Brian M. Andersen

Increasing the efficacy of tumor cell vaccines by enhancing cross priming

Cancer immunotherapy has been attempted for more than a century, and investment has intensified in the last 20 years. The complexity of the immune system is exemplified by the myriad of immunotherapeutic approaches under investigation. While anti-tumor immunity has been achieved experimentally with multiple effector cells and molecules, particular promise is shown for harnessing the CD8 T cell response. Tumor cell-based vaccines have been employed in hundreds of clinical trials to date and offer several advantages over subunit and peptide vaccines. However, tumor cell-based vaccines, often aimed at cross priming tumor-reactive CD8 T cells, have shown modest success in clinical trials. Here we review the mechanisms of cross priming and discuss strategies to increase the efficacy of tumor cell-based vaccines. A synthesis of recent findings on tissue culture conditions, cell death, and dendritic cell activation reveals promising new avenues for clinical investigation.

Vaccine Injection Site Matters: Qualitative and Quantitative Defects in CD8 T Cells Primed as a Function of Proximity to the Tumor in a Murine Glioma Model

Malignant gliomas are lethal brain tumors for which novel therapies are urgently needed. In animal models, vaccination with tumor-associated Ags efficiently primes T cells to clear gliomas. In clinical trials, cancer vaccines have been less effective at priming T cells and extending survival. Generalized immune suppression in the tumor draining lymph nodes has been documented in multiple cancers. However, a systematic analysis of how vaccination at various distances from the tumor (closest to farthest) has not been reported. We investigated how the injection site chosen for vaccination dictates CD8 T cell priming and survival in an OVA-transfected murine glioma model. Glioma-bearing mice were vaccinated with Poly:ICLC plus OVA protein in the neck, hind leg, or foreleg for drainage into the cervical, inguinal, or axillary lymph nodes, respectively. OVA-specific CD8 T cell number, TCR affinity, effector function, and infiltration into the brain decreased as the vaccination site approached the tumor. These effects were dependent on the presence of the tumor, because injection site did not appreciably affect CD8 T cell priming in tumor-free mice. Our data suggest the site of vaccination can greatly impact the effectiveness of cancer vaccines. Considering that previous and ongoing clinical trials have used a variety of injection sites, vaccination site is potentially a critical aspect of study design that is being overlooked.

Superior Efficacy of Tumor Cell Vaccines Grown In Physiologic Oxygen

Purpose: Atmospheric oxygen (∼20% O2) has been the universal condition employed to culture tumor cells used as vaccine antigen. We tested the hypothesis that reducing oxygen tension would increase the efficacy of tumor cell lysate vaccines. Experimental Design: GL261 glioma cells and EMT6 breast carcinoma cells were grown in 5% or 20% O2. Syngeneic tumor-bearing mice were vaccinated with these tumor cell lysates mixed with CpG oligodeoxynucleotides as an adjuvant. Tumor infiltrating T cells and apoptotic GL261 cells were quantified by immunohistochemistry. Tumor-reactive immunoglobulin was detected by Western blot. Ovalbumin and gp100-derived peptides were mixed with GL261 lysates as marker antigens to detect changes in presentation of exogenous antigen on MHC class I in vitro, and in vivo following adoptive transfer of gp100-specific CD8+ T cells. Results: Mice bearing orthotopic glioma and breast carcinoma survived significantly longer when vaccinated with 5% O2 lysates. Antigen-specific CTL activation was significantly enhanced following stimulation with lysates derived from GL261 cells grown in 5% O2 versus 20% O2 through a mechanism that involved enhanced cross-presentation of exogenous antigen on MHC I. Vaccination with 5% O2 GL261 cell lysates caused a significant increase in CTL proliferation, tumoricidal function, and trafficking into brain tumor sites, whereas 20% O2 lysate vaccines predominantly evoked an antibody response. Conclusions: Tissue culture oxygen functions as an “immunologic switch” by dictating the cellular and humoral immune responses elicited by tumor cell lysates. These results have profound implications for cancer vaccines that utilize tumor cells as the source of antigen. Clin Cancer Res; 16(19); 4800–8. ©2010 AACR.

Vaccination for Invasive Canine Meningioma Induces in Situ Production of Antibodies Capable of Antibody-Dependent Cell-Mediated Cytotoxicity

Malignant and atypical meningiomas are resistant to standard therapies and associated with poor prognosis. Despite progress in the treatment of other tumors with therapeutic vaccines, this approach has not been tested preclinically or clinically in these tumors. Spontaneous canine meningioma is a clinically meaningful but underutilized model for preclinical testing of novel strategies for aggressive human meningioma. We treated 11 meningioma-bearing dogs with surgery and vaccine immunotherapy consisting of autologous tumor cell lysate combined with toll-like receptor ligands. Therapy was well tolerated, and only one dog had tumor growth that required intervention, with a mean follow up of 585 days. IFN-γ-elaborating T cells were detected in the peripheral blood of 2 cases, but vaccine-induced tumor-reactive antibody responses developed in all dogs. Antibody responses were polyclonal, recognizing both intracellular and cell surface antigens, and HSP60 was identified as one common antigen. Tumor-reactive antibodies bound allogeneic canine and human meningiomas, showing common antigens across breed and species. Histologic analysis revealed robust infiltration of antibody-secreting plasma cells into the brain around the tumor in posttreatment compared with pretreatment samples. Tumor-reactive antibodies were capable of inducing antibody-dependent cell-mediated cytotoxicity to autologous and allogeneic tumor cells. These data show the feasibility and immunologic efficacy of vaccine immunotherapy for a large animal model of human meningioma and warrant further development toward human trials.

Oxygen Is a Master Regulator of the Immunogenicity of Primary Human Glioma Cells

With recent approval of the first dendritic cell (DC) vaccine for patient use, many other DC vaccine approaches are now being tested in clinical trials. Many of these DC vaccines employ tumor cell lysates (TL) generated from cells cultured in atmospheric oxygen (∼20% O₂) that greatly exceeds levels found in tumors in situ. In this study, we tested the hypothesis that TLs generated from tumor cells cultured under physiologic oxygen (∼5% O₂) would be more effective as a source for DC antigens. Gene expression patterns in primary glioma cultures established at 5% O₂ more closely paralleled patient tumors in situ and known immunogenic antigens were more highly expressed. DCs treated with TLs generated from primary tumor cells maintained in 5% O₂ took up and presented antigens to CD8 T cells more efficiently. Moreover, CD8 T cells primed in this manner exhibited superior tumoricidal activity against target cells cultured in either atmospheric 20% O₂ or physiologic 5% O₂. Together, these results establish a simple method to greatly improve the effectiveness of DC vaccines in stimulating the production of tumoricidal T cells, with broad implications for many of the DC-based cancer vaccines being developed for clinical application.

CD200 in CNS tumor-induced immunosuppression: the role for CD200 pathway blockade in targeted immunotherapy

BackgroundImmunological quiescence in the central nervous system (CNS) is a potential barrier to immune mediated anti-tumor response. One suppressive mechanism results from the interaction of parenchyma-derived CD200 and its receptor on myeloid cells. We suggest that CD200/CD200R interactions on myeloid cells expand the myeloid-derived suppressor cell (MDSC) population and that blocking tumor-derived CD200 will enhance the efficacy of immunotherapy.MethodsCD200 mRNA expression levels in human brain tumor tissue samples were measured by microarray. The amount of circulating CD200 protein in the sera of patients with brain tumors was determined by ELISA and, when corresponding peripheral blood samples were available, was correlated quantitatively with MDSCs. CD200-derived peptides were used as competitive inhibitors in a mouse model of glioblastoma immunotherapy.ResultsCD200 mRNA levels were measured in human brain tumors, with different expression levels being noted among the sub groups of glioblastoma, medulloblastoma and ependymoma. Serum CD200 concentrations were highest in patients with glioblastoma and correlated significantly with MDSC expansion. Similarly, in vitro studies determined that GL261 cells significantly expanded a MDSC population. Interestingly, a CD200R antagonist inhibited the expansion of murine MDSCs in vitro and in vivo. Moreover, inclusion of CD200R antagonist peptide in glioma tumor lysate-derived vaccines slowed tumor growth and significantly enhanced survival.ConclusionThese data suggest that CNS-derived tumors can evade immune surveillance by engaging CD200. Because of the homology between mouse and human CD200, our data also suggest that blockade of CD200 binding to its receptor will enhance the efficacy of immune mediated anti-tumor strategies for brain tumors.

Abstract B32: Preclinical testing of three immune adjuvants in vaccination therapy for invasive canine meningioma.

Malignant and atypical meningiomas are resistant to standard therapies, but therapeutic vaccines have not been tested preclinically or clinically. Pet dogs with naturally occurring meningioma are an underutilized model for aggressive human meningioma and an outstanding opportunity for assessing experimental therapeutic approaches. We treated 11 meningioma-bearing dogs with surgery and vaccine immunotherapy consisting of autologous tumor cell lysate combined with CpG or imiquimod. Interferon gamma-elaborating T cells were detected in the peripheral blood of two of cases, but vaccine-induced tumor-reactive antibody responses were found in the sera of all dogs. Systemic antibody responses were polyclonal, recognizing intracellular and cell surface antigens, and heat shock protein 60 was identified as one common antigen. Tumor-reactive antibodies bound allogeneic canine and human meningiomas, demonstrating common antigens across breed and species. Histological analysis revealed robust infiltration of antibody-secreting plasma cells into the brain around the tumor in treated compared to pre-treatment samples. Tumor-reactive antibodies were capable of inducing antibody dependent cell-mediated cytotoxicity to autologous and allogeneic tumor cells. Moreover, median survival for lysate/CpG and lysate/imiquimod vaccination was 646 days versus 222 days in historic surgery controls (p Citation Format: Brian M. Andersen, G Elizabeth Pluhar, Charles E. Seiler, Zhengming Xiong, Michelle R. Goulart, Matthew Gerry O9Sullivan, Matthew A. Hunt, Charles E. Schiaffo, David M. Ferguson, John R. Ohlfest. Preclinical testing of three immune adjuvants in vaccination therapy for invasive canine meningioma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B32.

Abstract B20: Immunotherapy as treatment for dogs with glioma.

Gliomas, the most invasive tumors of the brain, are a disparate group of oncological diseases for which there is currently no cure. Despite the use of combination therapy (surgery, radiation and chemotherapy) as the standard of care for patients, limited progress has been made in the control of the disease. Similar to that in humans, dogs develop spontaneous brain tumors that often carry a dismal prognosis regardless of therapeutic intervention. There is an obvious need for new therapies to treat dogs with brain tumors. Cancer immunotherapy, with the premise that tumor-reactive lymphocytes are able to infiltrate the brain to eradicate microscopic disease, is emerging as a promising strategy to treat glioma. To date, very little is known regarding canine glioma treatment outcome and there has been no reported randomized clinical trials examining the use of immunotherapy for treatment of glioma in dogs. A clinical trial using immunotherapy with autologous glioma cell lysate vaccines, combined with the TLR9 agonist CpG oligodeoxynucleotide to treat dogs with glioma, is underway in our laboratory. The objective of the treatment is to induce specific anti-tumor immunity in the brain and to prevent tumor recurrence. Our preliminary results have shown that vaccinated dogs exhibited extended survival when compared with historical controls. Based on western blot and flow cytometric analysis, this therapeutic approach induced tumor-reactive antibody responses with elevation of IgG antibody levels after vaccination. Thus, the examination of immune responses in therapeutic trials conducted in dogs with gliomas should greatly help predict outcomes of similar treatments in humans, and additionally provide an opportunity to accelerate the development of more efficient immunotherapeutic cancer vaccines for treatment of intracranial malignances. Citation Format: Michelle Goulart, Elizabeth Pluhar, Brian Andersen, Charles Seiler, Matthew Hunt, Gerry O9Sullivan, Zoe Zhang, John Ohlfest. Immunotherapy as treatment for dogs with glioma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B20.