TREM1 activation of myeloid cells promotes antitumor immunity.

Myeloid cells in the tumor microenvironment (TME) can exist in immunosuppressive and immunostimulatory states that impede or promote antitumor immunity, respectively. Blocking suppressive myeloid cells or increasing stimulatory cells to enhance antitumor immune responses is an area of interest for therapeutic intervention. Triggering receptor expressed on myeloid cells-1 (TREM1) is a proinflammatory receptor that amplifies immune responses. TREM1 is expressed on neutrophils, subsets of monocytes and tissue macrophages, and suppressive myeloid populations in the TME, including tumor-associated neutrophils, monocytes, and tumor-associated macrophages. Depletion or inhibition of immunosuppressive myeloid cells, or stimulation by TREM1-mediated inflammatory signaling, could be used to promote an immunostimulatory TME. We developed PY159, an afucosylated humanized anti-TREM1 monoclonal antibody with enhanced FcγR binding. PY159 is a TREM1 agonist that induces signaling, leading to up-regulation of costimulatory molecules on monocytes and macrophages, production of proinflammatory cytokines and chemokines, and enhancement of T cell activation in vitro. An antibody against mouse TREM1, PY159m, promoted antitumor efficacy in syngeneic mouse tumor models. These results suggest that PY159-mediated agonism of TREM1 on tumoral myeloid cells can promote a proinflammatory TME and offer a promising strategy for immunotherapy.

Hyperstabilization of T cell microvilli contacts by chimeric antigen receptors.

T cells typically recognize their ligands using a defined cell biology-the scanning of their membrane microvilli (MV) to palpate their environment-while that same membrane scaffolds T cell receptors (TCRs) that can signal upon ligand binding. Chimeric antigen receptors (CARs) present both a therapeutic promise and a tractable means to study the interplay between receptor affinity, MV dynamics and T cell function. CARs are often built using single-chain variable fragments (scFvs) with far greater affinity than that of natural TCRs. We used high-resolution lattice lightsheet (LLS) and total internal reflection fluorescence (TIRF) imaging to visualize MV scanning in the context of variations in CAR design. This demonstrated that conventional CARs hyper-stabilized microvillar contacts relative to TCRs. Reducing receptor affinity, antigen density, and/or multiplicity of receptor binding sites normalized microvillar dynamics and synapse resolution, and effector functions improved with reduced affinity and/or antigen density, highlighting the importance of understanding the underlying cell biology when designing receptors for optimal antigen engagement.

Reverse Translating Molecular Determinants of Anti-Programmed Death 1 Immunotherapy Response in Mouse Syngeneic Tumor Models.

Targeting the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) pathway with immunotherapy has revolutionized the treatment of many cancers. Somatic tumor mutational burden (TMB) and T-cell-inflamed gene expression profile (GEP) are clinically validated pan-tumor genomic biomarkers that can predict responsiveness to anti-PD-1/PD-L1 monotherapy in many tumor types. We analyzed the association between these biomarkers and the efficacy of PD-1 inhibitor in 11 commonly used preclinical syngeneic tumor mouse models using murinized rat anti-mouse PD-1 DX400 antibody muDX400, a surrogate for pembrolizumab. Response to muDX400 treatment was broadly classified into three categories: highly responsive, partially responsive, and intrinsically resistant to therapy. Molecular and cellular profiling validated differences in immune cell infiltration and activation in the tumor microenvironment of muDX400-responsive tumors. Baseline and on-treatment genomic analysis showed an association between TMB, murine T-cell-inflamed gene expression profile (murine-GEP), and response to muDX400 treatment. We extended our analysis to investigate a canonical set of cancer and immune biology-related gene signatures, including signatures of angiogenesis, myeloid-derived suppressor cells, and stromal/epithelial-to-mesenchymal transition/TGFß biology previously shown to be inversely associated with the clinical efficacy of immune checkpoint blockade. Finally, we evaluated the association between murine-GEP and preclinical efficacy with standard-of-care chemotherapy or antiangiogenic agents that previously demonstrated promising clinical activity, in combination with muDX400. Our profiling studies begin to elucidate the underlying biological mechanisms of response and resistance to PD-1/PD-L1 blockade represented by these models, thereby providing insight into which models are most appropriate for the evaluation of orthogonal combination strategies.

Targeting TREM2 on tumor-associated macrophages enhances immunotherapy.

Converting checkpoint inhibitor (CPI)-resistant individuals to being responsive requires identifying suppressive mechanisms. We identify TREM2+ tumor-associated macrophages (TAMs) as being correlated with exhausted CD8+ tumor-infiltrating lymphocytes (TILs) in mouse syngeneic tumor models and human solid tumors of multiple histological types. Fc domain-enhanced anti-TREM2 monoclonal antibody (mAb) therapy promotes anti-tumor immunity by elimination and modulation of TAM populations, which leads to enhanced CD8+ TIL infiltration and effector function. TREM2+ TAMs are most enriched in individuals with ovarian cancer, where TREM2 expression corresponds to disease grade accompanied by worse recurrence-free survival. In an aggressive orthotopic ovarian cancer model, anti-TREM2 mAb therapy drives potent anti-tumor immunity. These results highlight TREM2 as a highly attractive target for immunotherapy modulation in individuals who are refractory to CPI therapy and likely have a TAM-rich tumor microenvironment.

A T-cell-dependent antibody response study using a murine surrogate anti-PD-1 monoclonal antibody as an alternative to a non-human primate model.

The programmed cell death 1 (PD-1) pathway represents a major immune checkpoint which may be engaged by cells in a tumor microenvironment to overcome active T-cell immune surveillance. Pembrolizumab (Keytruda®) is a potent and highly selective humanized monoclonal antibody (mAb) of the IgG4/κ isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. The current work was focused on developing a mouse T-Dependent Antibody Response (TDAR) model using a murinized rat anti-mouse PD-1 antibody (muDX400; a rodent surrogate for pembrolizumab) to evaluate the potential impact of treatment with a PD-1 inhibitor on immune responses to an antigen challenge (e.g. HBsAg in Hepatitis B vaccine). Despite the lower binding affinity and T1/2 compared to pembrolizumab, ligand blocking data indicated muDX400 had appropriate pharmacological activity and demonstrated efficacy in mouse tumor models, thus was suitable for pharmacodynamic and vaccination studies in mice. In a vaccination study in which mice were concomitantly administered muDX400 and the Hepatitis B vaccine, muDX400 was well-tolerated and did not result in any immune-mediated adverse effects. The treatment with muDX400 was associated with a shift in the ratio between naive and memory cells in both CD4+ and CD8+ T-lymphocytes in the spleen but did not affect anti-HBsAg antibody response profile. The mouse TDAR model using the Hepatitis B vaccine and the surrogate anti-PD1 monoclonal antibody was a useful tool in the evaluation of the potential immune-mediated effects of pembrolizumab following vaccination and appears to be a suitable alternative for the nonhuman primate TDAR models utilized for other checkpoint inhibitors.

Tuning the Tumor Myeloid Microenvironment to Fight Cancer.

The tumor microenvironment (TME) of diverse cancer types is often characterized by high levels of infiltrating myeloid cells including monocytes, macrophages, dendritic cells, and granulocytes. These cells perform a variety of functions in the TME, varying from immune suppressive to immune stimulatory roles. In this review, we summarize the different myeloid cell populations in the TME and the intratumoral myeloid targeting approaches that are being clinically investigated, and discuss strategies that identify new myeloid subpopulations within the TME. The TME therapies include agents that modulate the functional activities of myeloid populations, that impact recruitment and survival of myeloid subpopulations, and that functionally reprogram or activate myeloid populations. We discuss the benefits, limitations and potential side effects of these therapeutic approaches.

Characterization of MK-4166, a Clinical Agonistic Antibody That Targets Human GITR and Inhibits the Generation and Suppressive Effects of T Regulatory Cells.

GITR is a T-cell costimulatory receptor that enhances cellular and humoral immunity. The agonist anti-mouse GITR antibody DTA-1 has demonstrated efficacy in murine models of cancer primarily by attenuation of Treg-mediated immune suppression, but the translatability to human GITR biology has not been fully explored. Here, we report the potential utility of MK-4166, a humanized GITR mAb selected to bind to an epitope analogous to the DTA-1 epitope, which enhances the proliferation of both naïve and tumor-infiltrating T lymphocytes (TIL). We also investigated the role of GITR agonism in human antitumor immune responses and report here the preclinical characterization and toxicity assessment of MK-4166, which is currently being evaluated in a phase I clinical study. Expression of human GITR was comparable with that of mouse GITR in tumor-infiltrating Tregs despite being drastically lower in other human TILs and in many human peripheral blood populations. MK-4166 decreased induction and suppressive effects of Tregsin vitro In human TIL cultures, MK-4166 induced phosphorylation of NFκB and increased expression of dual specificity phosphatase 6 (DUSP6), indicating that MK-4166 activated downstream NFκB and Erk signaling pathways. Furthermore, MK-4166 downregulated FOXP3 mRNA in human tumor infiltrating Tregs, suggesting that, in addition to enhancing the activation of TILs, MK-4166 may attenuate the Treg-mediated suppressive tumor microenvironment. Cancer Res; 77(16); 4378-88. ©2017 AACR.

Preclinical efficacy of the anti-hepatocyte growth factor antibody ficlatuzumab in a mouse brain orthotopic glioma model evaluated by bioluminescence, PET, and MRI.

PURPOSE: Ficlatuzumab is a novel therapeutic agent targeting the hepatocyte growth factor (HGF)/c-MET pathway. We summarize extensive preclinical work using this agent in a mouse brain orthotopic model of glioblastoma. EXPERIMENTAL DESIGN: Sequential experiments were done using eight- to nine-week-old nude mice injected with 3 × 10(5) U87 MG (glioblastoma) cells into the brain. Evaluation of ficlatuzumab dose response for this brain tumor model and comparison of its response to ficlatuzumab and to temozolamide were conducted first. Subsequently, various small-animal imaging modalities, including bioluminescence imaging (BLI), positron emission tomography (PET), and MRI, were used with a U87 MG-Luc 2 stable cell line, with and without the use of ficlatuzumab, to evaluate the ability to noninvasively assess tumor growth and response to therapy. ANOVA was conducted to evaluate for significant differences in the response. RESULTS: There was a survival benefit with ficlatuzumab alone or in combination with temozolamide. BLI was more sensitive than PET in detecting tumor cells. Fluoro-D-thymidine (FLT) PET provided a better signal-to-background ratio than 2[(18)F]fluoro-2-deoxy-d-glucose (FDG) PET. In addition, both BLI and FLT PET showed significant changes over time in the control group as well as with response to therapy. MRI does not disclose any time-dependent change. Also, the MRI results showed a temporal delay in comparison to the BLI and FLT PET findings, showing similar results one drug cycle later. CONCLUSIONS: Targeting the HGF/c-MET pathway with the novel agent ficlatuzumab appears promising for the treatment of glioblastoma. Various clinically applicable imaging modalities including FLT, PET, and MRI provide reliable ways of assessing tumor growth and response to therapy. Given the clinical applicability of these findings, future studies on patients with glioblastoma may be appropriate.

IL-10 elicits IFNγ-dependent tumor immune surveillance.

Tumor immune surveillance and cancer immunotherapies are thought to depend on the intratumoral infiltration of activated CD8(+) T cells. Intratumoral CD8(+) T cells are rare and lack activity. IL-10 is thought to contribute to the underlying immune suppressive microenvironment. Defying those expectations we demonstrate that IL-10 induces several essential mechanisms for effective antitumor immune surveillance: infiltration and activation of intratumoral tumor-specific cytotoxic CD8(+) T cells, expression of the Th1 cytokine interferon-γ (IFNγ) and granzymes in CD8(+) T cells, and intratumoral antigen presentation molecules. Consequently, tumor immune surveillance is weakened in mice deficient for IL-10 whereas transgenic overexpression of IL-10 protects mice from carcinogenesis. Treatment with pegylated IL-10 restores tumor-specific intratumoral CD8(+) T cell function and controls tumor growth.

Apoptosis-induced inhibition of CD1d-mediated antigen presentation: different roles for caspases and signal transduction pathways.

The stimulation of programmed cell death can either enhance or inhibit antigen presentation by classic major histocompatibility complex molecules. In the current study, we report that the induction of apoptosis by topoisomerase I inhibition or elevation of intracellular ceramide levels substantially impairs CD1d-mediated antigen presentation. In the former case, such a reduction occurred via the regulation of both the p38 mitogen-activated protein kinases and protein kinase C delta signal transduction pathways as well as the caspase cascade, whereas the latter was p38-(but not caspase)-dependent. Confocal microscopic analysis showed an altered intracellular distribution of CD1d following the inhibition topoisomerase I or by an increase in intracellular ceramide levels, that was prevented by p38 and caspase inhibitors. Thus, the induction of apoptosis in antigen presenting cells severely compromises CD1d-mediated antigen presentation by multiple mechanisms.

Importance of N-linked glycosylation in the functional expression of murine CD1d1.

The mouse CD1d1 glycoprotein is specialized in presenting lipid antigens to a novel class of T cells called natural killer T (NKT) cells. CD1d1 is predicted to contain five potential N-linked glycosylation sites (asparagine residues at positions 25, 38, 60, 128, and 183). Glycosylation has been shown to invariably affect the molecular and functional properties of various glycoproteins, and in the current report it was found that a conservative change of the individual endogenous asparagine residues in CD1d1 to glutamine differentially affected its functional expression. Although the maturation rate of the glycosylation mutants was comparable to that of wild type, they differed in their relative levels of surface expression and in their ability to stimulate NKT cells. Mutating all five glycosylation residues resulted in the absence of detectable CD1d1 expression, with a concomitant lack of NKT cell activation. Therefore, these results demonstrate that glycosylation plays a significant role in the functional expression of CD1d1.

Inhibiting TGF-beta signaling restores immune surveillance in the SMA-560 glioma model.

Transforming growth factor-beta (TGF-beta) is a proinvasive and immunosuppressive cytokine that plays a major role in the malignant phenotype of gliomas. One novel strategy of disabling TGF-beta activity in gliomas is to disrupt the signaling cascade at the level of the TGF-beta receptor I (TGF-betaRI) kinase, thus abrogating TGF-beta-mediated invasiveness and immune suppression. SX-007, an orally active, small-molecule TGF-betaRI kinase inhibitor, was evaluated for its therapeutic potential in cell culture and in an in vivo glioma model. The syngeneic, orthotopic glioma model SMA-560 was used to evaluate the efficacy of SX-007. Cells were implanted into the striatum of VM/Dk mice. Dosing began three days after implantation and continued until the end of the study. Efficacy was established by assessing survival benefit. SX-007 dosed at 20 mg/kg p.o. once daily (q.d.) modulated TGF-beta signaling in the tumor and improved the median survival. Strikingly, approximately 25% of the treated animals were disease-free at the end of the study. Increasing the dose to 40 mg/kg q.d. or 20 mg/kg twice daily did not further improve efficacy. The data suggest that SX-007 can exert a therapeutic effect by reducing TGF-beta-mediated invasion and reversing immune suppression. SX-007 modulates the TGF-beta signaling pathway and is associated with improved survival in this glioma model. Survival benefit is due to reduced tumor invasion and reversal of TGF-beta-mediated immune suppression, allowing for rejection of the tumor. Together, these results suggest that treatment with a TGF-betaRI inhibitor may be useful in the treatment of glioblastoma.

A role for natural killer T cells and CD1d molecules in counteracting suppression of hematopoiesis in mice induced by infection with murine cytomegalovirus.

OBJECTIVE: Infection of immunocompromised patients with cytomegalovirus (CMV), such as that occurring in patients undergoing hematopoietic stem cell transplantation, is a serious clinical problem. CMV infection has been reported to suppress hematopoiesis. In immunocompetent hosts CMV is controlled initially by the innate immune system, with CD1d molecules and natural killer T (NKT) cells playing a role in the antiviral immune response in several model systems. We hypothesized that CD1d and NKT cells are involved in protection of the hematopoietic modulating effects of CMV, and that adoptive transfer of NKT cells would protect against these infection-induced effects. METHODS: To address our hypothesis, we used a murine CMV (MCMV) infection model in CD1d(-/-), Jalpha18(-/-), and wild-type (WT) control mice of two different genetic strains each. RESULTS: Infection with MCMV was associated with significant suppression of absolute numbers and cell cycling status of myeloid progenitor cells (CFU-GM, BFU-E, CFU-GEMM) in the marrow and spleen, especially in CD1d(-/-) (lack both CD1d and NKT cells), and Jalpha18(-/-) (express CD1d but lack NKT cells) mice. Adoptive transfer of NKT cells into WT and Jalpha18(-/-) mice shortly before infection with MCMV counteracted myelosuppression. CONCLUSIONS: The results implicate NKT cells, and also likely CD1d, in protection of progenitor cells from MCMV-induced suppression and suggest that NKT cells may be of value in an adoptive transfer setting to treat CMV-induced perturbations of hematopoiesis in immunocompromised individuals. However, further studies are required to better understand the full consequences of adoptive transfer in these settings.

CD44 differentially activates mouse NK T cells and conventional T cells.

NK T (NKT) cells are an important component of the innate immune system and recognize the MHC class I-like CD1d molecule. NKT cells possess significant immunoregulatory activity due to their rapid secretion of large quantities of pro- and anti-inflammatory cytokines following CD1d-dependent stimulation. Because the innate immune system is programmed to respond to a multitude of diverse stimuli and must be able to quickly differentiate between pathogenic and endogenous signals, we hypothesized that, apart from stimulation via the TCR (e.g., CD1d-dependent activation), there must be multiple activation pathways that can be triggered through other cell surface receptors on NKT cells. Therefore, we analyzed the ability of CD44, a structurally diverse cell surface receptor expressed on most cells, to stimulate murine NKT cells, compared with conventional T cells. Stimulation of CD44 through Ab cross-linking or binding to its natural ligands hyaluronan and osteopontin induced NKT cells to secrete cytokines, up-regulate activation markers, undergo morphological changes, and resist activation-induced cell death, whereas conventional T cells only exhibited changes in morphology and protection from activation-induced cell death. This CD44-specific stimulation of NKT cells correlated with their ability to bind hyaluronan. Thus, fundamental differences in CD44 function between these lymphocyte subsets suggest an important biological role for CD44 in the innate immune response.

Inhibition of antitumor immunity by invariant natural killer T cells in a T-cell lymphoma model in vivo.

We have investigated the role of the host's CD1d-dependent innate antitumor immune response in a murine T-cell lymphoma model in vivo. We found that C57BL/6 wildtype (WT) mice inoculated with RMA/S cells transfected with murine CD1d1 died at the same rate as mice inoculated with vector-transfected cells. In contrast, natural killer T (NKT) cell-deficient CD1d or Jalpha18 knockout mice inoculated with CD1d-transfected RMA/S cells survived significantly longer than mice inoculated with vector-transfected RMA/S cells, implicating the involvement of invariant NKT (iNKT) cells in inhibiting antitumor activity in vivo. In contrast to the mutant mice, which produced more of the proinflammatory cytokines IFN-gamma and GM-CSF, WT mice produced significantly elevated amounts of IL-13. Antitumor activity in the knockout mice was not due to the development of CD1d-specific cytotoxic T lymphocytes or circulating antibodies. However, iNKT cell numbers were elevated in tumor-bearing mice. Thus, iNKT cells may be playing a negative role in the host's antitumor immune response against T-cell lymphomas in a CD1d-dependent manner.

Cell wall glycosphingolipids of Sphingomonas paucimobilis are CD1d-specific ligands for NKT cells.

The current consensus on characterization of NKT cells is based on their reactivity to the synthetic glycolipid, alpha-galactosylceramide (alpha-GalCer) in a CD1d-dependent manner. Because of the limited availability of alpha-GalCer, there is a constant search for CD1d-presented ligands that activate NKT cells. The alpha-anomericity of the carbohydrate is considered to be an important requisite for the CD1d-specific activation of NKT cells. The gram-negative, lipopolysaccharide-free bacterium Sphingomonas paucimobilis is known to contain glycosphingolipids (GSL) with alpha-anomeric sugars attached to the lipid chain. Here, we report that GSL extracted from this bacterium are able to stimulate NKT cells in a CD1d-specific manner. In addition, soluble CD1d-Ig dimers loaded with this lipid extract specifically bind to NKT cells (but not conventional T cells). Further studies on the S. paucimobilis GSL could potentially lead to other natural sources of CD1d-specific ligands useful for NKT cell analyses and aimed at identifying novel therapies for a variety of disease states.

Development of a quantitative cell-based intracellular ELISA for the screening of B cell hybridoma supernatants: a novel rapid assay to detect positive clones.

The primary screening of hybridoma clones secreting monoclonal antibodies (MAbs) requires the testing of a large number of hybridoma culture supernatants within a short time and is very labor-intensive. In addition, the type of antigen and its location in the cell have to be considered when selecting the appropriate screening procedure, but relatively few reagents are available for analyzing these molecules. We have developed an intracellular and cell surface ELISA technique for screening hybridoma supernatants that hastens the screening procedure of a large number of clones in a short period of time, as the supernatants of fused cells grown in 96-well plates are used directly in the assay. This novel screening technique is rapid, sensitive, specific, and applicable to MAbs specific for a wide variety of intracellular and/or cell surface proteins.

Myeloid marker expression on antiviral CD8+ T cells following an acute virus infection.

CD11b, CD11c, and F4/80 are normally used to define dendritic cell and/or macrophage populations. In this study, the expression of all three markers was observed on CD8(+) T cells following infection of mice with several distinct viruses. Using lymphocytic choriomeningitis virus as a model virus, it was found that relatively more CD11b(+)CD8(+) and CD11c(+)CD8(+) T cells were present in the periphery than in primary lymphoid organs; in contrast, the F4/80(+)CD8(+) T cell population was more prevalent in the spleen. All three myeloid markers were detected on virus-specific CTL. The expression of CD11b and CD11c on CD8(+) T cells correlated with their level of CTL activity, whereas the F4/80(+)CD8(+) T cell population increased after the peak of the CTL response but did not have higher CTL activity. These data suggest that there is a differential induction of CD11b, CD11c, and F4/80 on virus-specific CD8(+) T cells following an acute virus infection.

Defective presentation of the CD1d1-restricted natural Va14Ja18 NKT lymphocyte antigen caused by beta-D-glucosylceramide synthase deficiency.

Va14Ja18 natural T (NKT) cells play an immunoregulatory role, which is controlled by a self glycolipid(s) presented by CD1d. Although the synthetic antigen alpha-D-galactosylceramide (alpha-D-GalCer) stimulates all Va14Ja18 NKT cells, alpha-anomeric D-glycosylceramides are currently unknown in mammals. We have used beta-D-GalCer-deficient mice and beta-D-glucosylceramide (beta-D-GlcCer)-deficient cells to define the chemical nature of a natural NKT cell antigen. beta-D-GalCer-deficient mice exhibit normal NKT cell development and function, and cells from these animals potently stimulate NKT hybridomas. In striking contrast, the same hybridomas fail to react to CD1d1 expressed by a beta-D-GlcCer-deficient cell line. Importantly, human beta-D-GlcCer synthase cDNA transfer, and hence the biosynthesis of beta-D-GlcCer, restores the recognition of mutant cells expressing CD1d1 by the Va14Ja18 NKT hybridomas. Additionally, suppression of beta-D-GlcCer synthesis inhibits antigen presentation to Va14Ja18 NKT cells. The possibility that beta-D-GlcCer itself is the natural NKT cell antigen was excluded because it was unable to activate NKT hybridomas in a cell-free antigen-presentation assay. These findings suggest that beta-D-GlcCer may play an important role in generating and/or loading a natural Va14Ja18 NKT antigen.

CD1d-mediated antigen presentation to natural killer T (NKT) cells.

CD1d molecules are lipid antigen-presenting molecules. They are involved in presenting these antigens to a unique subpopulation of T cells called natural killer T (NKT) cells, which have the capacity to produce both T helper (Th) 1 and Th2 cytokines. Thus, it is possible that the antigens presented by CD1d and/or the level at which they are presented could have profound effects on the immunoregulation of autoimmune and infectious diseases, as well as cancer. Because of the ability of CD1d-binding ligands to modulate NKT cell responses, targeting CD1d-mediated antigen presentation as a novel approach for new therapies in these and other diseases holds great promise.