Annie Mirsoian

Aging predisposes to acute inflammatory induced pathology after tumor immunotherapy

Aging strongly promotes inflammation responses, which may predispose individuals after cancer therapies to lethal system toxicities and pathology that can be partially prevented by TNF blockade.

Adiposity induces lethal cytokine storm after systemic administration of stimulatory immunotherapy regimens in aged mice

William Murphy’s group at UC Davis previously found that systemic administration of stimulatory immunotherapy (IT) in aged mice resulted in the rapid induction of cytokine storm culminating in multi-organ pathology and rapid lethality. They now show that in addition to age, increased body fat is critical to this adverse reaction, as aged calorie-restricted mice demonstrate protection from IT-induced toxicity. In contrast, young obese mice succumb to cytokine storm, multi-organ pathology, and lethality after systemic IT administration.

Treatment of chronic graft-versus-host disease with bortezomib

Chronic graft-versus-host disease (cGVHD) following allogeneic hematopoietic stem cell transplantation (HSCT) has emerged as a predominant complication following HSCT and has a distinct etiology. We and others have previously demonstrated that bortezomib, a proteasome inhibitor, can prevent but not treat acute GVHD in mice. To assess the effects of bortezomib on cGVHD, a mouse minor histocompatibility antigen-mismatched strain combination was used to mimic clinical cGVHD sclerodermatous pathogenesis and phenotype. Treatment of ongoing cGVHD with bortezomib ameliorated cutaneous lesions, which were also associated with a reduction in total numbers of germinal center B cells and lower B-cell activating factor gene expression levels in cutaneous tissues. Importantly, lymphoma-bearing mice receiving allogeneic HSCT with bortezomib preserved graft-versus-tumor (GVT) effects. Based on these animal studies, we initiated an intrapatient dose escalation clinical trial in patients with extensive steroid-intolerant, dependent, or resistant cGVHD. Marked clinical improvement was observed in patients, which was also associated with reductions of peripheral B cells and minimal toxicity. These results indicate that bortezomib can be of significant use in the treatment of cGVHD and may also allow for maintenance of GVT. This trial was registered at www.clinicaltrials.gov as #NCT01672229.

Immunoediting and Antigen Loss: Overcoming the Achilles Heel of Immunotherapy with Antigen Non-Specific Therapies

Cancer immunotherapy has emerged as a mainstream therapy option in the battle against cancer. Pre-clinical data demonstrates the ability of immunotherapy to harness the immune system to fight disseminated malignancy. Clinical translation has failed to recapitulate the promising results of pre-clinical studies although there have been some successes. In this review we explore some of the short-comings of cancer immunotherapy that have limited successful clinical translation. We will give special consideration to what we consider the most formidable hurdle to successful cancer immunotherapy: tumor-induced immune suppression and immune escape. We will discuss the need for antigen-specific immune responses for successful immunotherapy but also consider the need for antigen specificity as an Achilles heel of immunotherapy given tumor heterogeneity, immune editing, and antigen loss. Finally, we will discuss how combinatorial strategies may overcome some of the pitfalls of antigen specificity and highlight recent studies from our lab which suggest that the induction of antigen non-specific immune responses may also produce robust anti-tumor effects and bypass the need for antigen specificity.

Blocking Indolamine-2,3-Dioxygenase Rebound Immune Suppression Boosts Antitumor Effects of Radio-Immunotherapy in Murine Models and Spontaneous Canine Malignancies

Purpose: Previous studies demonstrate that intratumoral CpG immunotherapy in combination with radiotherapy acts as an in-situ vaccine inducing antitumor immune responses capable of eradicating systemic disease. Unfortunately, most patients fail to respond. We hypothesized that immunotherapy can paradoxically upregulate immunosuppressive pathways, a phenomenon we term “rebound immune suppression,” limiting clinical responses. We further hypothesized that the immunosuppressive enzyme indolamine-2,3-dioxygenase (IDO) is a mechanism of rebound immune suppression and that IDO blockade would improve immunotherapy efficacy. Experimental Design: We examined the efficacy and immunologic effects of a novel triple therapy consisting of local radiotherapy, intratumoral CpG, and systemic IDO blockade in murine models and a pilot canine clinical trial. Results: In murine models, we observed marked increase in intratumoral IDO expression after treatment with radiotherapy, CpG, or other immunotherapies. The addition of IDO blockade to radiotherapy + CpG decreased IDO activity, reduced tumor growth, and reduced immunosuppressive factors, such as regulatory T cells in the tumor microenvironment. This triple combination induced systemic antitumor effects, decreasing metastases, and improving survival in a CD8+ T-cell–dependent manner. We evaluated this novel triple therapy in a canine clinical trial, because spontaneous canine malignancies closely reflect human cancer. Mirroring our mouse studies, the therapy was well tolerated, reduced intratumoral immunosuppression, and induced robust systemic antitumor effects. Conclusions: These results suggest that IDO maintains immune suppression in the tumor after therapy, and IDO blockade promotes a local antitumor immune response with systemic consequences. The efficacy and limited toxicity of this strategy are attractive for clinical translation. Clin Cancer Res; 22(17); 4328–40. ©2016 AACR.

Western Diet–Induced Dysbiosis in Farnesoid X Receptor Knockout Mice Causes Persistent Hepatic Inflammation after Antibiotic Treatment

Patients who have liver cirrhosis and liver cancer also have reduced farnesoid X receptor (FXR). The current study analyzes the effect of diet through microbiota that affect hepatic inflammation in FXR knockout (KO) mice. Wild-type and FXR KO mice were on a control (CD) or Western diet (WD) for 10 months. In addition, both CD- and WD-fed FXR KO male mice, which had hepatic lymphocyte and neutrophil infiltration, were treated by vancomycin, polymyxin B, and Abx (ampicillin, neomycin, metronidazole, and vancomycin). Mice were subjected to morphological analysis as well as gut microbiota and bile acid profiling. Male WD-fed FXR KO mice had the most severe steatohepatitis. FXR KO also had reduced Firmicutes and increased Proteobacteria, which could be reversed by Abx. In addition, Abx eliminated hepatic neutrophils and lymphocytes in CD-fed, but not WD-fed, FXR KO mice. Proteobacteria and Bacteroidetes persisted in WD-fed FXR KO mice even after Abx treatment. Only polymyxin B could reduce hepatic lymphocytes in WD-fed FXR KO mice. The reduced hepatic inflammation by antibiotics was accompanied by decreased free and conjugated secondary bile acids as well as changes in gut microbiota. Our data revealed that Lactococcus, Lactobacillus, and Coprococcus protect the liver from inflammation.

Bystander Activation and Anti-Tumor Effects of CD8+ T Cells Following Interleukin-2 Based Immunotherapy Is Independent of CD4+ T Cell Help

We have previously demonstrated that immunotherapy combining agonistic anti-CD40 and IL-2 (IT) results in synergistic anti-tumor effects. IT induces expansion of highly cytolytic, antigen-independent “bystander-activated” (CD8+CD44high) T cells displaying a CD25−NKG2D+ phenotype in a cytokine dependent manner, which were responsible for the anti-tumor effects. While much attention has focused on CD4+ T cell help for antigen-specific CD8+ T cell expansion, little is known regarding the role of CD4+ T cells in antigen-nonspecific bystander-memory CD8+ T cell expansion. Utilizing CD4 deficient mouse models, we observed a significant expansion of bystander-memory T cells following IT which was similar to the non-CD4 depleted mice. Expanded bystander-memory CD8+ T cells upregulated PD-1 in the absence of CD4+ T cells which has been published as a hallmark of exhaustion and dysfunction in helpless CD8+ T cells. Interestingly, compared to CD8+ T cells from CD4 replete hosts, these bystander expanded cells displayed comparable (or enhanced) cytokine production, lytic ability, and in vivo anti-tumor effects suggesting no functional impairment or exhaustion and were enriched in an effector phenotype. There was no acceleration of the post-IT contraction phase of the bystander memory CD8+ response in CD4-depleted mice. The response was independent of IL-21 signaling. These results suggest that, in contrast to antigen-specific CD8+ T cell expansion, CD4+ T cell help is not necessary for expansion and activation of antigen-nonspecific bystander-memory CD8+ T cells following IT, but may play a role in regulating conversion of these cells from a central memory to effector phenotype. Additionally, the expression of PD-1 in this model appears to be a marker of effector function and not exhaustion.

Late administration of murine CTLA-4 blockade prolongs CD8-mediated anti-tumor effects following stimulatory cancer immunotherapy

Abstract We have demonstrated that immunostimulatory therapies such as interleukin-2 (IL-2) and anti-CD40 (αCD40) can be combined to deliver synergistic anti-tumor effects. While this strategy has shown success, efficacy varies depending on a number of factors including tumor type and severe toxicities can be seen. We sought to determine whether blockade of negative regulators such as cytotoxic T lymphocyte antigen-4 (CTLA-4) could simultaneously prolong CD8+ T cell responses and augment T cell anti-tumor effects. We devised a regimen in which anti-CTLA-4 was administered late so as to delay contraction and minimize toxicities. This late administration both enhanced and prolonged CD8 T cell activation without the need for additional IL-2. The quality of the T cell response was improved with increased frequency of effector/effector memory phenotype cells along with improved lytic ability and bystander expansion. This enhanced CD8 response translated to improved anti-tumor responses both at the primary and metastatic sites. Importantly, toxicities were not exacerbated with combination. This study provides a platform for rational design of immunotherapy combinations to maximize anti-tumor immunity while minimizing toxicities.

Obesity and cancer immunotherapy toxicity

Within the past decade the usage of cancer immunotherapy has gained momentum, achieving successes within various advancedstaged malignancies and is beginning to be explored as a primary therapeutic rather than secondary to traditional cytoreductive therapies. Immunotherapeutic antitumor approaches have included systemic stimulatory therapies, blocking checkpoint inhibitory pathways, direct induction of T-cell stimulatory pathways and adoptive cell transfer strategies, among others [1]. However, the widespread application of immunotherapy has been limited by the induction of dose-limiting, and often times life-threatening, immune-related adverse reaction events (irAEs). These reactions are best exemplified by stimulatory systemic therapies, such as IL-2 and high-dose IFN, that can result in cytokine-release syndrome, multiorgan failure and require their usage to be limited to highly specialized centers capable of providing continuous care [2]. More recently, novel immunotherapies have similarly resulted in the induction of irAEs ranging from mild, requiring dose and scheduling adjustments, to life-threatening. Immune checkpoint blockade therapies such as anti-CTLA-4 and anti-PD-1 induce a generalized expansion and activation of T cells that can adversely result in autoimmunity. Toxicities with checkpoint blockade therapies have included dermatitis, enterocolitis, endocrinopathies and life-threatening liver toxicities, and/or pneumonitis [3–5]. Likewise, a Phase II clinical trial using agonistic CD137 antibody to overcome T-cell inhibition through direct binding and activation of 4-1BB co-stimulatory receptor resulted in grade 4 hepatitis that halted the continuance of the trial [6]. Recently, great promise in antitumor efficacy has been demonstrated through autologous infusion of engineered T cells that express chimeric antigen receptors (CARs). Low-dose administration of CAR-T cells expressing CD137 and targeting CD19 into three patients with chemotherapy-resistant chronic lymphocytic leukemia resulted in all three demonstrating strong antitumor responses with two patients having complete remission [7]. However, CAR-T-cell therapy is accompanied by cytokine release syndrome and/or macrophage activation syndrome that can culminate in the development of severe respiratory and cardiac dysfunction, requiring specialized care throughout treatment [7–9]. Of importance is the commonality that preclinical modeling has often failed to reveal toxicities that are observed in human clinical trials. The vast majority of preclinical studies make usage of young, lean, specific-pathogen free (SPF) inbred mouse models that are Obesity and cancer immunotherapy toxicity

Monoclonal antibody therapies targeting immune checkpoints induce fatal anaphylactic reactions in a murine model of breast cancer.

Co-inhibitory molecules such as PD1, PD-L1 and CTLA-4 are being increasingly used as targets of therapeutic intervention against cancer. The use of monoclonal antibodies targeting these immune checkpoints has been shown to promote anti-tumor immune responses clinically. While these promising results have led to a critical paradigm shift in treatments for cancer, these approaches are also plagued with limitations owing to cancer immune evasion mechanisms and adverse toxicities associated with continuous treatment. It has been difficult to reproduce and develop interventions to these findings preclinically for many reasons including species/age-related differences in expression of these markers, poor tumor modeling, and reagent xenogenicity. In this study, we investigated adverse effects in mice receiving repeated anti-PD1 (clone J43) or PD-L1 (clone 10F.9G2) monoclonal antibody (mAb) administration in the 4T1 mouse model of mammary carcinoma. Mice bearing day 14 syngeneic mammary carcinomas were treated with mAbs to PD1 or PD-L1 which are of rat or hamster origin respectively. Control mice received rat IgG or hamster IgG mAb. Repeat administration of 250 mg i.p. of anti-PD1 or 200 mg i.p. of anti-PD-L1 into tumor bearing mice led to mortality in 50-100% of treated mice within 10 days of the initiation of treatment whereas mice receiving the control mAbs exhibited complete survival at equivalent doses. These toxicities were observed within 1-5 hours of administration of the final dose. Consistent with anaphylaxis, symptoms including piloerection, periorbital puffiness and dyspnea were observed prior to mortality. Furthermore, spleens of mice treated with anti-PD1 were observed to be greater in size relative to untreated mice indicating increased myelopoiesis. Previously, 4T1 has been associated with high expression of granulocyte-colony stimulating factor (G-CSF) systemically resulting in increased extramedullary hematopoiesis and the accumulation of myeloid derived suppressor cells (MDSCs). Both G-CSF and MDSCs have been shown to play an exacerbating role in anaphylactic reactions. Our observations here suggest that the foreign nature of anti-PD1 and anti-PD-L1 along with their important role in checkpoint blockade combined with tumor-induced immune modulation may promote an anaphylaxis-like response in tumor-bearing mice. This study highlights the importance of species-specific mAbs for preclinical models as well as the interesting roles of both checkpoint blockade and tumor-dependent immunomodulation contributing to this particular class of cancer immunotherapy.