CD200AR Ligands for Cancer Immunotherapy

Abstract

In certain embodiments, the present invention provides a method of inhibiting PD-1 in a cell by administering a CD200 activating receptor ligand (CD200AR-L) to the cell. In certain embodiments, the present invention provides a method of increasing the efficacy of a tumor lysate vaccine in a mammal, comprising administering a CD200 activating receptor ligand (CD200AR-L) to the mammal prior to administration of the tumor lysate vaccine.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 792,311, filed January 14, 2019. The entire contents of the above-referenced applications are incorporated herein by reference. [Background technology]

[0002] Despite advances in cancer research, there are still no satisfactory treatments for many cancers. For example, malignant glioma is a devastating disease that affects more than 14,000 patients annually in the United States. Due to the ability of glioma cells to migrate several centimeters away from the bulk tumor cavity, current standard treatments have only provided modest improvements, with a 5-year survival rate of less than 30%. Patients with glioblastoma exhibit systemic immunosuppression that leads to impaired adaptive immune responses. These deficiencies result from the concentration of immunosuppressive factors secreted by the tumor, which suppress T-cell proliferation and cytotoxic function. Immunosuppression plays a key role in tumor progression in glioblastoma patients. If immunosuppression could be reversed and effective immune targeting could be enabled, glioma patients would experience little tumor progression and improved outcomes.

[0003] Thus, there is a need for new compositions and methods for treating cancer, in particular new compositions that reverse the immunologically suppressive microenvironment caused by tumors. Summary of the Invention

[0004] In certain embodiments, the present invention provides a method of inhibiting PD-1 in a cell by administering a CD200 activating receptor ligand (CD200AR-L) to the cell. In certain embodiments, the CD200AR-L is a peptidomimetic. In certain embodiments, the peptidomimetic comprises one or more D-isomer amino acids. In certain embodiments, the peptidomimetic comprises one or more unnatural amino acids.

[0005] The present invention provides, in certain embodiments, a method of enhancing the efficacy of a tumor lysate vaccine in a mammal, comprising administering a CD200 activating receptor ligand (CD200AR-L) to the mammal prior to administration of the tumor lysate vaccine. In certain embodiments, the tumor lysate is substantially free of CD200. As used herein, "substantially free" means that the material (e.g., tumor lysate) has a lower level of CD200 than untreated material, e.g., 1-100% less CD200. In certain embodiments, CD200 is removed by absorption using standard methods. [Brief description of the drawings]

[0006] [Figure 1A] Experimental model. CD200 and PD-L are secreted by tumor cells and bind to the CD200 inhibitory receptor (CD200R1) or PD-1 receptor on antigen-presenting cells, shutting down the immune system. Both CD200R1 and PD-1 crosstalk with each other through the SHIP signaling molecule and interact with the activating receptor signaling pathway (Figure 1a). In addition to the inhibitory receptors, the CD200 checkpoint has activating receptors with adjuvant-like properties that, when stimulated, overcome the inhibitory signals and activate immune cells. [Figure 1B] Experimental model. CD200 and PD-L are secreted by tumor cells and bind to the CD200 inhibitory receptor (CD200R1) or PD-1 receptor on antigen-presenting cells, shutting down the immune system. Targeting CD200 activation with CD200 inhibitory peptide ligand (CD200AR-L) subjugates the CD200R1 and PD-L pathways by downregulating CD200AR and PD-1 on immune cells (Figure 1b). In addition to inhibitory receptors, the CD200 checkpoint has activating receptors that have adjuvant-like properties when stimulated, overcoming the inhibitory signals and activating immune cells. [Figure 2A]Stimulates antigen presenting cells by targeting CD200 activating receptors. (FIG. 2A) Canine CD200 (amino acids 60-178) (SEQ ID NO: 11), human CD200 (amino acids 33-152) (SEQ ID NO: 12), and mouse CD200 (amino acids 33-152) (SEQ ID NO: 13) sequences showing the homology of different CD200 peptides. Canine P4 is sequence 147-161 of SEQ ID NO: 11. Human P1 is sequence 61-75 of SEQ ID NO: 12, human P2 is sequence 110-124 of SEQ ID NO: 12, human P3 is sequence 76-90 of SEQ ID NO: 12, and human P4 is sequence 121-135 of SEQ ID NO: 12. Mouse P1 is sequence 62-75 of SEQ ID NO: 13. [Figure 2B] CD14+ cells from healthy donors were pulsed with human peptides P1 (IVTWQKKKAVSPENM) (SEQ ID NO: 1), P2 (NITLEDEGCYMCLFN) (SEQ ID NO: 2), P3 (VTFSENHGVVIQPAY) (SEQ ID NO: 3) or P4 (CLFNTFGFGKISGTA) (SEQ ID NO: 4) and incubated for 48 hours. Non-pulsed (NP) cells were used as control. Supernatants were collected and analyzed for IL1β (Figure 2B). [Figure 2C] CD14+ cells from healthy donors were pulsed with human peptides P1 (IVTWQKKKAVSPENM) (SEQ ID NO: 1), P2 (NITLEDEGCYMCLFN) (SEQ ID NO: 2), P3 (VTFSENHGVVIQPAY) (SEQ ID NO: 3) or P4 (CLFNTFGFGKISGTA) (SEQ ID NO: 4) and incubated for 48 hours. Non-pulsed (NP) cells were used as control. Supernatants were collected and analyzed for TNFα (Figure 2C). [Figure 2D]Immature dendritic cells were pulsed with CMV antigens pp65±P1-P4. Cells were washed and added back with autologous T cells from CMV-positive and CMV-negative healthy donors and incubated for an additional 48 hours. Supernatants were harvested and analyzed for IFNα production. Error bars represent standard deviation (n=3 / group, *P<0.05, **P<0.005, ***P<0.0005 by t-test, *P<0.05 by two-way ANOVA). Data represent 3 separate healthy donors in each group. [Figure 3A] Alanine substitution enhances antigen-presenting cell stimulation. (Figure 3A) CD14+ cells were pulsed with equimolar ratios of peptides and incubated for 48 hours. Supernatants were harvested and analyzed for IFN-1β, IL-12p70, MIG, and TNFα content. (Figure 3B) In a separate experiment, cells were incubated for 1 hour. RNA was harvested and analyzed by NanoSight for immune-related transcriptional changes. Pulsed cells were normalized to non-pulsed (NP) controls to derive heatmaps, which were generated by using Java Treeview within nSolver. Cluster analysis of the 35 genes examined showed significant P1A8 (IVTWQKKAAVSPENM) (SEQ ID NO: 6), P2A0 (NITLEDEGCYMCLFN) (SEQ ID NO: 7), P2A5 (NITLADEGCYMCLFN) (SEQ ID NO: 8), P3A12 (VTFSENHGVVIAPAY) (SEQ ID NO: 9), and P4A10 (CLFNTFGFGAISGTA) (SEQ ID NO: 10) expression changes in one or more of the treated samples when compared to NP controls. D1-P3A12 was an outlier and was excluded from the analysis. Error bars represent standard deviation (n=3 / group, *P<0.05, **P<0.005, ***P<0.0005 by t-test, *P<0.05 by two-way ANOVA). [Figure 3B]Alanine substitution enhances antigen-presenting cell stimulation. (Figure 3A) CD14+ cells were pulsed with equimolar ratios of peptides and incubated for 48 hours. Supernatants were harvested and analyzed for IFN-1β, IL-12p70, MIG, and TNFα content. (Figure 3B) In a separate experiment, cells were incubated for 1 hour. RNA was harvested and analyzed by NanoSight for immune-related transcriptional changes. Pulsed cells were normalized to non-pulsed (NP) controls to derive heatmaps, which were generated by using Java Treeview within nSolver. Cluster analysis of the 35 genes examined showed significant P1A8 (IVTWQKKAAVSPENM) (SEQ ID NO: 6), P2A0 (NITLEDEGCYMCLFN) (SEQ ID NO: 7), P2A5 (NITLADEGCYMCLFN) (SEQ ID NO: 8), P3A12 (VTFSENHGVVIAPAY) (SEQ ID NO: 9), and P4A10 (CLFNTFGFGAISGTA) (SEQ ID NO: 10) expression changes in one or more of the treated samples when compared to NP controls. D1-P3A12 was an outlier and was excluded from the analysis. Error bars represent standard deviation (n=3 / group, *P<0.05, **P<0.005, ***P<0.0005 by t-test, *P<0.05 by two-way ANOVA). [Figure 4A] Targeting the CD200 activating receptor enhances dendritic cell maturation. CD14 purified cells were pulsed with GM-CSF+IL4 or equimolar ratios of peptides P1A8, P2A0, or P4A10 and incubated for 48 hours. Cells were harvested and phenotyped for CD80, CD86, and HLA-DR. [Figure 4B] Targeting the CD200 activating receptor enhances dendritic cell maturation. CD14 purified cells were pulsed with GM-CSF+IL4 plus equimolar ratios of peptides P1A8, P2A0, or P4A10 and incubated for 48 hours. Cells were harvested and phenotyped for CD80, CD86, and HLA-DR. [Figure 4C]Donor-derived immature dendritic cells were pulsed with an equimolar ratio of peptides P1A8, P2A0, or P4A10 to the CMV antigen pp65+ / -, with or without CMV. Cells were washed and autologous T cells were added back in and incubated for an additional 48 hours. Supernatants were analyzed for IFNγ production. [Figure 4D] Immature dendritic cells were pulsed with CMV antigen pp65+ / -P4A10. Cells were washed before adding back autologous T cells and further incubated for 48 hours. Supernatants were analyzed for IFNγ, IL-6, TNFα and RANTES content. Error bars represent standard deviation (n=3 healthy donors / group, *P<0.05, **P<0.005, ***P<0.0005 by t-test). Data represent three separate healthy donors. Black bars represent CMV negative donors. [Figure 5A] Treatment with CD200 inhibitors induces tumor regression in dogs. (FIG. 5A) Tumors were resected and dogs were vaccinated with autologous tumor lysate and canine-specific CD200AR-L and followed for progression-free survival (top black line) and overall survival (middle line with arrows). For comparison, a historical cohort treated with tumor lysate alone is shown (steep line on the left). Black arrows represent dogs currently in clinical trials. [Figure 5B] (Figures 5B, 5C) Following resection, (Figure 5B) there was an area of ​​residual tumor, (Figure 5C) showing regression 4 months after surgery. [Figure 5C] (Figures 5B, 5C) Following resection, (Figure 5B) there was an area of ​​residual tumor, (Figure 5C) showing regression 4 months after surgery. [Figure 5D] (Figures 5D, 5E) Images from one of five dogs that developed central leukoencephalopathy, characterized by T2 hyperintensities in periventricular white matter tracts and ventricular dilation (Figure 5D), which resolved after corticosteroid treatment (Figure 5E). [Figure 5E] (Figures 5D, 5E) Images from one of five dogs that developed central leukoencephalopathy, characterized by T2 hyperintensities in periventricular white matter tracts and ventricular dilation (Figure 5D), which resolved after corticosteroid treatment (Figure 5E). [Figure 5F] (Figures 5F-5I) However, this dog had tumor recurrence visible on MRI performed 2 months after corticosteroid treatment. [Figure 5G] (Figures 5F-5I) However, this dog had tumor recurrence visible on MRI performed 2 months after corticosteroid treatment. [Figure 5H] (Figures 5F-5I) However, this dog had tumor recurrence visible on MRI performed 2 months after corticosteroid treatment. [Figure 5I] (Figures 5F-5I) However, this dog had tumor recurrence visible on MRI performed 2 months after corticosteroid treatment. [Figure 5J] (Figures 5J-5M) Vaccination with CD200AR-L was resumed in this dog after recovery, and there was evidence of tumor regression 2 months after two doses of the vaccine. [Figure 5K] (Figures 5J-5M) Vaccination with CD200AR-L was resumed in this dog after recovery, and there was evidence of tumor regression 2 months after two doses of the vaccine. [Figure 5L] (Figures 5J-5M) Vaccination with CD200AR-L was resumed in this dog after recovery, and there was evidence of tumor regression 2 months after two doses of the vaccine. [Figure 5M] (Figures 5J-5M) Vaccination with CD200AR-L was resumed in this dog after recovery, and there was evidence of tumor regression 2 months after two doses of the vaccine. [Figure 5N] Serum CD200 (sCD200) levels were significantly associated with overall survival (*p=0.005). Survival groups were analyzed using the log-rank test for trend, Mantel-Cox, and Gehan-Breslow-Wilcoxon. [Figure 6A]CD200R1-related genes are associated with shorter overall survival in humans. (Figure 6A) Clusters of genes associated with overall survival were analyzed in patient tumor samples available in The Cancer Genome Atlas database. Gene Cluster Expression Summary Scores were used to identify the identified clusters in the figure by numbers. Significant in the Kaplan-Meier analysis are indicated with symbols and their associated p-values ​​are shown. [Figure 6B] Kaplan-Meier (KM) plot showing that patients who expressed high levels of the gene cluster containing CD200 (dashed lines) showed shorter overall survival than those who expressed low levels (solid lines). Comparison is made between the top quartile (Q1) versus the lowest quartile of the three (Q234). [Figure 6C] Kaplan-Meier (KM) plot showing that patients who expressed high levels of the gene cluster containing CD200 (dashed line) showed shorter overall survival than those who expressed low levels (solid line). Comparison is made between the two highest quartiles (Q12) versus the two lowest quartiles (Q34). [Figure 6D] Kaplan-Meier (KM) plot showing that patients who expressed high levels of the gene cluster containing CD200 (dashed lines) showed shorter overall survival than those who expressed low levels (solid lines). Comparison is made between the three upper quartiles (Q123) versus the lowest quartile (Q4). [Figure 6E]We provide a table showing whether CD200-associated genes are associated with survival, and used GCESS to analyze gene expression profiles of patient tumor samples in the TCGA dataset. This allowed us to identify a gene cluster that was consistently expressed across the dataset and associated with overall survival in an unbiased statistical analysis. CD200R1 expression was found within a large cluster of genes highly enriched for immune-related transcripts. Increased transcript levels of genes within this cluster were significantly associated with reduced survival. Patients who expressed high levels of the CD200R1-containing cluster (cluster 14) showed shorter overall survival times than patients who expressed low levels of the cluster. These results suggest the critical importance of CD200 / CD200R1 interactions in mediating an immunosuppressive glioblastoma microenvironment. [Figure 7A] CD200AR-L pulsing downregulates inhibitory CD200R1. Mouse CD11b cells were pulsed with mCD200AR-L, RNA was extracted, and analyzed for CD200R1 or PD-1 expression (Figures 7A and B). The experiments in Figure 7 demonstrated that CD200AR targets downregulate inhibitory CD200R1 and PD-1 in both mouse and human antigen-presenting cells, validating the crosstalk between CD200R1 and PD-1, and that the inhibitory CD200R1 and PD-1 pathways are interconnected with CD200AR4. Error bars represent standard deviation (n=3 / group, *P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005 by t-test). [Figure 7B]CD200AR-L pulsing downregulates inhibitory CD200R1. Mouse CD11b cells were pulsed with mCD200AR-L, RNA was extracted, and analyzed for CD200R1 or PD-1 expression (Figures 7A and B). The experiments in Figure 7 demonstrated that CD200AR targets downregulate inhibitory CD200R1 and PD-1 in both mouse and human antigen-presenting cells, validating the crosstalk between CD200R1 and PD-1, and that the inhibitory CD200R1 and PD-1 pathways are interconnected with CD200AR4. Error bars represent standard deviation (n=3 / group, *P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005 by t-test). [Figure 7C] To further these studies, CD11b cells were stimulated with OVA+ / -CD200AR-L, cells were washed, and purified T cells were added. After 24 hours of incubation, T cells were isolated and analyzed for CD200R1 or PD-1 expression (Figure 7C and D). The experiments in Figure 7 demonstrated that CD200AR targets downregulate inhibitory CD200R1 and PD-1 in both mouse and human antigen-presenting cells, validating the crosstalk between CD200R1 and PD-1, and that the inhibitory CD200R1 and PD-1 pathways are interconnected with CD200AR4. Error bars represent standard deviation (n=3 / group, *P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005 by t-test). [Figure 7D]To further these studies, CD11b cells were stimulated with OVA+ / -CD200AR-L, cells were washed, and purified T cells were added. After 24 hours of incubation, T cells were isolated and analyzed for CD200R1 or PD-1 expression (Figure 7C and D). The experiments in Figure 7 demonstrated that CD200AR targets downregulate inhibitory CD200R1 and PD-1 in both mouse and human antigen-presenting cells, validating the crosstalk between CD200R1 and PD-1, and that the inhibitory CD200R1 and PD-1 pathways are interconnected with CD200AR4. Error bars represent standard deviation (n=3 / group, *P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005 by t-test). [Figure 7E] To confirm whether these experiments translated to humans, CD14 cells were pulsed with our human CD200AR-L. After 24 hours of incubation, cells were stained with anti-CD200R1 and analyzed by flow cytometry (Figure 7E). The experiment in Figure 7 demonstrated that CD200AR targets downregulate inhibitors CD200R1 and PD-1 in both mouse and human antigen-presenting cells, validating the crosstalk between CD200R1 and PD-1, and that the inhibitory CD200R1 and PD-1 pathways are interconnected with CD200AR4. Error bars represent standard deviation (n=3 / group, *P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005 by t-test). [Figure 7F] The mean fluorescence intensity was analyzed for CD200R1 expression. The experiments in Figure 7 demonstrated that CD200AR targets downregulate inhibitors CD200R1 and PD-1 in both mouse and human antigen-presenting cells, validating the crosstalk between CD200R1 and PD-1, and the inhibitory CD200R1 and PD-1 pathways are interconnected with CD200AR4. Error bars represent standard deviation (n=3 / group, *P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005 by t-test). [Figure 7G]The mean fluorescence intensity was analyzed for PD-1 expression. The experiments in Figure 7 demonstrated that CD200AR targets inhibitors CD200R1 and downregulate PD-1 in both mouse and human antigen-presenting cells, validating the crosstalk between CD200R1 and PD-1, and the inhibitory CD200R1 and PD-1 pathways are interconnected with CD200AR4. Error bars represent standard deviation (n=3 / group, *P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005 by t-test). [Figure 8] Pulsing CD200AR-L conquers inhibitory PD-L1. Murine CD11b cells were activated with PMA and treated with PD-L1 or PD-L1+murine CD200AR-L. After 48 hours, supernatants were collected and analyzed for TNFα (Figure 8A), IL4 (Figure 8B), or IL5 (Figure 8C). These experiments demonstrated that pulsing cells with CD200AR-L conquers the inhibitory properties of PD-L1. [Figure 9] Vaccine schedule. Surgical debulking and post-op MRI were performed on day 0. Intradermal (ID) injection of CD200 inhibitor, followed by imiquimod application 6-24 hours later, and ID injection of a mixture of tumor lysate vaccine + CD200 inhibitor (downward pointing solid arrow) were started on day 10 and weekly for 3 weeks, then once every 4 weeks for 3 months, then repeated every 8 weeks for 1 year or until tumor recurrence or death. Brain MRI (upward pointing dashed arrow) was performed immediately after surgery and then every 4 months thereafter. [Figure 10] Experimental model. [Figure 11A] Targeting the CD200 activating receptor inhibits the effects of inhibitory PD-L1. Mouse splenocytes were activated with PMA, + / -PD-L1 or PD-L1+mCD200AR-L. After 48 h incubation, supernatants were analyzed for TNFα. Error bars are representative of n=3, *P<0.005. [Figure 11B]Targeting the CD200 activating receptor inhibits the effects of inhibitory PD-L1. Mouse splenocytes were activated with PMA, + / -PD-L1 or PD-L1+mCD200AR-L. After 48 h incubation, supernatants were analyzed for IL-4. Error bars are representative of n=3, *P<0.005. [Figure 11C] Targeting the CD200 activating receptor inhibits the effects of inhibitory PD-L1. Mouse splenocytes were activated with PMA, + / -PD-L1 or PD-L1+mCD200AR-L. After 48 h incubation, supernatants were analyzed for IL-5. Error bars are representative of n=3, *P<0.005. [Figure 12A] The effect of PD-L1 is blocked by knocking out CD200R1. Wild-type or CD200R1KO macrophages were pulsed with PD-L1 and analyzed for pSHP. [Figure 12B] The effect of PD-L1 is inhibited by knocking out CD200R1. Wild-type CD200R1KO cells were pulsed with LPS+ / -PD-L1. Error bars are representative of n=3, **P<0.001 by t-test. [Figure 13A] Targeting the CD200 activating receptor downregulates the expression of CD200R1 and PD-1. Mouse CD11b cells were pulsed with CD200AR-L and analyzed for CD200R1 transcription levels. Error bars are representative of n=3, *P<0.005, **P<0.0001, and ***P<0.00001. [Figure 13B] Targeting the CD200 activating receptor downregulates the expression of CD200R1 and PD-1. Mouse CD11b cells were pulsed with CD200AR-L and analyzed for PD-1 transcription levels. Error bars are representative of n=3, *P<0.005, **P<0.0001, and ***P<0.00001. [Figure 13C]Human CD14 cells were pulsed with human CD200AR-L and after 48 h of incubation, cells were analyzed by flow cytometry for expression of CD200R1. Error bars are representative of n=3, *P<0.005, **P<0.0001, and ***P<0.00001. [Figure 13D] Human CD14 cells were pulsed with human CD200AR-L and after 48 h of incubation, cells were analyzed by flow cytometry for expression of PD-1. Error bars are representative of n=3, *P<0.005, **P<0.0001, and ***P<0.00001. [Figure 13E] Murine CD11b cells from wild-type mice were pulsed with OVA + / - CD200AR-L. Cells were washed and purified CD3 cells were added to the CD11b cells for 24 hours. CD3 cells were purified and analyzed for CD200R1 transcript levels. Error bars are representative of n=3, *P<0.005, **P<0.0001, and ***P<0.00001. [Figure 13F] Murine CD11b cells from wild-type mice were pulsed with OVA + / - CD200AR-L. Cells were washed and purified CD3 cells were added to the CD11b cells for 24 hours. CD3 cells were purified and analyzed for PD-1 transcription levels. Error bars are representative of n=3, *P<0.005, **P<0.0001, and ***P<0.00001. [Figure 14A] CD200AR-L upregulates CTLA4 and PD-L1 expression. Splenocytes were pulsed with CD200, PD-L1+ / -mCD200AR-L. Cells were analyzed by flow cytometry for CD4+CTLA4+ populations. Error bars are representative of n=2, *P<0.005, **P<0.0001. [Figure 14B] CD200AR-L upregulates CTLA4 and PD-L1 expression. Splenocytes were pulsed with CD200, PD-L1+ / -mCD200AR-L. Cells were analyzed by flow cytometry for CD8+CTLA4+ populations. Error bars are representative of n=2, *P<0.005, **P<0.0001. [Figure 14C] CD200AR-L upregulates CTLA4 and PD-L1 expression. Splenocytes were pulsed with CD200, PD-L1+ / -mCD200AR-L. Cells were analyzed by flow cytometry for CD11b+PD-L1+ populations. Error bars are representative of n=2, *P<0.005, **P<0.0001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] The unprecedented success of cancer immunotherapy over the past decade is a direct result of the concept of immune surveillance, which has revolutionized the clinical management of malignancies that previously had poor prognoses. At the forefront of immunotherapy development are immune checkpoint inhibitors, which have seen enormous and unparalleled success in cancer therapy as a result of their broad biological activity across many histological tumor types, the durability of their responses, and the cures that have been observed even in metastatic and chemoresistant disease.

[0008] Among immune checkpoint blockade strategies, the two most prominent (in terms of clinical success to date) are targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and the interaction between programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1). However, the use of monotherapy is hindered due to the multiple mechanisms that tumors utilize to inhibit immune responses. This is particularly true for both high-grade (HGG) and low-grade (LGG) malignant brain tumors, such as gliomas, as tumors with low mutational burden and / or low immunogenicity may be inherently resistant to this form of therapy. Even for favorable tumor types, tumor diversity hampers immunotherapy. This may be related to redundancy in the set of activating and inhibitory molecules targeted by immune checkpoint inhibitors. Thus, multiple inhibitors have been used that significantly improve survival rates. Unfortunately, these therapies often cause significant immune-related adverse events, including death. Therefore, alternative approaches that target multiple checkpoints are essential.

[0009] Currently, research is focused on single inhibitors against PD-1, PD-L1 and CTLA4. The inventors have discovered that targeting the CD200 checkpoint overcomes the suppressive properties of the CD200, PD-1 and CTLA4 checkpoints. CD200 inhibitors are effective and safe checkpoint inhibitors for immunotherapy against solid tumors.

[0010] In certain embodiments, the present invention provides a method of inhibiting PD-1 in a cell by administering a CD200 activating receptor ligand (CD200AR-L) to the cell. In certain embodiments, the CD200AR-L is a peptidomimetic. In certain embodiments, the peptidomimetic comprises one or more D-isomer amino acids. In certain embodiments, the peptidomimetic comprises one or more unnatural amino acids.

[0011] In a specific embodiment, CD200AR-L is a canine-specific peptide (cCD200AR-L).

[0012] In certain embodiments, the cell is a cancer cell.

[0013] In certain embodiments, the cancer cells are glioblastoma cells.

[0014] In certain embodiments, the CD200AR-L is peptide P1A8, P2A0, P2A5, P3A12, or P4A10.

[0015] In a particular embodiment, the CD200AR-L is the peptide human P1A8.

[0016] In a specific embodiment, the CD200AR-L is the peptide human P2A0.

[0017] In a particular embodiment, the CD200AR-L is the peptide human P4A10.

[0018] In certain embodiments, the cell is a cancer cell.

[0019] In certain embodiments, the cancer cells are glioblastoma cells.

[0020] The present invention provides, in certain embodiments, a method of enhancing the efficacy of a tumor lysate vaccine in a mammal, comprising administering a CD200 activating receptor ligand (CD200AR-L) to the mammal prior to administration of the tumor lysate vaccine. In certain embodiments, the tumor lysate is substantially free of CD200. As used herein, "substantially free" means that the material (e.g., tumor lysate) has a lower level of CD200 than untreated material, e.g., 1-100% less CD200. In certain embodiments, CD200 is removed by absorption using standard methods.

[0021] In a specific embodiment, CD200AR-L is a human-specific peptide (hCD200AR-L).

[0022] In a specific embodiment, CD200AR-L is a canine-specific peptide (cCD200AR-L).

[0023] In certain embodiments, the cell is a cancer cell.

[0024] In certain embodiments, the cancer cells are glioblastoma cells.

[0025] In certain embodiments, the CD200AR-L is peptide P1A8, P2A0, P2A5, P3A12, or P4A10.

[0026] In a particular embodiment, the CD200AR-L is the peptide human P1A8.

[0027] In a specific embodiment, the CD200AR-L is the peptide human P2A0.

[0028] In a particular embodiment, the CD200AR-L is the peptide human P4A10.

[0029] In certain embodiments, the cell is a cancer cell.

[0030] In a specific embodiment, CD200AR-L is administered by local injection.

[0031] In certain embodiments, the tumor lysate vaccine is administered subcutaneously.

[0032] In certain embodiments, the tumor lysate vaccine is an autologous tumor lysate vaccine.

[0033] In a specific embodiment, the mammal is administered CD200AR-L 5-14 days after surgical removal of the tumor.

[0034] In a specific embodiment, the mammal is administered the CD200AR-L about 10 days after surgical removal of the tumor.

[0035] In a specific embodiment, (a) a species-specific CD200AR-L peptide is injected intradermally (ID); (b) 24 hours later, imiquimod is applied topically to the skin and allowed to absorb for 10-15 minutes; and (c) autologous tumor lysate mixed with CD200AR-L is injected ID.

[0036] In a particular embodiment, the species-specific CD200AR-L peptide is injected intradermally (ID) at a dosage of about 5 μg / kg.

[0037] In certain embodiments, imiquimod (1 packet) is applied topically to the skin in a dosage of 5% cream / 12.5 g.

[0038] In a specific embodiment, about 500 μg protein of autologous tumor lysate is mixed with about 5 μg / kg of CD200AR-L and injected intradermally.

[0039] In certain embodiments, the treatment regimen is repeated weekly for three weeks.

[0040] In certain embodiments, the treatment regimen is repeated weekly for three weeks, then once every four weeks for three months, and then every six to eight weeks as needed.

[0041] The term "mammal" as used herein refers to humans, higher non-human primates, rodents, livestock, cattle, horses, pigs, sheep, dogs and cats. In one embodiment, the subject is a human. In one embodiment, the subject is a dog.

[0042] In certain embodiments, the present invention provides the use of a pharmaceutical composition comprising a pharma- ceutically acceptable carrier or diluent, CD200AR-L as an active ingredient, and optionally a cancer vaccine (e.g., tumor lysate) as defined above for the use of a medicament for treating or for the manufacture of a disease or disorder resulting from abnormal cell growth, function, or behavior. In certain embodiments, the disease or disorder is cancer. In certain embodiments, the cancer is selected from solid tumors of the colon, breast, brain, liver, ovary, stomach, lung, and head and neck. In certain embodiments, the cancer is selected from glioblastoma, melanoma, prostate cancer, endometrial cancer, ovarian cancer, breast cancer, lung cancer, head and neck cancer, hepatocellular carcinoma, and thyroid cancer. In certain embodiments, the cancer is selected from breast, ovary, cervix, prostate, testis, genitourinary tract, esophagus, larynx, glioblastoma, neuroblastoma, stomach, skin, keratoacanthoma, lung, epidermoid carcinoma, large cell carcinoma, non-small cell lung cancer (NSCLC), small cell carcinoma, lung adenocarcinoma, bone, colon, adenoma, pancreas, adenocarcinoma, thyroid, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, testicular tumor, melanoma, sarcoma, bladder cancer, liver cancer and biliary tract, kidney cancer, bone marrow disorders, lymphatic system disorders, hairy cell, oral cavity and pharynx (oral cavity), lips, tongue, mouth, pharynx, small intestine, colorectum, large intestine, rectum, brain and central nervous system, Hodgkin's lymphoma, and leukemia.

[0043] The present invention provides, in certain embodiments, a method of treating a disease or disorder resulting from abnormal cell growth, function, or behavior, the method comprising administering to a patient in need thereof a therapeutic regimen comprising CD200AR-L and a cancer vaccine (e.g., tumor lysate) administered sequentially. In certain embodiments, the therapeutic regimen is administered at least twice. In certain embodiments, the administration comprises administering at least three doses of the therapeutic regimen. In certain embodiments, the administration comprises administering at least five doses of the therapeutic regimen. In certain embodiments, the administration comprises administering at least ten doses of the therapeutic regimen. In certain embodiments, the administration of at least two consecutive doses is separated by an interval of about one week or about one month. In certain embodiments, the method further comprises administering an adjuvant before, simultaneously with, or after administration of the therapeutic regimen.

[0044] In certain embodiments, the present invention comprises a method of reversing or modulating immunosuppression in a patient having a disease or disorder resulting from aberrant cell growth, function or behavior, the method comprising administering to a patient in need thereof a composition or treatment regimen described above.

[0045] In certain embodiments, the present invention includes a method of reversing or modulating immune suppression in the tumor microenvironment or sentinel lymph node in a patient with a disease or disorder resulting from abnormal cell growth, function or behavior, the method comprising administering to a patient in need thereof a composition or treatment regimen as described above. In certain embodiments, the disease or disorder is cancer. In certain embodiments, the cancer is selected from glioblastoma, melanoma, prostate cancer, endometrial cancer, ovarian cancer, breast cancer, lung cancer, head and neck cancer, hepatocellular carcinoma, and thyroid cancer.

[0046] In certain embodiments, the cancer is selected from breast, ovary, cervix, prostate, testis, genitourinary tract, esophagus, larynx, glioblastoma, neuroblastoma, stomach, skin, keratoacanthoma, lung, epidermoid carcinoma, large cell carcinoma, non-small cell lung cancer (NSCLC), small cell carcinoma, lung adenocarcinoma, bone, colon, adenoma, pancreas, adenocarcinoma, thyroid, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, testicular tumor, melanoma, sarcoma, bladder cancer, liver cancer and biliary tract, kidney cancer, bone marrow disorders, lymphatic system disorders, hairy cell, oral cavity and pharynx (oral cavity), lips, tongue, mouth, pharynx, small intestine, colorectum, large intestine, rectum, brain and central nervous system, Hodgkin's lymphoma, and leukemia.

[0047] In certain embodiments, the invention provides a method, wherein delivering comprises administering the composition to the animal intravenously.

[0048] In certain embodiments, the invention provides methods, wherein the composition is administered to the animal using a systemic pump.

[0049] The term "therapeutically effective amount," with respect to treating a disease state / condition, refers to an amount of a therapeutic agent that, when administered as a single dose or in multiple doses, is capable of having any detectable positive effect on any symptom, aspect, or characteristic of the disease state / condition. Such an effect need not be absolutely beneficial.

[0050] The terms "treat" and "treatment" refer to both therapeutic treatment and preventative or prophylactic measures, the purpose of which is to prevent or reduce undesirable physiological changes or disorders. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, a stabilized (i.e., not worsening) state of disease, slowing or slowing disease progression, amelioration or alleviation (partial or total) of pathology. "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already suffering from a condition or disorder, those susceptible to suffering from a condition or disorder, or those in whom a condition or disorder is to be prevented.

[0051] Cancer Vaccines As described herein, the cancer vaccine can be a tumor antigen vaccine. A tumor antigen vaccine is a vaccine made from cancer cells (e.g., tumor lysate), parts of cancer cells, or pure tumor antigens (materials isolated from tumor cells). A tumor antigen vaccine can stimulate the body's immune system to find and kill cancer cells. For example, a cancer vaccine can include glioma cancer cells, breast cancer cells, or other solid tumor cancer cells, or parts of these cells or antigens derived from these cells. In certain embodiments, a cancer vaccine includes a vaccine antigen, where a lysate from cultured tumor cells is the source of antigen.

[0052] Peptides and peptidomimetics In certain embodiments, CD200AR-L is a peptide having a length of 12 to 15 amino acids, for example, in certain embodiments, the peptide is 12, 13, 14, or 15 amino acids in length.

[0053] In a specific embodiment, the CD200AR-L is human CD200AR-L. P1: IVTWQKKKAVSPENM (SEQ ID NO: 1) P2: NITLEDEGCYMCLFN (SEQ ID NO: 2) P3: VTFSENHGVVIQPAY (SEQ ID NO: 3) P4: CLFNTFGFGKISGTA (SEQ ID NO: 4)

[0054] In a specific embodiment, the CD200AR-L is canine CD200AR-L. P4: CLFNTFGSGKISGTA (SEQ ID NO: 5)

[0055] In a specific embodiment, the CD200AR-L is mouse CD200AR-L. P1: VTWQKKKAVSPENM (SEQ ID NO: 17)

[0056] In certain embodiments, CD200AR-L is a non-naturally occurring peptide that is not a natural product. In certain embodiments, CD200AR-L described herein comprises significantly different characteristics (e.g., structure, function, and / or other properties) compared to naturally occurring peptides corresponding to domains of CD200. The purity of the peptides is greater than 95%, and each peptide is modified by N-terminal acetylation and C-terminal amidation to improve its stability.

[0057] In certain embodiments, the CD200AR-L is peptide P1A8, P2A0, P2A5, P3A12, or P4A10. P1A8 (IVTWQKKAAVSPENM) (SEQ ID NO: 6), P2A0 (NITLEDEGCYMCLFN) (SEQ ID NO: 7), P2A5 (NITLADEGCYMCLFN) (SEQ ID NO: 8), P3A12 (VTFSENHGVVIAPAY) (SEQ ID NO: 9), or P4A10 (CLFNTFGFGAISGTA) (SEQ ID NO: 10).

[0058] In certain embodiments, CD200AR-L is a peptidomimetic. As used herein, a "peptidomimetic" is a small protein-like chain that is chemically designed to mimic a peptide. In certain embodiments, a peptidomimetic is designed to have certain modulated molecular properties, such as improved stability (e.g., in vivo half-life) or biological activity, compared to the unmodified peptide. For example, a peptidomimetic can be generated by chemically modifying the peptides described herein. Chemical modifications include, but are not limited to, including D-isomer amino acids, β3 aza-amino acids, altered backbones, and / or incorporating unnatural amino acids. For example, these strategies are based on replacing natural (L-) amino acids with unnatural (D-) amino acids or β3 aza-amino acids. Other strategies include replacing amide bonds (-CONH-) with thioamides or N-methylated amides. Other methods involve changing the N- and C-terminal amino acids to amides, esters, or D-amino acids.

[0059] Thus, in certain embodiments, CD200AR-L is engineered to contain one or more D-isomer amino acids. In certain embodiments, CD200AR-L contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more D-isomer amino acids. In certain embodiments, about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% of the amino acids are D-isomer amino acids.

[0060] In certain embodiments, CD200AR-L is engineered to include one or more unnatural amino acids (e.g., dehydroalanine, homoserine, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma carboxyglutamate, hippuric acid, octahydroindole 2-carboxylic acid, statins, 1,2,3,4,tetrahydroisoquinoline-3 carboxylic acid, penicillamine, ornithine, citrulline, alpha methylalanine, parabenzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, and tertbutylglycine). In certain embodiments, the CD200 peptide inhibitor includes 12, 13, 14, or 15 unnatural amino acids. In certain embodiments, about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the amino acids are unnatural amino acids.

[0061] Using the CD200AR-L As described herein, CD200AR-L or compositions described herein comprising CD200AR-L can be used to reverse or modulate immune suppression. CD200 is an immune suppressive protein that negatively regulates immune cells bearing CD200R (e.g., suppresses antigen-specific CD8+ T cell responses). As described herein, "reversing or modulating immune suppression" refers to altering, interfering with, or decreasing the immune suppressive properties of CD200 protein and / or tumors. In certain embodiments, CD200 protein activity is reduced by 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% in mammals compared to activity against tumors in the absence of CD200 protein. For example, immune suppressive cells such as myeloid-derived suppressor cells and regulatory T cells show increased activity, while dendritic cells (DCs) appear to be impaired in tumors and sentinel lymph nodes of cancer patients. Thus, in certain embodiments, administration of CD200AR-L or a composition described herein comprising CD200AR-L can reduce the activity of immune suppressive cells, such as myeloid-derived suppressor cells and regulatory T cells, and / or increase the activity of DCs in the tumor microenvironment and / or sentinel lymph nodes. In certain embodiments, reversal or modulation can be confirmed by assessing a cytokine profile as described herein. (For example, cytokine profiles can be determined and compared before and after administration of CD200AR-L; see also the Examples.)

[0062] As referred to herein, the "tumor microenvironment" is the normal cells, molecules, and blood vessels that surround and supply tumor cells. Tumors can alter their microenvironment, which can affect tumor growth and spread.

[0063] As described herein, CD200AR-L can enhance the efficacy of a cancer vaccine (e.g., when administered simultaneously or sequentially). For example, in certain embodiments, CD200AR-L and a cancer vaccine can be in a combined formulation (i.e., a composition described herein) or can be in separate formulations for sequential or simultaneous administration.

[0064] As used herein, "enhancing efficacy" refers to a beneficial immune response generated by administering CD200AR-L and a cancer vaccine that is greater than the beneficial immune response generated by administering the cancer vaccine alone. In certain embodiments, administration of CD200AR-L and a cancer vaccine (e.g., a composition described herein) reduces tumor size (volume) in a mammal by 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, which is greater than the reduction from administration of the cancer vaccine alone. In certain embodiments, administration of CD200AR-L and a cancer vaccine (e.g., co-administration or sequential administration) results in a synergistic effect.

[0065] Pharmaceutical Compositions The present invention also provides, in certain embodiments, a pharmaceutical composition comprising a pharma- ceutically acceptable carrier or diluent and, as an active ingredient, a composition as described herein, hi certain embodiments, the composition is formulated for oral administration or injection.

[0066] The invention also provides, in certain embodiments, the compositions described herein for use in methods of treatment of the human or animal body by therapy.

[0067] The present invention also provides, in certain embodiments, the compositions described herein for use in medical therapy.

[0068] The present invention also provides, in certain embodiments, the compositions described herein for use in the treatment of a disease or disorder resulting from abnormal cell growth, function or behavior.

[0069] The present invention also provides, in certain embodiments, the use of the compositions described herein in the manufacture of a medicament for treating a disease or disorder resulting from abnormal cell growth, function or behavior. In certain embodiments, the disease or disorder is cancer. In certain embodiments, the cancer is selected from solid tumors of the colon, breast, brain, liver, ovary, stomach, lung, and head and neck. In certain embodiments, the cancer is selected from glioblastoma, melanoma, prostate cancer, endometrial cancer, ovarian cancer, breast cancer, lung cancer, head and neck cancer, hepatocellular carcinoma, and thyroid cancer. In certain embodiments, the cancer is selected from breast, ovary, cervix, prostate, testis, genitourinary tract, esophagus, larynx, glioblastoma, neuroblastoma, stomach, skin, keratoacanthoma, lung, epidermoid carcinoma, large cell carcinoma, non-small cell lung cancer (NSCLC), small cell carcinoma, lung adenocarcinoma, bone, colon, adenoma, pancreas, adenocarcinoma, thyroid, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, testicular tumor, melanoma, sarcoma, bladder cancer, liver cancer and biliary tract, kidney cancer, bone marrow disorders, lymphatic system disorders, hairy cell, oral cavity and pharynx (oral cavity), lips, tongue, mouth, pharynx, small intestine, colorectum, large intestine, rectum, brain and central nervous system, Hodgkin's lymphoma, and leukemia.

[0070] The present invention also provides, in certain embodiments, a method of treating a disease or disorder resulting from abnormal cell growth, function or behavior, comprising administering to a patient in need thereof a composition described herein. In certain embodiments, the disease or disorder is cancer. In certain embodiments, the cancer is selected from glioblastoma, melanoma, prostate cancer, endometrial cancer, ovarian cancer, breast cancer, lung cancer, head and neck cancer, hepatocellular carcinoma, and thyroid cancer. In certain embodiments, the cancer is selected from breast, ovary, cervix, prostate, testis, genitourinary tract, esophagus, larynx, glioblastoma, neuroblastoma, stomach, skin, keratoacanthoma, lung, epidermoid carcinoma, large cell carcinoma, non-small cell lung cancer (NSCLC), small cell carcinoma, lung adenocarcinoma, bone, colon, adenoma, pancreas, adenocarcinoma, thyroid, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, testicular tumor, melanoma, sarcoma, bladder cancer, liver cancer and biliary tract, kidney cancer, bone marrow disorders, lymphatic system disorders, hairy cell, oral cavity and pharynx (oral cavity), lips, tongue, mouth, pharynx, small intestine, colorectum, large intestine, rectum, brain and central nervous system, Hodgkin's lymphoma, and leukemia.

[0071] The invention also provides, in certain embodiments, a process for making a pharmaceutical composition comprising combining a composition described herein with a pharma- ceutically acceptable carrier.

[0072] The present invention also provides, in certain embodiments, a kit for treating cancer, the kit comprising: (a) a first pharmaceutical composition comprising a composition described herein; and (b) instructions for use.

[0073] The present invention further provides a nucleic acid sequence encoding the above-mentioned CD200AR-L peptide. The nucleic acid encoding the CD200AR-L peptide can be produced using methods well known in the art (see, for example, Sambrook and Russell, 2001).

[0074] To immunize a subject, the compositions are usually administered parenterally, by intramuscular or subcutaneous injection in a suitable vehicle, although other modes of administration, such as oral, intranasal, or intradermal delivery, are also acceptable.

[0075] The vaccine formulation contains an effective amount of the active ingredient in a vehicle, the effective amount being readily determined by one of skill in the art. The active ingredient may typically be from about 1% to about 95% (w / w) of the composition, or a higher or lower range, as appropriate. The amount administered will depend on factors such as the age, weight and health of the animal or human subject being considered for vaccination. This amount will also depend on the ability of the animal's immune system to synthesize antibodies, and the degree of protection desired. Effective dosages can be readily established by one of skill in the art through routine testing to establish dose-response curves. The subject is immunized by administering one or more doses of the biofilm peptide or fragment thereof. Multiple doses can be administered as necessary to maintain an immune state against the bacteria of interest.

[0076] Intranasal formulations may include vehicles that do not cause irritation to the nasal mucosa and do not significantly impair ciliary function. Diluents such as water, saline, or other known substances may be used with the present invention. Nasal formulations may also contain preservatives, such as, but not limited to, chlorobutanol and benzalkonium chloride. Surfactants may be present to enhance absorption of the target protein by the nasal mucosa. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented in the form of tablets or products in a dry state for reconstitution with water or other suitable vehicle before use. Such liquid preparations may include conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservatives.

[0077] To prepare a vaccine, the purified composition can be isolated, lyophilized, and stabilized. The composition can then be adjusted to an appropriate concentration, optionally combined with a suitable vaccine adjuvant, and packaged for use.

[0078] Adjuvants An "adjuvant" is any molecule or compound that non-specifically stimulates humoral and / or cellular immune responses. They are considered non-specific because they only produce an immune response in the presence of an antigen. Adjuvants allow for the use of much smaller doses of antigen and are essential for inducing a strong antibody response to soluble antigens. For example, when a therapeutic agent is administered in conjunction with an adjuvant, the therapeutic agent may be administered before, after, and / or simultaneously with the adjuvant. Adjuvants are known in the art and may include, but are not limited to, CpG oligonucleotides, poly:ICLC, and imiquimod.

[0079] Methods for producing tumor lysates Tumor lysate is prepared by extracting the sample of tumor to be treated from the subject.Tumor cells are then lysed.Methods for preparing effective tumor lysate include, but are not limited to, freeze-thawing, ultrasonication, microwave, boiling, high heat, detergent or chemical cell lysis, electricity or current-based lysis, and other physical methods such as extreme force.

[0080] In certain embodiments, for example when gliomas are being treated, glioma-specific receptors (or expression vectors encoding these proteins), EGF receptor VIII variant and IL-13 receptor alpha-2, may be added to the tumor lysate.

[0081] Formulations and Methods of Administration The vaccines and compositions of the present invention can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms adapted to the selected route of administration, i.e., oral, intranasal, intradermal, or parenteral, intravenous, intramuscular, topical, or subcutaneous routes.

[0082] Thus, the compounds of the invention may be administered systemically (e.g., orally) in combination with a pharma- ceutically acceptable vehicle, such as, for example, an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the food of the patient's diet. For therapeutic oral administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be from about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0083] Tablets, troches, pills, capsules, etc. may also contain the following: binders, such as gum tragacanth, acacia, corn starch, or gelatin, excipients, such as dicalcium phosphate, disintegrants, such as corn starch, potato starch, alginic acid, etc., lubricants, such as magnesium stearate, and sweeteners, such as sucrose, fructose, lactose, or aspartame, or flavorings, such as peppermint, wintergreen oil, or cherry flavoring, may be added. When the unit dosage form is a capsule, in addition to the above-mentioned types of materials, it may contain liquid carriers, such as vegetable oil or polyethylene glycol. Various other materials may be present as coatings or to modify the physical form of the solid unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, sugar, etc. Syrup or elixir may contain the active compound, sucrose or fructose as a sweetener, methyl and propylparabens as preservatives, dyes, and flavorings such as cherry or orange flavor.Of course, any material used to prepare any unit dosage form should be pharma-ceutically acceptable and substantially non-toxic in the amounts used.In addition, the active compound may be incorporated into sustained-release preparations and devices.

[0084] The active compound may also be administered intravenously or intraperitoneally by infusion or injection. A solution of the active compound or its salt may be prepared in water, optionally mixed with a non-toxic surfactant. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof, and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0085] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions, or sterile powders containing the active ingredient, optionally encapsulated in liposomes, suitable for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions. In all cases, the final dosage form must be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or vehicle may be, for example, a solvent or liquid dispersion medium, including water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by maintaining the required particle size in the case of dispersions, or by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, such as, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0086] Sterile injectable solution is prepared by incorporating the required amount of active compound in a suitable solvent, together with various other ingredients as listed above, as necessary, and then sterilizing by filtration.For the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, obtaining the powder of active ingredient and any additional desired ingredient present in the solution previously sterilized and filtered.For topical administration, the compounds of the present invention can be applied in pure form (i.e., when they are liquid).However, it is generally desirable to administer them to the skin as compositions or preparations, in combination with dermatologically acceptable carriers, which can be solid or liquid.

[0087] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, etc. Useful liquid carriers include water, alcohol or glycol, or water-alcohol / glycol blends, in which the compounds of the present invention can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Additional ingredients such as fragrances or antibacterial agents can be added to optimize the properties for a given use. The resulting liquid composition can be applied from absorbent pads, used to impregnate bandages and other wound dressings, or sprayed onto the affected area using pump-action or aerosol sprays.

[0088] Thickening agents such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses, or modified mineral materials may also be used with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like for direct application to the skin of the user.

[0089] Examples of useful dermatological compositions that can be used to deliver the compounds of the invention to the skin are known in the art, see, e.g., Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157), and Wortzman (U.S. Pat. No. 4,820,508).

[0090] Useful dosages of the compounds of the invention can be determined by comparing their in vitro and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; see, for example, U.S. Patent No. 4,938,949.

[0091] In general, the concentration of the compound(s) of the present invention in a liquid composition, such as a lotion, is about 0.1-25% by weight, preferably about 0.5-10% by weight, and in a semi-solid or solid composition, such as a gel or powder, is about 0.1-5% by weight, preferably about 0.5-2.5% by weight.

[0092] The amount of the compound, or an active salt or derivative thereof, required for therapeutic use will vary depending not only on the particular salt selected, but also on the route of administration, the nature of the condition being treated, and the age and condition of the patient, and is ultimately at the discretion of the attending physician or clinician.

[0093] In general, however, suitable dosages are within the range of about 0.5 to about 100 mg / kg per day, for example, about 10 to about 75 mg / kg of body weight per day, for example, 3 to about 50 mg / kg of recipient body weight per day, preferably within the range of 6 to 90 mg / kg / day, and most preferably within the range of 15 to 60 mg / kg / day.

[0094] The compound is conveniently administered in unit dosage form, for example containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently 50 to 500 mg of active ingredient per unit dosage form.

[0095] Ideally, the active ingredient should be administered to achieve a peak plasma concentration of the active compound of about 0.5 to about 75 μM, preferably about 1 to 50 μM, and most preferably about 2 to about 30 μM. This may be achieved, for example, by intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1 to 100 mg of the active ingredient. Desirable blood levels may be maintained by continuous infusion to provide about 0.01 to 5.0 mg / kg / hour, or by intermittent infusions containing about 0.4 to 15 mg / kg of the active ingredient(s).

[0096] The desired dose can be conveniently presented as a single dose or as a divided dose administered at appropriate intervals, for example, two, three, four or more sub-doses per day. The sub-doses themselves can be further divided, for example, by multiple separate loosely spaced administrations, for example, multiple inhalations from an inhaler, or by applying multiple drops to the eye.

[0097] In certain embodiments, the vaccines of the present invention reduce the size of a tumor in a subject by at least about 10% to 100% (tumor volume).

[0098] definition "Attached" refers to a bond or connection that can be covalent, for example, by chemical bonding, or non-covalent (e.g., ionic interactions, hydrophobic interactions, hydrogen bonds). Covalent bonds can be, for example, esters, ethers, phosphoesters, amides, peptides, imides, carbon-sulfur bonds, carbon-phosphorus bonds, etc. The term "attached" is broader than and includes terms such as "conjugated," "coupled," "fused," and "connected."

[0099] The present invention encompasses isolated or substantially purified protein (or peptide) compositions. In the context of the present invention, an "isolated" or "purified" polypeptide is a polypeptide that exists apart from its natural environment and is therefore not a product of nature. The polypeptide may exist in purified form or may exist in a non-native environment, such as, for example, a transgenic host cell. For example, an "isolated" or "purified" protein, or a biologically active portion thereof, is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Proteins that are substantially free of cellular material include protein or polypeptide preparations that contain less than about 30%, 20%, 10%, 5% (by dry weight) of contaminating proteins. When the protein of the present invention, or a biologically active portion thereof, is produced recombinantly, preferably the culture medium represents less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or non-proteins of the chemical of interest. Fragments and variants of the disclosed proteins or partial length proteins encoded thereby are also encompassed by the invention. By "fragment" or "portion" is meant a full-length or less than full-length amino acid sequence of a polypeptide or protein.

[0100] "Naturally occurring" is used to describe an entity that can be found in nature as distinct from something that occurs artificially. For example, a protein or nucleotide sequence present in an organism (including viruses) that can be isolated from a natural source and has not been intentionally modified by humans in the laboratory is naturally occurring.

[0101] The term "substantial identity" in the context of a peptide indicates that the peptide comprises a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, at least 90%, 91%, 92%, 93%, or 94%, or 95%, 96%, 97%, 98%, or 99% sequence identity to a reference sequence over a particular comparison window. Optimal alignment is performed using the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970). An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with an antibody raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by conservative substitutions.

[0102] For sequence comparison, typically, one sequence serves as a reference sequence to which test sequences are compared.When using sequence comparison algorithm, test sequences and reference sequences are input into computer, and sequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.The sequence comparison algorithm then calculates the percent sequence identity of test sequence(s) to reference sequence based on the designated program parameters.

[0103] The term "amino acid" includes residues of natural amino acids in either the D- or L-form (e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val), as well as unnatural amino acids (e.g., dehydroalanine, homoserine, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma carboxyglutamate, hippuric acid, octahydroindole 2-carboxylic acid, statins, 1,2,3,4 tetrahydroisoquinoline-3 carboxylic acid, penicillamine, ornithine, citrulline, alpha methylalanine, parabenzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, and tertbutylglycine). The term also includes natural and unnatural amino acids with conventional amino acid protecting groups (e.g., acetyl or benzyloxycarbonyl), as well as natural and unnatural amino acids protected at the carboxy terminus (e.g., as (C1-C6) alkyl, phenyl or benzyl esters or amides, or as α-methylbenzyl amides). Other suitable amino- and carboxy-protecting groups are known to those of skill in the art (see, for example, TW Greene, Protecting Groups In Organic Synthesis, Wiley: New York, 1981, and references cited therein). The term also includes natural and unnatural amino acids with cyclopropyl or ethyl side chains.

[0104] The present invention encompasses isolated or substantially purified protein compositions. In the context of the present invention, an "isolated" or "purified" polypeptide is a polypeptide that exists apart from its natural environment. The terms "polypeptide" and "protein" are used interchangeably herein. An isolated protein molecule may exist in purified form or may exist in a non-native environment, such as, for example, a transgenic host cell or a bacteriophage. For example, an "isolated" or "purified" protein, or a biologically active portion thereof, may be substantially free of other cellular material or culture medium if produced by recombinant techniques, or may be substantially free of chemical precursors or other chemicals if chemically synthesized. Proteins that are substantially free of cellular material include protein or polypeptide preparations that contain less than about 30%, 20%, 10%, 5% (by dry weight) of contaminating proteins. In certain embodiments, an "isolated" or "purified" protein may include a cell lysate. When the proteins of the invention, or biologically active portions thereof, are recombinantly produced, preferably the culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-proteins of the chemical of interest. Fragments and variants of the disclosed proteins or partial length proteins encoded thereby are also encompassed by the invention. By "fragment" or "portion" is meant the full length or less than the full length amino acid sequence of the protein.

[0105] By "variant" polypeptide is intended a polypeptide derived from a native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminus and / or C-terminus of the native protein, deletion or addition of one or more amino acids at one or more sites in the native protein, or substitution of one or more amino acids at one or more sites in the native protein. Such variants may, for example, form genetic polymorphisms or result from human manipulation. Methods for such manipulation are generally known in the art.

[0106] Thus, the polypeptide of the present invention may be altered in various ways, including amino acid substitution, deletion, truncation, and insertion. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a polypeptide may be prepared by mutations in DNA. Methods for mutagenesis and nucleotide sequence modification are well known in the art. See, e.g., Kunkel, Proc. Natl. Acad. Sci. USA, 82:488 (1985); Kunkel et al., Meth. Enzymol., 154:367 (1987); U.S. Pat. No. 4,873,192; Walker and Gaastra, Techniques in Mol. Biol. (MacMillan Publishing Co. (1983), and references cited therein. Guidance for suitable amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al., Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found. 1978). Conservative substitutions, such as exchanging one amino acid for another with similar properties, are preferred when little or no change in biological activity is desired.

[0107] Thus, the polypeptide of the present invention encompasses naturally occurring proteins as well as their variants and modifications. Such variants will still retain the desired activity. The deletion, insertion, and substitution of the polypeptide sequence encompassed herein is not expected to cause a fundamental change in the characteristics of the polypeptide. However, in cases where it is difficult to predict the exact effect of the substitution, deletion, or insertion before doing so, those skilled in the art will understand that the effect will be evaluated by routine screening assays.

[0108] Individual substitution deletions or additions that modify, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in a coding sequence are "conservatively modified mutations" in which the modification replaces the amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following five groups each contain amino acids that are conservative substitutions for one another: aliphatic: glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I); aromatic: phenylalanine (F), tyrosine (Y), tryptophan (W); sulfur-containing: methionine (M), cysteine ​​(C); basic: arginine (R), lysine (K), histidine (H); acidic: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q). In addition, individual substitutions, deletions or additions which change, add or delete a single amino acid or a small percentage of amino acids in a coding sequence are also "conservatively modified variations."

[0109] "Operably linked" refers to an association of molecules such that the function of one is affected by the other.

[0110] As used herein, the term "therapeutic agent" or "therapeutic complex" refers to any drug or material that has a beneficial effect on a mammalian recipient. Thus, "therapeutic agent" encompasses both therapeutic and prophylactic molecules having nucleic acid or protein components.

[0111] "Treating," as used herein, refers to ameliorating at least one symptom of, curing, and / or preventing the onset of a given disease or condition.

[0112] "Immune response" refers to a humoral and / or cellular immune response that results in activation or proliferation of B lymphocytes and / or T lymphocytes and / or antigen-presenting cells. However, in some cases, the immune response may be low intensity and only becomes detectable when using at least one substance according to the invention. "Immunogenic" refers to an agent used to stimulate the immune system of an organism so that one or more functions of the immune system are increased and directed towards the immunogenic agent. "Immunogenic polypeptide" is a polypeptide that induces a cellular and / or humoral immune response alone or linked to a carrier. Preferably, antigen-presenting cells may be activated.

[0113] A substance that "boosts" an immune response refers to a substance that, when compared to the same immune response measured without the addition of the substance, results in an observed immune response that is increased, enhanced, or deviated in some way with the addition of the substance. For example, cytotoxic T cell lytic activity can be measured in samples obtained with or without the use of a substance during immunization, for example, using a 51Cr release assay. The amount of substance that enhances CTL lytic activity compared to CTL lytic activity without the substance is said to be an amount sufficient to enhance the animal's immune response to the antigen. In certain embodiments, the immune response is enhanced by at least about two-fold, for example, about three-fold or more. The amount or type of cytokine secreted can also be altered. Alternatively, the amount of antibodies or subclasses thereof induced can be altered.

[0114] The term "immunize" or "immunization" or related terms refer to conferring the ability to mount a substantial immune response (including cellular immunity, such as antibodies and / or effector CTLs) against a target antigen or epitope. These terms do not require the production of complete immunity, but rather the generation of an immune response that is substantially greater than baseline. For example, a mammal may be considered to be immunized against a target antigen if a cellular and / or humoral immune response against the target antigen occurs following application of the methods of the invention.

[0115] The term "immunotherapeutic agent" refers to a composition for treating a disease, disorder, or condition. More specifically, the term is used to refer to a therapeutic method in which a beneficial immune response is generated by vaccination or by transfer of immune molecules. An "immunologically effective amount" refers to an amount of a composition that is sufficient to induce an immune response in an individual when introduced into that individual. In the context of active immunization, the term is synonymous with an "immunogenically effective amount." The amount of a composition required to be immunologically effective varies depending on many factors, including its composition, the presence of other components in the composition, the antigen, the route of immunization, the individual, previous immune or physiological state, etc.

[0116] The invention will now be illustrated by the following non-limiting examples. EXAMPLES

[0117] Example 1 Reversal of the CD200 immune checkpoint combined with autologous tumor vaccination for glioblastoma immunotherapy. Recent advances in immunotherapy include inhibition of immune checkpoint proteins in the tumor microenvironment and tumor lysate-based vaccination strategies. Here, we combined these approaches in a glioblastoma model. Administration of a synthetic peptide targeting the immune checkpoint protein CD200 improved the ability of antigen-presenting cells to induce T cell-mediated anti-glioblastoma effects. We found that local injection of a dog-specific CD200-directed peptide prior to subcutaneous delivery of an autologous tumor lysate vaccine in spontaneously developing canine gliomas extended survival compared to historical controls treated with autologous tumor lysate alone (median survival of 12.7 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 mount an immune response in the glioblastoma microenvironment enriched for the immunosuppressive CD200 protein. These results support the use of CD200 ligand as a novel glioblastoma immunotherapeutic agent.

[0118] Immunotherapy is the third most important advance in the history of cancer treatment. The clinical success of immune checkpoint blockade with monoclonal antibodies has led to an estimated 3000 immuno-oncology clinical trials. In contrast, the relevance of immune checkpoints for tumor vaccine-based therapeutic strategies has received less attention. In principle, the presence of immune checkpoint molecules in tumor vaccines would impair the ability of local antigen-presenting cells (APCs) to activate adaptive immune responses against tumor antigens, thereby compromising the efficacy of the treatment.

[0119] During adaptive immune responses, expression of select cell surface proteins downregulates or terminates immune activation. The observation that these proteins, known as immune checkpoint molecules, are expressed in solid tumors was a seminal discovery in immuno-oncology. These molecules interact with their cognate receptors on T lymphocytes to suppress signaling required for T cell activation upon antigen presentation, thereby allowing tumors to evade subsequent immune responses. These findings underlie efforts to develop immune checkpoint inhibitors to reverse immune suppression in the tumor microenvironment.

[0120] Here, we investigate whether inhibitors of the CD200 (OX2) immune checkpoint enhance the efficacy of autologous tumor lysate vaccines against glioblastoma. Glioblastoma is the most common form of primary adult brain cancer. The prognosis for patients with glioblastoma remains poor, with a median survival of 14.6 months after invasive surgical resection, chemotherapy, and radiation therapy. Although the central nervous system (CNS) was previously thought to be an immune-privileged site, subsequent studies have revealed that innate and adaptive immune responses play a key role in the host response to glioblastoma pathogenesis. This finding has led to the investigation of tumor vaccines as a therapeutic modality for glioblastoma. In this approach, antigens from resected tumors are injected to elicit an immune response against any remaining tumor cells. However, glioblastoma expresses several immunosuppressive checkpoint molecules in the form of both cell surface and soluble proteins that neutralize the host immune response against the tumor. In addition to immune suppression in the glioblastoma tumor microenvironment, these checkpoint molecules present a formidable challenge for tumor lysate-based vaccination strategies: the presence of one such checkpoint molecule, CD200 (also known as OX2), in the lysate impairs the ability of local APCs to mount adaptive immune responses against tumor antigens.

[0121] CD200 is an immune checkpoint protein related to the B7 family of costimulatory receptors required for T cell signaling. CD200-deficient mice exhibit an autoimmune phenotype, further substantiating the role of CD200 as an immune checkpoint protein. Importantly, CD200 is expressed in a wide range of cancers, including chronic lymphocytic leukemia (CLL), multiple myeloma, acute myeloid leukemia, melanoma, ovarian tumors, metastatic small cell carcinoma, and glioblastoma. CD200 is expressed on the tumor cell surface but is cleaved by metalloproteases such as ADAM28 (A Disintegrin And Metalloprotease enzyme 28). Notably, the level of soluble CD200 in plasma correlates with tumor burden and survival in CLL patients. The physical interaction between CD200 released by cancer and its inhibitory receptor (CD200R1) on APCs (Figures 1A and 1B) suppresses the secretion of pro-inflammatory cytokines, including interleukin 2 (IL2) and interferon gamma (IFNγ), and increases the production of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), resulting in impaired antitumor immune responses.

[0122] We previously demonstrated that CD200-CD200R1 interactions are central to maintaining the immunosuppressive microenvironment of glioblastoma and developed peptides that target these interactions. Subsequent studies revealed that these peptides mediate their effects not through the inhibitory CD200R1 but through a distinct CD200 activating receptor (CD200AR) (Figures 1A and 1B). These findings suggest that CD200-mimetic peptides modulate CD200AR. Furthermore, our previous mouse experiments suggest that immune stimulation can be achieved by administering CD200-mimetic peptides as ligands (CD200AR-L) that target CD200AR. We found compelling therapeutic effects of CD200-mimetic peptides and hoped to translate these findings into the clinical setting. Due to sequence divergence between the human and mouse CD200AR-L receptors, mouse peptides were not optimal for targeting the human CD200AR. Here, we describe the development of human-specific CD200AR-L and canine-specific CD200AR-L. We show that these peptides enhance the ability of human APCs to mount antitumor responses. Importantly, local injection of canine CD200AR-L prior to administration of a subcutaneous autologous tumor lysate vaccine significantly enhanced the efficacy of this vaccine in vivo in a canine spontaneous glioblastoma model.

[0123] method Peptide synthesis. Peptides were designed based on 1) regions of the CD200 protein previously shown to interact with CD200AR and 2) sequences showing homology between mouse, dog, and human CD200. Three 15 amino acid peptides (P1, P3, and P4) and one 14 amino acid peptide (P2) were synthesized (Thermo Fisher Scientific, Rockford, IL). P1: IVTWQKKKAVSPENM (SEQ ID NO: 1) P2: NITLEDEGCYMCLFN (SEQ ID NO: 2) P3: VTFSENHGVVIQPAY (SEQ ID NO: 3) P4: CLFNTFGFGKISGTA (SEQ ID NO: 4)

[0124] In a specific embodiment, the CD200AR-L is canine CD200AR-L. P4: CLFNTFGSGKISGTA (SEQ ID NO: 5)

[0125] In a specific embodiment, the CD200AR-L is mouse CD200AR-L. P1: VTWQKKKAVSPENM (SEQ ID NO: 17)

[0126] The purity of the peptides is greater than 95%, and each peptide is modified by N-terminal acetylation and C-terminal amidation to improve its stability.

[0127] TCGA Analysis. The Cancer Genome Atlas (TCGA) data portal (tcga-data.nci.nih.gov / tcga) was used to download survival time, death events, and RNAseq data for glioblastoma patients. Gene expression data were log-scaling using Cluster 3.0 (C Clustering Library 1.52). 2 Transformation and mean centering were calculated, followed by hierarchical mean linkage clustering using Pearson similarity criteria for all genes with standard deviation >1.0. Gene clusters with dendrogram node correlation >0.20 and at least 50 individual genes were identified and associations with outcome were generated. Associations between Gene Cluster Expression Summary Scores (GCESS) of CD200R1-containing clusters and outcome are reported based on Kaplan-Meier analysis of groups generated using quartiles of GCESS values. A list of genes colocalized in cluster 14 is included as CD200R1_cluster_gene list.xls.

[0128] Cytokine measurements. Human CD14 was detected using anti-CD14 beads (BD Biosciences, San Jose, CA). + cells (5×10 5 (1000 cells) were isolated from peripheral blood mononuclear cells (PBMCs) with typical recovery rates of >70% and purity of >90%. Cells were pulsed with 2 μM CD200AR-L peptide and incubated for 48 h, after which supernatants were analyzed by bead array (BD Biosciences, San Jose, CA) for cytokine production.

[0129] Dendritic cell differentiation. Cytomegalovirus (CMV) positive HLA-A2 + Lymphocyte packs were obtained from the American Red Cross and were cloned using CD14 + The cells were purified at approximately 1 × 10 8 CD14 + Cells were incubated at 37 °C in 5% CO 2 The cells were cultured in polystyrene tissue culture flasks at 37° C. for 12 h at 4° C. Both GM-CSF (25 ng / mL) and IL-4 (40 ng / mL) were added on days 3 and 5 to induce immature dendritic cells (iDCs).

[0130] CMV assay. iDC (5 × 10 5 iDCs) were pulsed with 10 μg of CMV antigen peptide pp65495-503 (NLVPMVATV) and cultured as described above. iDCs were then washed three times and cultured with 5 × 10 5 CD8 + T cells were incubated with pulsed iDCs. CMV-negative PBMCs were used as a negative control. Supernatants were collected after 48 hours of incubation and analyzed for IFNγ production by cytometric bead array (BD Biosciences).

[0131] Nanostring Gene Expression Analysis: CD14 +Total RNA from the cells was sent to New Zealand Genomics Limited (Dunedin, New Zealand) to measure the expression of genes differentially expressed during inflammation (nCounter GX, NanoString Technologies, Seattle, WA, USA). Briefly, total RNA was analyzed for CD14 expression using a tissue-adapted protocol (KingFisher Duo machine, Thermo Fisher Scientific, Waltham, Massachusetts). + RNA was extracted from cells (MagJET RNA kit, Thermo Fisher Scientific, Waltham, Massachusetts). RNA samples were then quantified (Qubit® 2.0 Fluorometer, Thermo Fisher Scientific) and subjected to RNA integrity analysis (2100 Bioanalyzer, Agilent Technologies, Santa Clara, CA). Probes for genes encoding CD44 (NM_001001392.1), NANOG (NM_024865.2), OCT4 (NM_002701.4), STAT3 (NM_139276.2), and the housekeeping genes glucuronidase beta (NM_000181.1), clathrin heavy chain (NM_4859.2), and hypoxanthine phosphoribosyltransferase 1 (NM_000194.1) were designed and manufactured by NanoString Technologies.

[0132] Expression data obtained with NanoString GX were analyzed using nSolver Analysis Software 3.0 (nanostring.com / products / nSolver) with default settings and normalized to housekeeping genes. Cluster analysis was performed using nSolver and heat maps were generated by using Java Treeview Version 1.1.6r4. Pathway analysis was performed using the PathCards Pathway Unification Database (pathcards.genecards.org). Student's t-test was used to determine significant differences (p<0.05).

[0133] Dog Study. Pet dogs with solitary intraaxial masses found by magnetic resonance imaging (MRI) were recruited in a pilot study to evaluate the effect of the dog-specific peptide. MRI was performed to find the cause of generalized seizure activity or behavioral changes, and antiepileptic drug therapy and corticosteroids were prescribed to control seizures and minimize cerebral edema, respectively. Additional inclusion criteria were: 1) tumor suitable for surgical resection, 2) informed consent provided by the dog's owner, 3) normal mental status at the time of surgery, and 4) no previous causal therapy for suspected glioma.

[0134] All dogs underwent surgical resection of the intraaxial mass for maximal tumor debulking. The excised tumors were immediately processed to generate autologous tumor lysates. Single cell suspensions from freshly minced tumor samples were cultured in 5% O for 1 h. 2Lysates were prepared by incubation at 37°C in 1000 mL of PBS. Cultured tumor cells were lysed by multiple freeze-thaw cycles and irradiated with 20 Gy. MRI was performed immediately after surgery to assess the extent of resection and to quantify the residual tumor volume. Tumor volume was measured using planimetry to outline the tumor periphery on each MR slice. Areas were calculated using Osirix software, and then areas were summed and multiplied by slice thickness + gap width. After recovery from anesthesia, all dogs were monitored for at least 24 hours in the intensive care unit. Antiepileptic drugs (phenobarbital, levetiracetam, and zonisamide, alone or in combination) were continued postoperatively, but corticosteroid administration was gradually tapered and discontinued within 10–14 days.

[0135] The first vaccination was administered 10 days after surgery, weekly for 3 weeks, then once every 4 weeks for 3 months, and then repeated every 6-8 weeks until tumor progression or death (Figure 9). The vaccine protocol was as follows: Canine-specific CD200AR-L peptide (5 μg / kg) was injected intradermally (ID). 24 hours later, imiquimod (1 packet 5% cream / 12.5 g) was applied topically to the skin and allowed to absorb for 10-15 minutes before autologous tumor lysate (~500 μg protein) mixed with CD200AR-L (5 μg / kg) was injected intradermally. Disease status was monitored by MRI and physical examination at 4, 8, and 12 months after surgery, and thereafter as needed for suspected tumor recurrence.

[0136] Surgical resection was defined as total resection if there was no residual contrast enhancement for gadolinium-enhancing lesions or no hyperintensity on T2-weighted FLAIR (fluid-attenuated inversion recovery) for nonenhancing lesions, near-total resection if less than 10% of the original tumor volume remained, or partial resection if there was more than 10% residual tumor volume. Clinical response on follow-up MRI was considered complete response (CR) if there was no evidence of tumor, partial response (PR) if there was a reduction in tumor volume of 65% or more, progressive disease (PD) if there was an increase in tumor volume of more than 40%, and stable disease if the response did not qualify as CR, PR, or PD as defined above. If dogs presented with recurrent or worsening neurological signs prior to the scheduled MRI, an MRI was performed.

[0137] result Design and Identification of Inhibitory Peptides Against CD200AR-L. Previous analysis of the regions of CD200 that interact with CD200AR-L revealed four regions that share significant homology between human, dog, and mouse (Figure 2a). We generated four CD200AR-L peptides, designated P1-4, that correspond to these four regions in human CD200. To determine whether these peptides would activate APCs, we used purified human CD14 peptides, as previously observed for mouse CD200 mimetic peptides. + Cells were pulsed with four different CD200AR-L peptides and the supernatants were analyzed for immune stimulatory cytokines (Fig. 2b, c). CD14 cells pulsed with P1, P2, P3, and P4, respectively, showed significantly higher levels of cytokines compared to unpulsed controls. +A statistically significant increase in IL-1β (p=0.0126, p=0.364, p=0.0022, p=0.0008) and TNFα (p=0.0146, p=0.0007, p=0.0022, and p=0.0082) was observed in the cells. To determine whether these peptides generate antigen-specific responses, a CMV model was used in which T cells from CMV-positive donors were primed with the CMV antigen pp65. Pulsing immature dendritic cells with the CMV antigen pp65+CD200AR-L peptide induced a significant antigen-specific response, i.e., the production of IFNγ, compared to pp65 alone (P1: p=0.034, P2: p=0.033, P3: p=0.0042, and P4: p=0.0202, Fig. 2d).

[0138] To generate peptides that maximally stimulate APCs, each amino acid in the four peptides was replaced with alanine, resulting in 61 alanine-substituted peptides. + Cells were pulsed with these peptides and the response was measured as cytokine release. + Five peptides that maximally stimulated the secretion of inflammatory cytokines from cells were selected: P1A8 (P1A8 = P1 peptide with an alanine substituted at the eighth amino acid), P2A0 (P1A0 = P2 peptide with no alanine substitution), P2A5, P3A12, and P4A10.

[0139] These peptides then bind to CD14 + The effect of these peptides on a broader set of immune stimulatory cytokines (including IL-12p70, MIG, and TNF) after they were used to pulse cells was analyzed (Figure 3a). Significant cytokine induction was observed after treatment with each of the five peptides. CD14 +To characterize the effects of these peptides on cells, RNaseq was performed. Treatment with P1A8, P2A0, or P4A10 induced a significant increase in mRNA expression of cytokines released in response to TNF signaling (Table 1, Figure 3a), consistent with our previous results that CD200 exposure suppresses TNF signaling in APCs and that a CD200 mimetic reverses this effect. These results were replicated using the NanoString platform designed to detect mRNA expression of TNF-regulated cytokines (Figure 3b). The three peptides (P1A8, P2A0, and P4A10) that most potently and consistently induced mRNA expression of TNF-regulated cytokines were selected for subsequent analysis. [Table 1-1] [Table 1-2]

[0140] Targeting the CD200 activating receptor enhances dendritic cell maturation. TNF-regulated cytokines (Table 1) are involved in dendritic cell maturation. Thus, the RNAseq results (Figure 3b) suggest that P1A8, P2A0, and P4A10 contribute to dendritic cell maturation. To demonstrate this, we isolated CD14 + Cells were isolated and pulsed with GM-CSF+IL4, P1A8, P2A0, or P4A10 27. Cells pulsed with P1A8, P2A0, and P4A10 expressed higher levels of the costimulatory molecules CD80, CD86, and HLA-DR than cells treated with GM-CSF+IL4 (p<0.0001, Fig. 4a). Furthermore, we observed synergistic upregulation of CD80 / 86 and HLA-DR when CD14+ cells were incubated with GM-CSF+IL4 and each peptide (p<0.0001) (Fig. 4b). These results suggest that P1A8, P2A0, and P4A10 enhanced the differentiation of CD14+ monocytes into iDCs.

[0141] To evaluate the effect of P1A8, P2A0, and P4A10 on antigen-specific responses, IFNγ release was measured in the following assay. iDCs were pulsed with the CMV antigen pp65 with or without equimolar amounts of P1A8, P2A0, or P4A10. Autologous T cells were then added and incubated with iDCs for 48 h, after which IFNγ production was measured in the supernatant. Exposure to pp65 increased T cell IFNγ production approximately 4-fold (Figure 4c). Co-treatment of iDCs with pp65 and P1A8, P2A0, or P4A10 increased IFNγ release an additional 2- to 3-fold over pp65 alone. These data indicate that P1A8, P2A0, or P4A10 enhanced the ability of dendritic cells to induce antigen-specific responses from human T cells. Treatment of iDCs with P4A10, the most potent of the three peptides, enhanced T cell secretion of IFNγ, IL6, TNFα, and RANTES by 5.4-, 5.6-, 16.6-, and 16.3-fold, respectively. A scrambled peptide control failed to enhance pp65 responses (Figure 4d). These results suggest that P4A10 enhances the ability of APCs to induce T cell-mediated immune responses.

[0142] CD200AR-L peptide enhances the efficacy of tumor lysate vaccination in a spontaneous canine glioma model. Our laboratory has a long-standing interest in the development of autologous tumor lysate vaccination as a strategy to treat glioblastoma. We hypothesize that the presence of immune checkpoint proteins in such lysates may impair the efficacy of this therapy. Because the human P4A10 peptide shares high homology with the canine peptide (Figure 2a), we hypothesized that subcutaneous injection of canine CD200AR-L prior to the introduction of autologous tumor lysate would enhance vaccine efficacy in a canine model of spontaneous glioblastoma. To test this hypothesis, 21 dogs with spontaneous glioblastoma were treated with tumor resection and vaccination consisting of canine CD200AR-L + autologous tumor lysate (Figure 9). Tumor progression and overall survival were compared to historical data from a previous cohort of dogs treated with autologous tumor lysate alone after tumor resection. The extent of resection for these two cohorts was comparable. A 25-month progression-free survival rate of 67% was observed in dogs receiving canine CD200AR-L (Figure 5a). Death was due to tumor recurrence in 33% (7 / 21) of dogs, and 33% (7 / 21) of dogs died from causes other than tumor progression or were euthanized. Two dogs without postmortem examination were presumed to have died from tumor progression. In contrast, tumor progression was the cause of death in 100% (15 / 150) of dogs in the tumor lysate only treated group. The median survival time for dogs treated with canine CD200AR-L administration prior to autologous tumor lysate vaccination was 12.7 months. This survival time compared favorably to a survival time of 6.36 months for dogs treated with tumor lysate vaccination alone (Figure 5a). One dog with tumor recurrence 18 months (548 days) after surgery was treated with a second surgery followed by autologous tumor lysate vaccine co-administered with canine CD200AR-L and survived an additional 9.93 months for a total of 28 months. This dog was not included in Figure 5a.

[0143] Interestingly, six dogs had up to 50% residual tumor after surgery, but tumor regression was seen on MRI obtained 4 months after coadministration of CD200AR-L and tumor lysate vaccine (Figure 5b, c). Five dogs developed cerebral leukoencephalopathy characterized by T2 hyperintensities of periventricular white matter tracts and ventricular dilatation. However, these signs disappeared after corticosteroid treatment (Figure 5d and e). In one dog with severe clinical signs, vaccine therapy was also discontinued, and although the T2 hyperintensities disappeared, tumor recurrence was noted on MRI performed 2 months later (Figure 5f-5i). Vaccine and peptide therapy was resumed when the dog recovered, and tumor regression was again seen at 2 months (Figure 5J-5M).

[0144] Because serum CD200 levels have previously been shown to be associated with tumor burden and overall survival in ependymoma patients, we collected and analyzed serum from 21 canine subjects. Consistent with previous studies, we found a significant association between serum CD200 levels and overall survival (Fig. 5n). Complete blood counts and serum chemistry profiles revealed no obvious evidence of treatment-related adverse effects. These data suggest the potential utility of serum CD200 as a companion biomarker for P4A10-based therapeutic strategies.

[0145] T lymphocytes primed with P4A10 downregulate the expression of CD200R1. To investigate whether CD200-associated genes are associated with survival, we used GCESS24 to analyze gene expression profiles of patient tumor samples in the TCGA dataset. This allowed us to identify a gene cluster that was consistently expressed across the dataset and associated with overall survival in an unbiased statistical analysis. CD200R1 expression was found within a large cluster of genes highly enriched for immune-related transcripts. Increased transcript levels of genes within this cluster were significantly associated with reduced survival (Figures 6a, 6e). Patients who expressed high levels of the CD200R1-containing cluster (cluster 14) showed shorter overall survival times than patients who expressed low levels of the cluster (Figures 6b-d). These results suggest the critical importance of CD200 / CD200R1 interactions in mediating an immunosuppressive glioblastoma microenvironment.

[0146] The efficacy of the P4A10+ autologous tumor lysate strategy in this context raises an interesting and challenging problem. T cells that respond to APC presentation after stimulation express the inhibitory CD200R129. Thus, the immunostimulatory effect of these primed T cells is expected to be blunted upon entering the glioblastoma microenvironment, which is rich in soluble and tumor-expressed CD200. However, we found that CD200AR-L priming prior to antigen presentation to T cells suppressed the expression of inhibitory CD200R1 in both APCs (p=0.0001) and T cells (p=0.009) in our mouse model (Fig. 7a, b), thereby rendering these cells resistant to the effects of CD200 in the glioblastoma tumor microenvironment. Supporting this hypothesis, we found that P4A10 priming prior to antigen presentation suppressed CD200R1 expression (Fig. 7d). Such suppression was not observed in mock-treated cells. Similar downregulation of PD-1 was observed in APCs (p=0.005, FIG. 7c). These results indicate that immune suppression in the CNS can be overcome through immune modulation occurring outside the CNS.

[0147] Further experiments were performed in which human CD14 cells were pulsed with human CD200AR-L (P1A8) and non-pulsed cells were used as a control. Cells were isolated and analyzed for levels of PD-1 by flow cytometry (Fig. 7e). Mean fluorescence intensity was analyzed for CD200R1 (Fig. 7f) and PD-1 expression (Fig. 7g). These experiments demonstrated that CD200AR-L downregulates the inhibitory PD-1 receptor (Fig. 7g).

[0148] Consideration Most immune checkpoint inhibitors are developed with the goal of reversing immune suppression in the tumor microenvironment. Here, we explored an alternative paradigm to explore the efficacy of immune checkpoint reversal at the site of autologous tumor vaccination. Our previous work provided compelling data that the presence of CD200 immune checkpoint protein in tumor lysates suppresses the ability of local APCs to activate recruited T cells and elicit effective antitumor immune responses. Based on this observation, we investigated whether P4A10, a peptide that targets CD200-mediated immune suppression, could reverse the immunosuppressive effects of CD200. Our results showed that P4A10 enhances the ability of APCs to induce antigen-specific responses from human T cells. Notably, CD200AR-L priming prior to antigen presentation to T cells suppresses the expression of inhibitory CD200R1, thereby rendering these cells resistant to the effects of CD200 in the glioblastoma tumor microenvironment. Intradermal injection of these immune stimulatory molecules minimizes the potential for systemic toxicity associated with systemic administration of immune modulators.

[0149] Mouse brain tumor models have provided valuable insights into the pathogenesis of glioblastoma, but therapies that show great promise in these models often fail in clinical translation. Without consensus on the optimal therapeutic model, several expert panels have been convened by the United States National Institutes of Health to address these failures. In the absence of such consensus, therapeutic testing in animals other than mice must be considered before designing clinical trials. Our canine studies are particularly relevant in this context. Glioblastoma frequently occurs naturally in certain breeds of dogs, including boxers, French bulldogs, and English bulldogs. This naturally occurring canine "model" is an attractive platform for characterizing the immunological effects of CD200. 1) the tumors display histological features very similar to those observed in human glioblastoma, 2) the tumors arise and grow in an immune microenvironment without experimental manipulation, 3) there is considerable similarity in immune cell populations in histopathological samples of human and canine glioblastoma, 4) like human glioblastoma, canine glioblastoma is typically detected at a late stage when tumor burden causes neurological deficits, and 5) there is significant homology between human and canine CD200. Taken in this context, the efficacy of canine CD200AR-L in the canine model provides strong support for future human clinical trials.

[0150] In current clinical practice, the assessment of treatment response is mainly based on the evaluation of glioblastoma appearance on MRI. This practice is problematic because MRI is costly and the resolution of MR images is insufficient to detect tumor growth at the cellular level. Therefore, there is a critical need for minimally invasive biomarkers to assess glioblastoma growth. In many embodiments, the biological properties of CD200 make it well suited as a biomarker of tumor burden. Although CD200 is a cell surface glycoprotein, most of the CD200 expressed in glioblastoma is released as soluble factor 10. An independent group, including the data presented in this manuscript, determined that the level of soluble CD200 in the serum of cancer patients correlates with tumor burden, which was supported by the data presented in this manuscript (Figure 5n). Overall, these results support serum CD200 as a potential "liquid biopsy" platform for glioblastoma monitoring.

[0151] In summary, our studies provide data supporting the therapeutic efficacy of subcutaneous administration of immunostimulatory peptides directed at interfering with the biological function of the CD200 immune checkpoint prior to tumor vaccination as a novel therapy for glioblastoma.

[0152] Example 2 CD200AR-L subjugates PD-L1 inhibitory signals. In these experiments, mouse CD11b cells were stimulated with PMA and pulsed with PD-L1 with or without mouse CD200AR-L. These experiments demonstrated that PD-L1 suppresses the activation of CD11b cells, but that CD200AR-L overcomes the inhibitory effect of PD-L1 (Figures 8A-8C).

[0153] Example 3 Effect of inhibitory molecules (CD200 and PD-L1) on T cell inhibition We propose that soluble CD200 and PD-L1 inhibit T cell activation through multiple checkpoints (Figure 10). Therefore, we investigated the effect of multiple inhibitory molecules (CD200 and PD-L1) on T cell inhibition. To achieve this, we pulsed whole splenocytes with CD200 or PD-L1 and demonstrated upregulation of CTLA4 on CD4 cells after CD200 treatment. In addition, upregulation of PD-L1 on CD11b and upregulation of CTLA4 on T cells was observed after PD-L1 treatment. Since CTLA4 and PD-L1 propagate distinct inhibitory signals, we hypothesized that PD-L1 inhibits T cell activation via 1) direct CD200 and PD-L1 on T cells, and 2) direct PD-L1 (on APC) / PD-I (on APC) producing cytokines that upregulate CTLA4, explaining the enhanced response seen with the combination of α-PD-1 and α-CTLA4 treatment. However, none of them inhibit the effect of CD200 on T cells.

[0154] CD200 and PD-1 pathways crosstalk, allowing CD200AR-L to override the inhibitory PD-L1. We established that treatment of animals with mouse P1A12 (VTWQKKKAVSPANM, SEQ ID NO: 15) and canine P4A10 CD200AR-L (CLFNTFGSGAISGTA, SEQ ID NO: 16) resulted in significant survival benefit in both mouse and canine glioma models. These results led to the hypothesis that CD200 and PD-1 may share a common signaling pathway. To assess the effect of CD200AR-L on PD-1 / PD-L1, splenocytes were activated with PMA+ / -PD-L1 or PD-L1+CD200AR-L and supernatants were analyzed for TNFα, IL-4, and IL-5 production (Figures 11A-11C). As observed with CD200 protein (Figure 11B), PD-L1 induced an immunosuppressive environment as measured by cytokine production, but PD-L1 reduced PMA-induced production of TNFα, IL-4, and IL-5, which was restored by addition of CD200AR-L. These experiments led to the hypothesis that the CD200 and PD-1 pathways are connected.

[0155] CD200AR-L downregulates the expression of CD200R1 and PD-1. To further explore the mechanism by which CD200AR-L prevents PD-L1 inhibition, CD11b cells were pulsed with CD200AR-L and the transcription levels of CD200R1 and PD-1 were analyzed (Figures 12A-12B). Both inhibitory receptors were downregulated by CD200AR-L. In addition, pulsing human CD14 cells with human CD200AR-L resulted in the same inhibition (Figures 12C-12D). CD11b cells from wild-type mice were pulsed with ovalbumin (OVA) containing the well-characterized protein SIINFEKL (SEQ ID NO: 14) to elucidate antigen-specific responses. OVA+ / -CD200AR-L was washed off and CD3 cells were added to CD11b cells. These experiments demonstrated that CD200AR-L can regulate the expression of CD200R1 and PD-1 on antigen-presenting cells (CD11b and human CD14) and T cells, indicating that different checkpoints are interconnected.

[0156] CD200AR-L downregulates the expression of CD200R1 and PD-1. To further explore the mechanism by which CD200AR-L prevents PD-L1 inhibition, CD11b cells were pulsed with CD200AR-L and the transcription levels of CD200R1 and PD-1 were analyzed (Figures 13A-13B). Both inhibitory receptors were downregulated by CD200AR-L. In addition, pulsing human CD14 cells with human CD200AR-L produced the same inhibition (Figures 13C-13D). CD11b cells from wild-type mice were pulsed with ovalbumin (OVA) containing the well-characterized protein SIINFEKL (SEQ ID NO: 14) to elucidate antigen-specific responses. OVA+ / -CD200AR-L was washed off and CD3 cells were added to CD11b cells. These experiments demonstrated that CD200AR-L can regulate the expression of CD200R1 and PD-1 on antigen-presenting cells (CD11b and human CD14) and T cells, indicating that different checkpoints are interconnected.

[0157] CD200AR-L inhibits CTLA-4 upregulation. Next, we examined the effect of CD200AR-L on T cell inhibition. In these experiments, splenocytes were pulsed with CD200 or PD-L1, revealing upregulation of CTLA4 on CD4 cells, but not on CD8 cells (Figure 14A). In contrast to CD200, PD-L1 upregulated CTLA4 on both CD4 and CD8 T cells (Figure 14A). Furthermore, PD-L1, but not CD200, upregulated the expression of PD-L1 on CD11b cells (Figure 14C), which may be mediated through soluble PD-L1 / PD-1 (on CD11b) interaction. Because CTLA4 and PD-L1 propagate distinct inhibitory signals, it is hypothesized that PD-L1 inhibits T cell activation by two distinct mechanisms: 1) through direct PD-L1 (on APCs) / PD-I (on T cells) interaction and through cytokine production following binding of PD-L1 to PD-1 on CD11b cells, which would explain the enhanced responses seen with combined α-PD-1 and α-CTLA4 therapy.

[0158] While the above specification and examples fully disclose and enable the present invention, they are not intended to limit the scope of the invention, which is defined by the claims appended hereto.

[0159] All publications, patents, and patent applications are incorporated herein by reference. In the foregoing specification, the invention has been described with respect to specific embodiments thereof, and numerous details have been set forth for purposes of illustration, but it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that the specific details described herein may be modified considerably without departing from the underlying principles of the invention.

[0160] In the context of describing the present invention, the use of the terms "a" and "an" as well as "the" and similar referents should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including but not limited to"), unless otherwise indicated. The recitation of ranges of values ​​herein is intended merely to serve as a shorthand method of individually referring to each individual value falling within the range, unless otherwise indicated herein, and each individual value is incorporated herein as if it were individually recited herein. All methods described herein may be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "for example") provided herein is intended merely to better illuminate the invention, and does not limit the scope of the invention unless specifically claimed otherwise. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0161] Embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect that those skilled in the art will employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context. In certain embodiments, for example, the following are provided: (Item 1) A method of inhibiting PD-1 in a cell, comprising or consisting of administering to said cell a CD200 activating receptor ligand (CD200AR-L). (Item 2) The method of claim 1, wherein the CD200AR-L is a human-specific peptide (hCD200AR-L). (Item 3) The method of claim 1, wherein the CD200AR-L is a canine-specific peptide (cCD200AR-L). (Item 4) 4. The method according to any one of items 1 to 3, wherein the cells are cancer cells. (Item 5) 5. The method of claim 4, wherein the cancer cells are glioblastoma cells. (Item 6) The method according to any one of items 1 to 5, wherein the CD200AR-L is peptide P1A8, P2A0, P2A5, P3A12, or P4A10. (Item 7) 6. The method according to any one of items 1 to 5, wherein said CD200AR-L is peptide human P1A8. (Item 8) 6. The method according to any one of items 1 to 5, wherein said CD200AR-L is peptide human P2A0. (Item 9) 6. The method according to any one of items 1 to 5, wherein said CD200AR-L is peptide human P4A10. (Item 10) 10. The method according to any one of items 1 to 9, wherein the cells are cancer cells. (Item 11) 11. The method of claim 10, wherein the cancer cells are glioblastoma cells. (Item 12) A method for increasing the effectiveness of a tumor lysate vaccine in a mammal, comprising or consisting of administering a CD200 activating receptor ligand (CD200AR-L) to the mammal prior to administration of the tumor lysate vaccine. (Item 13) The method of claim 12, wherein the CD200AR-L is a human-specific peptide (hCD200AR-L). (Item 14) The method of claim 12, wherein the CD200AR-L is a canine-specific peptide (cCD200AR-L). (Item 15) 15. The method according to any one of items 12 to 14, wherein the cells are cancer cells. (Item 16) 6. The method of claim 5, wherein the cancer cells are glioblastoma cells. (Item 17) 17. The method of any one of items 12 to 16, wherein the CD200AR-L is peptide P1A8, P2A0, P2A5, P3A12, or P4A10. (Item 18) 17. The method according to any one of items 12 to 16, wherein said CD200AR-L is peptide human P1A8. (Item 19) 17. The method according to any one of items 12 to 16, wherein said CD200AR-L is peptide human P2A0. (Item 20) 17. The method according to any one of items 12 to 16, wherein said CD200AR-L is peptide human P4A10. (Item 21) 21. The method according to any one of items 12 to 20, wherein the cells are cancer cells. (Item 22) 22. The method of any one of items 12 to 21, wherein the CD200AR-L is administered by local injection. (Item 23) 23. The method according to any one of items 12 to 22, wherein the tumor lysate vaccine is administered subcutaneously. (Item 24) 24. The method according to any one of items 12 to 23, wherein the tumor lysate vaccine is an autologous tumor lysate vaccine. (Item 25) 25. The method of any one of items 12 to 24, wherein the mammal is administered the CD200AR-L 5 to 14 days after surgical removal of a tumor. (Item 26) 25. The method of any one of items 12 to 24, wherein the mammal is administered the CD200AR-L about 10 days after surgical removal of a tumor. (Item 27) 24. The method according to any one of items 12 to 23, comprising: (a) intradermally (ID) injection of a species-specific CD200AR-L peptide; (b) 24 hours later, topically applying imiquimod to the skin and allowing it to absorb for 10 to 15 minutes; and (c) intradermally injecting autologous tumor lysate mixed with CD200AR-L. (Item 28) 28. The method of claim 27, wherein the species-specific CD200AR-L peptide is injected intradermally (ID) at a dosage of about 5 μg / kg. (Item 29) 29. The method according to item 27 or 28, wherein the imiquimod (1 packet) is applied topically to the skin in a dosage of 5% cream / 12.5g. (Item 30) 30. The method according to any one of items 27 to 29, wherein about 500 μg protein of the autologous tumor lysate is mixed with about 5 μg / kg CD200AR-L and injected intradermally. (Item 31) 31. The method of any one of items 27 to 30, wherein the treatment regimen is repeated weekly for three weeks. (Item 32) 32. The method of claim 31, wherein the treatment regimen is repeated weekly for 3 weeks, then once every 4 weeks for 3 months, and then every 6 to 8 weeks as needed.

Claims

1. A pharmaceutical composition for use in the treatment of cancer, comprising (i) a pharma- ceutically acceptable carrier or diluent and (ii) a peptide, wherein the peptide is P1A8 (SEQ ID NO: 6), P2A5 (SEQ ID NO: 8), P3A12 (SEQ ID NO: 9), or P4A10 (SEQ ID NO: 10).

2. The pharmaceutical composition for use according to claim 1 , wherein the cancer is glioblastoma.

3. 3. The pharmaceutical composition for use according to claim 1 or 2, wherein the peptide is P1A8 (SEQ ID NO: 6).

4. 3. The pharmaceutical composition for use according to claim 1 or 2, wherein the peptide is P2A5 (SEQ ID NO: 8).

5. 3. The pharmaceutical composition for use according to claim 1 or 2, wherein the peptide is P3A12 (SEQ ID NO: 9).

6. 3. The pharmaceutical composition for use according to claim 1 or 2, wherein the peptide is P4A10 (SEQ ID NO: 10).

7. A composition for use in enhancing the effectiveness of a tumor lysate vaccine in a mammal, said composition comprising a peptide, said peptide being P1A8 (SEQ ID NO:6), P2A5 (SEQ ID NO:8), P3A12 (SEQ ID NO:9), or P4A10 (SEQ ID NO:10), and said composition is for administration to the mammal prior to the tumor lysate vaccine.

8. The composition of claim 7, wherein the peptide is P1A8 (SEQ ID NO: 6).

9. The composition of claim 7, wherein the peptide is P2A5 (SEQ ID NO: 8).

10. The composition of claim 7, wherein the peptide is P3A12 (SEQ ID NO: 9).

11. The composition of claim 7, wherein the peptide is P4A10 (SEQ ID NO: 10).

12. The composition of any one of claims 7 to 11, wherein the composition is administered by local injection.

13. The composition of any one of claims 7 to 12, wherein the tumor lysate vaccine is administered subcutaneously.

14. The composition of any one of claims 7 to 13, wherein the tumor lysate vaccine is an autologous tumor lysate vaccine.

15. The composition of any one of claims 7 to 14, wherein the composition is for administration 5 to 14 days after surgical removal of a tumor in the mammal.

16. The composition of any one of claims 7 to 14, wherein the composition is for administration about 10 days after surgical removal of a tumor in the mammal.

17. said composition being mixed with said tumor lysate vaccine and injected intradermally 24 hours prior to topical application of imiquimod to the skin; The composition of any one of claims 7 to 13, wherein the tumor lysate vaccine is an autologous tumor lysate vaccine and imiquimod is allowed to absorb for 10 to 15 minutes.

18. The composition of claim 17, wherein the composition is for intradermal injection at a dosage of about 5 μg / kg of peptide.

19. 19. The composition of claim 17 or 18, wherein the imiquimod is for topical application to the skin in a dosage of 5% (w / w) cream / 12.5g.

20. The composition of any one of claims 17 to 19, wherein the autologous tumor lysate vaccine comprises about 500 μg of protein.

21. The composition of any one of claims 17 to 20, wherein the intradermal injection and topical application are performed weekly for three weeks.

22. 22. The composition of claim 21, wherein the intradermal injection and topical application is performed weekly for 3 weeks, then once every 4 weeks for 3 months, then once every 6-8 weeks.

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