination in experimental autoimmune encephalomyelitis.

We adopted a genetic approach to test the importance of edited GluR2-free, Ca(2+)-permeable, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in the pathophysiology of experimental autoimmune encephalomyelitis, an inflammatory demyelinative disorder resembling multiple sclerosis. Initial studies showed that oligodendroglial lineage cells from mice lacking functional copies of the gene encoding the GluR3 AMPA receptor subunit (Gria3) had a diminished capacity to assemble edited GluR2-free AMPA receptors, and were resistant to excitotoxicity in vitro. Neurological deficits and spinal cord demyelination elicited by immunization with myelin oligodendrocyte glycoprotein peptide were substantially milder in these Gria3 mutant mice than in their wild-type littermates. These results support the hypothesis that oligodendroglial excitotoxicity mediated by AMPA receptors that do not contain edited GluR2 subunits contributes to demyelination in experimental autoimmune encephalomyelitis, and suggest that inhibiting these Ca(2+)-permeable AMPA receptors would be therapeutic in multiple sclerosis.

Astrogliosis in EAE spinal cord: derivation from radial glia, and relationships to oligodendroglia.

A prominent feature of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) is the accumulation of enlarged, multipolar glial fibrillary acidic protein (GFAP) and brain lipid binding protein (BLBP) immunoreactive astroglia within and at the margins of the inflammatory demyelinative lesions. Whether this astrogliosis is due to both astroglial hyperplasia and hypertrophy or solely to astroglial hypertrophy is controversial. We now report that coincident with the first appearance of inflammation and clinical deficits in mice with myelin oligodendrocyte glycoprotein peptide (MOG peptide)-induced EAE, the radially oriented, bipolar, GFAP, and BLBP positive cells (adult radial glia) present in normal spinal cord white matter undergo mitosis and phenotypic transformation to hypertrophic astroglia. To facilitate visualization of relationships between these hypertrophic astroglia and dying and regenerating oligodendroglia, we used mice that express enhanced green fluorescent protein (EGFP) in cells of the oligodendroglial lineage. During the first week after onset of illness, markedly swollen EGFP+ cells without processes were seen within lesions, whereas EGFP+ cells that expressed immunoreactive cleaved caspase-3 were uncommon. These observations support the hypothesis that necrosis contributes to oligodendroglial loss early in the course of EAE. Later in the illness, EGFP+ cells accumulated amongst hypertrophic astroglia at the margins of the lesions, while the lesions themselves remained depleted of oligodendroglia, suggesting that migration of oligodendroglial lineage cells into the lesions was retarded by the intense perilesional gliosis.

Modulation of sciatic nerve expression of class 3 semaphorins by nerve injury.

Beginning with the unexpected finding by cDNA array analysis that neuropilin-2 is induced in sciatic nerve distal to a transection, we document, for the first time, up-regulation in the axotomized adult peripheral nervous system of class 3 semaphorins and their receptors, which are known to play prominent roles in axonal guidance during neural development. Previously, we described the use of cDNA arrays to screen for novel peripheral nervous system axotomy-induced candidate neurotrophic proteins. A novel finding of that prior study was substantial induction of neuropilin 2 (NP2) mRNA in the axotomized nerve segments. Following up on that initial observation, we have now used real-time quantitative reverse transcription-polymerase chain reaction to demonstrate induction of genes encoding neuropilin 1 (NP1), which, like NP2, serves as a coreceptor for members of the class 3 semaphorin family of axonal guidance molecules and of five of the six known class 3 semaphorins (Sema3A, Sema3B, Sema3C, Sema3E, and Sema3F, but not Sema3D) in crushed or transected sciatic nerves.