Treating gliomas with calcitonin gene-related peptide antagonist

Abstract

Disclosed herein are methods for treating gliomas in a subject, that involve administering to the subject a therapeutically effective amount of a calcitonin gene-related peptide (CGRP) antagonist. For example, the method can involve treating the subject with checkpoint immunotherapy, determining that the subject is refractory to the checkpoint immunotherapy, and then administering to the subject a therapeutically effective amount of the CGRP.

Description

TREATING GLIOMAS WITH CALCITONIN GENE-RELATED PEPTIDE ANTAGONISTCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 583,404, filed September 18, 2023, and U.S. Provisional Application No. 63 / 565,187, filed March 14, 2024, which are hereby incorporated herein by reference in their entireties.BACKGROUND OF THE INVENTION

[0002] Gliomas are the most common type of central nervous system tumor in adults. Low-grade glioma (LGG) is classified into grade 2 and grade 3 with isocitrate dehydrogenase (IDH) mutations occurring in the majority of these tumors. Although the prognosis of LGG is relatively better than that of high-grade gliomas (HGGs), nearly 70% of LGG patients are prone to eventually develop into Grade 4 tumors which are characterized by higher malignancy and invasiveness. The standard of care for LGG includes surgical resection, radiotherapy, and chemotherapy. However, the existing treatments still fail to significantly improve the survival rate of patients who transition to HGGs. Several studies suggest that changes in the tumor microenvironment (TME), especially in the immune compartment may play a fundamental role in cancer development. Thus, changes in the immune compartment may also play a role in facilitating the transition from LGG to HGG. Indeed, a study confirmed that changes in the molecular determinant of the immune cells are associated with the prognosis of LGG.

[0003] Glioblastoma, high grade glioma (grade IV) is the most prevalent primary malignant brain tumor in adults. Despite maximal surgical resection and chemo-radiation, the prognosis remains poor, glioblastoma patients remain refractory to immune checkpoint blockade therapy (ICBs). Immune suppressive networks of tumor-associated macrophages and microglia, which constitute the majority of the immune population in glioblastoma tumor microenvironment (TME), impede immunotherapy. Investigation of the cross-talk between various cell types and immune cells in the TME is critical for understanding these immune suppressive circuits..SUMMARY OF THE INVENTION

[0004] Neurons are a unique and critical component of glioblastoma TME. Although, neuro-immune interactions play a fundamental role in shaping immune responses in various other tissue types the role of such communications in regulating anti-tumor immune responses is only beginning to be uncovered. A recent study indicated that sensory neurons induce T cell exhaustion in melanoma. It was hypothesize here that CGRP produced by the neurons in the TME of glioblastoma acts locally on the immune cells that express CGRPreceptors and these receptor-ligand interactions shape the immune-landscape of glioblastoma.

[0005] Therefore, disclosed herein is a method for treating gliomas, such as a glioblastoma or low-grade glioma (LGG), in a subject that involves administering to the subject a therapeutically effective amount of a calcitonin gene-related peptide (CGRP) antagonist.

[0006] In some embodiments, the CGRP antagonist blocks the binding of CGRP to Receptor Activity Modifying Protein 1 (RAMP1). For example, in some embodiments, the CGRP antagonist is an antibody or antibody fragment that binds CGRP or RAMP1 . In some embodiments, the CGRP antagonist is a bi-specific antibody or antibody fragment that binds PD-1 and CGRP or RAMP1 . In some embodiments, the CGRP antagonist is selected from the group consisting of Erenumab (AMG-334), Eptinezumab (ALD403), Fremanezumab (TEV-48125), and Galcanezumab (LY2951742). In some embodiments, the CGRP antagonist is selected from the group consisting of Ubrogepant, Rimegepant (BMS-927711), Atogepant (AGN-241689), Telcagepant (MK-0974), Olcegepant (BIBN-4096BS), Bl 44370, MK-3207, and SB-268262.

[0007] In some embodiments, the subject is refractory to checkpoint immunotherapy, such as an anti-PD-1 immunotherapy, anti-PD-L1 immunotherapy, anti-CTLA-4 immunotherapy, or a combination thereof. In some embodiments, the method further involves administering to the subject a checkpoint inhibitor, such as an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, or a combination thereof.

[0008] In some embodiments, the method involves treating the subject with checkpoint immunotherapy; determining that the subject is refractory to the checkpoint immunotherapy; and administering to the subject a therapeutically effective amount of the CGRP antagonist.

[0009] In some embodiments, the CGRP antagonist is given at a dose lower than is used to treat migraines when used alone or when used in combination with a checkpoint immunotherapy.

[0010] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF FIGURES

[0011] FIGs. 1A and 1B show neuro-immune units in glioblastoma. FIG. 1A shows multiplexed immunofluorescent analysis of nuclei, Iba (macrophages) and NF (neurons) in human IDHwt glioblastoma tumor tissue. Scale bar 20 pm. FIG. 1 B shows Harmony- corrected UMAP plots of CD45+ immune cells from human newly diagnosed glioblastomatumors (left) Violin plots showing relative expression of RAMP1 on glioblastoma infiltrating immune cell populations as shown in the clusters in the UMAP.

[0012] FIGs. 2A and 2B show blocking CGRP-RAMP1 axis leads to improved survival in glioblastoma. GL261 -luc cells were implanted in C57BL / 6 mice. On days 7, 9, 1 1 , 13, 15, and 17 post implantation mice were treated with vehicle control or anti-CGRP i.v and monitored for body weight (FIG. 2A) (n=5, p<0.001 ***, simple non-linear regression) and survival (FIG. 2B) (n=5, p<0.05; Logrank Mantel-Cox test).

[0013] FIGs. 3A to 3D show blocking CGRP-RAMP1 axis reduces the immune- suppressive features of macrophages in glioblastoma TME. glioblastoma bearing WT mice treated with vehicle control (Ctrl) or anti-CGRP on days 7, 9, 1 1 , and 13 post implantation, were sacrificed on day15 and tumors were harvested. Representative flow cytometry data (FIG. 3A) and summary plots of the frequency of Arg 1 + macrophages and mean fluorescence intensity (MFI) of Arg1 on macrophages (gated on CD45hlCD1 1 b+F4 / 80+CD64+) (FIG. 3B). Representative flow cytometry data (FIG. 3C) and summary plots of the frequency of CD38+macrophages and MFI of CD38 on macrophages (FIG. 3D) (n=5-6, *p< 0.05, **p<0.01 , unpaired Student’s t-test, Mean ± SEM are shown).

[0014] FIGs. 4A and 4B show blocking CGRP-RAMP1 axis increases the abundance and effector function of CD8+ T cells in glioblastoma TME. glioblastoma bearing WT mice treated with vehicle control (Ctrl) or anti-CGRP on days 7, 9, 1 1 , and 13 post implantation were sacrificed on day15 and tumors were harvested. FIG. 4A shows summary plots of the frequency (left) and absolute number (right) of CD8+TILs. FIG. 4B shows representative flow cytometry data and summary plots of the frequency of CD8+IFNg+TILs. (n=5-6, *p< 0.05, **p<0.01 , ***p<0.001 , unpaired Student’s t-test Mean ± SEM are shown).

[0015] FIG. 5 shows RAMP1 is highly expressed by the myeloid cells in GBM TME. FIG. 5 contains dot plots showing the expression of neuropeptide receptor expression in the immune infiltrates of GBM TME of newly diagnosed GBM patients.

[0016] FIGs. 6A to 6C contain heatmaps showing differentially expressed genes between the RAMP1hiand RAMP110myeloid cells in the GBM TME. FIG. 6A shows differentially expressed genes between RAMP1h' and RAMP110cells in GBM macrophages. FIG. 6B shows differentially expressed genes between RAMP1hiand RAMP110cells in GBM microglia. FIG. 6C shows differentially expressed genes between RAMP1hiand RAMP110cells in GBM dendritic cells.

[0017] FIG. 7 shows CGRP receptors are expressed in higher abundance in glioblastoma tumor tissue compared to that in normal tissue. Expression of RAMP1 and CALCRL, the receptor components of CGRP receptor was analyzed between GBM tumor tissue (n=163 from TCGA) and normal tissue (n=207 from TCGA normal and GTEx data) using GEPIA.

[0018] FIGs. 8A and 8B show CGRP signature is highly present in the myeloid cells in the TME of glioblastoma. FIG. 8A shows the myeloid cells infiltrating glioblastoma tumor microenvironment. FIG. 8B shows projection of CGRP signature onto scRNA-Seq dataset of glioblastoma patients.

[0019] FIGs. 9A and 9B show RAMP1himacrophages are associated with lower survival in GBM patients. The correlation of RAMPI hi macrophages with the overall survival of GBM patients shown in Kaplan-Meier curve from CGGA (FIG. 9A) and TCGA (FIG. 9B) databases. A two-sided log-rank test was used to assess statistical significance.

[0020] FIGs. 10A and 10B show glioblastoma induces CGRP production from trigeminal ganglion and dural nociceptors. FIG. 10A contains bar graphs representing the expression level of CALCA in the trigeminal ganglia of mice with sham surgery or mice bearing tumors on Day 7(left) and Day 14 (right) post-surgery. FIG. 10B contains bar graphs representing the expression level of CGRP in the dural explants of mice with sham surgery or mice bearing tumors on Day 7(left) and Day 14 (right) post-surgery. (n=5-8, *p< 0.05, **p<0.01 , unpaired Student’s t-test, mean ± SEM are shown).

[0021] FIGs. 1 1 shows CGRP level is lowered in the CSF of glioblastoma bearing mice. Bar graphs shows CGRP levels in the CSF on Day 7 (left) and Day 14 (left) postsurgery in the CSF of mice that underwent sham surgery or from tumor bearing mice. (n=5- 8, *p< 0.05, **p<0.01 , unpaired Student’s t-test, mean ± SEM are shown).

[0022] FIGs. 12A and 12B show blocking CGRP-RAMP1 axis leads to improved survival in GBM. GL261-luc cells were implanted in C57BL / 6 mice. On days 7, 9, 1 1 , 13, 15, and 17 post implantation mice were treated with vehicle control or anti-CGRP i.v. and monitored for body weight (FIG. 12A) (n=5, p<0.001 ***, simple non-linear regression) and survival (FIG. 12B) (n=5, p<0.05; Logrank Mantel-Cox test).

[0023] FIGs. 13A to 13D show blocking CGRP-RAMP1 axis reduces the immune- suppressive features of macrophages in GBM TME. GB bearing WT mice treated with vehicle control (Ctrl) or anti-CGRP on days 7, 9, 1 1 , and 13 post implantation, were sacrificed on day15 and tumors were harvested. Representative flow cytometry data (FIG. 13A) and summary plots of the frequency of Arg1+macrophages and mean fluorescence intensity (MFI) of Arg1 on macrophages (gated on CD45h'CD1 1 b+F4 / 80+CD64+) (FIG. 13B). Representative flow cytometry data (FIG. 13C) and summary plots of the frequency of CD38+macrophages and MFI of CD38 on macrophages (FIG. 13D) (n=5-6, *p< 0.05, **p<0.01 , unpaired Student’s t-test, Mean ± SEM are shown)

[0024] FIGs. 14A and 14B show blocking CGRP-RAMP1 axis alters the phenotype of microglia in GBM TME GB bearing WT mice treated with vehicle control (Ctrl) or anti- CGRP on days 7, 9, 1 1 , and 13 post implantation, were sacrificed on day15 and tumors were harvested. Representative flow cytometry data and summary plots of the frequency ofKi67+microglia and mean fluorescence intensity (MFI) of Ki67+(FIG. 14A) and PD1 (FIG. 14B) on microglia (gated on CD45loCD11 b+F4 / 80+CX3CR1+). (n=5-6, *p< 0.05, **p<0.01 , unpaired Student’s t-test, Mean ± SEM are shown)

[0025] FIGs. 15A and 15B show blocking CGRP-RAMP1 axis increases the abundance and effector function of CD8+T cells in GBM TME. GBM bearing WT mice treated with vehicle control (Ctrl) or anti-CGRP on days 7, 9, 11 , and 13 post implantation were sacrificed on day15 and tumors were harvested. FIG. 15A shows summary plots of the frequency (left) and absolute number (right) of CD8+TILs. FIG. 15B contains representative flow cytometry data and summary plots of the frequency of CD8+IFNg+TILs. (n=5-6, *p< 0.05, **p<0.01 , ***p<0.001 , unpaired Student’s t-test Mean ± SEM are shown).

[0026] FIG. 16 shows CGRP receptors are expressed in higher abundance in low grade glioma (LGG) tumor tissue compared to that in normal tissue. Expression of RAMP1 (left) and CALCRL (right), the receptor components of CGRP receptor was analyzed between low grade glioma (LGG) tumor tissue (n=163 from TCGA) and normal tissue (n=207 from GTex) using GEPIA.

[0027] FIGA. 17A to 17C show ablation of dural nociceptors reduce the progression of GB. Relative expression of TRVP1 (FIG. 17A) and CALCA (FIG. 17B) genes in TGs post RTX treatment. FIG. 17C shows GL261 cells were implanted into the right brain hemisphere of C57BL / 6 mice treated previously with vehicle (CTRL) and RTX and monitored for survival (n=5-6, **p<0.01, ****p<0.0001).

[0028] FIGs. 18A to 18C show ablation of nociceptors shapes anti-tumor immune responses in Brain and Meninges. GB bearing C57BL / 6 mice treated previously with vehicle control (Ctrl) or RTX were sacrificed on day15 post tumor implantation. Summaries of flow cytometry plots = of the frequency and MFI of Arg1+ macrophages, CD38+ macrophages, CD86+ macrophages & Ki67 (FIG. 18A) in tumor. Summaries of flow cytometry plots of the frequency and MFI of Arg1+ macrophages, CD38+ macrophages, CD86+ macrophages & Ki67 (FIG. 18B) in meninges (FIG. 18C). (n=5-6 *p< 0.05, **p<0.01 , ***p<0.001 , unpaired Student’s t-test Mean ± SEM).

[0029] FIGs. 19A and 19B show ablation of nociceptors shapes T cell compartment. Representative flow cytometry plots and summary plots of the frequency and MFI of GZMB+ CD8+ T cells, TCF-1+ CD8+ T cells, Ki67+ CD8+ T cells & TIM3+ CD8+ T cells (A) in the tumor bearing brain hemisphere. Representative flow cytometry plots and summary plots of the frequency and MFI of GZMB+ CD8+ T cells, TCF-1+ CD8+ T cells, Ki67+ CD8+ T cells & TIM3+ CD8+ T cells (B) in meninges. Representative flow cytometry plots and summary plots of the frequency and MFI of FoxP3+ CD4+ T cells (C) in the tumor bearing brain hemisphere. (n=5-6 *p< 0.05, **p<0.01 , ***p<0.001 , unpaired Student’s t-test Mean ± SEM). (n=5-6 *p< 0.05, **p<0.01 , ***p<0.001 , unpaired Student’s t-test Mean ± SEM).

[0030] FIG. 20 shows macrophages are the strongest interactors with dural nociceptors. A heatmap of the interaction strength between TG neurons37 (senders) and dural immune cells36 (receivers) calculated. Color saturation represents the communication probability between the senders and receivers calculated by CELLCHAT.

[0031] FIG. 21 shows CGRP receptor RAMP1 is highly expressed in myeloid cell subsets of GB TME. UMAP showing the expression of sensory neuropeptide receptor expression in the immune infiltrates of GB TME of newly diagnosed GB patients.

[0032] FIGs. 22A and 22B show glioblastoma (GB) activates dural nociceptors. Mice were implanted with GL261 , CT2A or M005 or PBS (sham surgery). Ipsilateral TGs and meningeal explants were harvested on Day 7. Expression of CALCA in the TGs was detected by qPCR and (FIG. 22A) production of CGRP in the intracranial meningeal explant as detected by ELISA (FIG. 22B). (n=5-9 *p< 0.05, ****p<0.0001 , unpaired Student’s t-test, Mean ± SEM).

[0033] FIG. 23 shows CGRP is more abundant in the serum of Glioma patients. Estimation of CGRP in Human serum Samples from normal versus Glioma patients.

[0034] FIG. 24 is a Volcano plot showing the differential expression of genes in human glioma tumor-associated macrophages (TAMs) categorized by CGRP signaling (CGRPAsighi vs. CGRPAsiglo). The x-axis represents log2fold change, while the y-axis shows the — log10of the p-value.

[0035] FIGs. 25A to 25E show blocking CGRP-RAMP1 axis reduces the immune suppressive features of macrophages in GBM TME. GBM bearing WT mice treated with vehicle control (Ctrl) or anti-CGRP on days 7,9,11&13 post implantation were sacrificed on day15, and tumors were harvested. FIG. 25A shows representative flow cytometry data and FIG. 25B contains summary plots of the frequency of Arg1+ macrophages and mean fluorescence intensity (MFI) of Arg1 on macrophages (gated on CD45hiCD11b+F4 / 80+CD64+). FIG. 25C shows representative flow cytometry data and FIG. 25D contains summary plots of the frequency of CD38+ macrophages and MFI of CD38 on macrophages. FIG. 25E shows the frequency of CD38+Arg1+ cells (n=5-6, *p< 0.05, **p< 0.05).

[0036] FIGs. 26A to 26C show blocking CGRP-RAMP1 axis increases the abundance and effector function of CD8+ T cells in GB TME. GB bearing WT mice treated with vehicle control (Ctrl) or anti-CGRP on days 7,9,11 and 13 post implantation were sacrificed on day15 and tumors were harvested. FIG. 26A contains summary plots of the frequency (left) and absolute number (right) of CD8+TILS. FIG. 26B contains representative flow cytometry data and summary plots of the frequency of CD8+IFNg+ TILs. FIG. 26C shows composition of T-cell compartments post treatment with anti-CGRP. (n=5-6, **p<0.01 ,***p<0.001 , unpaired Student’s t-test Mean ± SEM are shown).

[0037] FIG. 27 shows abrogation of CGRP-RAMP1 axis enriches for TAMs with more pro-inflammatory features. Shown is a bar plot representing the normalized enrichment scores (NES) of gene sets in TAMs treated with BIBN4096 compared to control.

[0038] FIG. 28 shows dual treatment with anti-CGRP and peptide vaccine improves survival of GB bearing mice. GL261-luc cells were implanted in right cerebral hemisphere of C57BL / 6 mice. On day 4 peptide vaccine and on days 7, 9, 11 , 13, 15, 17, 19, 21 & 23 post implantation mice were treated with vehicle control or anti CGRP i.v or peptide vaccine or dual treatment and monitored for survival (n=5-6, *p<0.05 *p<0.01 Logrank Mantel-Cox test).

[0039] FIG. 29 shows blocking the CGRP-RAMP1 axis in macrophages and granulocytes improved survival. GL261 cells were implanted in right cerebral hemisphere of C57BL / 6 mice and monitored for survival (n=5-6, *p<0.05 Logrank Mantel-Cox test).

[0040] FIGs. 30A to 30D show mice lacking RAMP1 expression selectively on macrophages and granulocytes increases the abundance and effector function of CD8+ T cells in GB. GB bearing control (Ctrl, Ramp1fl / fl) or cKO (Ramp1fl / flLysmCre+) mice were sacrificed on day15 and tumors were harvested. FIG. 30A contains summary plots of the frequency (left) and absolute number (right) of CD8+TILS. FIG. 26B shows the ratio of CD8T cells and Tregs. FIG. 30C contains summary plots of the frequency of CD8+GZMB+ TILs. FIG. 30D contains summary plots of the frequency of CD8+Perforin+ TILs. (n=5-6, **p<0.01 , ***p<0.001 , unpaired Student’s t-test Mean ± SEM are shown).DETAILED DESCRIPTION

[0041] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0042] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to thosedescribed herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0044] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0045] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0046] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

[0047] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0048] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.

[0049] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.CGRP antagonists

[0050] In some embodiments, the CGRP antagonist is Erenumab, which is used to treat migraines at 70 mg or 140 mg subcutaneously once a month. Therefore, in some embodiments, Erenumab is used to in the disclosed methods at a dosage of 70 mg or 140 mg subcutaneously once a month. In some embodiments, Erenumab is used in the disclosed methods at a dosage of 35 mg, 105 mg, or 210 mg subcutaneously once a month.

[0051] In some embodiments, the CGRP antagonist is Fremanezumab , which is used to treat migraines at 225 mg subcutaneously once a month or 675 mg subcutaneously once every three months. Therefore, in some embodiments, Fremanezumab is used to in the disclosed methods at a dosage of 225 mg subcutaneously once a month or 675 mg subcutaneously once every three months. In some embodiments, Fremanezumab is used in the disclosed methods at a dosage of 112.5 mg subcutaneously once a month, 450 mg subcutaneously every two months, or 900 mg subcutaneously every three months.

[0052] In some embodiments, the CGRP antagonist is Galcanezumab, which is used to treat migraines at an initial dose or 240 mg subcutaneously once and at a maintenance dose of 120 mg subcutaneously monthly; or which is used to treat migraines at 300 mg (3 consecutive subcutaneous injections of 100 mg) at the onset of the cluster period and then monthly until the end of the cluster period. Therefore, in some embodiments, Galcanezumab is used to in the disclosed methods at a dosage of 120, 240, or 300 mg monthly. In some embodiments, Galcanezumab is used in the disclosed methods at a dosage of 100 mg, 200 mg, or 220 mg subcutaneously once a month.

[0053] In some embodiments, the CGRP antagonist is Eptinezumab, which is used to treat migraines at an initial dose of 100 mg via intravenous infusion every 3 months and at a maintenance dose of 100 mg or 300 mg via intravenous infusion every 3 months. Therefore, in some embodiments, Eptinezumab is used to in the disclosed methods at a dosage of 100 or 300 mg via intravenous infusion every 3 months. In some embodiments, Eptinezumab is used in the disclosed methods at a dosage of 200 mg or 400 mg intravenous infusion every three months.

[0054] In some embodiments, the CGRP antagonist is Ubrogeapnt, which is used to treat migraines at an initial dose: 50-100 mg orally, once, and then after 2 hours at a second dose if needed with a maximum dose of 200 mg in a 24-hour period. Therefore, in some embodiments, Ubrogeapnt is used to in the disclosed methods at a dosage of 50-200 mg orally.

[0055] In some embodiments, the CGRP antagonist is Rimegepant sulfate, which is used to treat migraines at 75 mg taken orally as needed. Therefore, in some embodiments, Rimegepant sulfate is used to in the disclosed methods at a dosage of 75 mg orally. In someembodiments, Rimegepant sulfate is used in the disclosed methods at a dosage of 75 or 150 mg taken orally every 1 , 3, 4, 5 days.

[0056] In some embodiments, the CGRP antagonist is Atogepant, which is used to treat migraines at 10 mg, 30 mg or 60 mg taken orally once daily with or without food. Therefore, in some embodiments, Atogepant is used to in the disclosed methods at a dosage of 10 mg, 30 mg or 60 mg taken orally once daily. In some embodiments, Atogepant is used in the disclosed methods at a dosage of 20, 40, 50, 80, or 90 mg taken daily.Checkpoint Inhibitor

[0057] The disclosed methods can involve administering to the subject a checkpoint inhibitor. The two known inhibitory checkpoint pathways involve signaling through the cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed-death 1 (PD-1) receptors. These proteins are members of the CD28-B7 family of cosignaling molecules that play important roles throughout all stages of T cell function. The PD-1 receptor (also known as CD279) is expressed on the surface of activated T cells. Its ligands, PD-L1 (B7-H1 ; CD274) and PD-L2 (B7-DC; CD273), are expressed on the surface of APCs such as dendritic cells or macrophages. PD-L1 is the predominant ligand, while PD-L2 has a much more restricted expression pattern. When the ligands bind to PD-1 , an inhibitory signal is transmitted into the T cell, which reduces cytokine production and suppresses T-cell proliferation. Checkpoint inhibitors include, but are not limited to antibodies that block PD-1 (Nivolumab (BMS-936558 or MDX1106), CT-011 , MK-3475), PD-L1 (MDX-1105 (BMS-936559), MPDL3280A, MSB0010718C), PD-L2 (rHlgM12B7), CTLA-4 (Ipilimumab (MDX-010), Tremelimumab (CP- 675,206)), IDO, B7-H3 (MGA271), B7-H4, TIM3, LAG-3 (BMS-986016).

[0058] Pembrolizumab is currently used to treat cancer at a dosage of 200 mg intravenously every 3 weeks or 400 mg intravenously every 6 weeks. In some embodiments, Pembrolizumab is used in the disclosed methods at a dose of 100, 200, 300, 400 mg intravenously every 2, 3, 4, 5, 6, 7, 8 weeks.

[0059] Nivolumab is currently used to treat cancer at a dosage of 240 mg intravenously every 2 weeks or 480 mg intravenously every 4 weeks. In some embodiments, Nivolumab is used in the disclosed methods at a dose of 240 or 480 mg intravenously every 3, 4, 5, 6 weeks.

[0060] Ipilimumab is currently used to treat cancer at a dosage of 1 mg / kg IV every 3 weeks for up to 4 doses. In some embodiments, Ipilimumab is used in the disclosed methods at a dose of 0.5 mg / kg IV every 3 weeks for up to 4 doses, 0.75 mg / kg IV every 3 weeks for up to 4 doses, 0.75 mg / kg IV every 4 weeks for up to 4 doses, or 1 mg / kg IV every 4 weeks for up to 4 doses.Embodiments

[0061] Embodiment 1. A method for treating a glioma in a subject refractory to checkpoint immunotherapy, comprising administering to the subject a therapeutically effective amount of a calcitonin gene-related peptide (CGRP) antagonist.

[0062] Embodiment 2. The method of embodiment 1 , wherein the CGRP antagonist blocks the binding of CGRP to Receptor Activity Modifying Protein 1 (RAMP1).

[0063] Embodiment 3. The method of embodiment 2, wherein the CGRP antagonist is an antibody or antibody fragment that binds CGRP or RAMP1 .

[0064] Embodiment 4. The method of embodiment 3, wherein the CGRP antagonist is a bi-specific antibody or antibody fragment that binds PD-1 and CGRP or RAMP1.

[0065] Embodiment 5. The method of embodiment 3, wherein the CGRP antagonist is selected from the group consisting of Erenumab (AMG-334), Eptinezumab (ALD403), Fremanezumab (TEV-48125), and Galcanezumab (LY2951742).

[0066] Embodiment 6. The method of embodiment 1 , wherein the CGRP antagonist is selected from the group consisting of Ubrogepant, Rimegepant (BMS-927711), Atogepant (AGN-241689), Telcagepant (MK-0974), Olcegepant (BIBN-4096BS), Bl 44370, MK-3207, and SB-268262.

[0067] Embodiment 7. The method of any one of embodiments 1 to 6, comprising (a) treating the subject with checkpoint immunotherapy; (b) determining that the subject is refractory to the checkpoint immunotherapy; and(c) administering to the subject a therapeutically effective amount of the CGRP antagonist.

[0068] Embodiment 8. The method of any one of embodiments 1 to 7, wherein the checkpoint immunotherapy is anti-PD1 immunotherapy.

[0069] Embodiment 9. The method of any one of embodiments 1 to 8, further comprising administering to the subject a checkpoint inhibitor.

[0070] Embodiment 10. The method of embodiment 9, wherein the checkpoint inhibitor comprises an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, or a combination thereof.

[0071] Embodiment 11 . The method of any one of embodiments 1 to 10, wherein the CGRP antagonist is given at a dose lower than is used to treat migraines.

[0072] Embodiment 12. The method of any one of embodiments 1 to 11 , wherein the glioma is a glioblastoma.

[0073] Embodiment 13. The method of any one of embodiments 1 to 11 , wherein the glioma is low-grade glioma (LGG).

[0074] Embodiment 14. The method of any one of embodiments 1 to 13, further comprising detecting in a tumor tissue from the subject elevated expression levels comparedto a control of RAMP1 , calcitonin receptor-like receptor (CALCRL), or a combination thereof prior to treatment with the CGRP antagonist.

[0075] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.EXAMPLESExample 1:Background and Significance

[0076] Glioblastoma is the most prevalent primary malignant brain tumor in adults. Despite maximal surgical resection and chemo-radiation, the prognosis remains poor. Glioblastoma patients remain refractory to immune checkpoint blockade therapy (ICBs). Immune suppressive networks of tumor-associated macrophages and microglia, which constitute the majority of the immune population in glioblastoma tumor microenvironment (TME), impede immunotherapy. Investigation of the cross-talk between various cell types and immune cells in the TME is critical for understanding these immune suppressive circuits.

[0077] Neurons are a unique and critical component of glioblastoma TME.Communication between neurons and cancer cells via secreted growth factors or direct electrochemical synapses is an important integrant of brain cancer pathophysiology. Although bona fide neuron-to-cancer cell synapses is critical in the central nervous system, neurons may play a fundamental role in the initiation, maintenance and progression of brain cancer via paracrine regulation of the immune cells. The importance of neuronal regulation of immune responses was shown in extra CNS tumor such as in primary melanoma where it was demonstrated that neo-innervation of the tumors by sensory neurons (nociceptors) affected cancer immunosurveillance. Specifically, they showed that calcitonin gene-related peptide (CGRP) directly increased the frequency of exhausted CD8+ T cells. Highlighting the importance of CGRP in regulating tumor growth, another study showed that CGRP produced by sensory neurons induced cytoprotective autophagy in cancer cells and that blocking CGRP signaling enhanced anti-tumor efficacy of nutrient-starvation therapies in murine models of head and neck cancer. These studies establish the nociceptors as a critical component of TME. Of note, in murine model of glioblastoma, blocking the CGRP pathway improved survival by shaping the immune-landscape of the TME across multiple immunologic nodes. Specifically, anti-CGRP treatment reduced the immune-suppressive property of the macrophages and increased the number and effector function of CD8+ T cells in the TME. Next, the expression of RAMP1 , the receptor of CGRP, was analyzed by mining publicly available scRNA Seq dataset of tumor infiltrating immune cells of newly diagnosed glioblastoma patients. RAMP1 was expressed at a very low level in CD8+ T cellsand expressed highly on the myeloid cells suggesting that these cells are the main target cells of CGRP in this tumor type. The paucity of T cells and the presence of a network of immune suppressive myeloid cells in the glioblastoma tumor microenvironment (TME) is a major limitation in developing an effective immunotherapy against glioblastoma.

[0078] Given that myeloid cells constitute the most abundant population of the glioblastoma infiltrating immune cells, this example unravels a fundamental immune suppressive signaling pathway such that targeting this pathway will significantly improve the outcome of glioblastoma patients by augmenting anti-tumor immunity. Given that CGRP antagonist is an FDA approved drug, the mechanistic insights gained from this study can lead to repurposing of the drug for the benefit of glioblastoma patients. Targeting neuropeptides, such as CGRP, to induce robust anti-tumor immune responses is a novel treatment strategy which could either be used as a monotherapy or in conjunction with ICB therapy to benefit cancer patients. Further, the knowledge gained from the study could be leveraged to use CGRP as serum biomarker for disease progression and also as a prognostic factor, thus underscoring the clinical application of the study.ResultsNeuro-lmmune units in glioblastoma

[0079] Neurons are juxtaposed with macrophages in the TME of glioblastoma suggesting that there is a direct cross-talk between these two cell types in the TME. Given that neurons regulate the function of immune cells via neuropeptides, we examined the expression level of neuropeptide receptors on the scRNA Seq dataset of tumor infiltrating immune cells of newly diagnosed glioblastoma patients. We found that receptor for CGRP was highly expressed on the myeloid cells (macrophages and dendritic cells (DCs)). Together these data further support a connection between active CGRP-RAMP1 signaling and myeloid cell dysfunction in glioblastoma.

[0080] Blocking CGRP-RAMP1 axis leads to improved survival in glioblastoma.

[0081] To examine the effect of CGRP-RAMP1 signaling on tumor progression an orthotopic model of glioblastoma (GL261-luc) was used wherein stereotactic implantation of glioblastoma cancer cells was performed into the brains of C57BL / 6 (WT) mice. Next, vehicle control (Ctrl) or anti-CGRP small molecule antagonist was administered on Day 7, 9, 11 , 13, and 17 post implantation and body weight and survival were monitored as a measure of tumor progression. Blocking the CGRP-RAMP1 axis significantly improved survival.Blocking CGRP-RAMP1 axis reduces the immune-suppressive features of macrophages in glioblastoma TME.

[0082] Glioblastoma bearing mice were administered anti-CGRP small molecule antagonist on Day 7, 9, 11 , and 13 post implantation. Mice were sacrificed and tumors harvested on Day 15. Blocking CGRP signaling reduced the frequency and expression of theimmune suppressive enzyme Arg1 and the ectoenzyme CD38 in macrophages. Arg1 metabolizes the amino acid (aa) arginine and depletion of this aa in the TME dampens the function of T cells. CD38 is an ectozyme that converts NAD+ to ADP-ribose (ADPR) and cADPR, which play an important role in the regulation of intracellular Ca2+, and signal transduction pathways. Of note, CD38 expression mediates suppression via adenosine receptor signaling on cytotoxic T cells. Thus, disrupting the CGRP-RAMP1 axis reduces the immune-suppressive feature of macrophages in the TME and may lead to shifting the phenotype of the macrophages to a more activated status which culminates in improved anti-tumor immune responses.Blocking CGRP-RAMP1 axis increases the abundance and effector function of CD8+ T cells in glioblastoma TME

[0083] Anti-CGRP or vehicle control treated glioblastoma bearing WT mice were sacrificed on day 15 and tumors were harvested. Analysis of the T cell compartment revealed that blocking the CGRP pathway leads to a significant increase in the frequency and absolute number of CD8+ T cells in the TME. The functionality of the CD8+ T cells was further examined showing that blocking the CGRP-RAMP1 axis leads to an increase in the frequency of IFN-g producing CD8+ T cells. Thus, blocking the CGRP-RAMP1 axis can augment antitumor immunity by regulating immune responses at multiple nodes.Example 2:

[0084] Several studies suggest that changes in the tumor microenvironment (TME), especially in the immune compartment may play a fundamental role in cancer development (Suarez-Carmona, M., et al. Mol Oncol. 2017 11 (7):805-823; Chen, D. S. & Mellman, I. Nature. 2017 541 (7637) :321-330). Thus, changes in the immune compartment may also play a role in facilitating the transition from LGG to HGG. Indeed, a study confirmed that changes in the molecular determinant of the immune cells are associated with the prognosis of LGG. CALCRL (gene that encodes CLR, a component of CGRP receptor) has also been shown to be associated with worse prognosis in LGG patients (Han, T. et al. Front Pharmacol. 2021 12:748993). Calcitonin gene related peptide (CGRP) is a 37 amino acid neuropeptide produced mostly by the nociceptors and has potent immunomodulatory capacity. CGRP mediates its effect by acting though a receptor heterodimer comprising of receptor activity modifying protein (RAMP1) and CLR. Using the TCGA dataset RAMP1 and CALCRL were shown to be expressed at a significantly higher level in the LGG tumor tissue compared to the normal tissue. Thus, ablation of the CGRP signaling pathway may improve the prognosis of high risk LGG patients.

[0085] Further, a vast majority of all newly diagnosed gliomas are classified as glioblastoma (GB), (Grade 4 glioma), a highly aggressive form of glioma with a poor prognosis. Despite multimodal treatment strategies such as maximum safe surgicalresection and chemo-radiation, the survival rate is low, with patients having 15-18 months of median survival post diagnosis and a 5-year survival rate of less than 5%. The clinical treatment stages for glioblastoma are newly diagnosed GBM (ndGB) and recurrent GBM (rGB). ndGB is usually treated with radiation and temozolomide (TMZ), an alkylating drug that triggers DNA damage in cancer cells. rGB treatment involves the use of CCNU (Lomustine), another alkylating agent and an inhibitor against vascular endothelial growth factor-A (VEGF-A) called bevacizumab, which inhibits angiogenesis within the TME.

[0086] Immune checkpoint blockade (ICB), a therapy which has been successful in treating other forms of cancer, is currently under investigation for treatment of GBM. However, recent advanced-phase clinical trials have failed to generate improved patient outcomes (Rocha Pinheiro, S. L. et al. World J Clin Oncol. 2023 14(4): 138- 159), suggesting that additional studies are necessary to improve our understanding of this immunomodulatory therapeutic platform and achieve meaningful results for treating GBM. For GBM, immunosuppressive forces generated by the tumor microenvironment (TME) create a protective shield to avoid immune rejection and instead promote tumor growth. Thus, a deeper understanding of the regulation of immune responses in GBM is critical for delineating and reprogramming these suppressive immune circuits.

[0087] Neurons are a unique and critical component of GB TME. Communication between neurons and cancer cells via secreted growth factors or direct electrochemical synapses is an important integrant of brain cancer pathophysiology (Venkatesh, H. S. et al. Nature 2019 573:539-545; Venkataramani, V. et al. Nature 2019 573:532-538; Venkatesh, H. S. et al. Cell. 2015 161 (4):803- 16). Although bona fide neuron-to-cancer cell synapses is critical in the central nervous system, neurons may play a fundamental role in the initiation, maintenance and progression of brain cancer via paracrine regulation of the immune cells. The importance of neuronal regulation of immune responses was shown in extra CNS tumor such as in primary melanoma where it was demonstrated that neo-innervation of the tumors by sensory neurons (nociceptors) affected cancer immunosurveillance (Balood, M. et al. Nature 2022611 :405-412). Specifically, they showed that CGRP directly increased the frequency of exhausted CD8+ T cells. Highlighting the importance of CGRP in regulating tumor growth, another study showed that CGRP produced by sensory neurons induced cytoprotective autophagy in cancer cells and that blocking CGRP signaling enhanced antitumor efficacy of nutrient-starvation therapies in murine models of head and neck cancer (Zininga, T. et al. Molecules. 201823(11):2846). These studies establish the nociceptors as a critical component of TME. RAMP1 and CALCRL were shown using the TCGA dataset to be expressed at a significantly higher level in the tumor in the GB tumor tissue compared to the normal tissue. Of note, in murine model of GB, blocking the CGRP pathway improved survival by shaping the immune-landscape of the TME across multiple immunologic nodes.Specifically, anti-CGRP treatment reduced the immune-suppressive property of the macrophages and increased the number and effector function of CD8+ T cells in the TME. The expression of RAMP1 , the receptor of CGRP was next analyzed by mining publicly available scRNA Seq dataset of tumor infiltrating immune cells of newly diagnosed GBM patients (Zhang, P. et al. Nat Commun. 2019 10(1):3635). RAMP1 was expressed at a very low level in CD8+ T cells and expressed highly on the myeloid cells suggesting that these cells are the main target cells of CGRP in this tumor type.ResultsCGRP receptor is highly expressed in glioblastoma TME infiltrating myeloid cells

[0088] A meta-analysis was performed by combining the scRNA-Seq of 54 newly diagnosed glioblastoma patients, 166615 cells from four independent studies (GSE131928, GSE138794, GSE163120, GSE182109). As previously observed, the myeloid population particularly the microglia and macrophages are the most abundant cell types in the tumor microenvironment of glioblastoma. In fact, macrophages and microglia make up 80-85% of the total immune cells of the glioblastoma TME. The expression of neuropeptide receptors was analyzed in this meta-data, demonstrating that the myeloid cells almost exclusively and very highly express RAMP1 (receptor activity modifying protein 1) (Fig. 5). RAMP1 is a single transmembrane domain protein that provides ligand specificity to bind CGRP.CGRP-RAMP1 axis alters the transcriptome profile of myeloid cells in the Glioblastoma TME

[0089] 1st quartile expression level was used to split the dataset by RAMP1 expression and delineate RAMP1hiand RAMP110cells. In order to study the pathways that altered by CGRP-RAMP1 axis the top 50 differentially expressed genes (DEGs) were analyzed between RAMP1hiand RAMP110macrophages, microglia, and dendritic cells (DCs). In the macrophages lower RAMP1 expression was associated with higher expression of heat-shock proteins, DNAJA4,HSPA5, DNAJB1 , HSP90AA1 (Fig. 6A). HSPs act as potent immune modulators that can actively shape immune responses in the tumor microenvironment (Zininga, T. et al. Molecules. 201823(11):2846). Interestingly, lower RAMP1 expression was associated with higher expression of NEAT which is a IncRNA that plays an important role in activation of inflammasome in macrophages (Zhang, P. et al. Nat Commun. 2019 10(1):3635) and regulation of inflammation (Pan, Y. et al. J Inflamm Res. 2022 15:557-571). Further, lower RAMP1 is associated with lower expression of tumor supportive or immune-suppressive molecules such as PTN, PTPRZ1 (Shi, Y. et al. Nat Commun. 2017 8:15080), CHI3L1 (Chen, A. et al. J Clin Invest. 2021 131 (16):e147552)), TREM2 (Katzenelenbogen, Y. et al. Cell. 2020 182(4):872-885.e19), CD9 and SPP1 (Shurin, M. R. J Clin Invest. 2018 128(12):5209-5212).

[0090] Similarly, in the microglia, lower RAMP1 expression was associated with higher expression of heat-shock proteins HSPB1 , HSPA5, HSPD1 , HSP6A, HSPA1 B, DNAJB1 , HSPH1 , and HSP90AA1. Additionally, lower RAMP1 expression was associated with lower expression of SPP1 , CD9 and TREM2 (Fig. 6B). Similarly, in the DCs lower RAMP1 expression was associated with higher expression of heat-shock proteins HSPA1 A, DNAJB1 , HSPA1 B, HSPA6, and HSP90AA1 (Fig. 6C).

[0091] CGRP receptors are expressed in higher abundance in glioblastoma tumor tissue compared to that in normal tissue.

[0092] The expression levels of RAMP1 and CALCRL were analyzed in the glioblastoma tumor tissue and normal using an online tool GEPIA (Li, C., et al. Nucleic Acids Res. 2021 49(W1):W242-W246). Both genes are expressed at significantly higher level in the tumor tissue (Fig. 7).

[0093] CGRP signature is highly present in the myeloid cells in the TME of glioblastoma

[0094] Given that RAMP1 was highly expressed by the GB infiltrating myeloid cells, a curated list of CGRP induced signatures was projected onto scRNA-Seq dataset of myeloid cells of GB patients to determine if the CGRP pathway is active in these cell populations. Indeed, the CGRP-RAMP1 pathway was active on the GB infiltrating myeloid cells (Fig. 8).

[0095] RAMP1himacrophages is associated with lower survival in GBM patients.

[0096] Data of CGGA (Fig. 9A) and TCGA (Fig. 9B) patients with GB grade IV, Wildtype IDH1 were used for cibersort analysis. Patients were sorted based on mean Macrophage RAMP+cell content wherein less than mean Macrophage RAMP1+cell content is designated as Low Macrophage RAMP1highand greater than mean Macrophage RAMP1+cell content is designated as high Macrophage RAMP1high. Using these criteria overall survival of these patients were analyzed. Higher abundance of RAMP1highmacrophages is associated with reduced OS.Glioblastoma induces CGRP production from trigeminal ganglion and dural nociceptors

[0097] To determine whether GB induces CGRP release, mice were either subjected to sham surgery where sterile saline was stereotactically injected to the right cerebral hemisphere of the mice or GL261-Luc tumor cells were implanted. On Days 7 and 14 post implantation mice were sacrificed and Trigeminal ganglion and meningeal explants were used to determine the level of CGRP between the two groups of mice. Increased levels of CALCA (gene that encodes CGRP) were observed in the TGs (Fig. 10A) and increased level of soluble CGRP were observed in the dural explants (Fig. 10B) tumor bearing mice.CGRP level is lowered in the CSF of glioblastoma bearing mice

[0098] The level of CGRP was measured in the cerebrospinal fluid (CSF) the two groups of mice as described in Fig. 10 on Day 7 and day 14 post implantation. The level of CGRP was significantly reduced in the CSF of tumor bearing mice (Fig. 11). This was surprising given that the dural nociceptors of tumor bearing mice has higher expression of CGRP. It is plausible that the high abundance of macrophages and microglia that express RAMP1 sequester CGRP and hence reduces its availability in the CSF.Blocking CGRP-RAMP1 axis leads to improved survival in GBM

[0099] To examine the effect of CGRP-RAMP1 signaling on tumor progression an orthotopic model of GB (GL261-luc) was used wherein stereotactic implantation of GB cancer cells was performed into the brains of C57BL / 6 (WT) mice. Next, vehicle control (Ctrl) or anti-CGRP small molecule antagonist was administered on Day 7, 9, 11 , 13, and 17 post implantation and body weight and survival were monitored as a measure of tumor progression. Blocking the CGRP-RAMP1 axis significantly improved survival (Fig. 12).Blocking CGRP-RAMP1 axis reduces the immune-suppressive features of macrophages in GBM TME

[0100] GB bearing mice were administered anti-CGRP small molecule antagonist on Day 7, 9, 11 , and 13 post implantation. Mice were sacrificed and tumors harvested on Day 15. Blocking CGRP signaling reduced the frequency and expression of the immune suppressive enzyme Arg1 and the ectoenzyme CD38 in macrophages (Fig. 13). Arg1 metabolizes the amino acid (aa) arginine and depletion of this aa in the TME dampens the function of T cells. CD38 is an ectozyme that converts NAD+ to ADP-ribose (ADPR) and cADPR, which play an important role in the regulation of intracellular Ca2+, and signal transduction pathways (Malavasi, F. et al. Physiol Rev. 2008 88(3):841-86). Of note, CD38 expression mediates suppression via adenosine receptor signaling on cytotoxic T cells (Chen, L. et al. Cancer Discov. 2018 8(9): 1156-1175). Thus, disrupting the CGRP-RAMP1 axis reduces the immune-suppressive feature of macrophages in the TME and may lead to shifting the phenotype of the macrophages to a more activated status which culminates in improved anti-tumor immune responses.Blocking CGRP-RAMP1 axis alters the phenotype of microglia in GBM TME.

[0101] GBM bearing mice were administered anti-CGRP small molecule antagonist on Day 7, 9, 11 , and 13 post implantation. Mice were sacrificed and tumors harvested on Day 15. Blocking CGRP signaling increased the proliferation of microglia (Fig. 14A) and increased the frequency of PD1+ microglia (Fig. 14C). The expression of PD1 on the microglia was also enhanced post CGRP blockade. Of note, no changes were observfed in the expression level of MHCII and PDL1 on microglia post treatment (Figs. 14B, 14D). Thesedata indicate that abrogating the CGRP-RAMP1 axis profoundly alters the phenotype of microglia.Blocking CGRP-RAMP1 axis increases the abundance and effector function of CD8+ T cells in GBM TME

[0102] Anti-CGRP or vehicle control treated GBM bearing WT mice were sacrificed on day 15 and tumors were harvested. Analysis of the T cell compartment revealed that blocking the CGRP pathway leads to a significant increase in the frequency and absolute number of CD8+ T cells in the TME (Fig. 15). The functionality of the CD8+ T cells was further examined, showing that blocking the CGRP-RAMP1 axis leads to an increase in the frequency of IFN-g producing CD8+ T cells (Fig. 15). Thus, blocking the CGRP-RAMP1 axis can augment antitumor immunity by regulating immune responses at multiple nodes.CGRP receptors are expressed in higher abundance in low grade glioma tumor tissue compared to that in normal tissue

[0103] The expression of RAMP1 and CALCRL in the LGG tumor tissue and normal was analyzed using an online tool GEPIA (Li, C., et al. Nucleic Acids Res. 2021 49(W1):W242-W246). The tumor tissue has higher expression of both the genes examined compared to the normal tissue (Fig. 16).Example 3:Ablation of nociceptors improves survival of GB bearing mice.

[0104] 4-week-old C57BL / 6 mice were treated with RTX or vehicle control subcutaneously above the cranial suture and GB cell-line, ablation of sensory neurons was confirmed by checking the expression of Trpvl (Fig. 16A) and CALCA (Fig. 16B) genes in TGs at one week post RTX treatment. GL261 was sterotactically implanted 4 weeks after RTX treatment in the right hemisphere of the brain and monitored for disease progression and survival to examine the role of nociceptors on GB progression. We found that ablation of nociceptors increases survival of GB bearing mice (Fig. 16C).

[0105] Thus, this study is a paradigm of integrative physiology, aiming to decipher the molecular determinants of tumor-induced pro-tumorigenic crosstalk between two fundamentally discreet physiological units: the nervous and immune systems, which ultimately will lead to the discovery of novel therapeutic targets and treatment strategies for the benefit of GB patients.Ablation of nociceptors reduces the immune-suppressive features of Macrophages in the meninges and brain tumor tissue

[0106] RTX or vehicle treated GB bearing mice were sacrificed and intra-cranial meninges and tumor-bearing brain hemisphere were harvested on day 15 post implantation. Immune phenotyping was performed with high dimensional multi-spectral flow cytometry. We found that ablation of nociceptors reduced the immune-suppressive features ofmacrophages (CD11b+F4 / 80+) by reducing the frequency and expression of the immune suppressive enzyme Arg1 and the ectoenzyme CD38 in macrophages, in both the anatomical locations examined (Fig 18A, 18B). Arg1 metabolizes the amino acid (aa) arginine and depletion of this aa in the TME dampens the function of T cells. CD38 is an ectozyme that dampens the function of cytotoxic T cells via adenosine receptor in the TME.Ablation of nociceptors improves the effector function of CD8+ T cells in the meninges and brain tumor tissue

[0107] Next, we examined the T cell compartment in both the meninges and the brain tumor tissue. We found that ablation of nociceptors dramatically reshaped the CD8+ T cell compartment; specifically nociceptor ablation led to i) increased the abundance of GranzymeB expression in CD8+ T cells conferring them with better cytotoxic capacities ii) increased the frequency of Ki67+ T cells demonstrating increased proliferation iii) increased the abundance of CD8+ PD1+Tcf1+T cells (stem-like T cells). These cell types are the key mediators of immunotherapy and positively corelate with better prognosis in cancer patients iv) decrease in the expression of coinhibitory molecule Tim3 (Fig. 19A & 19B). V) decrease the frequency of Tregs in brain tumor tissue (Fig. 19C). Thus, nociceptor ablation promotes effector T cell differentiation which culminates in improved anti-tumor immune responses.Macrophages are the strongest interactors with nociceptors

[0108] To predict all possible interactions between each dural immune cell type and TG neuronal cell types, as well as their interaction strength, we utilized the CELLCHAT R package. We complemented the human dura immune scRNA-seq9 and human TG neuron snRNA-seq data. We focused primarily on interactions between immune cells as receivers and neurons as the source and quantified the total interaction strength, among all ligandreceptor pairs summed, we found that macrophages are the strongest overall interactors with dural nociceptors (Fig. 20).CGRP receptor RAMP1 is highly expressed by the myeloid cell subsets in GB TME

[0109] Given that nociceptor regulate the function of immune cells via neuropeptides, we screened for nociceptor-derived neuropeptides by using a human trigeminal ganglion cell atlas (TGCA) (Yang et al., 2022) and found 36 neuropeptides (Fig. 21). To examine the role of nociceptors-derived neuropeptide in regulating immune cells we examined the expression level of the receptors of those 36 neuropeptides on scRNA Seq atlas we generated by performing a meta-analysis by combining the scRNA-Seq of 54 newly diagnosed glioblastoma patients, 166615 cells from four independent studies (GSE131928, GSE138794, GSE163120, GSE182109). We found that receptor for CGRP, receptor activity modifying protein 1 (RAMP1), was highly expressed on the myeloid cells (macrophages and dendritic cells (DCs)) (Fig. 21). These data further support a connection between activeCGRP-RAMP1 signaling and myeloid cell functional regulation in GBM. RAMP1 is a single transmembrane domain protein that provides ligand specificity to bind CGRP.Glioblastoma induces CGRP production from trigeminal ganglion and dural nociceptors

[0110] Nociceptors are the chief source of CGRP in trigeminal ganglion and we confirmed that CALCA was specifically expressed in sensory neuron populations using TGCA (Fig. 22A & 22B). To examine the effect of GB on nociceptors, we performed stereotactic implantation of PBS or GB cell-lines (GL261 , CT2A or M005) into the right hemisphere of the brains of C57BL / 6 mice as previously described. Activation of nociceptors causes the release of the neuropeptide CGRP, hence we measured CGRP from tumor associated and sham operated ipsilateral TGs and intra-cranial meningeal explants as a measure of nociceptor activation. Ipsilateral TGs and intra-cranial meningeal explants were harvested on day 7 post implantation. RNA was extracted from the TGs and qPCR was performed to examine the expression of CGRP (encoded by CALCA). CGRP in the meningeal explant was measured by ELISA as per manufacturer’s protocol (Novus Biologicals). We found that GB induces higher CGRP production in both the ipsilateral TGs (Fig. 22C) and intra-cranial meningeal explants (Fig. 6D).Human Serum CGRP

[0111] Next, we measured the CGRP level in normal human (control) and glioma patient’s serum and we found higher CGRP levels in the glioma patients compared to control (Fig. 23).Human CGRPsighi TAMs exhibit more features of immune suppression

[0112] Figure 24 illustrates the differential gene expression between two subsets of tumor-associated macrophages (TAMs) from human gliomas, classified by their CGRP signaling levels (CGRPAsighi vs. CGRPAsiglo). Human CGRPsighi TAMs exhibit more features of immune suppression.Blocking CGRP-RAMP1 axis reduces the immune-suppressive features of macrophages in GBM TME

[0113] GBM bearing mice were administered either vehicle control (Ctrl) or CGRP- receptor antagonist on Days 7,9,11&13 post implantation. Mice were sacrificed and tumors harvested on day 15. Blocking CGRP-signaling reduced the frequency and expression of the immune suppressive Arg1 (Fig. 25A & 25B) and the ectoenzyme CD38 in macrophages (CD45hiCD11 b+F4 / 80+CD64+) (Fig. 25C & 25D). The frequency of the dual positive population also decreased (Fig. 25E). Arg1 metabolizes the amino acid (aa) arginine and depletion of this aa in the TME dampens the function of T cells. CD38 is an ectozyme that dampens the function of cytotoxic T cells via adenosine receptor in the TME. Thus,disrupting the CGRP-RAMP1 axis reduces the immune-suppressive feature of macrophages in the TME which culminates in improved anti-tumor immune responses.Blocking CGRP-RAMP1 axis increases the abundance and effector function of CD8+ T cells in GB TME

[0114] CGRP-receptor antagonist or Ctrl treated GB bearing WT mice were sacrificed on day 15 and tumors were harvested. Tumor associated lymphocytes (TILs) were stimulated with PMA / ionomycin for cytokine analysis. Blocking the CGRP pathway leads to a significant increase in the abundance (Fig. 26A) and effector function (IFNg) of CDS+TILs (Fig. 26B). scRNA-seq analysis showed the composition of T-cell compartments post treatment with anti- CGRP were changed (Fig. 26C). Thus, blocking the CGRP-RAMP1 axis can augment antitumor immunity by regulating immune responses at multiple nodes.Abrogation of CGRP-RAMP1 axis enriches for TAMs with more pro-inflammatory features

[0115] UMAP plots illustrating the clustering of monocyte / macrophage populations in control and anti-CGRP (BIBN4096) treated conditions. The plots show distinct shifts in the distribution of clusters following CGRP inhibition, with changes in cell populations indicating differential phenotypic profiles. The color gradient represents varying levels of cellular expression profiles across different clusters (Fig. 27). the pathway enrichment analysis comparing gene sets in tumor-associated macrophages (TAMs) under conditions with and without CGRP inhibition (using the antagonist BIBN4096). The analysis identifies significant pathways related to immune responses, particularly those involving interferon signaling and inflammatory responses, that are influenced by CGRP signaling inhibition (Fig. 28-30D). The Enriched GO terms pathways & GSEA analysis showing that Blockade of CGRP-RAMP1 axis improves IFN sensitivity in macrophages.Dual treatment with CGRP-RAMP1 axis blockade and peptide vaccination leads to improved survival in GB

[0116] To examine the effect of CGRP-RAMP1 signaling on tumor progression we used an orthotopic model of GB wherein we performed stereotactic implantation of GBM cancer cells (GL261) into the brains of C57BL / 6 (WT) mice as previously described13. We treated with either a CGRP antagonist Olcegepant, (Tocris, USA) (anti-CGRP) (1 mg / kg) on Days 7,9,11 ,13,15 & 17, a peptide vaccine (Immunogenic Luc peptide 50 pg / mouse & Poly l:C 100 pg / mouse) on day 4, or both post implantation. Survival was monitored as a measure of tumor progression. We found that dual treatment with anti- CGRP and peptide vaccination significantly improved survival of tumor bearing mice compared to individual treatments (Fig. 28)Mice lacking RAMP1 expression selectively on macrophages and granulocytes improved survival of GB-bearing mice

[0117] CGRP signaling has divergent effects on different myeloid cell subsets; dampening macrophage function while enhancing the sentinel function of DCs, we generated a conditional knockout (cKO) mice model lacking RAMP1 expression selectively on macrophages and granulocytes by using Ramp1fl / flLysMCre+cKO. We performed stereotactic implantation of GBM cancer cells (GL261) into the brains of Ramp1fl / flLysMCre+cKO and control littermates and survival were monitored. Ramp1fl / flLysMCre+cKO mice improved survival compared to control littermate of GB-bearing mice (Fig. 29).Mice lacking RAMP1 expression selectively on macrophages and granulocytes increases the abundance and effector function of CD8+ T cells in GB

[0118] GB bearing control (Ctrl, Ramp1fl / fl) or cKO (Ramp1fl / flLysmCre+) mice were sacrificed on day 15 and tumors were harvested. Tumor associated lymphocytes (TILs) were stimulated with PMA / ionomycin for cytokine analysis. Blocking the CGRP pathway leads to a significant increase in the abundance (Fig. 30A & 30B) and effector function (GZMB, Fig. 30C and Perforin, Fig. 14D) of CD8+TILS. Thus, mice lacking RAMP1 expression selectively on macrophages and granulocytes increases the abundance and effector function of CD8+ T cells in GB.

[0119] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0120] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS1. A method for treating a glioma in a subject refractory to checkpoint immunotherapy, comprising administering to the subject a therapeutically effective amount of a calcitonin gene-related peptide (CGRP) antagonist.

2. The method of claim 1 , wherein the CGRP antagonist blocks the binding of CGRP to Receptor Activity Modifying Protein 1 (RAMP1).

3. The method of claim 2, wherein the CGRP antagonist is an antibody or antibody fragment that binds CGRP or RAMP1 .

4. The method of claim 3, wherein the CGRP antagonist is a bi-specific antibody or antibody fragment that binds PD-1 and CGRP or RAMP1.

5. The method of claim 3, wherein the CGRP antagonist is selected from the group consisting of Erenumab (AMG-334), Eptinezumab (ALD403), Fremanezumab (TEV-48125), and Galcanezumab (LY2951742).

6. The method of claim 1 , wherein the CGRP antagonist is selected from the group consisting of Ubrogepant, Rimegepant (BMS-927711), Atogepant (AGN-241689), Telcagepant (MK-0974), Olcegepant (BIBN-4096BS), Bl 44370, MK-3207, and SB-268262.

7. The method of claim 1 , comprising(a) treating the subject with checkpoint immunotherapy;(b) determining that the subject is refractory to the checkpoint immunotherapy; and(c) administering to the subject a therapeutically effective amount of the CGRP antagonist.

8. The method of claim 1 , wherein the checkpoint immunotherapy is anti-PD1 immunotherapy.

9. The method of claim 1 , further comprising administering to the subject a checkpoint inhibitor.

10. The method of claim 9, wherein the checkpoint inhibitor comprises an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, or a combination thereof.11 . The method of claim 1 , wherein the CGRP antagonist is given at a dose lower than is used to treat migraines.

12. The method of claim 1 , wherein the glioma is a glioblastoma.

13. The method of claim 1 , wherein the glioma is low-grade glioma (LGG).

14. The method of claim 1 , further comprising detecting in a tumor tissue from the subject elevated expression levels compared to a control of RAMP1 , calcitonin receptor-like receptor (CALCRL), or a combination thereof prior to treatment with the CGRP antagonist.

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