There are over 400 ongoing clinical trials using tumor-derived vaccines. This approach is especially attractive for many types of brain tumors, including glioblastoma, yet so far the clinical response is highly variable. One contributor to poor response is CD200, which acts as a checkpoint blockade, inducing immune tolerance. We demonstrate that, in response to vaccination, glioma-derived CD200 suppresses the anti-tumor immune response. In contrast, a CD200 peptide inhibitor that activates antigen-presenting cells overcomes immune tolerance. The addition of the CD200 inhibitor significantly increased leukocyte infiltration into the vaccine site, cytokine and chemokine production, and cytolytic activity. Our data therefore suggest that CD200 suppresses the immune system's response to vaccines, and that blocking CD200 could improve the efficacy of cancer immunotherapy.

Recent advances in immunotherapy have included inhibition of immune checkpoint proteins in the tumor microenvironment and tumor lysate-based vaccination strategies. We combined these approaches in pet dogs with high-grade glioma. Administration of a synthetic peptide targeting the immune checkpoint protein, CD200, enhanced the capacity of antigen-presenting cells to prime T-cells to mediate an anti-glioma response. We found that in canine spontaneous gliomas, local injection of a canine-specific, CD200-directed peptide before subcutaneous delivery of an autologous tumor lysate vaccine prolonged survival relative to a historical control treated with autologous tumor lysate alone (median survivals of 12.7 months and 6.36 months, respectively). Antigen-presenting cells and T-lymphocytes primed with this peptide suppressed their expression of the inhibitory CD200 receptor, thereby enhancing their ability to initiate immune reactions in a glioblastoma microenvironment replete with the immunosuppressive CD200 protein. These results support consideration of a CD200 ligand as a novel glioblastoma immunotherapeutic agent.

Numerous therapies aimed at driving an effective anti-glioma response have been employed over the last decade; nevertheless, survival outcomes for patients remain dismal. This may be due to the expression of immune-checkpoint ligands such as PD-L1 by glioblastoma (GBM) cells which interact with their respective receptors on tumor-infiltrating effector T cells curtailing the activation of anti-GBM CD8 + T cell-mediated responses. Therefore, a combinatorial regimen to abolish immunosuppression would provide a powerful therapeutic approach against GBM. We developed a peptide ligand (CD200AR-L) that binds an uncharacterized CD200 immune-checkpoint activation receptor (CD200AR). We sought to test the hypothesis that CD200AR-L/CD200AR binding signals via he DAP10&12 pathways through in vitro studies by analyzing transcription, protein, and phosphorylation, and in vivo loss of function studies using inhibitors to select signaling molecules. We report that CD200AR-L/CD200AR binding induces an initial activation of the DAP10&12 pathways followed by a decrease in activity within 30 min, followed by reactivation via a positive feedback loop. Further in vivo studies using DAP10&12KO mice revealed that DAP10, but not DAP12, is required for tumor control. When we combined CD200AR-L with an immunestimulatory gene therapy, in an intracranial GBM model in vivo, we observed increased median survival, and long-term survivors. These studies are the first to characterize the signaling pathway used by the CD200AR, demonstrating a novel strategy for modulating immune checkpoints for immunotherapy currently being analyzed in a phase I adult trial.

Purpose: Advances in immunotherapy have revolutionized care for some cancer patients. However, current checkpoint inhibitors are associated with significant toxicity and yield poor responses for patients with central nervous system tumors, calling into question whether cancer immunotherapy can be applied to glioblastoma multiforme. We determined that targeting the CD200 activation receptors (CD200AR) of the CD200 checkpoint with a peptide inhibitor overcomes tumor-induced immunosuppression. We have shown the clinical efficacy of the peptide inhibitor in a trial in companion dogs with spontaneous high-grade glioma; adding the peptide to autologous tumor lysate vaccines significantly increased overall survival relative to tumor lysate alone (median survival of 12.7 and 6.36 months, respectively).Experimental design: This study was developed to elucidate the mechanism of the CD200ARs and develop a humanized peptide inhibitor. We developed macrophage cell lines with each of four CD200ARs knocked out to determine their binding specificity and functional responses. Using proteomics, we developed humanized peptide inhibitors to explore their effects on cytokine/chemokine response, dendritic cell maturation and CMV pp65 antigen response in CD14 + cells. GMP-grade peptide was further validated for activity. Results:We demonstrated that the peptide specifically targets the CD200AR complex to induce an immune response. Moreover, we developed and validated our humanized peptides for inducing chemokine response, stimulating immature dendritic cell differentiation and significantly enhancing an antigen-specific response. We also determined that the use of the peptide downregulated the expression of CD200 inhibitory and PD-1 receptors. Conclusion:These results support consideration of a CD200 peptide ligand as a novel platform for immunotherapy against multiple cancers including glioblastoma multiforme.Translational relevance. This report evaluates the ability to modulate the CD200 immune checkpoint by employing synthetic peptides directed as ligands to its paired immune activation receptor. We previously reported the presence of CD200 in the sera and tumor vasculature of patients with glioblastoma multiforme (GBM). We have also shown that a canine CD200 activation receptor ligand extends the lives of companion dogs with high grade glioma. The data we present here show that the human peptide ligand (hCD200ARL) directed to the CD200 activation receptor on CD14+ cells activates immune upregulation through induction of a cytokine response and dendritic cell differentiation. In addition, hCD200ARL downregulates the inhibitory CD200 and PD-1 receptors. These findings provide a basis to evaluate hCD200ARL as a novel immune therapy for patients with GBM. Downregulation of PD-1 suggests that hCD200ARL may also obviate the need for PD1 and PD-L1 directed therapies for GBM and other malignancies.

Extracellular vesicles (EVs) may be used as a non-invasive screening platform to discover markers associated with early diagnosis, prognosis, and treatment response. Such an approach is invaluable for diseases such as glioblastoma, for which only a few non-invasive diagnostic or prognostic markers are available. We used mass spectrometry to analyze proteomics profiles of EVs derived from four glioblastoma cell lines and human primary astrocytes (HPAs) and found that SRPX is the only protein enriched in the majority of glioblastoma EVs that was absent in the HPA-derived EVs. Then, we evaluated the relationship between SRPX protein expression and tumor grade using immunohistochemical staining (IHC) and performed colony formation and viability assays to analyze the possible function of SRPX in glioblastoma. SRPX mRNA and protein expression were associated with tumor grade. Moreover, temozolomide (TMZ)-resistant tumor tissues showed highly positive SRPX staining, compared to all other tumor grades. Additionally, glioblastoma cells displayed enhanced SRPX gene expression when exposed to TMZ. Knockdown of SRPX gene expression via siRNA inhibited cell viability. Taken together, the results of this study suggest that SRPX can be used as a novel tumor marker for diagnostic and prognostic purposes and can also be a therapeutic target for glioblastomas.