Katherine A. Murphy

Immunogenicity of murine solid tumor models as a defining feature of in vivo behavior and response to immunotherapy

Immune profiling has been widely used to probe mechanisms of immune escape in cancer and identify novel targets for therapy. Two emerging uses of immune signatures are to identify likely responders to immunotherapy regimens among individuals with cancer and to understand the variable responses seen among subjects with cancer in immunotherapy trials. Here, the immune profiles of 6 murine solid tumor models (CT26, 4T1, MAD109, RENCA, LLC, and B16) were correlated to tumor regression and survival in response to 2 immunotherapy regimens. Comprehensive profiles for each model were generated using quantitative reverse transcriptase polymerase chain reaction, immunohistochemistry, and flow cytometry techniques, as well as functional studies of suppressor cell populations (regulatory T cells and myeloid-derived suppressor cells), to analyze intratumoral and draining lymphoid tissues. Tumors were stratified as highly or poorly immunogenic, with highly immunogenic tumors showing a significantly greater presence of T-cell costimulatory molecules and immune suppression in the tumor microenvironment. An absence of tumor-infiltrating cytotoxic T lymphocytes and mature dendritic cells was seen across all models. Delayed tumor growth and increased survival with suppressor cell inhibition and tumor-targeted chemokine+/−dendritic cells vaccine immunotherapy were associated with high tumor immunogenicity in these models. Tumor MHC class I expression correlated with the overall tumor immunogenicity level and was a singular marker to predict immunotherapy response with these regimens. By using experimental tumor models as surrogates for human cancers, these studies demonstrate how select features of an immune profile may be utilized to identify patients most likely to respond to immunotherapy regimens.

An in vivo immunotherapy screen of costimulatory molecules identifies Fc-OX40L as a potent reagent for the treatment of established murine gliomas

Purpose: We tested the combination of a tumor lysate vaccine with a panel of costimulatory molecules to identify an immunotherapeutic approach capable of curing established murine gliomas. Experimental Design: Glioma-bearing mice were primed with a tumor lysate vaccine, followed by systemic administration of the following costimulatory ligands: OX40L, CD80, 4-1BBL, and GITRL, which were fused to the Fc portion of human immunoglobulin. Lymphocytes and mRNA were purified from the brain tumor site for immune monitoring studies. Numerous variations of the vaccine and Fc-OX40L regimen were tested alone or in combination with temozolomide. Results: Lysate vaccinations combined with Fc-OX40L led to the best overall survival, yielding cure rates of 50% to 100% depending on the timing, regimen, and combination with temozolomide. Cured mice that were rechallenged with glioma cells rejected the challenge, showing immunologic memory. Lymphocytes isolated from the draining lymph nodes of vaccine/Fc-OX40L–treated mice had superior tumoricidal function relative to all other groups. Vaccine/Fc-OX40L–treated mice exhibited a significant increase in proliferation of brain-infiltrating CD4 and CD8 T cells, as indicated by Ki67 staining. Fc-OX40L had single-agent activity in transplanted and spontaneous glioma models, and the pattern of inflammatory gene expression in the tumor predicted the degree of therapeutic response. Conclusions: These data show that Fc-OX40L has unique and potent activity against experimental gliomas and warrants further testing. Clin Cancer Res; 18(17); 4657–68. ©2012 AACR.

CD8+ T Cell–Independent Tumor Regression Induced by Fc-OX40L and Therapeutic Vaccination in a Mouse Model of Glioma

Despite the growing number of preclinical and clinical trials focused on immunotherapy for the treatment of malignant gliomas, the prognosis for this disease remains grim. Although some promising advances have been made, the immune response stimulated as a result of immunotherapeutic protocols has been inefficient at complete tumor elimination, primarily due to our lack of understanding of the necessary effector functions of the immune system. We previously demonstrated that a tumor lysate vaccine/Fc-OX40L therapy is capable of inducing enhanced survival and tumor elimination in the GL261 mouse glioma model. The following experiments were performed to determine the mechanism(s) of action of this therapy that elicits a potent antitumor immune response. The evidence subsequently outlined indicates a CD8+ T cell–independent and CD4+ T cell–, NK cell–, and B cell–dependent means of prolonged survival. CD8+ T cell–independent tumor clearance is surprising considering the current focus of many cancer immunotherapy protocols. These results provide evidence for CD8+ T cell–independent means of antitumor response and should lead to additional examination of the potential manipulation of this mechanism for future treatment strategies.

Gut Microbial Membership Modulates CD4 T Cell Reconstitution and Function after Sepsis

Transient lymphopenia is one hallmark of sepsis, and emergent data indicate the CD4 T cell compartment in sepsis survivors is numerically and functionally altered (when examined at the Ag-specific level) compared with nonseptic control subjects. Previous data from our laboratory demonstrated Ag-independent, lymphopenia-induced homeostatic proliferation to be a contributing mechanism by which CD4 T cells numerically recover in sepsis survivors. However, we reasoned it is also formally possible that some CD4 T cells respond directly to Ag expressed by gut-resident microbes released during polymicrobial sepsis. The effect of gut microbiome leakage on CD4 T cells is currently unknown. In this study, we explored the number and function of endogenous CD4 T cells specific for segmented filamentous bacterium (SFB) after cecal ligation and puncture (CLP)-induced sepsis using mice that either contained or lacked SFB as a normal gut-resident microbe. Interestingly, SFB-specific CD4 T cells underwent Ag-driven proliferation in CLP-treated SFB+, but not in SFB−, mice. Moreover, CLP-treated SFB+ mice showed resistance to secondary lethal infection with recombinant SFB Ag-expressing virulent Listeria (but not wild-type virulent Listeria), suggesting the CLP-induced polymicrobial sepsis primed for a protective response by the SFB-specific CD4 T cells. Thus, our data demonstrate that the numerical recovery and functional responsiveness of Ag-specific CD4 T cells in sepsis survivors is, in part, modulated by the intestinal barrier’s health discreetly defined by individual bacterial populations of the host’s microbiome.

Triptolide enhances the tumoricidal activity of TRAIL against renal cell carcinoma

Renal cell carcinoma (RCC) is resistant to traditional cancer therapies, and metastatic RCC (mRCC) is incurable. The shortcomings in current therapeutic options for patients with mRCC provide the rationale for the development of novel treatment protocols. Tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) has proven to be a potent inducer of tumor cell death in vitro and in vivo, and a number of TRAIL death receptor agonists (recombinant TRAIL or TRAIL death receptor‐specific mAb) have been developed and tested clinically. Unfortunately the clinical efficacy of TRAIL has been underwhelming and is likely due to a number of possible mechanisms that render tumors resistant to TRAIL, prompting the search for drugs that increase tumor cell susceptibility to TRAIL. The objective of this study was to determine the effectiveness of combining the diterpene triepoxide triptolide, or its water‐soluble prodrug, Minnelide, with TRAIL receptor agonists against RCC in vitro or in vivo, respectively. TRAIL‐induced apoptotic death of human RCC cells was increased in the presence of triptolide. The triptolide‐induced sensitization was accompanied by increased TRAIL‐R2 (DR5) and decreased heat shock protein 70 expression. In vivo treatment of mice bearing orthotopic RCC (Renca) tumors showed the combination of Minnelide and agonistic anti‐DR5 mAb significantly decreased tumor burden and increased animal survival compared to either therapy alone. Our data suggest triptolide/Minnelide sensitizes RCC cells to TRAIL‐induced apoptosis through altered TRAIL death receptor and heat shock protein expression.

Polymeric nanoparticles encapsulating novel TLR7/8 agonists as immunostimulatory adjuvants for enhanced cancer immunotherapy

Cytotoxic T lymphocytes (CTLs) play a major role in cancer immunotherapy because of their ability to directly kill tumor cells and secrete tumor suppressive cytokines. Anticancer vaccines aim to provoke tumor-specific CTL responses, which require activation of antigen presenting cells (APCs) including dendritic cells (DCs) and macrophages. Therefore, a potent immunostimulatory adjuvant capable of activating APCs is an essential component of anticancer vaccines. In this study, we introduce novel TLR 7/8 bi-specific agonists that significantly enhance cytokine secretion compared to TLR7 mono-selective compounds. Encapsulation of these TLR 7/8 agonists in poly(lactide-co-glycolide) (PLGA) nanoparticles increased the co-stimulatory molecule expression and antigen presentation via MHC I by DCs compared to the soluble agonist. When administered subcutaneously, these nanoparticles migrated to draining lymph node and triggered DC activation and expansion. This lead to expansion of antigen-specific CD8 T cells and enhanced CTL response, which resulted in significant prophylactic and therapeutic efficacy in melanoma, bladder and renal cell carcinoma tumor models. Importantly, our studies demonstrate significant reductions in systemic metastasis with the nanoparticle vaccine. Our results suggest novel TLR 7/8 agonist-encapsulated nanoparticles are potent immunostimulatory adjuvants for cancer immunotherapy.

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects > 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

CD8 T Cell-Independent Antitumor Response and Its Potential for Treatment of Malignant Gliomas.

Malignant brain tumors continue to represent a devastating diagnosis with no real chance for cure. Despite an increasing list of potential salvage therapies, standard-of-care for these patients has not changed in over a decade. Immunotherapy has been seen as an exciting option, with the potential to offer specific and long lasting tumor clearance. The “gold standard” in immunotherapy has been the development of a tumor-specific CD8 T cell response to potentiate tumor clearance and immunological memory. While many advances have been made in the field of immunotherapy, few therapies have seen true success. Many of the same principles used to develop immunotherapy in tumors of the peripheral organs have been applied to brain tumor immunotherapy. The immune-specialized nature of the brain should call into question whether this approach is appropriate. Recent results from our own experiments require a rethinking of current dogma. Perhaps a CD8 T cell response is not sufficient for an organ as immunologically unique as the brain. Examination of previously elucidated principles of the brain’s immune-specialized status and known immunological preferences should generate discussion and experimentation to address the failure of current therapies.

Exploiting natural anti-tumor immunity for metastatic renal cell carcinoma

Clinical observations of spontaneous disease regression in some renal cell carcinoma (RCC) patients implicate a role for tumor immunity in controlling this disease. Puzzling, however, are findings that high levels of tumor infiltrating lymphocytes (TIL) are common to RCC. Despite expression of activation markers by TILs, functional impairment of innate and adaptive immune cells has been consistently demonstrated contributing to the failure of the immune system to control RCC. Immunotherapy can overcome the immunosuppressive effects of the tumor and provide an opportunity for long-term disease free survival. Unfortunately, complete response rates remain sub-optimal indicating the effectiveness of immunotherapy remains limited by tumor-specific factors and/or cell types that inhibit antitumor immune responses. Here we discuss immunotherapies and the function of multiple immune system components to achieve an effective response. Understanding these complex interactions is essential to rationally develop novel therapies capable of renewing the immune systems ability to respond to these tumors.

Cutting Edge: Elevated Leptin during Diet-Induced Obesity Reduces the Efficacy of Tumor Immunotherapy

Various malignancies are reproducibly cured in mouse models, but most cancer immunotherapies show objective responses in a fraction of treated patients. One reason for this disconnect may be the use of young, lean mice lacking immune-altering comorbidities present in cancer patients. Although many cancer patients are overweight or obese, the effect of obesity on antitumor immunity is understudied in preclinical tumor models. We examined the effect of obesity on two immunotherapeutic models: systemic anti–CTLA-4 mAb and intratumoral delivery of a TRAIL-encoding adenovirus plus CpG. Both therapies were effective in lean mice, but neither provided a survival benefit to diet-induced obese BALB/c mice. Interestingly, tumor-bearing leptin-deficient (ob/ob) obese BALB/c mice did respond to treatment. Moreover, reducing systemic leptin with soluble leptin receptor:Fc restored the antitumor response in diet-induced obese mice. These data demonstrate the potential of targeting leptin to improve tumor immunotherapy when immune-modulating comorbidities are present.