Smita Mauze

Interleukin-10 (IL-10) in Experimental Visceral Leishmaniasis and IL-10 Receptor Blockade as Immunotherapy

ABSTRACT Interleukin-10 (IL-10) is thought to promote intracellular infection, including human visceral leishmaniasis, by disabling Th1 cell-type responses and/or deactivating parasitized tissue macrophages. To develop a rationale for IL-10 inhibition as treatment in visceral infection, Th1 cytokine-driven responses were characterized in Leishmania donovani-infected BALB/c mice in which IL-10 was absent or overexpressed or its receptor (IL-10R) was blockaded. IL-10 knockout and normal mice treated prophylactically with anti-IL-10R demonstrated accelerated granuloma assembly and rapid parasite killing without untoward tissue inflammation; IL-12 and gamma interferon mRNA expression, inducible nitric oxide synthase reactivity, and responsiveness to antimony chemotherapy were also enhanced in knockout mice. In IL-10 transgenic mice, parasite replication was unrestrained, and except for antimony responsiveness, measured Th1 cell-dependent events were all initially impaired. Despite subsequent granuloma assembly, high-level infection persisted, and antimony-treated transgenic mice also relapsed. In normal mice with established infection, anti-IL-10R treatment was remarkably active, inducing near-cure by itself and synergism with antimony. IL-10s deactivating effects regulate outcome in experimental visceral leishmaniasis, and IL-10R blockade represents a potential immuno- and/or immunochemotherapeutic approach in this infection.

CD38+ CD45RB(low) CD4+ T cells: a population of T cells with immune regulatory activities in vitro.

An antibody reactive with CD38 revealed both phenotypic and functional heterogeneity amongst CD45RBlow cells. Functional analysis of the CD38+ and CD38− fractions showed that the latter contained T cells which responded to recall antigens and produced high levels of cytokine in response to polyclonal stimulation. In contrast, the CD38+ population failed to proliferate or to produce detectable levels of cytokines. Despite appearing unresponsive, the CD38+ population significantly inhibited anti‐CD3‐induced proliferation and cytokine secretion by the reciprocal CD38− population. Immune suppression required stimulation through the TCR and was dependent on a physical interaction between regulatory and responding CD4+ populations. It did not involve killing of the responding T cells or secretion of IL‐10 or TGF‐β. Despite some similarities there is no direct correlation between the in vitro suppression characteristic of the CD38+ CD45RBlow subset and in vivo suppression which has been shown to be mediated by unseparated CD45RBlow CD4+ T cells. However, these results demonstrate that two functionally distinct subsets of T cells reside within the antigen‐exposed or CD45RBlow CD4+ T cell population and are thus generated in vivo: (1) conventional memory T cells which proliferate and secrete cytokines in response to activation and (2) a population of regulatory T cells which inhibit T cell activation in vitro. Antibodies reactive with CD38 may provide a useful tool with which to study the role of these T cell subsets in the induction and regulation of the immune response.

Serial backcross mapping of multiple loci associated with resistance to Leishmania major in mice.

Resistance or susceptibility of inbred mouse strains to the parasite Leishmania major correlates with CD4+ T cell responses of the Th1 or Th2 subsets, respectively. To evaluate the genetic basis for this difference, resistant B10.D2 mice were backcrossed onto susceptible BALB/c mice for five generations with selection for resistance. Candidate resistance loci were identified by high frequency of heterozygosity in resistant N5 backcross mice. Loci on chromosomes 6, 7, 10, 11, 15, and 16 were associated with resistance, demonstrating the multigenic nature of this phenotype. The presence of all six loci was not necessary to confer resistance and no single locus was required. Rather, a variety of combinations of these loci may be capable of interacting to confer resistance.

Experimental Leishmania major infection in mice: role of IL-10.

L. major infection of mice induces polarized Th1 and Th2 responses that are correlated with healing of the infection (Th1) or a fatal disease (Th2). The Th subset specific cytokines, IFNγ and IL‐4, themselves were shown to be important factors for the differentiation into the Th1 and Th2 pathways during infection. We studied the role of the Th2 cytokine IL‐10 during leishmania infection: removal of endogenous IL‐10 by anti‐IL‐10 treatment did not alter the Th2 cytokine pattern in non‐healer mice nor did it modulate DTH reactivity, IgE production or fatal disease progression, but partially blocked the IFNγ inhibiting effect of rIL‐4 in healer mice. During chronic infection similar amounts of IL‐10 were produced in both healer and non‐healer mice. However, at early time‐points during infection IL‐10 production was significantly higher in the non‐healer Th2 responder animals. IL‐10 production in vitro caused significant inhibition of in vitro IFNγ production. In conclusion IL‐10, unlike IL‐4 and IFNγ, does not seem to play a readily detectable role in the Th subset differentiation during L. major infection. However, the high production of IL‐10 early during infection in non‐healer mice and inhibition of leishmania–specific IFNγ production may contribute to drive the immune response towards a Th2 response.

Dual roles for regulatory T cell depletion and co-stimulatory signaling in agonistic GITR targeting for tumor immunotherapy

Agonistic monoclonal antibodies (mAb) targeting the T-cell receptor coregulatory molecule GITR exert potent therapeutic activities in preclinical tumor models. Although anti-GITR mAb are thought to act by depleting and destabilizing the intratumoral T regulatory cell (Treg) population, the precise mechanism of action is obscure. Here, we addressed this issue using a Treg fate-mapping approach, which revealed that Treg loss was primarily due to cell depletion, with minimal evidence of Treg conversion to a non-Foxp3-expressing population. Further characterization of persisting Tregs following anti-GITR mAb treatment showed that a highly activated subpopulation of CD44hiICOShi intratumoral Tregs were preferentially targeted for elimination, with the remaining Tregs exhibiting a less suppressive phenotype. With these changes in the Treg population, intratumoral CD8+ T cells acquired a more functional phenotype characterized by downregulation of the exhaustion markers PD-1 and LAG-3. This reversal of CD8+ T-cell exhaustion was dependent on both agonistic GITR signaling and Treg depletion, as neither mechanism by itself could fully rescue the exhaustion phenotype. Tests of anti-human GITR antibody MK-4166 in a humanized mouse model of cancer mimicked many of the effects of anti-mouse GITR mAb in syngeneic tumor models, decreasing both Treg numbers and immune suppressor phenotype while enhancing effector responsiveness. Overall, our results show how anti-GITR mAb shifts Treg populations to enable immune attack on tumors, with clinical implications for molecular markers to modify emerging treatments. Cancer Res; 77(5); 1108-18. ©2016 AACR.

Leishmania major infection in interleukin-4 and interferon-gamma depleted mice.

The outcome of experimental Leishmania major infection in mice is closely correlated with the type of CD4+ helper T cell (Th) response. Whereas a Th1 response is host protective, a Th2 response leads to a disseminated, fatal course of disease. Previous studies in this murine model have shown, that the two prominent Th1 and Th2 cytokines, interferon (IFN)‐γ and interleukin (IL)‐4, themselves play a major role in the determination of the resulting Th response. Treatment of susceptible mouse strains (BALB/c) with anti‐IL‐4 induces a Th1 response, allowing the animals to cure the infection. Treatment of resistant strains (e.g. C3H/HeN) with anti‐IFN‐γ induces a Th2 response with dissemination of the disease. In this report, we investigated the course of infection and Th response in susceptible and resistant mice treated with anti‐IL‐4 and anti‐IFN‐γ simultaneously. Both mouse strains showed an initial exacerbation of the disease and an overall reduced cytokine response early after infection. Later during infection both strains had a strong Th1 response that was resulting in cure of disease in C3H/HeN mice. BALB/c mice however, could not control the spread of infection despite the strong Th1 response.

The Role of Anti-Drug Antibodies in the Pharmacokinetics, Disposition, Target Engagement, and Efficacy of a GITR Agonist Monoclonal Antibody in Mice

Administration of biologics to enhance T-cell function is part of a rapidly growing field of cancer immunotherapy demonstrated by the unprecedented clinical success of several immunoregulatory receptor targeting antibodies. While these biologic agents confer significant anti-tumor activity through targeted immune response modulation, they can also elicit broad immune responses potentially including the production of anti-drug antibodies (ADAs). DTA-1, an agonist monoclonal antibody against GITR, is a highly effective anti-tumor treatment in preclinical models. We demonstrate that repeated dosing with murinized DTA-1 (mDTA-1) generates ADAs with corresponding reductions in drug exposure and engagement of GITR on circulating CD3+ CD4+ T cells, due to rapid hepatic drug uptake and catabolism. Mice implanted with tumors after induction of preexisting mDTA-1 ADA show no anti-tumor efficacy when given 3 mg/kg mDTA-1, an efficacious dose in naive mice. Nonetheless, increasing mDTA-1 treatment to 30 mg/kg in ADA-positive mice restores mDTA-1 exposure and GITR engagement on circulating CD3+ CD4+ T cells, thereby partially restoring anti-tumor efficacy. Formation of anti-mDTA-1 antibodies and changes in drug exposure and disposition does not occur in GITR−/− mice, consistent with a role for GITR agonism in humoral immunity. Finally, the administration of muDX400, a murinized monoclonal antibody against the checkpoint inhibitor PD-1, dosed alone or combined with mDTA-1 did not result in reduced muDX400 exposure, nor did it change the nature of the anti-mDTA-1 response. This indicates that anti-GITR immunogenicity may not necessarily impact the pharmacology of coadministered monoclonal antibodies, supporting combination immunomodulatory strategies.

Abstract 4521: Evaluation of the relationship between serum exposure, receptor (GITR) availability and tumor suppression following administration of the anti-GITR antibody DX400 in mouse syngeneic tumor models

GITR is a type I transmembrane protein of the tumor necrosis factor receptor superfamily which is expressed primarily on T lymphocytes and natural killer cells. Ligation of GITR on activated T cells provides a costimulatory signal that positively modulates antigen-specific T cell responses, leading to enhanced cellular and humoral immunity. The anti-GITR antibody DX400 is a murinized agonistic monoclonal antibody that targets mouse GITR, and has shown tumor growth inhibition in mouse syngeneic tumor models. In this study we examined the pharmacokinetic/pharmacodynamics (PK/PD) properties of DX400. Studies were conducted to examine the potential relationships between anti-GITR antibody serum exposure (PK) and receptor availability (PD) on the relevant T-cell subsets. The concentrations of DX400 in serum were determined using an electrochemiluminescence (ECL) assay, and the availability of receptor (GITR) was determined using flow cytometry. Non-linear PK properties for DX400 were observed over the dose range examined. In line with changes in serum concentration-time profiles for the antibody, a dose dependent effect in receptor availability was also observed. The relationships between receptor availability, serum concentrations of DX400 and tumor suppression were described by a mechanistic PK/PD model. This analysis provided an estimated potency (EC50) value of 4.2 ng/mL for blood receptor engagement on T-cell subsets. Citation Format: Ayse Meric Ovacik, Natalie Shinsky-Bjorde, Douglas Hodges, Svetlana Antonenko, Roanna Ueda, Smita Mauze, Danling Gu, Derek Wiswell, Shuli Zhang, Amy Beebe, Mohammad Tabrizi. Evaluation of the relationship between serum exposure, receptor (GITR) availability and tumor suppression following administration of the anti-GITR antibody DX400 in mouse syngeneic tumor models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4521. doi:10.1158/1538-7445.AM2015-4521