A BCMA/CD16A bispecific innate cell engager for the treatment of multiple myeloma.

Despite the recent progress, multiple myeloma (MM) is still essentially incurable and there is a need for additional effective treatments with good tolerability. RO7297089 is a novel bispecific BCMA/CD16A-directed innate cell engager (ICE®) designed to induce BCMA+ MM cell lysis through high affinity binding of CD16A and retargeting of NK cell cytotoxicity and macrophage phagocytosis. Unlike conventional antibodies approved in MM, RO7297089 selectively targets CD16A with no binding of other Fcγ receptors, including CD16B on neutrophils, and irrespective of 158V/F polymorphism, and its activity is less affected by competing IgG suggesting activity in the presence of M-protein. Structural analysis revealed this is due to selective interaction with a single residue (Y140) uniquely present in CD16A opposite the Fc binding site. RO7297089 induced tumor cell killing more potently than conventional antibodies (wild-type and Fc-enhanced) and induced lysis of BCMA+ cells at very low effector-to-target ratios. Preclinical toxicology data suggested a favorable safety profile as in vitro cytokine release was minimal and no RO7297089-related mortalities or adverse events were observed in cynomolgus monkeys. These data suggest good tolerability and the potential of RO7297089 to be a novel effective treatment of MM patients.

Peripheral T cell expansion predicts tumour infiltration and clinical response.

Despite the resounding clinical success in cancer treatment of antibodies that block the interaction of PD1 with its ligand PDL11, the mechanisms involved remain unknown. A major limitation to understanding the origin and fate of T cells in tumour immunity is the lack of quantitative information on the distribution of individual clonotypes of T cells in patients with cancer. Here, by performing deep single-cell sequencing of RNA and T cell receptors in patients with different types of cancer, we survey the profiles of various populations of T cells and T cell receptors in tumours, normal adjacent tissue, and peripheral blood. We find clear evidence of clonotypic expansion of effector-like T cells not only within the tumour but also in normal adjacent tissue. Patients with gene signatures of such clonotypic expansion respond best to anti-PDL1 therapy. Notably, expanded clonotypes found in the tumour and normal adjacent tissue can also typically be detected in peripheral blood, which suggests a convenient approach to patient identification. Analyses of our data together with several external datasets suggest that intratumoural T cells, especially in responsive patients, are replenished with fresh, non-exhausted replacement cells from sites outside the tumour, suggesting continued activity of the cancer immunity cycle in these patients, the acceleration of which may be associated with clinical response.

CD96 functions as a co-stimulatory receptor to enhance CD8+ T cell activation and effector responses.

CD96 is a member of the poliovirus receptor (PVR, CD155)-nectin family that includes T cell Ig and ITIM domain (TIGIT) and CD226. While CD96, TIGIT, and CD226 have important roles in regulating NK cell activity, and TIGIT and CD226 have also been shown to regulate T cell responses, it is unclear whether CD96 has inhibitory or stimulatory function in CD8+ T cells. Here, we demonstrate that CD96 has co-stimulatory function on CD8+ T cells. Crosslinking of CD96 on human or mouse CD8+ T cells induced activation, effector cytokine production, and proliferation. CD96 was found to transduce its activating signal through the MEK-ERK pathway. CD96-mediated signaling led to increased frequencies of NUR77- and T-bet-expressing CD8+ T cells and enhanced cytotoxic effector activity, indicating that CD96 can modulate effector T cell differentiation. Antibody blockade of CD96 or genetic ablation of CD96 expression on CD8+ T cells impaired expression of transcription factors and proinflammatory cytokines associated with CD8+ T cell activation in in vivo models. Taken together, CD96 has a co-stimulatory role in CD8+ T cell activation and effector function.

Regulation of Tumor-Associated Myeloid Cell Activity by CBP/EP300 Bromodomain Modulation of H3K27 Acetylation.

Myeloid-derived suppressor cells (MDSCs) are found in most cancer malignancies and support tumorigenesis by suppressing immunity and promoting tumor growth. Here we identify the bromodomain (BRD) of CBP/EP300 as a critical regulator of H3K27 acetylation (H3K27ac) in MDSCs across promoters and enhancers of pro-tumorigenic target genes. In preclinical tumor models, in vivo administration of a CBP/EP300-BRD inhibitor (CBP/EP300-BRDi) alters intratumoral MDSCs and attenuates established tumor growth in immunocompetent tumor-bearing mice, as well as in MDSC-dependent xenograft models. Inhibition of CBP/EP300-BRD redirects tumor-associated MDSCs from a suppressive to an inflammatory phenotype through downregulation of STAT pathway-related genes and inhibition of Arg1 and iNOS. Similarly, CBP/EP300-BRDi decreases differentiation and suppressive function of human MDSCs in vitro. Our findings uncover a role of CBP/EP300-BRD in intratumoral MDSCs that may be targeted therapeutically to boost anti-tumor immunity.

CD226 regulates natural killer cell antitumor responses via phosphorylation-mediated inactivation of transcription factor FOXO1.

Natural killer (NK) cell recognition of tumor cells is mediated through activating receptors such as CD226, with suppression of effector functions often controlled by negative regulatory transcription factors such as FOXO1. Here we show that CD226 regulation of NK cell cytotoxicity is facilitated through inactivation of FOXO1. Gene-expression analysis of NK cells isolated from syngeneic tumors grown in wild-type or CD226-deficient mice revealed dysregulated expression of FOXO1-regulated genes in the absence of CD226. In vitro cytotoxicity and stimulation assays demonstrated that CD226 is required for optimal killing of tumor target cells, with engagement of its ligand CD155 resulting in phosphorylation of FOXO1. CD226 deficiency or anti-CD226 antibody blockade impaired cytotoxicity with concomitant compromised inactivation of FOXO1. Furthermore, inhibitors of FOXO1 phosphorylation abrogated CD226-mediated signaling and effector responses. These results define a pathway by which CD226 exerts control of NK cell responses against tumors.

Notch ligand Delta-like 4 induces epigenetic regulation of Treg cell differentiation and function in viral infection.

Notch ligand Delta-like ligand 4 (DLL4) has been shown to regulate CD4 T-cell differentiation, including regulatory T cells (Treg). Epigenetic alterations, which include histone modifications, are critical in cell differentiation decisions. Recent genome-wide studies demonstrated that Treg have increased trimethylation on histone H3 at lysine 4 (H3K4me3) around the Treg master transcription factor, Foxp3 loci. Here we report that DLL4 dynamically increased H3K4 methylation around the Foxp3 locus that was dependent upon upregulated SET and MYDN domain containing protein 3 (SMYD3). DLL4 promoted Smyd3 through the canonical Notch pathway in iTreg differentiation. DLL4 inhibition during pulmonary respiratory syncytial virus (RSV) infection decreased Smyd3 expression and Foxp3 expression in Treg leading to increased Il17a. On the other hand, DLL4 supported Il10 expression in vitro and in vivo, which was also partially dependent upon SMYD3. Using genome-wide unbiased mRNA sequencing, novel sets of DLL4- and Smyd3-dependent differentially expressed genes were discovered, including lymphocyte-activation gene 3 (Lag3), a checkpoint inhibitor that has been identified for modulating Th cell activation. Together, our data demonstrate a novel mechanism of DLL4/Notch-induced Smyd3 epigenetic pathways that maintain regulatory CD4 T cells in viral infections.

GNE-781, A Highly Advanced Potent and Selective Bromodomain Inhibitor of Cyclic Adenosine Monophosphate Response Element Binding Protein, Binding Protein (CBP).

Inhibition of the bromodomain of the transcriptional regulator CBP/P300 is an especially interesting new therapeutic approach in oncology. We recently disclosed in vivo chemical tool 1 (GNE-272) for the bromodomain of CBP that was moderately potent and selective over BRD4(1). In pursuit of a more potent and selective CBP inhibitor, we used structure-based design. Constraining the aniline of 1 into a tetrahydroquinoline motif maintained potency and increased selectivity 2-fold. Structure-activity relationship studies coupled with further structure-based design targeting the LPF shelf, BC loop, and KAc regions allowed us to significantly increase potency and selectivity, resulting in the identification of non-CNS penetrant 19 (GNE-781, TR-FRET IC50 = 0.94 nM, BRET IC50 = 6.2 nM; BRD4(1) IC50 = 5100 nΜ) that maintained good in vivo PK properties in multiple species. Compound 19 displays antitumor activity in an AML tumor model and was also shown to decrease Foxp3 transcript levels in a dose dependent manner.

Notch Ligand Delta-like 4 Promotes Regulatory T Cell Identity in Pulmonary Viral Infection.

Regulatory T (Treg) cells establish tolerance, prevent inflammation at mucosal surfaces, and regulate immunopathology during infectious responses. Recent studies have shown that Delta-like ligand 4 (Dll4) was upregulated on APC after respiratory syncytial virus (RSV) infection, and its inhibition leads to exaggerated immunopathology. In the present study, we outline the role of Dll4 in Treg cell differentiation, stability, and function in RSV infection. We found that Dll4 was expressed on CD11b+ pulmonary dendritic cells in the lung and draining lymph nodes in wild-type BALB/c mice after RSV infection. Dll4 neutralization exacerbated RSV-induced disease pathology, mucus production, group 2 innate lymphoid cell infiltration, IL-5 and IL-13 production, as well as IL-17A+ CD4 T cells. Dll4 inhibition decreased the abundance of CD62LhiCD44loFoxp3+ central Treg cells in draining lymph nodes. The RSV-induced disease was accompanied by an increase in Th17-like effector phenotype in Foxp3+ Treg cells and a decrease in granzyme B expression after Dll4 blockade. Finally, Dll4-exposed induced Treg cells maintained the CD62LhiCD44lo central Treg cell phenotype, had increased Foxp3 expression, became more suppressive, and were resistant to Th17 skewing in vitro. These results suggest that Dll4 activation during differentiation sustained Treg cell phenotype and function to control RSV infection.

IL-27R-mediated regulation of IL-17 controls the development of respiratory syncytial virus-associated pathogenesis.

IL-27 is a heterodimeric cytokine composed of the subunits p28 and Epstein-Barr virus induced gene (EBI)-3 and is known for its effects on T-cell function and differentiation. IL-27 signals through the widely expressed IL-27 receptor (IL-27R), composed of the ligand-specific IL-27Rα chain and gp130. Engagement of the IL-27R activates STAT1 signaling, induces the expression of the type 1 helper T-cell (Th1) cytokine, interferon γ, and suppresses the differentiation of Th2 and Th17 cells. This study investigates the role of IL-27 signaling in respiratory syncytial virus (RSV) infection using IL-27Rα-deficient mice (IL-27rKO). Analysis of lungs from RSV-infected IL-27rKO mice showed exacerbation of mucus secretion compared with wild type, as well as enhanced expression of Muc5ac and Gob5 mRNA, markers of goblet cell metaplasia/hyperplasia. When compared with wild-type mice, RSV-challenged IL-27rKO mice had enhanced expression of Th17-associated cytokine IL-17a and an imbalance between Th1 and Th2 cytokine levels. Neutralization of IL-17 in RSV-infected IL-27rKO mice resulted in a significant decrease in the pulmonary mucus response and inhibition of the Th2-associated cytokines. Interestingly, IL-17 blockage led to an increase in the expression of IL-27 subunits p28 and EBI-3 in the lungs and lymph nodes of RSV-infected mice. Thus, IL-27 functions as a regulatory cytokine during RSV pathogenesis by suppressing the development of Th17 cells, but it also appears to be regulated by IL-17 induced by the virus.

IPS-1 signaling has a nonredundant role in mediating antiviral responses and the clearance of respiratory syncytial virus.

The cytosolic RNA helicases melanoma differentiation-associated gene 5 and retinoic acid-inducible gene-I and their adaptor IFN-ß promoter stimulator (IPS-1) have been implicated in the recognition of viral RNA and the production of type I IFN. Complementing the endosomal TLR, melanoma differentiation-associated gene 5, and retinoic acid-inducible gene-I provides alternative mechanisms for viral detection in cells with reduced phagocytosis or autophagy. The infection route of respiratory syncytial virus (RSV)-via fusion of virus particles with the cell membrane-points to IPS-1 signaling as the pathway of choice for downstream antiviral responses. In the current study, viral clearance and inflammation resolution were indeed strongly affected by the absence of an initial IPS-1-mediated IFN-ß response. Despite the blunted inflammatory response in IPS-1-deficient alveolar epithelial cells, pulmonary macrophages, and CD11b(+) dendritic cells (DC), the lungs of RSV-infected IPS-1-knockout mice showed augmented recruitment of inflammatory neutrophils, monocytes, and DC. Interestingly, pulmonary CD103(+) DC could functionally compensate for IPS-1 deficiency with the upregulation of certain inflammatory cytokines and chemokines, possibly via TLR3 and TLR7 signaling. The increased inflammation and reduced viral clearance in IPS-1-knockout mice was accompanied by increased T cell activation and IFN-γ production. Experiments with bone marrow chimeras indicated that RSV-induced lung pathology was most severe when IPS-1 expression was lacking in both immune and nonimmune cell populations. Similarly, viral clearance was rescued upon restored IPS-1 signaling in either the nonimmune or the immune compartment. These data support a nonredundant function for IPS-1 in controlling RSV-induced inflammation and viral replication.