Despite enhanced therapies against primary breast carcinoma (BC), intracranial metastases remain incurable and are associated with a decreased quality of life. To address this unmet medical need, we developed a new therapy (ARL200CNS), representing a pivotal advancement in the field of cancer immunotherapy. ARL200CNS binds to a complex of activation receptors (CD200AR), priming a durable antitumor response when given with an antigen source such as tumor lysates, while simultaneously downregulating the inhibitory CD200 receptor (CD200R1), PD-1/PD-L1, and CTLA4. Moreover, ARL200CNS has demonstrated clinical and immunological responses and extended survival early in a phase 1 trial treating adults with recurrent glioblastoma. Although ARL200CNS educes anti-glioma and anti-breast cancer responses within the CNS, it exhibited little efficacy against primary BC. In contrast, the peptide ARL200Breast elicited an extra-CNS anti-BC response and failed against CNS tumors. However, despite the treatment location, both peptides induce a durable memory response. We hypothesize that targeting unique CD200AR complexes drives specific immune responses required for a peripheral or CNS anti-BC response. We revealed that ARL200CNS primarily binds to the CD200AR2&3 complex, signaling through a specific pathway downregulating NFkB and enhancing translocation of NFAT for the activation of an antitumor response. We now know that ARL200Breast does not signal through this pathway, however, ARL200Breast downregulates CD200R1, PD-1, and PD-L1 while eliciting a durable peripheral anti-BC response. Our overall objective is to demonstrate that the combination of radiotherapy and ARL200 enhances the elimination of tumor cells extending the durability of treatment. We are partnering with OX2 Therapeutics to elucidate immune responses elicited by the peptides in the periphery and CNS, filling the gap in knowledge involving the mechanism of and underlying the therapeutic effects of ARL200 with radiotherapy, enabling a vertical step to further clinical translation.

High-grade gliomas are a major health challenge with poor prognosis and high morbidity. Immune-checkpoint inhibitors (ICI) have emerged as promising therapeutic options for several malignancies yet show little efficacy against central nervous system (CNS) tumors. CD200 is a newly recognized immune checkpoint that modulates immune homeostasis. CD200 protein is expressed by a variety of cells, including immune cells and stromal cells, and is overexpressed by many tumors. The shedding of CD200 from tumor cells can create an immunosuppressive environment that dampens anti-tumor immunity by modulating cytolytic activity and cytokine expression both within and outside the tumor microenvironment (TME). While it is well-accepted that CD200 induces a pro-tumorigenic environment through its ability to suppress the immune response, we sought to determine the role of glioma-specific expression of CD200. We show that CD200 is expressed across glioma types, is shed from tumor cells, and increases over time in the serum of patients undergoing immunotherapy. Using CD200 knockout (KO) glioma models, we demonstrated that glioma cell-derived CD200 promotes tumor growth in vivo and in vitro. Notably, CD200 KO gliomas are spontaneously rejected by their host, a process that required a fully functional immune system, including NK and T-cells. Moreover, we report that glioma-derived or brain-injected soluble CD200 contributes to the suppression of antigen-specific CD8 T-cells in the draining lymph nodes (dLNs). Our work provides new mechanistic insights regarding CD200-mediated immunosuppression by gliomas. Statement of significance:We demonstrate mechanisms of the druggable glioma-derived CD200 checkpoint on tumor growth and immune suppression.

Although patients benefit from immune checkpoint inhibition (ICI) therapy in a broad variety of tumors, resistance may arise from immune suppressive tumor microenvironments (TME), which is particularly true of hepatocellular carcinoma (HCC). Since oncolytic viruses (OV) can generate a highly immune-infiltrated, inflammatory TME, OVs could potentially restore ICI responsiveness via recruitment, priming, and activation of anti-tumor T cells. Here we find that on the contrary, an oncolytic vesicular stomatitis virus, expressing interferon-ß (VSV-IFNß), antagonizes the effect of anti-PD-L1 therapy in a partially anti-PD-L1-responsive model of HCC. Cytometry by Time of Flight shows that VSV-IFNß expands dominant anti-viral effector CD8 T cells with concomitant relative disappearance of anti-tumor T cell populations, which are the target of anti-PD-L1. However, by expressing a range of HCC tumor antigens within VSV, combination OV and anti-PD-L1 therapeutic benefit could be restored. Our data provide a cautionary message for the use of highly immunogenic viruses as tumor-specific immune-therapeutics by showing that dominant anti-viral T cell responses can inhibit sub-dominant anti-tumor T cell responses. However, through encoding tumor antigens within the virus, oncolytic virotherapy can generate anti-tumor T cell populations upon which immune checkpoint blockade can effectively work.

Glioblastoma (GBM) is one of the most lethal central nervous systems (CNS) tumours in adults. As supplements to standard of care (SOC), various immunotherapies improve the therapeutic effect in other cancers. Among them, tumour vaccines can serve as complementary monotherapy or boost the clinical efficacy with other immunotherapies, such as immune checkpoint blockade (ICB) and chimeric antigen receptor T cells (CAR-T) therapy. Previous studies in GBM therapeutic vaccines have suggested that few neoantigens could be targeted in GBM due to low mutation burden, and single-peptide therapeutic vaccination had limited efficacy in tumour control as monotherapy. Combining diverse antigens, including neoantigens, tumour-associated antigens (TAAs), and pathogen-derived antigens, and optimizing vaccine design or vaccination strategy may help with clinical efficacy improvement. In this review, we discussed current GBM therapeutic vaccine platforms, evaluated and potential antigenic targets, current challenges, and perspective opportunities for efficacy improvement.

ObjectivesDespite surgical resection, chemoradiation, and targeted therapy, brain tumors remain a leading cause of cancer-related death in children. Immunotherapy has shown some promise and is actively being investigated for treating childhood brain tumors. However, a critical step in advancing immunotherapy for these patients is to uncover targets that can be effectively translated into therapeutic interventions.MethodsIn this study, our team performed a transcriptomic analysis across pediatric brain tumor types to identify potential targets for immunotherapy. Additionally, we assessed components that may impact patient response to immunotherapy, including the expression of genes essential for antigen processing and presentation, inhibitory ligands and receptors, interferon signature, and overall predicted T cell infiltration.ResultsWe observed distinct expression patterns across tumor types. These included elevated expression of antigen genes and antigen processing machinery in some tumor types while other tumors had elevated inhibitory checkpoint receptors, known to be associated with response to checkpoint inhibitor immunotherapy.ConclusionThese findings suggest that pediatric brain tumors exhibit distinct potential for specific immunotherapies. We believe our findings can guide investigators in their assessment of appropriate immunotherapy classes and targets in pediatric brain tumors.

Abstract Immune-checkpoint inhibitors, such as anti-PD1 and anti-CTLA4, have emerged as promising therapeutic options for several malignancies yet show little efficacy against malignant brain cancers. CD200 is a newly recognized immune-checkpoint which is expressed by a marid of cell types, modulating immune homeostasis through multiple receptors. There is currently limited information regarding CD200 effects in brain tumors. Furthermore, while it is well accepted that CD200 binding to its inhibitory receptor induces a pro-tumorigenic environment through its ability to suppress immune responses, there are increasing evidence that CD200 could also have anti-tumor characteristics. Here we evaluated the role of tumor-derived CD200 on anti-glioma immunity. We demonstrate that CD200 is expressed across glioma types, is shed from tumor cells, and increases over time in serum of patients undergoing immunotherapy. Transcriptomic analysis of CD200 knockout (KO) glioma models reveals that glioma-derived CD200 significantly modifies the glioma tumor microenvironment (TME), not only through immune regulation but also through immune-independent pathways, such as tumor metabolism. Furthermore, we show that CD200 KO gliomas have reduced proliferation and are rejected by their hosts. Downstream analysis revealed that rejection of CD200 KO gliomas relied on a functional adoptive immune system, while decreased proliferation was linked to reduced CXCL10 production by the CD200 KO gliomas. Additionally, we demonstrate that glioma-mediated CD200 strongly suppresses anti-glioma NK cell function within the tumor microenvironment (TME), while secreted CD200 inhibits the priming of antigen-specific CD8 T cells in the lymphatic. Notably, NK cells were essential for the initial rejection of CD200 KO gliomas, while CD8 T cells played a critical role in establishing durable anti-tumor responses. Our work provides new mechanistic insights on glioma-derived CD200-mediated immunosuppression and an untapped potential for targeting CD200 by immunotherapies for malignant gliomas.

PDF file, 380K, Histological Analysis of secondary malignancies.

Supplementary Tables 1-3, Figures 1-6 and Methods from Oxygen Is a Master Regulator of the Immunogenicity of Primary Human Glioma Cells

<p>PDF file - 63K, Table that summarizes the immune monitoring carried out, by dog.</p>

PDF file, 301K, Bioluminescence imaging to track tumor progression over time.

<p>PDF file - 75K, Contains descriptions of two methods used to acquire clinical histology and T cell response data.</p>

Abstract Immunotherapy has revolutionized clinical management of a select group of malignancies by offering a long-term, durable treatment response. Unfortunately, only a small percentage of brain cancers respond. The combination of multiple checkpoint inhibitors may result in serious immune-related adverse events. We have developed a Pan-Immune Checkpoint Ligand that simultaneously controls multiple immune checkpoints. The CD200 immune checkpoint modulates the immune system through a single inhibitory receptor (CD200R1) and multiple activation receptors (CD200ARs). We developed a peptide ligand, CD200AR-L, that targets the CD200ARs, which results in activation of the immune system and suppression of the inhibitory effects of CD200 and other immune checkpoints. Treatment of high-grade glioma in companion dogs with autologous tumor lysate vaccinations and CD200AR-L resulted in a two-year progression-free survival rate of 20% with no significant adverse events. We believe this response is due to the ability of CD200AR-L to modulate multiple immune checkpoints through shared signaling molecules in both the CD200 and PD-1/PD-L1 checkpoint pathways. Our preliminary data demonstrate that CD200R1 and PD-1 mediated immune checkpoint signaling is through SHIP1. CD200AR-L overcomes the immunosuppressive effects of the tumor-derived CD200 and PD-L1 by downregulating these checkpoints on both antigen-presenting cells (APC) and T-cells. CD200AR-L also downregulates PD-1 on APCs and inhibits the upregulation of PD-1 and CTLA4 on T cells. This translational research has led to the initiation of a phase I dose-escalation clinical trial for recurrent glioblastoma in adults (NCT04642937) and the writing of an IND for a pediatric HGG/DIPG trial.

High-grade glioma is an aggressive cancer that occurs naturally in pet dogs. Canine high-grade glioma (cHGG) is treated with radiation, chemotherapy or surgery, but has no curative treatment. Within the past eight years, there have been advances in our imaging and histopathology standards as well as genetic charactereization of cHGG. However, there are only three cHGG cell lines publicly available, all of which were derived from astrocytoma and established using methods involving expansion of tumour cells in vitro on plastic dishes. In order to provide more clinically relevant cell lines for studying cHGG in vitro, the goal of this study was to establish cHGG patient-derived lines, whereby cancer cells are expanded in vivo by injecting cells into immunocompromized laboratory mice. The cells are then harvested from mice and used for in vitro studies. This method is the standard in the human field and has been shown to minimize the acquisition of genetic alterations and gene expression changes from the original tumour. Through a multi-institutional collaboration, we describe our methods for establishing two novel cHGG patient-derived lines, Boo-HA and Mo-HO, from a high-grade astrocytoma and a high-grade oligodendroglioma, respectively. We compare our novel lines to G06-A, J3T-Bg, and SDT-3G (traditional cHGG cell lines) in terms of proliferation and sensitivity to radiation. We also perform whole genome sequencing and identify an NF1 truncating mutation in Mo-HO. We report the characterization and availability of these novel patient-derived lines for use by the veterinary community.

<p>PDF file - 63K, Table that summarizes the immune monitoring carried out, by dog.</p>

PDF file, 210K, In vitro activity assay of costimulatory molecules tested.

<p>PDF file - 72K, Table that contains clinical information on the dogs enrolled in the vaccination cohort arm of the study.</p>

PDF file - 75K, Contains descriptions of two methods used to acquire clinical histology and T cell response data.

In multiple models of oncolytic virotherapy, it is common to see an early anti-tumor response followed by recurrence. We have previously shown that frontline treatment with oncolytic VSV-IFN-β induces APOBEC proteins, promoting the selection of specific mutations that allow tumor escape. Of these mutations in B16 melanoma escape (ESC) cells, a C-T point mutation in the cold shock domain-containing E1 (CSDE1) gene was present at the highest frequency, which could be used to ambush ESC cells by vaccination with the mutant CSDE1 expressed within the virus. Here, we show that the evolution of viral ESC tumor cells harboring the escape-promoting CSDE1C-T mutation can also be exploited by a virological ambush. By sequential delivery of two oncolytic VSVs in vivo, tumors which would otherwise escape VSV-IFN-β oncolytic virotherapy could be cured. This also facilitated the priming of anti-tumor T cell responses, which could be further exploited using immune checkpoint blockade with the CD200 activation receptor ligand (CD200AR-L) peptide. Our findings here are significant in that they offer the possibility to develop oncolytic viruses as highly specific, escape-targeting viro-immunotherapeutic agents to be used in conjunction with recurrence of tumors following multiple different types of frontline cancer therapies.

AbstractPurpose: Advances in immunotherapy have revolutionized care for some patients with cancer. 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 (CD200AR-L) overcomes tumor-induced immunosuppression. We have shown the clinical efficacy of the CD200AR-L in a trial in companion dogs with spontaneous high-grade glioma. Addition of the peptide to autologous tumor lysate vaccines significantly increased the median overall survival to 12.7 months relative to tumor lysate vaccines alone, 6.36 months. 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 response. Using proteomics, we developed humanized CD200AR-L to explore their effects on cytokine/chemokine response, dendritic cell maturation and CMV pp65 antigen response in human CD14+ cells. GMP-grade peptide was further validated for activity. Results: We demonstrated that the CD200AR-L specifically targets a CD200AR complex. Moreover, we developed and validated a humanized CD200AR-L for inducing chemokine response, stimulating immature dendritic cell differentiation and significantly enhanced an antigen-specific response, and determined that the use of the CD200AR-L downregulated the expression of CD200 inhibitory and PD-1 receptors. Conclusions: These results support consideration of a CD200AR-L as a novel platform for immunotherapy against multiple cancers including glioblastoma multiforme.