Compositions and methods for treating autoimmune diseases and cancers

Abstract

To provide compositions and methods for treating autoimmune diseases and cancers.SOLUTION: The present invention provides methods and compositions for treating a cancer, methods for increasing an immune response to a tumor, methods for treating an autoimmune disease, and methods for decreasing an inflammation response in a subject in need thereof by modulating the expression and / or activity of Siglec 15 and / or its binding ligands. A method of treating a cancer in a subject in need of treatment of the cancer comprises administering to the subject an effective amount of a modulator of Siglec 15, where the modulator decreases the expression and / or activity of Siglec 15 in the subject, thereby treating the cancer in the subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 253,437, filed November 10, 2015, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Background of the Invention Cancer is known to be one of the leading causes of death in industrialized countries. Cancer is caused by the progressive growth of the progeny of a single transformed cell. Cancer treatment requires the removal or destruction of all malignant cells without causing patient death. An attractive way to achieve this is to induce an immune response against the tumor that distinguishes between tumor cells and their normal cellular counterparts. Indeed, the immune system has great potential to specifically destroy tumors without toxicity to normal tissues. The immune system's natural ability to detect and destroy abnormal cells may prevent the development of many cancers. Furthermore, the immune system's long-term memory may prevent cancer recurrence.

[0003] However, cancer cells can sometimes avoid detection and destruction by the immune system by reducing the expression of tumor antigens on the surface of the cancer cells, making it difficult for the immune system to detect the cancer cells. Alternatively, cancer cells may express proteins on their surface that induce the inactivation of immune cells or induce cells in the surrounding environment to release substances that suppress the immune response and promote the growth and survival of tumor cells. Therefore, there is still a need to identify new molecules that modulate the immune response to tumors in order to develop new immunotherapeutic agents (Sznol M and Chen L, Clin Cancer Res, 19(5):1021-1034, 2013).

[0004] The cell membrane of immune cells is covered with a dense coating of various glycans, including sialic acid, which are recognized by various glycan-binding proteins. Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of type I membrane proteins that regulate the function of cells in the innate and adaptive immune systems through glycan recognition (Kameda Y et al., J. Bone Miner. Res., 2013, 28(12):24463-75. Siglecs proteins contain sialic acid-binding components and Ig-like molecules. They are involved in cell adhesion interactions, neuronal and brain function, and neuronal development. There are two smaller members of the Siglec family: Siglec14 and Siglec15. Both of these molecules have small intracellular and transmembrane domains that can be phosphorylated. Due to their size, they are small enough to transmit signals across the cell membrane. These Siglec members are found in both mice and humans and are highly evolutionarily conserved between species.

[0005] Previous microarray data have shown that Siglec15 is present in peripheral myeloid cells, macrophages, and monocytes. Siglec15 is generally expressed on monocytes, macrophages, dendritic cells, B cells, and osteoclasts. Siglec15 expression in monocytes, myeloid cells, and B cells can be induced and upregulated by RANK ligand and M-CSF (Stuible M et al., J Biol Chem., 2014, 289(10):6498-512; Takamiya R et al., Glycobiology., 2013, 23(2):178-87). Siglec-15-deficient mice exhibit mild osteopetrosis due to impaired osteoclast development (Kameda Y et al., J. Bone Miner. Res., 28(12):24463-75, 2013). Osteoclast differentiation and bone resorption are also impaired. Although the role of Siglec15 in modeling has been extensively examined, little is known about the immunological functions of Siglec15 (Stuible M et al., J Biol Chem., 2014, 289(10):498-512). Recent studies have demonstrated the expression and co-localization of Siglec15 and CD68 in lung tumors, rectal adenocarcinoma, and hepatocellular carcinoma, which were also found to be expressed in macrophages, further supporting the expression of Siglec15 in myeloid cells (Takamiya R et al., Glycobiology., 2013, 23(2):178-87). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Sznol M and Chen L, Clin Cancer Res, 19(5):1021-1034, 2013 [Non-patent document 2] Kameda Y et al., J. Bone Miner. Res., 2013, Vol. 28(12): 24463-75 [Non-patent document 3] Stuible M et al., J Biol Chem., 2014, 289(10):6498-512 [Non-patent document 4] Takamiya R et al., Glycobiology., 2013;23(2):178-87 Summary of the Invention [Problem to be solved by the invention]

[0007] Thus, there is a need in the art for the identification of additional molecules that modulate the immune response to tumors in order to broaden and capitalize on the success of anti-tumor and autoimmune immunotherapies. [Means for solving the problem]

[0008] The present invention is based, at least in part, on the discovery that Siglec15 plays an essential role in suppressing the immune response to cancer, thus providing a rationale for manipulating the Siglec15 pathway for the future development of cancer therapeutics. In particular, it has been discovered that reducing Siglec15 expression reduces tumor size and prolongs survival in mice bearing brain tumors. Furthermore, brain inflammation was accelerated by blocking the interaction between Siglec15 and its ligand in a mouse model of brain inflammation, and by inhibiting Siglec15 activity by abolishing Siglec15 expression in Siglec15 knockout mice.

[0009] Thus, the present invention provides methods for treating cancer or increasing an immune response to cancer by inhibiting or suppressing the expression and / or activity of Siglec15 and / or its binding ligands, such as MAG, LRRC4C, and / or sialyl-Tn. In another aspect, the present invention includes methods for treating autoimmune disease or reducing inflammatory responses in a subject by increasing the expression and / or activity of Siglec15 and / or its binding ligands, such as MAG, LRRC4C, and / or sialyl-Tn.

[0010] In one aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a Siglec15 modulator, wherein the modulator reduces the expression and / or activity of Siglec15 in the subject, thereby treating the cancer in the subject.

[0011] In some embodiments, the modulator is a small molecule inhibitor of Siglec15, an antagonist antibody to Siglec15 or an antigen-binding fragment thereof, a recombinant Siglec15 fusion protein, or a combination thereof. In another embodiment, the Siglec15 fusion protein is selected from the group consisting of a fusion protein, an inhibitory peptide or a nucleic acid that targets Siglec15. In another embodiment, the Siglec15 fusion protein is a Siglec15-Fc fusion protein.

[0012] In some embodiments, the modulator blocks the interaction between Siglec15 and a binding ligand. In some embodiments, the binding ligand is selected from the group consisting of MAG and LRRC4C. In some embodiments, the modulator is a mutant Siglec15 protein having a single substitution mutation at residue 143. In other embodiments, the modulator is a mutant Siglec15 protein having a deletion of the IgV domain. In other embodiments, the binding ligand is sialyl-Tn. In some embodiments, the modulator is a soluble sialyl-Tn molecule. In other embodiments, the modulator is an antibody or antigen-binding fragment thereof that specifically binds to sialyl-Tn.

[0013] In some embodiments, the modulator reduces the expression and / or activity levels of MAG and / or LRRC4C. In other embodiments, the modulator reduces the expression and / or activity levels of a protein containing sialyl-Tn. In some embodiments, the modulator is selected from the group consisting of a small molecule inhibitor of MAG, an antagonist antibody to MAG or an antigen-binding fragment thereof, a recombinant MAG fusion protein, an inhibitory peptide or nucleic acid targeting MAG, a small molecule inhibitor of LRRC4C, an antagonist antibody to LRRC4C or an antigen-binding fragment thereof, a recombinant LRRC4C fusion protein, or an inhibitory peptide or nucleic acid targeting LRRC4C.

[0014] In some embodiments, the cancer is selected from the group consisting of brain cancer, lung cancer, pancreatic cancer, melanoma cancer, breast cancer, ovarian cancer, renal cell carcinoma, rectal adenocarcinoma, hepatocellular carcinoma, and Ewing's sarcoma. In other embodiments, the brain cancer is glioblastoma. In some embodiments, the cancer is colon cancer, endometrioid cancer, renal cancer (e.g., papillary renal cell carcinoma, renal clear cell carcinoma), liver cancer, thyroid cancer, lung cancer (e.g., lung adenocarcinoma, lung squamous cell carcinoma), head and neck cancer, breast cancer, cervical cancer, prostate cancer, bladder cancer, glioblastoma, rectal cancer, or bile duct cancer. In one embodiment, the cancer is brain cancer. In one embodiment, the cancer is glioblastoma. In one embodiment, the cancer is not a blood-borne cancer. In one embodiment, the cancer is not leukemia. In one embodiment, the cancer is not acute myeloid leukemia (AML).

[0015] In some embodiments, the subject is not yet afflicted with an autoimmune disease.

[0016] In other embodiments, the subject is a human.

[0017] In another aspect, the present invention provides a method for reducing tumor size in a subject in need thereof, comprising administering to the subject an effective amount of a Siglec15 modulator, wherein the modulator reduces the expression and / or activity of Siglec15, thereby reducing the tumor size in the subject.

[0018] In some embodiments, the modulator is selected from the group consisting of a small molecule inhibitor of Siglec15, an antagonist antibody to Siglec15 or an antigen-binding fragment thereof, a recombinant Siglec15 fusion protein, or an inhibitory peptide or nucleic acid that targets Siglec15. In some embodiments, the Siglec15 fusion protein is a Siglec15-Fc fusion protein.

[0019] In some embodiments, the modulator blocks the interaction between Siglec15 and a binding ligand. In some embodiments, the binding ligand is selected from the group consisting of MAG and LRRC4C. In other embodiments, the binding ligand is sialyl-Tn. In some embodiments, the modulator is a mutant Siglec15 protein having a single substitution mutation at residue 143. In other embodiments, the modulator is a mutant Siglec15 protein having a deletion of the IgV domain.

[0020] In some embodiments, the modulator reduces the expression and / or activity of MAG and LRRC4C. In some embodiments, the modulator is selected from the group consisting of a small molecule inhibitor of MAG, an antagonist antibody to MAG or an antigen-binding fragment thereof, a recombinant MAG fusion protein, an inhibitory peptide or nucleic acid targeting MAG, a small molecule inhibitor of LRRC4C, an antagonist antibody to LRRC4C or an antigen-binding fragment thereof, a recombinant LRRC4C fusion protein, or an inhibitory peptide or nucleic acid targeting LRRC4C.

[0021] In some embodiments, the tumor is associated with a cancer selected from the group consisting of brain cancer, lung cancer, pancreatic cancer, melanoma cancer, breast cancer, ovarian cancer, renal cell carcinoma, rectal adenocarcinoma, hepatocellular carcinoma, and Ewing's sarcoma. In some embodiments, the brain cancer is glioblastoma. In some embodiments, the cancer is colon cancer, endometrioid cancer, renal cancer (e.g., papillary renal cell carcinoma, renal clear cell carcinoma), liver cancer, thyroid cancer, lung cancer (e.g., lung adenocarcinoma, lung squamous cell carcinoma), head and neck cancer, breast cancer, cervical cancer, prostate cancer, bladder cancer, glioblastoma, rectal cancer, or bile duct cancer. In one embodiment, the cancer is brain cancer. In one embodiment, the cancer is glioblastoma. In one embodiment, the cancer is not a blood-borne cancer. In one embodiment, the cancer is not leukemia. In one embodiment, the cancer is not acute myeloid leukemia (AML).

[0022] In some embodiments, the subject is not yet afflicted with an autoimmune disease.

[0023] In other embodiments, the subject is a human.

[0024] In one aspect, the present invention provides a method for prolonging survival of a subject having cancer, comprising administering to the subject an effective amount of a Siglec15 modulator, wherein the modulator reduces the expression and / or activity of Siglec15, thereby prolonging survival of the subject.

[0025] In some embodiments, the modulator is selected from the group consisting of a small molecule inhibitor of Siglec15, an antagonist antibody to Siglec15 or an antigen-binding fragment thereof, a recombinant Siglec15 fusion protein, or an inhibitory peptide or nucleic acid that targets Siglec15. In some embodiments, the Siglec15 fusion protein is a Siglec15-Fc fusion protein.

[0026] In some embodiments, the modulator blocks the interaction between Siglec15 and a binding ligand. In other embodiments, the binding ligand is selected from the group consisting of MAG and LRRC4C. In other embodiments, the binding ligand is sialyl-Tn. In some embodiments, the modulator is a mutant Siglec15 protein having a single substitution mutation at residue 143. In other embodiments, the modulator is a mutant Siglec15 protein having a deletion of the IgV domain.

[0027] In some embodiments, the modulator reduces the expression and / or activity of MAG and LRRC4C. In some embodiments, the modulator is selected from the group consisting of a small molecule inhibitor of MAG, an antagonist antibody to MAG or an antigen-binding fragment thereof, a recombinant MAG fusion protein, an inhibitory peptide or nucleic acid targeting MAG, a small molecule inhibitor of LRRC4C, an antagonist antibody to LRRC4C or an antigen-binding fragment thereof, a recombinant LRRC4C fusion protein, or an inhibitory peptide or nucleic acid targeting LRRC4C. are selected.

[0028] In some embodiments, the cancer is selected from the group consisting of brain cancer, lung cancer, pancreatic cancer, melanoma cancer, breast cancer, ovarian cancer, renal cell carcinoma, rectal adenocarcinoma, hepatocellular carcinoma, and Ewing's sarcoma. In some embodiments, the brain cancer is glioblastoma. In some embodiments, the cancer is colon cancer, endometrioid cancer, renal cancer (e.g., papillary renal cell carcinoma, renal clear cell carcinoma), liver cancer, thyroid cancer, lung cancer (e.g., lung adenocarcinoma, lung squamous cell carcinoma), head and neck cancer, breast cancer, cervical cancer, prostate cancer, bladder cancer, glioblastoma, rectal cancer, or bile duct cancer. In one embodiment, the cancer is brain cancer. In one embodiment, the cancer is glioblastoma. In one embodiment, the cancer is not a blood-borne cancer. In one embodiment, the cancer is not leukemia. In one embodiment, the cancer is not acute myeloid leukemia (AML).

[0029] In some embodiments, the subject is not yet afflicted with an autoimmune disease.

[0030] In other embodiments, the subject is a human.

[0031] In one aspect, the present invention provides a method for increasing an immune response against a tumor in a subject in need thereof, the method comprising administering to the subject an effective amount of a Siglec15 modulator, wherein the modulator reduces the expression and / or activity of Siglec15, thereby increasing the immune response against the tumor in the subject.

[0032] In some embodiments, the modulator is selected from the group consisting of a small molecule inhibitor of Siglec15, an antagonist antibody to Siglec15 or an antigen-binding fragment thereof, a recombinant Siglec15 fusion protein, or an inhibitory peptide or nucleic acid that targets Siglec15. In some embodiments, the Siglec15 fusion protein is a Siglec15-Fc fusion protein.

[0033] In some embodiments, the modulator blocks the interaction between Siglec15 and a binding ligand. In some embodiments, the binding ligand is selected from the group consisting of MAG and LRRC4C. In other embodiments, the binding ligand is sialyl-Tn. In some embodiments, the modulator is a mutant Siglec15 protein having a single substitution mutation at residue 143. In other embodiments, the modulator is a mutant Siglec15 protein having a deletion of the IgV domain.

[0034] In some embodiments, the modulator reduces the expression and / or activity of MAG and LRRC4C. In some embodiments, the modulator is selected from the group consisting of a small molecule inhibitor of MAG, an antagonist antibody to MAG or an antigen-binding fragment thereof, a recombinant MAG fusion protein, an inhibitory peptide or nucleic acid targeting MAG, a small molecule inhibitor of LRRC4C, an antagonist antibody to LRRC4C or an antigen-binding fragment thereof, a recombinant LRRC4C fusion protein, or an inhibitory peptide or nucleic acid targeting LRRC4C.

[0035] In some embodiments, the tumor is associated with a cancer selected from the group consisting of brain cancer, lung cancer, pancreatic cancer, melanoma cancer, breast cancer, ovarian cancer, renal cell carcinoma, rectal adenocarcinoma, hepatocellular carcinoma, and Ewing's sarcoma. In some embodiments, the brain cancer is glioblastoma. In some embodiments, the cancer is colon cancer, endometrioid carcinoma, renal cancer (e.g., papillary renal cell carcinoma, renal clear cell carcinoma), liver cancer, thyroid cancer, lung cancer (e.g., lung adenocarcinoma, lung squamous cell carcinoma), head and neck cancer, breast cancer, cervical cancer, prostate cancer, bladder cancer, glioblastoma, rectal cancer, or bile duct cancer. In one embodiment, the cancer is brain cancer. In one embodiment, the cancer is glioblastoma. In embodiments, the cancer is not a blood-borne cancer. In one embodiment, the cancer is not a leukemia. In one embodiment, the cancer is not acute myeloid leukemia (AML).

[0036] In some embodiments, the subject is not yet afflicted with an autoimmune disease.

[0037] In other embodiments, the subject is a human.

[0038] In another aspect, the present invention provides a method of treating an autoimmune disease in a subject in need thereof, comprising administering to the subject an effective amount of a Siglec15 modulator, wherein the modulator increases the expression and / or activity of Siglec15 in the subject, thereby treating the autoimmune disease in the subject.

[0039] In some embodiments, the modulator is selected from the group consisting of a small molecule activator of Siglec15, an agonist antibody or antigen-binding fragment thereof of Siglec15, or a protein or nucleic acid that activates transcription and / or translation of Siglec15.

[0040] In some embodiments, the modulator promotes the interaction between Siglec15 and a binding ligand. In some embodiments, the binding ligand is selected from the group consisting of MAG and LRRC4C. In other embodiments, the binding ligand is sialyl-Tn. In some embodiments, the modulator increases the expression and / or activity of MAG and LRRC4C.

[0041] In some embodiments, the modulator is selected from the group consisting of a small molecule activator of MAG, an agonistic antibody to MAG or an antigen-binding fragment thereof, proteins and nucleic acids that activate the transcription and / or translation of MAG, a small molecule activator of LRRC4C, an agonistic antibody to LRRC4C or an antigen-binding fragment thereof, or proteins and nucleic acids that activate the transcription and / or translation of LRRC4C.

[0042] In other embodiments, the modulator is selected from the group consisting of a MAG protein, a nucleic acid encoding a MAG protein, an LRRC4C protein, or a nucleic acid encoding an LRRC4C protein. In one embodiment, the modulator is a synthetic sialyl-Tn or a synthetic peptide having a sialyl-Tn.

[0043] In some embodiments, the autoimmune disease is an inflammatory brain disease. In other embodiments, the inflammatory brain disease is multiple sclerosis. In other embodiments, the inflammatory brain disease is experimental autoimmune encephalomyelitis (EAE). In other embodiments, the subject is a human.

[0044] In one aspect, the present invention provides a method for treating an autoimmune disease in a subject in need thereof, comprising administering to the subject an effective amount of Siglec15 protein or a nucleic acid encoding Siglec15 protein, thereby treating the autoimmune disease in the subject.

[0045] In some embodiments, the Siglec15 protein is selected from the group consisting of a full-length Siglec15 protein, a functional fragment of Siglec15, or the IgV domain of Siglec15.

[0046] In some embodiments, the autoimmune disease is an inflammatory brain disease. In other embodiments, the inflammatory brain disease is multiple sclerosis. In other embodiments, the inflammatory brain disease is experimental autoimmune encephalomyelitis (EAE). In some embodiments, the subject is a human.

[0047] In another aspect, the present invention provides a method for reducing a brain inflammatory response in a subject in need thereof, comprising administering to the subject an effective amount of a Siglec15 modulator, wherein the modulator increases the expression and / or activity of Siglec15, thereby reducing the brain inflammatory response in the subject.

[0048] In some embodiments, the modulator is selected from the group consisting of a small molecule activator of Siglec15, an agonist antibody or antigen-binding fragment thereof of Siglec15, or a protein or nucleic acid that activates transcription and / or translation of Siglec15.

[0049] In some embodiments, the modulator promotes the interaction between Siglec15 and a binding ligand. In some embodiments, the binding ligand is selected from the group consisting of MAG and LRRC4C. In other embodiments, the binding ligand is sialyl-Tn. In other embodiments, the modulator increases the expression and / or activity of MAG and LRRC4C.

[0050] In some embodiments, the modulator is selected from the group consisting of a small molecule activator of MAG, an agonistic antibody to MAG or an antigen-binding fragment thereof, proteins and nucleic acids that activate the transcription and / or translation of MAG, a small molecule activator of LRRC4C, an agonistic antibody to LRRC4C or an antigen-binding fragment thereof, or proteins and nucleic acids that activate the transcription and / or translation of LRRC4C.

[0051] In other embodiments, the modulator is selected from the group consisting of a MAG protein, a nucleic acid encoding a MAG protein, an LRRC4C protein, or a nucleic acid encoding an LRRC4C protein.

[0052] In other embodiments, the brain inflammatory response is associated with multiple sclerosis. In some embodiments, the brain inflammatory response is associated with experimental autoimmune encephalomyelitis (EAE). In some embodiments, the subject is a human.

[0053] In one aspect, the present invention provides a method for reducing a brain inflammatory response in a subject in need thereof, the method comprising administering to the subject an effective amount of Siglec15 protein or a nucleic acid encoding a Siglec15 protein, thereby reducing the brain inflammatory response in the subject.

[0054] In some embodiments, the Siglec15 protein is selected from the group consisting of a full-length Siglec15 protein, a functional fragment of Siglec15, or the IgV domain of Siglec15.

[0055] In some embodiments, the brain inflammatory response is associated with multiple sclerosis. In some embodiments, the brain inflammatory response is associated with experimental autoimmune encephalomyelitis (EAE). In some embodiments, the subject is a human.

[0056] In another aspect, the present invention provides a method for identifying a compound useful for treating an autoimmune disease or cancer in a subject, the method comprising the steps of providing a test compound, determining the effect of the test compound on Siglec15 expression and / or activity, and selecting a compound that modulates Siglec15 expression and / or activity, thereby identifying a compound useful for treating an autoimmune disease or cancer in the subject.

[0057] In some embodiments, increased Siglec15 expression and / or activity indicates that the compound is useful for treating autoimmune diseases, while in other embodiments, decreased Siglec15 expression and / or activity indicates that the compound is useful for treating cancer.

[0058] In one aspect, the present invention provides a method for identifying a compound useful for increasing an immune response against a tumor in a subject in need thereof, the method comprising the steps of providing a test compound, determining the effect of the test compound on the expression and / or activity of Siglec15, and selecting a compound that reduces the expression and / or activity of Siglec15, thereby identifying a compound useful for increasing an immune response against the tumor in the subject.

[0059] In another aspect, the present invention provides a method for identifying a compound useful for reducing a brain inflammatory response in a subject in need thereof, the method comprising the steps of providing a test compound, determining the effect of the test compound on Siglec15 expression and / or activity, and selecting a compound that increases Siglec15 expression and / or activity, thereby identifying a compound useful for reducing a brain inflammatory response in the subject.

[0060] In one aspect, the present invention provides Siglec15 modulators that modulate the interaction between Siglec15 and MAG, LRRC4C, and / or sialyl-Tn. In some embodiments, the modulator binds to the IgV domain of Siglec15. In other embodiments, the modulator binds to an epitope comprising residue 143 of Siglec15.

[0061] In some embodiments, the modulator is an inhibitor of Siglec15. In other embodiments, the inhibitor is selected from the group consisting of a small molecule inhibitor of Siglec15, an antagonist antibody to Siglec15 or an antigen-binding fragment thereof, a recombinant Siglec15 fusion protein, or an inhibitory peptide or nucleic acid that targets Siglec15.

[0062] In some embodiments, the modulator is an activator of Siglec15. In other embodiments, the activator is selected from the group consisting of a small molecule activator of Siglec15, an agonist antibody or antigen-binding fragment thereof of Siglec15, or a protein or nucleic acid that activates transcription and / or translation of Siglec15.

[0063] The invention is illustrated by the following figures and detailed description, which do not limit the scope of the invention as defined in the claims. In certain embodiments, for example, the following are provided: (Item 1) A method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a Siglec15 modulator, wherein the modulator reduces the expression and / or activity of Siglec15 in the subject, thereby treating cancer in the subject. (Item 2) The method of item 1, wherein the modulator is selected from the group consisting of a small molecule inhibitor of Siglec15, an antagonist antibody to Siglec15 or an antigen-binding fragment thereof, a recombinant Siglec15 fusion protein, or an inhibitory peptide or nucleic acid targeting Siglec15. (Item 3) The Siglec15 fusion protein is a Siglec15-Fc fusion protein. The method according to item 2, (Item 4) 2. The method of claim 1, wherein the modulator blocks the interaction between Siglec15 and a binding ligand. (Item 5) 5. The method of claim 4, wherein the binding ligand is selected from the group consisting of MAG, LRRC4C, and sialyl-Tn. (Item 6) 5. The method of claim 4, wherein the modulator is a mutant Siglec15 protein having a single substitution mutation at residue 143. (Item 7) 5. The method of claim 4, wherein the modulator is a mutant Siglec15 protein having a deletion of the IgV domain. (Item 8) 2. The method of item 1, wherein the modulator reduces the expression and / or activity levels of MAG and LRRC4C. (Item 9) 9. The method of claim 8, wherein the modulator is selected from the group consisting of a small molecule inhibitor of MAG, an antagonist antibody to MAG or an antigen-binding fragment thereof, a recombinant MAG fusion protein, an inhibitory peptide or nucleic acid targeting MAG, a small molecule inhibitor of LRRC4C, an antagonist antibody to LRRC4C or an antigen-binding fragment thereof, a recombinant LRRC4C fusion protein, or an inhibitory peptide or nucleic acid targeting LRRC4C. (Item 10) 2. The method of item 1, wherein the cancer is selected from the group consisting of brain cancer, lung cancer, pancreatic cancer, melanoma cancer, breast cancer, ovarian cancer, renal cell carcinoma, rectal adenocarcinoma, hepatocellular carcinoma, and Ewing's sarcoma. (Item 11) Item 11. The method of item 10, wherein the brain cancer is glioblastoma. (Item 12) Item 10. The method of item 1, wherein the subject is a human. (Item 13) A method for reducing tumor size in a subject in need thereof, comprising administering to the subject an effective amount of a Siglec15 modulator, wherein the modulator reduces the expression and / or activity of Siglec15, thereby reducing the tumor size in the subject. (Item 14) 14. The method of claim 13, wherein the modulator is selected from the group consisting of a small molecule inhibitor of Siglec15, an antagonist antibody to Siglec15 or an antigen-binding fragment thereof, a recombinant Siglec15 fusion protein, or an inhibitory peptide or nucleic acid targeting Siglec15. (Item 15) Item 15. The method of item 14, wherein the Siglec15 fusion protein is a Siglec15-Fc fusion protein. (Item 16) 14. The method of claim 13, wherein the modulator blocks the interaction between Siglec15 and a binding ligand. (Item 17) 17. The method of claim 16, wherein the binding ligand is selected from the group consisting of MAG, LRRC4C, and sialyl-Tn. (Item 18) 17. The method of claim 16, wherein the modulator is a mutant Siglec15 protein having a single substitution mutation at residue 143. (Item 19) 17. The method of claim 16, wherein the modulator is a mutant Siglec15 protein having a deletion of the IgV domain. (Item 20) 14. The method of claim 13, wherein the modulator decreases the expression and / or activity of MAG, LRRC4C, or sialyl-Tn. (Item 21) 21. The method of claim 20, wherein the modulator is selected from the group consisting of a small molecule inhibitor of MAG, an antagonist antibody to MAG or an antigen-binding fragment thereof, a recombinant MAG fusion protein, an inhibitory peptide or nucleic acid targeting MAG, a small molecule inhibitor of LRRC4C, an antagonist antibody to LRRC4C or an antigen-binding fragment thereof, a recombinant LRRC4C fusion protein, or an inhibitory peptide or nucleic acid targeting LRRC4C. (Item 22) Item 14. The method of item 13, wherein the tumor is associated with a cancer selected from the group consisting of brain cancer, lung cancer, pancreatic cancer, melanoma cancer, breast cancer, ovarian cancer, renal cell carcinoma, rectal adenocarcinoma, hepatocellular carcinoma, and Ewing's sarcoma. (Item 23) 23. The method of claim 22, wherein the brain cancer is glioblastoma. (Item 24) Item 14. The method of item 13, wherein the subject is a human. (Item 25) A method for prolonging the survival of a subject having cancer, comprising administering to the subject an effective amount of a Siglec15 modulator, wherein the modulator reduces the expression and / or activity of Siglec15, thereby prolonging the survival of the subject. (Item 26) 26. The method of claim 25, wherein the modulator is selected from the group consisting of a small molecule inhibitor of Siglec15, an antagonist antibody to Siglec15 or an antigen-binding fragment thereof, a recombinant Siglec15 fusion protein, or an inhibitory peptide or nucleic acid targeting Siglec15. (Item 27) 27. The method of claim 26, wherein the Siglec15 fusion protein is a Siglec15-Fc fusion protein. (Item 28) 26. The method of claim 25, wherein the modulator blocks the interaction between Siglec15 and a binding ligand. (Item 29) 29. The method of claim 28, wherein the binding ligand is selected from the group consisting of MAG, LRRC4C, and sialyl-Tn. (Item 30) 29. The method of claim 28, wherein the modulator is a mutant Siglec15 protein having a single substitution mutation at residue 143. (Item 31) 29. The method of claim 28, wherein the modulator is a mutant Siglec15 protein having a deletion of the IgV domain. (Item 32) the modulator reduces the expression and / or activity of MAG and LRRC4C. Item 26. The method according to item 25. (Item 33) 33. The method of claim 32, wherein the modulator is selected from the group consisting of a small molecule inhibitor of MAG, an antagonist antibody to MAG or an antigen-binding fragment thereof, a recombinant MAG fusion protein, an inhibitory peptide or nucleic acid targeting MAG, a small molecule inhibitor of LRRC4C, an antagonist antibody to LRRC4C or an antigen-binding fragment thereof, a recombinant LRRC4C fusion protein, or an inhibitory peptide or nucleic acid targeting LRRC4C. (Item 34) 26. The method of item 25, wherein the cancer is selected from the group consisting of brain cancer, lung cancer, pancreatic cancer, melanoma cancer, breast cancer, ovarian cancer, renal cell carcinoma, rectal adenocarcinoma, hepatocellular carcinoma, and Ewing's sarcoma. (Item 35) 35. The method of claim 34, wherein the brain cancer is glioblastoma. (Item 36) 26. The method of claim 25, wherein the subject is a human. (Item 37) A method for increasing an immune response against a tumor in a subject in need thereof, comprising administering to the subject an effective amount of a Siglec15 modulator, wherein the modulator reduces the expression and / or activity of Siglec15, thereby increasing the immune response against the tumor in the subject. (Item 38) 38. The method of claim 37, wherein the modulator is selected from the group consisting of a small molecule inhibitor of Siglec15, an antagonist antibody to Siglec15 or an antigen-binding fragment thereof, a recombinant Siglec15 fusion protein, or an inhibitory peptide or nucleic acid targeting Siglec15. (Item 39) 39. The method of claim 38, wherein the Siglec15 fusion protein is a Siglec15-Fc fusion protein. (Item 40) 38. The method of claim 37, wherein the modulator blocks the interaction between Siglec15 and a binding ligand. (Item 41) 41. The method of claim 40, wherein the binding ligand is selected from the group consisting of MAG, LRRC4C, and sialyl-Tn. (Item 42) 41. The method of claim 40, wherein the modulator is a mutant Siglec15 protein having a single substitution mutation at residue 143. (Item 43) 41. The method of claim 40, wherein the modulator is a mutant Siglec15 protein having a deletion of the IgV domain. (Item 44) 38. The method of item 37, wherein the modulator reduces the expression and / or activity of MAG and LRRC4C. (Item 45) 45. The method of claim 44, wherein the modulator is selected from the group consisting of a small molecule inhibitor of MAG, an antagonist antibody to MAG or an antigen-binding fragment thereof, a recombinant MAG fusion protein, an inhibitory peptide or nucleic acid targeting MAG, a small molecule inhibitor of LRRC4C, an antagonist antibody to LRRC4C or an antigen-binding fragment thereof, a recombinant LRRC4C fusion protein, or an inhibitory peptide or nucleic acid targeting LRRC4C. method. (Item 46) 38. The method of item 37, wherein the tumor is associated with a cancer selected from the group consisting of brain cancer, lung cancer, pancreatic cancer, melanoma cancer, breast cancer, ovarian cancer, renal cell carcinoma, rectal adenocarcinoma, hepatocellular carcinoma, and Ewing's sarcoma. (Item 47) Item 47. The method of item 46, wherein the brain cancer is glioblastoma. (Item 48) 38. The method of claim 37, wherein the subject is a human. (Item 49) A method for treating an autoimmune disease in a subject in need thereof, comprising administering to the subject an effective amount of a Siglec15 modulator, wherein the modulator increases the expression and / or activity of Siglec15 in the subject, thereby treating the autoimmune disease in the subject. (Item 50) 50. The method of claim 49, wherein the modulator is selected from the group consisting of a small molecule activator of Siglec15, an agonist antibody of Siglec15 or an antigen-binding fragment thereof, or a protein or nucleic acid that activates transcription and / or translation of Siglec15. (Item 51) 50. The method of claim 49, wherein the modulator promotes the interaction between Siglec15 and a binding ligand. (Item 52) 52. The method of claim 51, wherein the binding ligand is selected from the group consisting of MAG, LRRC4C, and sialyl-Tn. (Item 53) 50. The method of item 49, wherein the modulator increases the expression and / or activity of MAG and LRRC4C. (Item 54) 54. The method of claim 53, wherein the modulator is selected from the group consisting of a small molecule activator of MAG, an agonistic antibody to MAG or an antigen-binding fragment thereof, proteins and nucleic acids that activate the transcription and / or translation of MAG, a small molecule activator of LRRC4C, an agonistic antibody to LRRC4C or an antigen-binding fragment thereof, or proteins and nucleic acids that activate the transcription and / or translation of LRRC4C. (Item 55) 54. The method of item 53, wherein the modulator is selected from the group consisting of a MAG protein, a nucleic acid encoding a MAG protein, an LRRC4C protein, or a nucleic acid encoding an LRRC4C protein. (Item 56) 50. The method of claim 49, wherein the autoimmune disease is an inflammatory brain disease. (Item 57) 57. The method of claim 56, wherein the inflammatory brain disease is multiple sclerosis. (Item 58) 50. The method of claim 49, wherein the subject is a human. (Item 59) A method for treating an autoimmune disease in a subject in need thereof, comprising administering to the subject an effective amount of Siglec15 protein or a nucleic acid encoding Siglec15 protein, thereby treating the autoimmune disease in the subject. (Item 60) 60. The method of item 59, wherein the Siglec15 protein is selected from the group consisting of a full-length Siglec15 protein, a functional fragment of Siglec15, or the IgV domain of Siglec15. (Item 61) 60. The method of claim 59, wherein the autoimmune disease is an inflammatory brain disease. (Item 62) Item 62. The method of item 61, wherein the inflammatory brain disease is multiple sclerosis. (Item 63) 60. The method of claim 59, wherein the subject is a human. (Item 64) A method for reducing a brain inflammatory response in a subject in need thereof, comprising administering to the subject an effective amount of a Siglec15 modulator, wherein the modulator increases the expression and / or activity of Siglec15, thereby reducing the brain inflammatory response in the subject. (Item 65) 65. The method of item 64, wherein the modulator is selected from the group consisting of a small molecule activator of Siglec15, an agonist antibody of Siglec15 or an antigen-binding fragment thereof, or a protein and nucleic acid that activates transcription and / or translation of Siglec15. (Item 66) 65. The method of claim 64, wherein the modulator promotes the interaction between Siglec15 and a binding ligand. (Item 67) 67. The method of claim 66, wherein the binding ligand is selected from the group consisting of MAG, LRRC4C, and sialyl-Tn. (Item 68) 65. The method of item 64, wherein the modulator increases the expression and / or activity of MAG and LRRC4C. (Item 69) 69. The method of claim 68, wherein the modulator is selected from the group consisting of a small molecule activator of MAG, an agonistic antibody to MAG or an antigen-binding fragment thereof, proteins and nucleic acids that activate the transcription and / or translation of MAG, a small molecule activator of LRRC4C, an agonistic antibody to LRRC4C or an antigen-binding fragment thereof, or proteins and nucleic acids that activate the transcription and / or translation of LRRC4C. (Item 70) 69. The method of item 68, wherein the modulator is selected from the group consisting of a MAG protein, a nucleic acid encoding a MAG protein, an LRRC4C protein, or a nucleic acid encoding an LRRC4C protein. (Item 71) 65. The method of claim 64, wherein the brain inflammatory response is associated with multiple sclerosis. (Item 72) Item 65. The method of item 64, wherein the subject is a human. (Item 73) A method for reducing a brain inflammatory response in a subject in need thereof, comprising administering to the subject an effective amount of Siglec15 protein or a nucleic acid encoding Siglec15 protein, thereby reducing the brain inflammatory response in the subject. (Item 74) The Siglec15 protein is selected from the group consisting of a full-length Siglec15 protein, a functional fragment of Siglec15, or the IgV domain of Siglec15. Item 73. The method according to item 73. (Item 75) 74. The method of claim 73, wherein the brain inflammatory response is associated with multiple sclerosis. (Item 76) 74. The method of claim 73, wherein the subject is a human. (Item 77) 1. A method for identifying a compound useful for treating an autoimmune disease or cancer in a subject, comprising: providing a test compound; determining the effect of the test compound on Siglec15 expression and / or activity; selecting a compound that modulates the expression and / or activity of Siglec15, thereby identifying a compound useful for treating an autoimmune disease or cancer in the subject. (Item 78) 78. The method of item 77, wherein an increase in Siglec15 expression and / or activity indicates that the compound is useful for treating an autoimmune disease. (Item 79) 78. The method of item 77, wherein a decrease in the expression and / or activity of Siglec15 indicates that the compound is useful for treating cancer. (Item 80) 1. A method for identifying a compound useful for increasing an immune response against a tumor in a subject in need thereof, comprising: providing a test compound; determining the effect of the test compound on Siglec15 expression and / or activity; selecting a compound that reduces the expression and / or activity of Siglec15, thereby identifying a compound useful for increasing the immune response against the tumor in the subject. (Item 81) 1. A method for identifying a compound useful for reducing a brain inflammatory response in a subject in need thereof, comprising: providing a test compound; determining the effect of the test compound on Siglec15 expression and / or activity; selecting a compound that increases the expression and / or activity of Siglec15, thereby identifying a compound useful for reducing brain inflammatory responses in the subject. (Item 82) A Siglec15 modulator that modulates the interaction between Siglec15 and MAG, LRRC4C, and sialyl-Tn. (Item 83) 83. The modulator of item 82, which binds to the IgV domain of Siglec15. (Item 84) 83. The modulator of item 82, which binds to an epitope comprising residue 143 of Siglec15. (Item 85) 83. The modulator of item 82, which is an inhibitor of Siglec15. (Item 86) The inhibitor may be a small molecule inhibitor of Siglec15, an antagonism to Siglec15, or the like. 86. The modulator of claim 85, wherein the modulator is selected from the group consisting of an anti-Siglec15 antibody or antigen-binding fragment thereof, a recombinant Siglec15 fusion protein, or an inhibitory peptide or nucleic acid targeting Siglec15. (Item 87) 83. The modulator according to item 82, which is an activator of Siglec15. (Item 88) 88. The modulator of item 87, wherein the activator is selected from the group consisting of a small molecule activator of Siglec15, an agonist antibody or antigen-binding fragment thereof of Siglec15, or a protein and nucleic acid that activates transcription and / or translation of Siglec15. (Item 89) 2. The method of claim 1, wherein the subject does not already suffer from an autoimmune disease. (Item 90) 14. The method of claim 13, wherein the subject does not already suffer from an autoimmune disease. (Item 91) 26. The method of claim 25, wherein the subject does not already have an autoimmune disease. (Item 92) 38. The method of claim 37, wherein the subject does not already have an autoimmune disease. [Brief explanation of the drawings]

[0064] [Figure 1A-B] Figure 1A shows the RNA expression pattern of Siglec15 in various mouse tissues using RT-PCR. Figure 1B shows the RNA expression pattern of Siglec15 in human tissues using microarray analysis. Siglec15 is generally expressed on monocytes, macrophages, dendritic cells, B cells, and osteoclasts. [Figure 1C] Figure 1C shows a schematic representation of the expression pattern of Siglec15 based on microarray analysis. Siglec15 is generally expressed on monocytes, macrophages, dendritic cells, B cells, and osteoclasts.

[0065] [Figure 2] FIG. 2 shows Siglec15 expression levels in human cancers compared with normal tissues.

[0066] [Figure 3A] FIG. 3A shows the expression of human Siglec15 in human cancer cell lines as determined using NCI60 microarray data (BioGPS; arbitrary units). [Figure 3B] FIG. 3B shows the expression of human Siglec15 in human cancer cell lines, as determined using FACS.

[0067] [Figure 4A] Figure 4A shows the interaction between Siglec15 and its binding ligands, myelin-associated glycoprotein (MAG) and leucine-rich repeat-containing 4C (LRRC4C). The interaction between Siglec15 and MAG or LRRC4C is well conserved between mice and humans. [Figure 4B] FIG. 4B shows the binding between sialyl-Tn antigen and Siglec15-mIg fusion protein or control mIg, as determined using an Octet streptavidin biosensor.

[0068] [Figure 5-1] Figure 5 shows the mRNA expression of MAG in normal tissues and cancers. MAG expression is enriched in brain or brain-related tumors. [Figure 5-2] Figure 5 shows the mRNA expression of MAG in normal tissues and cancers. MAG expression is enriched in brain or brain-related tumors. [Figure 5-3] Figure 5 shows the mRNA expression of MAG in normal tissues and cancers. MAG expression is enriched in brain or brain-related tumors.

[0069] [Figure 6-1]Figure 6 shows the mRNA expression of LRRC4C in normal tissues and cancers. LRRC4C expression is upregulated in cancers such as brain cancer, breast cancer, ovarian cancer, renal cell carcinoma, and Ewing's sarcoma. [Figure 6-2] Figure 6 shows the mRNA expression of LRRC4C in normal tissues and cancers. LRRC4C expression is upregulated in cancers such as brain cancer, breast cancer, ovarian cancer, renal cell carcinoma, and Ewing's sarcoma. [Figure 6-3] Figure 6 shows the mRNA expression of LRRC4C in normal tissues and cancers. LRRC4C expression is upregulated in cancers such as brain cancer, breast cancer, ovarian cancer, renal cell carcinoma, and Ewing's sarcoma.

[0070] [Figure 7] Figure 7 shows a schematic of the binding domains for Siglec15-MAG or Siglec15-LRRC4C interactions. In particular, the IgV domain of Siglec15 is required for interactions with MAG and LRRC4C. The R143A mutation in the IgV domain prevented binding to either MAG or LRRC4C, suggesting that both ligands bind to residue 143 in the IgV domain of Siglec15.

[0071] [Figure 8] Figure 8A shows the level of H-thymidine incorporation in PBMCs stimulated with the anti-CD3 antibody OKT3 and exposed to immobilized human Siglec15 or control IgG. Figure 8B shows the level of luciferase activity observed in Jurkat NF-AT reporter cells after incubation with mock plasmid (Mock), full-length FASLG (FASLG), full-length Siglec15 (Siglec15 FL), a construct encoding the Siglec15 ectodomain and B7-H6 transmembrane domain (Siglec15 ATM), or 293T.m.OKT3 cells overexpressing LRRC4C.

[0072] [Figure 9]Figure 9A shows the level of H-thymidine incorporation in mouse splenocytes stimulated with immobilized anti-CD3 and either immobilized mouse Siglec-15-mIgG fusion protein (S15-mIg) or control IgG (mIg). Figure 9B shows the level of H-thymidine incorporation in mouse splenocytes stimulated with immobilized anti-CD3 and either soluble mouse Siglec-15-mIgG fusion protein (S15-mIg) or control IgG (mIg). Figure 9C shows the level of H-thymidine incorporation in activated splenocytes from OT-1 transgenic mice cocultured with 293T-KbOVA cells overexpressing full-length mouse Siglec15 (KbOVA-S15) or mock control (KbOVA-control). Figure 9D shows the level of Siglec15 expression in three 293T-KbOVA cell lines overexpressing mouse Siglec15. Figure 9E shows the levels of IFN-γ present in the supernatant of activated splenocytes from OT-1 transgenic mice cocultured with 293T-KbOVA cells with elevated levels of Siglec15 expression (as determined in Figure 9D), and Figure 9F shows the levels of TNF-α present in the supernatant of activated splenocytes from OT-1 transgenic mice cocultured with 293T-KbOVA cells with elevated levels of Siglec15 expression.

[0073] [Figure 10] Figure 10A shows the dose-response of OT-1 T cell killing of 293T-KbOVA target cells, as demonstrated by an adhesion-based cytotoxicity assay (ACEA Biosciences). Figure 10B compares the adhesion signals of 293T-KbOVA cells overexpressing Siglec15 (293T-KbOVA-S15) with those of mock-transfected 293T-KbOVA cells (293T-KbOVA-control) in the presence of increasing amounts of OT-1 T cells. The ratio indicates the proportion of OT-1 T cells to 293T-KbOVA cells.

[0074] [Figure 11]Figure 11A is a schematic diagram of the OT-1 in vivo activation model described in Example 8. Figure 11B shows the percentage of OT-1 cells in the total CD8 T cell population in the blood (left panel) and spleen (right panel) of wild-type (WT) or Siglec15 whole-body knockout (KO) mice after intravenous transfer of splenocytes from OT-1 / RagKO mice. The percentage of OT-1 cells was monitored by FACS staining using OT-1 tetramer. Figure 11C shows the proliferation of blood OT-1 cells on day 5, determined by anti-EdU staining and calculated as the percentage of EdU-positive OT-1 cells / total OT-1-positive cells. Figure 11D shows apoptosis in splenocytes isolated and cultured overnight without stimulation and stained for Annexin V. Apoptosis was calculated as the percentage of Annexin V-positive OT-1 cells / total OT-1-positive cells.

[0075] [Figure 12] Figure 12A shows the percentage of OT-1 cells in the total CD8+ T cell population in the blood of mice at various time points after intravenous transfer of splenocytes from OT-1 / RagKO mice according to the schedule described in Figure 11A. WT, wild-type mice; KO, Siglec15 whole-body knockout mice; LysM-Cre KO, macrophage-specific Siglec15 knockout mice. Figure 12B shows the levels of IL-10 in the plasma of whole-body KO and LysM-Cre KO mice compared with wild-type mice, assessed during the OT-1 T cell response.

[0076] [Figure 13] FIG. 13 shows the percentages of myeloid cells, CD8+ cells, and CD4+ cells in the blood of 11-month-old wild-type (WT) or Siglec15 knockout (S15KO) mice.

[0077] [Figure 14] FIG. 14 shows cytokine levels in the supernatants of macrophages co-cultured with 293T cells overexpressing mock plasmid (control), full-length LRRC4C, or Siglec15.

[0078] [Figure 15] Figure 15 shows that the Siglec15 antibody S15m02 acted as a blocking antibody that prevented Siglec15 from binding to MAG or LRRC4C, whereas the Siglec15 antibody S15m03 did not affect the interaction between Siglec15 and MAG or LRRC4C.

[0079] [Figure 16] FIG. 16 shows that addition of S15m02, a blocking antibody against Siglec15, caused EAE mice to develop more severe disease symptoms than their counterparts injected with control mAb or receiving S15m03 antibody.

[0080] [Figure 17] FIG. 17 shows that addition of Siglec15-Fc fusion protein to EAE (experimental autoimmune encephalomyelitis) mice led to enhanced inflammation in those mice.

[0081] [Figure 18] Figure 18 shows that Siglec15 knockout (KO) mice (top line) exhibited more severe EAE disease symptoms than wild-type (WT) mice (bottom line), thereby demonstrating an inhibitory role for Siglec15 in regulating brain inflammatory responses.

[0082] [Figure 19]Figure 19A shows EAE clinical scores in WT mice immunized with MOG peptide on days 0 and 1, boosted with pertussis toxin, and then treated with 100 μg of Siglec15-mIg fusion protein (Siglec15-mIg) or control mIg (left panel), or Siglec15-hIg fusion protein (Siglec15-hIg) or control hIg (right panel) twice a week for a total of four doses starting on day 6. Figure 19B shows H-thymidine incorporation in splenocytes obtained from mice treated with control mIg on day 12 and restimulated with MOG peptide (60 μg / ml) for 3 days in the presence of 5 μg / ml of Siglec15-mIg (S15-mIg) or control mIg (mIg).

[0083] [Figure 20] FIG. 20 shows that blocking Siglecs by Siglec15-Fc treatment significantly reduced tumor size in glioblastoma-bearing mice.

[0084] [Figure 21] FIG. 21 shows that blocking Siglecs by Siglec15-Fc treatment significantly prolonged survival of glioblastoma-bearing mice.

[0085] [Figure 22-1] FIG. 22 shows the synergistic effect of Siglec15-Fc and anti-PDL1 treatment on reducing tumor size and promoting survival benefit in glioblastoma-bearing mice. [Figure 22-2] FIG. 22 shows the synergistic effect of Siglec15-Fc and anti-PDL1 treatment on reducing tumor size and promoting survival benefit in glioblastoma-bearing mice.

[0086] [Figure 23A]FIG. 23A shows luciferase activity in the brains of wild-type (WT) or Siglec15 knockout (Siglec15 KO) mice intracranially inoculated with GL261 glioblastoma cells containing a luciferase reporter on day 0. [Figure 23B] FIG. 23B shows images of luciferase activity in the brains of WT and KO mice 13 and 18 days after inoculation with GL261 cells. [Figure 23C] FIG. 23C shows the survival curves of WT and KO mice inoculated with GL261.

[0087] [Figure 24A] FIG. 24A shows the percentage and total number of CD8 T cells in the brain or spleen of both groups (WT and KO) on day 14. [Figure 24B] FIG. 24B shows the percentage and total number of CD4 T cells in the brain or spleen of both groups (WT and KO) on day 14. [Figure 24C-1] FIG. 24C shows the percentage and total number of dendritic cells (DCs), macrophages, and microglia in the brain or spleen of both groups (WT and KO) on day 14. [Figure 24C-2] FIG. 24C shows the percentage and total number of dendritic cells (DCs), macrophages, and microglia in the brain or spleen of both groups (WT and KO) on day 14. [Figure 24D] Figure 24D shows the percentage and total number of IFN-γ positive CD8 or CD4 T cells in brain lymphocytes from tumor-bearing WT or KO mice obtained on day 14 and restimulated overnight with GL-261 tumor cells.

[0088] [Figure 25] Figure 25A shows Siglec15 expression in MC38-S15- and MC38-S15+ cells as determined by FACS staining using an anti-Siglec15 monoclonal antibody (Figure 25A). Figure 25B shows tumor growth of MC38-S15- and MC38-S15+ cells after subcutaneous inoculation in B6 mice (Figure 25B).

[0089] [Figure 26] Figure 26 shows the average tumor size in B6 mice inoculated with the MC38-S15+ stable cell line and treated with a control antibody, anti-Siglec15 antibody m01, or Siglec15-mIg fusion protein. The average tumor size in each group is shown.

[0090] [Figure 27] Figure 27A summarizes the proposed mechanism of action of Siglec15 functioning as a ligand, and Figure 27B summarizes the proposed mechanism of action of Siglec15 functioning as a receptor. DETAILED DESCRIPTION OF THE INVENTION

[0091] Detailed Description of the Invention The present invention is based, at least in part, on the discovery that Siglec15 plays an essential role in suppressing the immune response to cancer, thus providing a rationale for manipulating the Siglec15 pathway for the future development of cancer therapeutics. In particular, it has been discovered that reducing Siglec15 expression reduces tumor size and prolongs survival in mice bearing brain tumors. Furthermore, inhibiting Siglec15 activity by blocking the interaction between Siglec15 and its ligands in a mouse model of brain inflammation and by abolishing Siglec15 expression in Siglec15 knockout mice exacerbated brain inflammation. Thus, the present invention provides methods for treating cancer, autoimmune diseases, regulating immune responses to cancer, and regulating inflammatory responses in subjects by modulating the expression and / or activity of Siglec15 and its binding partners in the same pathway.

[0092] I. Definition In order to make the present invention easier to understand, certain terms are first defined. Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. The meaning and scope of the terms should be clear, but in the event of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definition. The recitation of ranges of values ​​herein, unless otherwise specified herein, merely serves as a shorthand way of individually referring to each of the separate values ​​listed or falling within the range, and each separate value is incorporated herein as if it were individually listed.

[0093] In the following description, for purposes of explanation, specific numbers, materials, and configurations are set forth to provide a thorough understanding of the present invention. However, it will be apparent to those skilled in the art that the present invention can be practiced without these specific details. In some instances, well-known features may be omitted or simplified so as not to obscure the present invention. Furthermore, in this specification, reference to phrases such as "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Appearances of phrases such as "in one embodiment" in various places in this specification do not necessarily all refer to the same embodiment.

[0094] The use of the terms "a," "an," and "the" and similar referents, in describing the present invention (particularly in connection with the claims which follow), should be construed to encompass both the singular and the plural (i.e., one or more) unless otherwise specified herein or clearly contradicted by context. By way of example, "an element" means one element or more than one element. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise specified.

[0095] As used herein, the term "Siglec15" is also known as sialic acid-binding Ig-like lectin 15, CD33 antigen-like 3, CD33 molecule-like 3, CD33L3, and HsT1361, and is a member of the lectin family of proteins containing a sialic acid-binding component and an Ig-like molecule. The sequence of human Siglec15 mRNA can be found, for example, in GenBank accession number GI:225637536 (NM_213602.2; SEQ ID NO:1). The sequence of human Siglec15 polypeptide sequence can be found, for example, in GenBank accession number GI:4 7106069 (NP_998767.1 SEQ ID NO:2).

[0096] As used herein, the term "MAG," also known as myelin-associated glycoprotein, sialic acid-binding Ig-like lectin 4A, SIGLEC4A, or GMA, is a type I membrane protein and a member of the immunoglobulin superfamily. MAG is a functional receptor for NOGO-66 and is thought to be involved in neuronal myelination. MAG is also present in myeloid cells and, in particular, microglia. MAG knockout (KO) mice exhibit phagocytosis problems. MAG binds to sialylated glycoconjugates and mediates certain myelin-neuron cell interactions. Three alternatively spliced ​​transcripts encoding different isoforms have been described for this gene. The sequence of human MAG mRNA can be found, for example, in GenBank accession GI:312836849 (NM_002361.3; SEQ ID NO: 3). The sequence of the human MAG polypeptide sequence can be found, for example, in GenBank Accession No. GI:11225258 (NP_002352.1 SEQ ID NO:4).

[0097] As used herein, the term "LRRC4C" is also known as leucine-rich repeat (LRR)-containing 4C, netrin-G1 ligand, NGL1, or KIAA1580. LRRC4C is an LRR family molecule associated with neuronal growth, dendritogenesis, and axonal elongation. LRRC4C is primarily localized on the postsynaptic side of excitatory synapses and interacts with the presynaptic ligand netrin-G1 to regulate excitatory synapse formation. The sequence of human LRRC4C mRNA can be found, for example, in GenBank accession number GI:385724810 (NM_020929.2; SEQ ID NO:5). The sequence of human LRRC4C polypeptide can be found, for example, in GenBank accession number GI:51317373 (NP_065980.1 SEQ ID NO:6).

[0098] As used herein, the term "sialyl-Tn," also known as "STn," "sialyl-Tn antigen," and "Neu5Acα2-6GalNAcα-O-Ser / Thr," refers to a sialic acid-substituted form of the Tn antigen. The Tn antigen, as an O-glycan, is an N-acetylgalactosamine (GalNAc) oligosaccharide linked to serine or threonine by a glycosidic bond. Sialyl-Tn is a truncated O-glycan containing sialic acid α-2,6 linked to GalNAc α-O-Ser / Thr. Sialyl-Tn expression is caused by activation of an aberrant glycosylation pathway and typically occurs in tumor cells. Sialyl-Tn biosynthesis is associated with the expression of sialyltransferase ST6GalNAc1 and mutations in COSMC (core 1 β3-Gal-T specific molecular chaperone). Sialyl-Tn expression has been reported in over 80% of human cancers, including gastric, colon, breast, lung, esophageal, prostate, and endometrial cancers, and is associated with poor prognosis (Munkley, Int. J. Mol. Sci. ( (2016), Volume 17(3): 275 pages).

[0099] It should be noted that throughout, unless otherwise specified, the names of molecules, e.g., Siglec15, MAG, or LLRC4C, include both the gene and the protein. Thus, the term "Siglec15," when used in reference to a molecule, includes both the Siglec15 protein and the Siglec15 gene.

[0100] The term "Siglec15 level" encompasses the level of Siglec15 mRNA or cDNA, and / or protein concentration, expression, activity, function, or stability of Siglec15 protein, DNA, mRNA, or cDNA. In one embodiment, the term "level" as used herein refers to the measurable quantity of Siglec15. The amount can be either (a) an absolute amount, measured in molecules, moles, or weight per unit volume or cell, or (b) a relative amount, measured, for example, by densitometry analysis.

[0101] As used herein, "Siglec15 modulator" refers to any compound or molecule that modulates Siglec15 mRNA expression and / or protein expression; and / or Siglec15 mRNA and / or protein stability; and / or Siglec15 biological activity. Modulators may directly or indirectly modulate Siglec15 expression and / or activity. Siglec15 modulators act directly on Siglec15, e.g., antibodies that bind to Siglec15 and inhibit or activate its function. In another embodiment, Siglec15 modulators act indirectly on Siglec15 (e.g., through another molecule, e.g., a binding partner of Siglec15), resulting in an increase or decrease in Siglec15 activity. Exemplary agents suitable for use in the methods of the present invention include: Agents suitable for use in the methods of the present invention include interfering nucleic acid molecules (e.g., antisense RNA, sdRNA, and siRNA), intracellular antibodies, recombinant fusion proteins, inhibitory peptides, or small molecules. Agents suitable for use in the methods of the present invention are discussed in detail below.

[0102] As used herein, the various forms of the term "modulate" include stimulation (e.g., increasing or upregulating a particular response or activity) and inhibition (e.g., decreasing or downregulating a particular response or activity). In some embodiments, a modulator is an inhibitor of Siglec15. In some embodiments, a modulator is an activator of Siglec15.

[0103] As used herein, the term "inhibit" refers to a decrease in the expression, stability, and / or biological activity of Siglec15. For example, the term "inhibit" refers to the ability to decrease or downregulate the expression, stability, and / or activity of Siglec15 as described herein.

[0104] As used herein, "stimulate" refers to increasing the expression, stability, and / or biological activity of Siglec15. For example, the term "stimulate" refers to the ability to increase or upregulate the expression, stability, and / or activity of Siglec15 as described herein.

[0105] As used herein, "inhibitor of Siglec15 activity" or "Siglec15 antagonist" includes any molecule that partially or completely blocks, inhibits, or neutralizes a biological activity mediated by Siglec15. In some embodiments, the Siglec15 antagonist inhibits a Siglec15 polypeptide. In other embodiments, the Siglec15 antagonist inhibits a ligand of Siglec15, such as MAG, LRRC4C, or sialyl-Tn.

[0106] An "agonist of Siglec15 activity" or "Siglec15 agonist," as used herein, is an agent that partially or fully stimulates or inhibits the biological activity of an antigen to which it binds. Exemplary agonists of Siglec15 activity include any molecule that binds to Siglec15 and, upon binding, stimulates signaling through Siglec15, mimicking the interaction of Siglec15 with its natural ligand, e.g., MAG, LRRC4C, or sialyl-Tn. Other exemplary agonists of Siglec15 activity bind to and signal through a Siglec15 ligand, e.g., MAG, LRRC4C, or sialyl-Tn, mimicking the interaction of that ligand with Siglec15.

[0107] The term "antibody," as used herein, refers to an immunoglobulin molecule composed of four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) and interspersed, more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR1, CDR2, FR3, CDR3, FR4.

[0108] The term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion"), as used herein, refers to a portion of a full-length antibody, generally the target-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments. The phrase "functional fragment" of an antibody refers to a compound that shares qualitative biological activity with a full-length antibody. For example, a functional fragment of an antagonist anti-Siglec15 antibody may bind to Siglec15 so as to block, inhibit, or neutralize Siglec15-mediated biological activity. As used herein, "functional fragment" refers to Fv, scFv, diabody, F(ab)2, and F(ab')2 fragments with respect to antibodies. An "Fv" fragment is the smallest antibody fragment that contains a complete target recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, noncovalent association (VH-VL dimer). In this configuration, the three CDRs of each variable domain interact to define a target-binding site on the surface of the VH-VL dimer. An scFv contains one heavy-chain variable domain and one light-chain variable domain connected by a linker peptide sized to allow the VH and VL domains to interact to form the target-binding site. Collectively, the six CDRs confer target-binding specificity to the antibody or antibody fragment. However, even a single variable domain (or half of an Fv, containing only three target-specific CDRs) can have the ability to recognize and bind to a target, albeit with lower affinity than the entire binding site.

[0109] The term "antagonist antibody" or "blocking antibody," as used herein, refers to an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Exemplary antagonist antibodies substantially or completely inhibit the biological activity of the antigen.

[0110] The term "agonist antibody," as used herein, refers to an antibody that stimulates or enhances the biological activity of the antigen to which it binds. Exemplary agonist antibodies stimulate the transmission of a signal through the antigen upon binding, mimicking the interaction of the antigen with a natural ligand.

[0111] The term "subject," as used herein, refers to an animal, e.g., a mammal, including a primate (e.g., a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (e.g., a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, and a whale), a bird (e.g., a duck or a goose), The term "human" is used to refer to animals such as sharks and other mammals. In some embodiments, the subject is a human, for example, a human being treated or evaluated for a disease, disorder, or condition, a human being at risk for a disease, disorder, or condition, a human being with a disease, disorder, or condition, and / or a human being treated for a disease, disorder, or condition described herein. In some embodiments, the subject does not yet suffer from an autoimmune disease. In one embodiment, the subject is approximately 1 year old, approximately 2 years old, approximately 3 years old, approximately 4 years old, approximately 5 years old, approximately 6 years old, approximately 7 years old, approximately 8 years old, approximately 9 years old, or approximately 10 years old. In another embodiment, the subject is approximately 5-10 years old, approximately 10-15 years old, approximately 15-20 years old, approximately 20-25 years old, approximately 25-30 years old, approximately 30-35 years old, approximately 35-40 years old, approximately 40-45 years old, approximately 45-50 years old, approximately 50-55 years old, approximately 55-60 years old, approximately 60-65 years old, approximately 65-70 years old, approximately 70-75 years old, approximately 75-80 years old, approximately 80-85 years old, approximately 85-90 years old, approximately 90-95 years old, approximately 95-100 years old. Values ​​and ranges interspersed within the ranges listed above are also intended to be part of the present invention. Additionally, ranges of values ​​using a combination of any of the values ​​listed above as upper and / or lower limits are intended to be included.

[0112] As used herein, the terms "treating" or "treatment" refer to a beneficial or desired result, including, but not limited to, alleviation or reversal of one or more symptoms, whether detectable or undetectable, a decrease in the severity of the disorder, a state in which the disorder is stable (i.e., not worsening), or an improvement or alleviation of the disorder. "Treatment" also includes prolonging survival as compared to expected survival in the absence of treatment.

[0113] As used herein, the term "effective amount" refers to the amount of a therapy that is sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof, inhibit or prevent the progression of a disorder, cause regression of a disorder, inhibit or prevent the recurrence, development, onset, or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., a prophylactic or therapeutic agent). An effective amount may require more than one dose.

[0114] II. The Methods of the Invention. The present invention is based, at least in part, on the discovery that Siglec15 plays an essential role in immune regulation. Siglec15 has been extensively studied in osteoclast differentiation, where it plays a non-immunological role. In addition to its role in osteoclast differentiation, Siglec15, as described herein, regulates immune responses by suppressing the activity of immune cells, including T cells and macrophages. Thus, upregulation of Siglec15 in cancer cells or tumor microenvironments can suppress a subject's immune response to cancer. Furthermore, deficiency of Siglec15 can contribute to autoimmune-related conditions.

[0115] Without wishing to be bound by theory, it has been proposed that Siglec15 may fulfill its immunomodulatory function by acting as a ligand and / or receptor (Figure 27). Siglec15 is expressed in myeloid cells and is overexpressed in the brain microenvironment, cancer cells, and tumor microenvironment. When Siglec15 is overexpressed in the brain, cancer cells, or tumor microenvironment, it may function as a ligand, where it may affect T cell responses directly or indirectly through myeloid cells. Both T cells and myeloid cells may express receptor(s) involved in the immune suppressive function of Siglec15 (Figure 27A). Additionally or alternatively, expression of Siglec15 ligands in the brain microenvironment, cancer cells, or tumor microenvironment may allow Siglec15 to act as a receptor. Signal transduction through Siglec15 can be induced by the expression of IL-10 and TGF-beta (Figure 27B). Thus, Siglec15 may indirectly affect myeloid cell:T cell interactions or T cell responses through immunosuppressive cytokines such as IL-10 and TGF-beta.

[0116] In various aspects, the present invention is directed to methods of modulating immune function by promoting or inhibiting the activity of Siglec15.

[0117] A. A method of increasing an immune response by decreasing the expression and / or activity of Siglec15. In one aspect, the invention provides a method for increasing an immune or inflammatory response in a subject by administering to the subject an effective amount of a Siglec15 modulator, wherein the modulator reduces Siglec15 expression and / or activity in the subject. In one embodiment, the method can be used to increase the number of T cells, e.g., CD4 T cells and / or CD8 T cells, in the subject. In another embodiment, the method can be used to increase the activity of T cells, e.g., CD4 T cells and / or CD8 T cells, in the subject.

[0118] The above-mentioned method can be used to treat disorders that may be beneficial to increase or strengthen immune response in a subject.For example, the Siglec15 regulator that reduces the expression and / or activity of Siglec15 can be used to treat cancer in a subject that needs to be treated.Such method can include administering an effective amount of Siglec15 regulator to a subject that needs to be administered, and the regulator reduces the expression level and / or activity level of Siglec15 in the subject, thereby treating cancer in the subject.

[0119] In another aspect, the present invention provides a method for reducing tumor size in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of a Siglec15 modulator, wherein the modulator reduces the expression and / or activity level of Siglec15 in the subject, thereby reducing tumor size in the subject.

[0120] In one aspect, the invention features a method for prolonging survival of a subject in need thereof, the method including administering to the subject in need thereof an effective amount of a modulator of Siglec15, where the modulator reduces expression and / or activity levels of Siglec15 in the subject, thereby prolonging survival of the subject.

[0121] In another aspect, the invention features a method for increasing an anti-tumor immune response in a subject in need thereof, the method including administering to the subject in need thereof an effective amount of a Siglec15 modulator, wherein the modulator reduces the expression and / or activity level of Siglec15 in the subject, thereby increasing the anti-tumor immune response in the subject.

[0122] Modulators of Siglec15 suitable for use in the methods of the present invention include any compound or molecule capable of modulating Siglec15 expression and / or activity, e.g., Siglec15 mRNA expression and / or protein expression; Siglec15 mRNA and / or protein stability; and / or Siglec15 biological activity. Modulators can directly or indirectly modulate Siglec15 expression and / or activity. In some embodiments, the modulator is a compound or molecule capable of modulating Siglec15 expression and / or activity. The expression and / or activity of Siglec15 is inhibited. The inhibitory modulator of Siglec15 can act directly on Siglec15, for example, an antagonist antibody that binds to Siglec15 and inhibits its function. Alternatively, the inhibitory modulator of Siglec15 acts indirectly on Siglec15 (for example, through another molecule, for example, a binding partner of Siglec15), resulting in reduced activity. Exemplary modulators suitable for use in the methods of the present invention include small molecule inhibitors, antagonist antibodies or antigen-binding fragments thereof, recombinant fusion proteins (e.g., soluble Siglec15 fusion proteins, e.g., soluble Siglec15-Fc), inhibitory peptides, or interfering nucleic acid molecules (e.g., antisense RNA, sdRNA, and siRNA). Modulators suitable for use in the methods of the present invention are discussed in detail below.

[0123] The Siglec15 modulator of the present invention can also block the interaction between Siglec15 and its binding ligand. Siglec15 binding ligands can be identified by any method known in the art. For example, protein-protein interactions between Siglec15 and its binding ligand can be identified by co-immunoprecipitation, in which the binding of a pair of proteins of interest is determined in vitro by the formation of a co-precipitate with an antibody. Alternatively, yeast two-hybrid or phage display techniques can be used to screen for Siglec15 binding ligands. In some embodiments, chemical cross-linking assays followed by mass spectrometry can be used to identify interacting proteins.

[0124] In some embodiments, binding ligands of Siglec15 suitable for use in the present invention can be identified by receptor array technology. Receptor array technology is a well-established technique for screening counter-receptors of target proteins (Zhu Y et al., Nat Commun, 4:2043, 2013; Yao S et al., Immunity, 34:2043, 2013). 5:729-740, 2011). Briefly, receptor arrays comprise a solid support structure containing multiple wells, each containing cells transfected with a gene encoding a receptor protein. As previously described, a target gene (encoding a secreted protein) or an extracellular domain of a target gene (encoding a transmembrane protein, e.g., an immunomodulator) is genetically fused with a tag gene (mouse IgG2a Fc, human IgG1 Fc, FLAG, or 6xHIS) to prepare a fusion gene (Chapoval, AI et al., Mol Biotechnol, 21(3):259-264, 2011). (2002). Once individual fusion genes are transfected into 293T cells, purified recombinant fusion proteins are used to screen against a receptor array. Binding of target proteins, e.g., immunomodulators identified based on the methods of the present invention, to the transfected 293T cells is detected by applying a fluorescently labeled secondary antibody against the tag, screened using the Applied Biosystems 8200 Cellular Detection System, and analyzed by CDS 8200 software. The entire contents of the aforementioned references are incorporated herein by reference.

[0125] In some embodiments, the binding ligand of Siglec15 is MAG. In other embodiments, the binding ligand of Siglec15 is LRRC4C. In other embodiments, the binding ligand of Siglec15 is sialyl-Tn.

[0126] The binding site on Siglec15 for its ligand, e.g., MAG, LRRC4C, and / or sialyl-Tn, can be determined by any method known in the art. For example, Siglec15 mutant proteins with domain deletions can be produced and purified using standard methods known in the art. In some embodiments, a modulator suitable for use in the methods of the present invention is a mutant Siglec15 protein with reduced activity in ligand binding. For example, a modulator may have a single deletion at residue 143. It may be a mutant Siglec protein having a substitution mutation.In another embodiment, a modulator suitable for use in the methods of the invention is a mutant Siglec15 protein having a deletion of the IgV domain.

[0127] Modulators of Siglec15 suitable for use in the methods of the present invention can increase the immune response against tumors by inhibiting activities involved in the Siglec15 pathway, for example, by reducing the expression and / or activity of any binding ligand of Siglec15, such as MAG, LRRC4C, or sialyl-Tn. In some embodiments, modulators suitable for use in the methods of the present invention are small molecule inhibitors of MAG or LRRC4C, antagonistic antibodies or antigen-binding fragments thereof against MAG or LRRC4C, naturally inhibitory recombinant MAG fusion proteins, naturally inhibitory recombinant LRRC4C fusion proteins, or inhibitory proteins or nucleic acids targeting MAG or LRRC4C. In other embodiments, the modulator is an antibody or antigen-binding fragment thereof that specifically binds to sialyl-Tn. Such antibodies or antigen-binding fragments can bind to sialyl-Tn and block the association of sialyl-Tn with Siglec15. In other embodiments, the modulator is a soluble sialyl-Tn molecule, optionally coupled to a scaffold, e.g., a scaffold peptide. Modulators suitable for use in the methods of the invention are discussed in detail below.

[0128] The above-mentioned method can be used to treat the disorders that are considered beneficial to increase or strengthen immune response in subjects.Such disorders include, but are not limited to, cancer.The administration of Siglec15 regulator that reduces the expression and / or activity of Siglec15 can be used to, for example, stimulate immune response against cancer (for example, stimulate T cell response against cancer), reduce tumor size, and / or prolong the survival of cancer subjects.

[0129] As described herein, the term "cancer" refers to one of a group of diseases caused by the uncontrolled, abnormal growth of cells, which can spread to adjacent tissues or other parts of the body.Cancer cells can form solid tumors, in which cancer cells are clustered together or exist as dispersed cells, as in leukemia.Cancer types suitable for treatment by reducing the expression or activity of Siglec15 include, but are not limited to, solid tumors and / or blood cancers.In one embodiment, the cancer is of epithelial origin. Exemplary cancer types that can be treated by the aforementioned methods include, but are not limited to, adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain / CNS tumors, breast cancer, Castleman's disease, cervical cancer, colon / rectal cancer, uterine cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal cancer, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor. In some embodiments, the cancer is selected from the group consisting of brain cancer, lung cancer, pancreatic cancer, melanoma cancer, breast cancer, ovarian cancer, renal cell carcinoma, rectal adenocarcinoma, hepatocellular carcinoma, and Ewing's sarcoma.

[0130] In some embodiments, the cancer is colon cancer, endometrioid cancer, renal cancer (e.g., papillary renal cell carcinoma, renal clear cell carcinoma), liver cancer, thyroid cancer, lung cancer (e.g., lung adenocarcinoma, lung squamous cell carcinoma), head and neck cancer, breast cancer, cervical cancer, prostate cancer, bladder cancer, glioblastoma, rectal cancer, or bile duct cancer. In one embodiment, the cancer is brain cancer. In one embodiment, the cancer is glioblastoma. In one embodiment, the cancer is not a blood-borne cancer. In one embodiment, The cancer is not a leukemia. In one embodiment, the cancer is not acute myeloid leukemia (AML).

[0131] Use of an "effective amount" of a modulator of the present invention (and therapeutic compositions containing such a modulator) refers to an amount effective at dosages necessary to achieve the desired result and for periods of time necessary to achieve the desired result. For example, an effective amount of a modulator may vary depending on factors such as the disease state, age, sex, reproductive status, and weight, as well as the ability of the agent to elicit a desired response in an organism. Dosage regimens can be adjusted to produce the optimal response. For example, several divided doses can be provided daily, or the dose can be proportionally reduced as indicated by the exigencies of the situation.

[0132] "Treat," as used herein, refers to an intervention that benefits a patient, e.g., a patient suffering from or at risk of developing a disease. Treating includes actions taken and refraining from taking to improve the patient's condition, e.g., to alleviate one or more symptoms or to delay the onset or progression of the disease.

[0133] In one embodiment, the present invention relates to a combination therapy for treating cancer in which a Siglec15 modulator that reduces Siglec15 expression and / or activity is administered to a subject in combination with a checkpoint inhibitor, e.g., a PD-L1 antagonist or a PD-1 antagonist. PD-1 is a checkpoint protein on T cells that prevents T cells from attacking cells in the body that express PD-L1. Some cancer cells overexpress PD-L1, allowing them to evade detection by T cells. PD-L1 and PD-1 inhibitors can boost the immune response against cancer and can synergistically promote tumor cell killing when used in conjunction with an antagonist of Siglec15 activity. Exemplary anti-PD-L1 inhibitory antibodies that can be used in conjunction with antagonists of Siglec15 activity include, but are not limited to, atezolizumab (Genentech), avelumab (Pfizer), and durvalumab (AstraZeneca). Exemplary anti-PD-1 inhibitory antibodies that can be used in conjunction with antagonists of Siglec15 activity include, but are not limited to, pembrolizumab (Merck) and nivolumab (Bristol-Myers Squibb).

[0134] In some embodiments, the method further comprises screening a patient with cancer for upregulation of Siglec15 in the cancer or tumor microenvironment. For example, in one embodiment, a biological sample containing cancer cells is obtained from the subject, Siglec15 expression is determined, and compared with an appropriate control, such as a comparable sample obtained from a normal subject, a reference value representing the expression level in a normal subject, etc. Upregulation of Siglec15 in a biological sample containing cancer cells indicates that the subject would benefit from an agent that reduces the expression or activity of Siglec15.

[0135] In some embodiments, the methods described above may include a screening step to exclude patients who still have an autoimmune disease from treatment with a Siglec15 modulator. In other embodiments, Siglec15 modulators suitable for use in the methods of the invention do not cause a nonspecific autoimmune disease in the subject. Subjects receiving treatment with a Siglec15 modulator can be selected so that they do not naturally develop an autoimmune disease.

[0136] B. A method of reducing inflammation by increasing the expression and / or activity of Siglec15. Another aspect of the present invention provides a method for reducing an undesirable immune or inflammatory response in a subject by administering to the subject an effective amount of a Siglec15 modulator, wherein the modulator increases Siglec15 expression and / or activity in the subject. In one embodiment, the method can be used to reduce the number of T cells, e.g., CD4 T cells and / or CD8 T cells, in the subject. In another embodiment, the method can be used to reduce the activity of T cells, e.g., CD4 T cells and / or CD8 T cells, in the subject.

[0137] The above-mentioned method can be used to treat disorders that would be beneficial to reduce immune response or inflammatory response in a subject.For example, a Siglec15 regulator that increases the expression and / or activity of Siglec15 can be used to treat autoimmune disease in a subject that needs to be treated.The method includes administering an effective amount of a Siglec15 regulator to a subject, and the regulator increases the expression and / or activity of Siglec15 in the subject, thereby treating the autoimmune disease in the subject.

[0138] Another aspect of the present invention provides a method for treating an autoimmune disease in a subject in need thereof, comprising administering to the subject an effective amount of Siglec15 protein or a nucleic acid encoding a Siglec15 protein, thereby treating the autoimmune disease in the subject.

[0139] In one aspect, the invention features a method for reducing a brain inflammatory response in a subject in need thereof, the method including administering to the subject an effective amount of a Siglec15 modulator, wherein the modulator increases the expression and / or activity level of Siglec15, thereby reducing the brain inflammatory response in the subject.

[0140] In another aspect, the present invention provides a method for reducing a brain inflammatory response in a subject in need thereof, comprising administering to the subject an effective amount of Siglec15 protein or a nucleic acid encoding a Siglec15 protein, thereby reducing the brain inflammatory response in the subject.

[0141] Modulators of Siglec15 suitable for use in the aforementioned methods include any compound or molecule capable of positively regulating Siglec15 expression and / or activity, for example, by modulating Siglec15 mRNA expression and / or protein expression; Siglec15 mRNA and / or protein stability; and / or Siglec15 biological activity. Modulators can directly or indirectly modulate Siglec15 expression and / or activity. In some embodiments, modulators increase Siglec15 expression and / or activity. Stimulatory modulators of Siglec15 can act directly on Siglec15, for example, an agonistic antibody that binds to Siglec15 and stimulates its function. In other embodiments, stimulatory modulators of Siglec15 act indirectly on Siglec15 (e.g., through another molecule, e.g., a binding partner of Siglec15), resulting in increased activity. Exemplary modulators suitable for use in the methods of the present invention include small molecule activators, agonist antibodies or antigen-binding fragments thereof, or proteins and nucleic acids that activate the transcription and / or translation of Siglec15. Alternatively, modulators that increase the expression and / or activity of Siglec15 for use in the methods of the present invention include Siglec15 protein or nucleic acids encoding Siglec15 protein. In some embodiments, the Siglec15 protein is selected from the group consisting of a full-length Siglec15 protein, a functional fragment of Siglec15, or the IgV domain of Siglec15. Suitable modulators for use in the methods are discussed in detail below.

[0142] The Siglec15 modulators of the present invention can also promote the interaction between Siglec15 and binding ligands. Siglec15 binding ligands can be identified by any method known in the art. For example, protein-protein interactions between Siglec15 and binding ligands can be identified by co-immunoprecipitation assays, in which the binding of a pair of proteins of interest is determined in vitro by the formation of co-precipitates with antibodies. Alternatively, yeast two-hybrid assays or phage display assays can be used to screen for binding ligands to Siglec15. In some embodiments, chemical cross-linking assays followed by mass spectrometry can be used to analyze interacting proteins.

[0143] In some embodiments, binding ligands of Siglec15 suitable for use in the present invention can be identified by receptor array technology as described herein.

[0144] In some embodiments, the binding ligand of Siglec15 is MAG. In other embodiments, the binding ligand of Siglec15 is LRRC4C. In other embodiments, the binding ligand of Siglec15 is sialyl-Tn.

[0145] Exemplary Siglec15 modulators suitable for use in practicing certain embodiments of the methods described herein can reduce inflammation by stimulating activity involved in the Siglec15 pathway, e.g., by increasing the expression and / or activity of Siglec15 binding ligands, such as MAG, LRRC4C, and / or sialyl-Tn. In some embodiments, a modulator suitable for use in the methods of the invention is a small molecule activator of MAG or LRRC4C, an agonistic antibody or antigen-binding fragment thereof against MAG or LRRC4C, or a protein or nucleic acid that activates the transcription and / or translation of MAG or LRRC4C. In other embodiments, a modulator suitable for use in the methods of the invention is a MAG protein or a nucleic acid encoding a MAG protein. In another embodiment, a modulator suitable for use in the methods of the invention is an LRRC4C protein or a nucleic acid encoding an LRRC4C protein. In one embodiment, the modulator is a synthetic sialyl-Tn molecule. For example, sialyl-Tn can be administered alone or coupled to a scaffold molecule, e.g., conjugated via covalent or non-covalent bonding. In one embodiment, the modulator is a synthetic peptide having a sialyl-Tn. Sialyl-Tn can bind to and activate Siglec15, acting as an agonist of Siglec15 activity. Modulators that increase Siglec15 expression and / or activity suitable for use in the methods of the present invention are discussed in detail below.

[0146] The diseases that can be treated using the method of the present invention include, but are not limited to, autoimmune diseases or cancer. "Treat" refers to any type of treatment that benefits patients, for example, patients who suffer from disease or are at risk of developing disease. Treating includes taking or refraining from taking actions to improve the patient's condition, for example, to alleviate one or more symptoms, or to delay the onset or progression of disease.

[0147] As used herein, an "autoimmune disease" is a disease that results from an abnormal immune response of the body to substances and tissues normally present in the body, including, but not limited to, rheumatoid arthritis (RA), juvenile chronic arthritis (JCA), thyroiditis, graft-versus-host disease (GVHD), scleroderma, diabetes, Graves' disease, allergies, e.g., Examples of autoimmune diseases include, but are not limited to, acute or chronic immune diseases associated with allogeneic transplants such as kidney, heart, bone marrow, liver, pancreas, small intestine, lung, and skin transplants. In some embodiments, the autoimmune disease is an inflammatory brain disease. In other embodiments, the inflammatory brain disease is multiple sclerosis. In other embodiments, the inflammatory brain disease is experimental autoimmune encephalomyelitis (EAE).

[0148] III. Pharmaceutical Preparations Pharmaceutical formulations containing Siglec15 or Siglec15 modulators of the present invention can be prepared by mixing the protein or nucleic acid having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, 16th ed., Osol, A. (1980)) to form an aqueous solution, lyophilized formulation or other dry formulation. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, histidine, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polymers proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as Tween™, Pluronics™, or polyethylene glycol (PEG).

[0149] The preparations of the present invention may contain more than one active compound as needed for the specific indications to be treated.For example, when treating autoimmune diseases, the preparations containing Siglec15 or Siglec15 modulators of the present invention can be combined with drugs known to treat autoimmune diseases, such as methylprednisolone, Kenalog, Medrol, prednisolone, Cortef, hydrocortisone, cortisone, triamcinolone acetonide, Celeston Solspan, methylprednisolone acetate, Orapred ODT, Veripred 20, Solu-Medrol or methylprednisolone sodium. When treating cancer, a formulation containing Siglec15 or a Siglec15 modulator of the present invention may be administered in combination with a drug known to treat cancer, such as abiraterone acetate, ABITREXATE (methotrexate), ABRAXANE (paclitaxel-albumin-stabilized nanoparticles), ADCETRIS (brentuximab vedotin), Ado-trastuzumab emtansine, ADRIAMYCIN (doxorubicin hydrochloride), ADRUCIL (fluorouracil), afatinib dimaleate, AFINITOR (everolimus), ALDAR A (Imiquimod), aldesleukin, alemtuzumab, ALIMTA (pemetrexed disodium), ALOXI (palonosetron hydrochloride), AMBOCHLORIN (chlorambucil), aminolevulinic acid, anastrozole, aprepitant, AREDIA (pamidronate disodium), ARIMIDEX (anastrozole), AROMASIN (exemestane), ARRANON (nelarabine), arsenic trioxide, ARZERRA (ofatumumab), asparaginase Erwinia chrysanthemi, AVASTIN (bevacizumab), Xitinib, azacitidine, bendamustine hydrochloride, bevacizumab, bexarotene, BE XXAR (tositumomab and I-131 iodine tositumomab), bleomycin, bortezomib, BOSULIF (bosutinib), cabazitaxel, cabozantinib-S-malate, CAMPATH (alemtuzumab), CAMPTOSAR (irinotecan hydrochloride), capecitabine, carboplatin, carfilzomib, CEENU (lomustine), CERUBIDINE (daunorubicin hydrochloride), cetuximab, chlorambucil, cisplatin, CLAFEN (cyclophosphamide), clofarabine, COMETRIQ (cabozantinib-S- Malate), COSMEGEN (dactinomycin), crizotinib, cyclophosphamide, CYFOS (ifosfamide), cytarabine, dabrafenib, dacarbazine, DACOGEN (decitabine), dactinomycin, dasatinib, daunorubicin hydrochloride, decitabine, degarelix, denileukin diftitox, denosumab, dexrazoxane hydrochloride, docetaxel, doxorubicin hydrochloride, EFUDEX (fluorouracil), ELITEK (rasburicase), ELLENCE (epirubicin hydrochloride), ELOXATIN (oxaliplatin) Platinum), Eltrombopag olamine, EMEND (Aprepitant), Enzalutamide, Epirubicin hydrochloride, ERBITUX (Cetuximab), Eribulin mesylate, ERIVEDGE (Vismodegib), Erlotinib hydrochloride, ERWINAZE (Asparaginase Erwinia chrysanthemum), Etoposide, Everolimus, EVISTA (Raloxifene hydrochloride), Exemestane, FARESTON (Toremifene), FASLODEX (Fulvestrant), FEMARA (Letrozole), Filgrastim, FLUDARA (Ribozyme), Fludarabine phosphate), fludarabine phosphate, FLUOROPLEX (fluorouracil), fluorouracil, folinic acid, FOLOTYN (pralatrexate), fulvestrant, gefitinib, gemcitabine hydrochloride, gemtuzumab ozogamicin, GEMZAR (gemcitabine hydrochloride), GILOTRIF (afatinib dimaleate), GLEEVEC (imatinib mesylate), HALAVEN (eribulin mesylate), HERCEPTIN (trastuzumab), HYCAMTIN (topotecan hydrochloride), ibritumomab tiuxetan,ICLUSIG (ponatinib hydrochloride), ifosfamide, imatinib mesylate, imiquimod, INLYTA (axitinib), Intron A (recombinant interferon alfa-2b), iodine-131 tositumomab and tositumomab, ipilimumab, IRESSA (gefitinib), irinotecan hydrochloride, ISTODAX (romidepsin), ixabepilone, JAKAFI (ruxolitinib phosphate) ), JEVTANA (cabazitaxel), Kadcyla (Ado-trastuzumab emtansine), Keoxifen (raloxifene hydrochloride), KEPIVANCE (palifermin), KYPROLIS (carfilzomib), lapatinib ditosylate, lenalidomide, letrozole, leucovorin calcium, leuprolide acetate, lomustine, LUPRON (leuprolide acetate, MARQIBO (vincristine sulfate liposome), MATULANE (procarbazine hydrochloride), mechlorethamine hydrochloride, MEGACE (megestrol acetate), megestrol acetate, MEKINIST (trametinib), mercaptopurine, mesna, METHAZOLASTONE (temozolomide), methotrexate, mitomycin, MOZOBIL (plelixafor), MUSTARGEN (mechlorethamine hydrochloride), MUTAMYCIN (mitomycin C), MYLOSAR (azacitidine), MYLOTARG (gemtuzumab ozogamicin), nanoparticle paclitaxel (paclitaxel albumin-stabilized nanoparticles), NAVELBINE (vinorelbine tartrate), nelarabine, NEOSAR (cyclophosphamide), NEUPOGEN (filgrastim), NEXAVAR (sorafenib) tosylate), nilotinib, NOLVADEX (tamoxifen citrate), NPLATE (romiplostim), ofatumumab, omacetaxine mepesuxinate, ONCASPAR (peguaspargase), ONTAK (denileukin diftitox), oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, palifermin, palonosetron hydrochloride, pamidronate disodium, panitumumab, pazopanib hydrochloride, pegaspargase, peginterferon alfa-2b, PEG-Intron (peginterferon alfa-2b), pemetrexed disodium, pertuzumab,PLATINOL (cisplatin), , PLATINOL-AQ (cisplatin), plerixafor, pomalidomide, POMALYST (pomalidomide), ponatinib hydrochloride, pralatrexate, prednisone, procarbazine hydrochloride, PROLEUKIN (aldesleukin), PROLIA (denosumab), PROMACTA (eltrombopag olamine), PROVENGE (sipuleucel-T), PURINETHOL (mercaptopurine), radium-223 dichloride, raloxifene hydrochloride, Rasburicas, recombinant interferon alfa-2b, regorafenib, Revlimid (lenalidomide), RHEUMATREX (methotrexate), rituximab, romidepsin, romiplostim, rubidomycin (daunorubicin hydrochloride), ruxolitinib Phosphat), Sipuleucel T, sorafenib tosylate, SPRYCEL (dasatinib), STIVARGA (regorafenib), sunitinib malate, SUTENT (sunitinib malate), SYLATRON (peginterferon alfa-2b), SYNOVIR (thalidomide), SYNRIBO (omacetaxine mepesuxinate), TAFINLAR (dabrafenib), tamoxifen citrate, TARABINE PFS (cytarabine), TARCEVA (erlotinib hydrochloride), TARGRETIN (bexarotene), TASIGNA (nilotinib), TAXOL (paclitaxel), TAXOTERE (docetaxel), TEMODAR (temozolomide), temozolomide, temsirolimus, thalidomide, TOPOSAR (etoposide), topotecan hydrochloride, toremifene, TORISEL (temsirolimus), tositumomab and I-131 iodine tositumomab Mab, TOTECT (dexrazoxane hydrochloride), trametinib, trastuzumab, TREANDA (bendamustine hydrochloride), TRISENOX (arsenic trioxide), TYKERB (lapatinib ditosylate), vandetanib, VECTIBIX (panitumumab), VelP, VELBAN (vinblastine sulfate), VELCADE (bortezomib), VELSAR (vinblastine sulfate), vemurafenib, VEPESID (etoposide), VIAD UR (leuprolide acetate), VIDAZA (azacitidine), vinblastine sulfate, vincristine sulfate, vinorelbine tartrate, vismodegib, VORAXAZE (glucarpidase), vorinostat, VOTRIENT (pazopanib hydrochloride), WELLCOVORIN (leucovorin calcium), XALKORI (crizotinib), Xeloda (capecitabine), XGEVA (denosumab), XOFIGO (radium-223 dichloride), XTA May be combined with NDI (enzalutamide), YERVOY (ipilimumab), ZALTRAP (Ziv-aflibercept), ZELBORAF (vemurafenib), ZEVALIN (ibritumomab tiuxetan), ZINECARD (dexrazoxane hydrochloride), Ziv-aflibercept, zoledronic acid, ZOLINZA (vorinostat), ZOMETA (zoledronic acid), and ZYTIGA (abiraterone acetate).In one embodiment, the Siglec15 modulator may be combined with a PD-L1 antagonist, such as an anti-PD-L1 antagonist antibody or antigen-binding portion thereof. In another embodiment, the Siglec15 modulator may be combined with a PD-1 antagonist, such as an anti-PD-1 antagonist antibody or antigen-binding portion thereof.

[0150] The active ingredient can also be packaged in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, prepared by, for example, coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, edited by Osol, A. (1980).

[0151] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0152] Generally, the components of the composition are supplied either separately or mixed together to form a unit dosage form as a dry lyophilized powder or a water-free concentrate in a sealed container such as an ampule or sachet indicating the quantity of active agent.If the administration mode is injection, the composition can be dispensed into an infusion bottle containing sterile pharmaceutical-grade water or saline.If the administration mode is by injection, an ampoule of sterile water for injection or saline can be provided, so that the components can be mixed before administration.In an alternative embodiment, one or more of the pharmaceutical compositions of the present invention are supplied in liquid form in a sealed container indicating the quantity and concentration of the agent.

[0153] The active agent can be incorporated into a pharmaceutical composition suitable for parenteral administration, typically prepared as an injectable solution. The injectable solution can be in either liquid or lyophilized form, contained in flint or amber vials, ampoules, or prefilled syringes. The liquid or lyophilized dosage form may further comprise a buffer (e.g., L-histidine, sodium succinate, sodium citrate, sodium or potassium phosphate, sodium chloride), a cryoprotectant (e.g., sucrose, trehalose, or lactose), a bulking agent (e.g., mannitol), a stabilizer (e.g., L-methionine, glycine, arginine), or an adjuvant (e.g., hyaluronidase).

[0154] The compositions of the present invention, e.g., Siglec15 or Siglec15 modulators, may be in various forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as solutions (e.g., injection and infusion solutions), microemulsions, dispersions, liposomes or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Pharmaceutical compositions containing Siglec15 or Siglec15 modulators described herein can be formulated for administration to specific tissues. For example, in certain embodiments, it may be desirable to administer Siglec15 or Siglec15 modulators to connective tissue, brain tissue, and / or tumor sites in various organs.

[0155] Siglec15 and Siglec15 modulators suitable for use in the methods of the present invention can be administered by any suitable means, including parenteral administration (e.g., injection, infusion), subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration, and, if desired, intralesional administration, e.g., intratumoral administration, for localized treatment. Parenteral infusions include intravenous, intraarterial, intraperitoneal, intramuscular, intradermal, or subcutaneous administration. Furthermore, Siglec15 and Siglec15 modulators can be suitably administered by pulse infusion, particularly tapering doses. Dosing can be by injection, such as intravenous or subcutaneous injection. The route of administration can be selected depending on various factors, such as whether the administration is short-term or chronic. Other administration methods are contemplated, including local administration, particularly transdermal, transmucosal, rectal, oral, or local administration, for example, via a catheter placed near the desired site. Injection, particularly intravenous, is important.

[0156] In some embodiments, the methods of the present invention comprise administering to a subject a therapeutically effective amount of Siglec15 as described herein. In some embodiments, the methods of the present invention comprise administering to a subject a therapeutically effective amount of a Siglec15 modulator. The therapeutically effective amount of Siglec15 or a Siglec15 modulator is an amount sufficient to treat a disease, e.g., an autoimmune disease or cancer, in a subject. Therapeutically effective dosages of Siglec15 or a Siglec15 modulator as described herein will vary somewhat between subjects and depend on factors such as the age, weight, and condition of the subject and the route of delivery. Such dosages can be determined according to procedures known to those skilled in the art. In an exemplary embodiment, a therapeutically effective amount of a Siglec15 antagonist is administered to a subject in an amount sufficient to treat a disease, e.g., an autoimmune disease or cancer, in a subject in an amount sufficient to treat a disease, e.g., an autoimmune disease or cancer. Therapeutically effective dosages of Siglec15 or a Siglec15 modulator as described herein will vary somewhat between subjects and will depend on factors such as the age, weight, and condition of the subject and the route of delivery. Such dosages can be determined according to procedures known to those skilled in the art. In an exemplary embodiment, a therapeutically effective amount of a Siglec15 antagonist is administered to a subject in an amount sufficient to treat a disease, e.g., an autoimmune disease or cancer, in ... It is an amount effective to decrease the level or activity of Siglec15. In another exemplary embodiment, a therapeutically effective amount of a Siglec15 agonist is an amount effective to increase the level or activity of Siglec15 in a subject.

[0157] In one embodiment, the therapeutic methods described herein are practiced on humans.

[0158] IV. Siglec15 Modulators for Use in the Methods of the Invention As described above, reduced expression of Siglec15 reduces tumor size and prolongs survival in mice bearing brain tumors, while reduced expression and / or activity of Siglec15 exacerbates brain inflammation, suggesting that Siglec15 plays an essential role as an immunoregulatory molecule in regulating immune responses to cancer and inflammation. Thus, molecules that modulate, e.g., inhibit or activate, the expression and / or activity of Siglec15 and / or molecules that modulate, e.g., inhibit or activate, the expression and / or activity of Siglec15's binding partners, e.g., MAG or LRRC4C, are useful in the methods of the present invention. Exemplary modulators can modulate, e.g., inhibit or promote, the association between Siglec15 and a Siglec15 ligand (e.g., MAG, LRRC4C, or sialyl-Tn). Inhibitory modulators (i.e., inhibitors) or activating modulators (i.e., activators) can be nucleic acids, polypeptides, antibodies, or small molecule compounds. In some embodiments, inhibitors or activators function at the level of transcription, mRNA stability, translation, protein stability / degradation, protein modification, and protein binding.

[0159] A. Inhibitory Modulators In some embodiments, the modulator for use in the methods of the present invention is an inhibitory modulator. In some embodiments, the inhibitory modulator is a small molecule, e.g., a small molecule inhibitor of Siglec15, or a small molecule inhibitor of a binding ligand of Siglec15, e.g., MAG, LRRC4C, and / or sialyl-Tn. In other embodiments, the inhibitory modulator for use in the methods of the present invention is an intracellular binding molecule that specifically inhibits the expression, stability, and / or activity of Siglec15 and / or its binding ligand, e.g., MAG, LRRC4C, and / or sialyl-Tn. In other embodiments, the inhibitory modulator is an antagonist antibody or antigen-binding fragment thereof against Siglec15 and / or its binding ligand, e.g., MAG or LRRC4C. In some embodiments, the inhibitory modulator is a recombinant fusion protein against Siglec15 and / or its binding ligand, e.g., MAG or LRRC4C. In other embodiments, the Siglec15 fusion protein is a Siglec15-Fc fusion protein. In some embodiments, inhibitory modulators for use in the methods of the invention are nucleic acid molecules that act to specifically reduce the expression, stability, and / or activity of Siglec15 and / or its binding ligands, such as MAG or LRRC4C.

[0160] As used herein, the term "intracellular binding molecule" is intended to include a molecule that acts intracellularly to inhibit the expression or activity of a protein by binding to the protein or a nucleic acid (e.g., an mRNA molecule) encoding the protein.

[0161] The Siglec15 modulators of the present invention may modulate, e.g., block, the interaction between Siglec15 and a binding ligand. In some embodiments, the Siglec15 binding ligand is selected from the group consisting of MAG and LRRC4C. In some embodiments, the modulator is a mutant Siglec15 protein having a single substitution mutation at residue 143. In other embodiments, the modulator is a mutant Siglec15 protein having a deletion of the IgV domain. In some embodiments, the modulator is a mutant Siglec15 protein having a deletion of the IgV domain. The modulator of Siglec15 binds to the IgV domain of Siglec15. In other embodiments, the modulator of Siglec15 binds to residue 143 of Siglec15. In some embodiments, the modulator of Siglec15 binds to a region within a ligand of Siglec15, e.g., MAG or LRRC4C, where the interaction between Siglec15 and its ligand occurs.

[0162] i. Inhibitory nucleic acids In one embodiment, the method described herein involves using an inhibitory nucleic acid to target Siglec15 and / or its binding ligand, such as MAG or LRRC4C. The nucleic acid inhibitor may encode a small interfering RNA (e.g., an RNAi agent) that targets Siglec15 and / or its binding ligand, such as MAG or LRRC4C, and inhibits its expression or activity. The term "RNAi agent" refers to an RNA or its analog that has sufficient sequence complementarity to a target RNA to induce RNA interference. Examples also include DNA that can be used to generate RNA.

[0163] RNA interference: RNA interference (RNAi) refers to a sequence-specific or selective process by which target molecules (e.g., target genes, proteins, or RNAs) are down-regulated. Generally, interfering RNAs ("RNAi") are double-stranded small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), or single-stranded microRNAs (miRNAs), which can lead to catalytic degradation of specific mRNAs and can also be used to reduce or inhibit gene expression. RNA interference (RNAi) is a process by which double-stranded RNA (dsRNA) induces sequence-specific regulation of gene expression in animal and plant cells, as well as in bacteria (Aravin and Tuschl, FEBS Lett., 26:5830-5855). 840, 2005; Herbert et al., Curr. Opin. Biotech., 19:500-505 Page, 2008; Sharp, Genes Dev., 15:485-490, 2001; Valencia-Sanchez et al., Genes Dev., 20:515-524, 2006). mammalian cells In the present study, RNAi is mediated by 21-nucleotide (nt) duplexes of small interfering RNA (siRNA) (Chiu et al., Mol. Cell. 10:549-561, 2002). by RNA (miRNA), functional short hairpin RNA (shRNA), or other dsRNA expressed in vivo using a DNA template with an RNA polymerase II or III promoter (Zeng et al., Mol. Cell. 9:1327-1333, 2002; Paddison et al., Genes Dev. 16:948-958, 2002). Denti et al., Mol. Ther., 10:191-199, 2004; Lee et al., Nature Biotechnol., 20:500-505, 2002; Paul et al., Nature Biotechnol. 20:505-508, 2002; Rossi, Human Gene Ther., 19:313 ~317, 2008; Tuschl, T., Nature Biotechnol., 20:440-448, 2002; Yu et al., Proc. Natl. Acad. Sci. USA, 99(9):6047-6052, 2002), can be induced.

[0164] siRNA molecule: The term "short interfering RNA" or "siRNA" (also known as "small interfering RNA") refers to an RNA agent, preferably a double-stranded agent, that is about 10 to 50 nucleotides in length, preferably about 15 to 25 nucleotides in length, more preferably about 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, the strand optionally having overhanging ends containing, for example, one, two, or three overhanging nucleotides (or nucleotide analogs), and that is capable of inducing or mediating RNA interference. Naturally occurring siRNAs are generated by the cellular RNAi machinery from longer dsRNA molecules (e.g., >25 nucleotides in length).

[0165] Generally, the methods described herein can use dsRNA molecules, each consisting of 16 to 30 nucleotides, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, where one strand is substantially identical to a target region in an mRNA, e.g., at least 80% (or higher, e.g., 85%, 90%, 95%, or 100%), with, e.g., 3, 2, 1, or 0 mismatched nucleotides, and the other strand is complementary to the first strand. dsRNA molecules can also be chemically synthesized, or, for example, shRNAs can be transcribed from a DNA template in vitro or in vivo. dsRNA molecules can be designed using any method known in the art. Negative control siRNAs should not have significant sequence complementarity to the appropriate genome. Such a negative control can be designed by randomly scrambling the nucleotide sequence of the selected siRNA, and performing a homology search to ensure that the negative control lacks homology to any other gene in the appropriate genome.Furthermore, the negative control siRNA can be designed by introducing one or more base mismatches into the sequence.

[0166] The methods described herein can use both siRNA and modified siRNA derivatives, for example, siRNA modified to change the specificity and / or pharmacokinetics of the composition, for example, to increase the half-life in the body, for example, cross-linked siRNA.Therefore, the present invention includes a method for administering siRNA derivatives, including siRNAs having two complementary strands of nucleic acid, thus cross-linking the two strands.Oligonucleotide modifications include, but are not limited to, 2'-O-methyl, 2'-fluoro, 2'-O-methyoxyethyl, and phospho. Examples of suitable thioribose include thiothioate, boranophosphate, and 4'-thioribose (Wilson and Keefe, Curr. Opin. Chem. Biol., 10:607-614, 2006; Prakash et al., J. Med. Chem., 48:4247-4253, 2005; Soutschek et al., Nature, 432:173-178, 2004).

[0167] In some embodiments, the siRNA derivative has a biotin molecule (e.g., photocleavable biotin), a peptide (e.g., Tat peptide), a nanoparticle, a peptidomimetic, an organic compound (e.g., a dye such as a fluorescent dye), or a dendrimer at the 3' end. By modifying the siRNA derivative in this way, the cellular uptake or cell targeting activity of the resulting siRNA derivative can be improved compared to the corresponding siRNA, which is useful for tracking the siRNA derivative within cells, or the stability of the siRNA derivative can be improved compared to the corresponding siRNA.

[0168] The inhibitory nucleic acid compositions can be unconjugated or can be conjugated to another moiety, such as a nanoparticle, to enhance the properties of the composition, e.g., absorption, efficacy, bioavailability, and / or pharmacokinetic parameters such as half-life. Conjugation can be by methods known in the art, e.g., Lambert et al., Drug Deliv. Rev.: Vol. 47(1): pp. 99-112, 2001; Fattal et al., J. Control Release, Vol. 53(1-3): pp. 137-43, 1998; Schwab et al., Ann. Oncol. Vol. 5 Suppl 4:55-8, 1994; and Godard et al., Eur. J. Biochem., 232(2):404-10, 1995). The inhibitory nucleic acid molecule can also be labeled using any method known in the art, for example, the nucleic acid composition can be labeled with a fluorophore, such as Cy3, fluorescein, or rhodamine. Labeling can be accomplished using kits, such as the SILENCER™ siRNA labeling kit. siRNA can be synthesized using a PCR kit (Ambion). 3 H, 32 It can be radiolabeled using P, or other suitable isotopes.

[0169] siRNA Delivery: siRNA can be directly delivered in saline or other vehicles to silence target genes in tissues such as the eye, lung, and central nervous system (Bitko et al., Nat. Med., 11:50-55 (2005); Shen et al., Gene Ther., 13:225-234 (2006); Thakker et al., Proc. Natl. Acad. Sci. USA (2004)). In adult mice, efficient delivery of siRNA can be achieved by a "high-pressure" delivery technique in which a large volume of siRNA-containing solution is rapidly injected (within 5 seconds) into the animal via the tail vein (Lewis, Nature Genetics, 32:107-1109). 08, 2002).

[0170] Liposomes and nanoparticles can also be used to deliver siRNA to animals. Delivery methods using liposomes, such as stable nucleic acid-lipid particles (SNALP), dioleoylphosphatidylcholine (DOPC)-based delivery systems, and lipoplexes, such as Lipofectamine 2000 and TransIT-TKO, have been shown to effectively silence target mRNA (de Fougerolles, Human Gene Ther., 19:125-130). 132 (2008); Landen et al., Cancer Res., 65:6910-6918 (2005); Luo et al., Mol. Pain, 1:29 (2005); Zimmermann et al., Nature , 441:111-114 (2006)). Conjugating siRNA to peptides, RNA aptamers, antibodies, or polymers, such as dynamic polyconjugates, cyclodextrin-based nanoparticles, atelocollagen, and chitosan, can improve siRNA stability and / or uptake. (Howard et al., Mol. Ther., 14:476-484 (2006); Hu-Lieskovan et al., Cancer Res., 65:8984-8992 (2005); Kumar et al., Nature, 448:39-43; McNamara et al., Nat. Biotechnol., 24:1005-1015 (2006)). 2007); Rozema et al., Proc. Natl. Acad. Sci. USA, 104:12982-1 2987 (2007); Song et al., Nat. Biotechnol., 23:709-717 ( 2005); Wolfrum et al., Nat. Biotechnol., 25:1149-1157 (2007).

[0171] Using a virus-mediated delivery mechanism, for example, recombinant adenoviruses carrying siRNA under the transcriptional control of an RNA Pol II promoter can be generated, thereby inducing specific silencing of targeted genes through the expression of siRNA. Infecting HeLa cells with these recombinant adenoviruses reduces endogenous target gene expression. Injecting recombinant adenoviral vectors into transgenic mice expressing the siRNA target gene reduces target gene expression in vivo. Ibid. In animal models, whole embryo electroporation can efficiently deliver synthetic siRNA to postimplantation mouse embryos (Calegari et al., Proc. Natl. Acad. Sci. USA, 99(22):14236-40 (2002)).

[0172] Use of engineered RNA precursors to induce RNAi: As described herein, engineered RNA precursors are introduced into cells or whole organisms, resulting in the production of desired siRNA molecules.Then, these siRNA molecules associate with the endogenous protein components of the RNAi pathway to bind to and target specific mRNA sequences for cleavage, destabilization, and / or translation inhibition destruction.In this way, the mRNA targeted by the siRNA generated from engineered RNA precursors is depleted from cells or organisms, thereby reducing the concentration of the protein coded by that mRNA in cells or organisms.

[0173] Antisense: An "antisense" nucleic acid may comprise a nucleotide sequence complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to a target mRNA sequence. The antisense nucleic acid may be complementary to the entire coding strand of a target sequence, or to only a portion thereof (e.g., the coding region of a target gene). In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding a selected target gene (e.g., the 5' untranslated region and 3' untranslated region).

[0174] Antisense nucleic acid can be designed to be complementary to the entire coding region of target mRNA, or it can be an oligonucleotide that is antisense to only a part of the coding region or non-coding region of target mRNA.For example, antisense oligonucleotide can be complementary to the region around the translation start site of target mRNA, for example, between the -10 region and +10 region of the nucleotide sequence of the target gene of interest.Antisense oligonucleotide can be, for example, about 7 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 45 nucleotides, about 50 nucleotides, about 55 nucleotides, about 60 nucleotides, about 65 nucleotides, about 70 nucleotides, about 75 nucleotides, about 80 nucleotides or more in length.

[0175] The antisense nucleic acid of the present invention can be constructed by using chemical synthesis and enzyme ligation reaction, using procedures known in the art.For example, antisense nucleic acid (for example, antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides that are designed to increase the biological stability of the molecule or increase the physical stability of the duplex formed between antisense nucleic acid and sense nucleic acid, for example, phosphorothioate derivatives and acridine-substituted nucleotides can be used.Antisense nucleic acid can also be produced biologically using an expression vector that nucleic acid is subcloned in antisense direction (i.e., the RNA transcribed from inserted nucleic acid is antisense direction to the target nucleic acid of interest, as further described in the following section).

[0176] Based on the sequences disclosed herein for Siglec15 and / or its binding ligands, e.g., MAG and LRRC4C, one of skill in the art can readily select and synthesize any number of antisense molecules suitable for use in accordance with the present invention. For example, a "gene walk" can be prepared comprising a series of 15-30 nucleotide oligonucleotides spanning the length of the target nucleic acid, which can then be tested for inhibition of target gene expression. Optionally, 5-10 nucleotide gaps can be left between oligonucleotides to reduce the number of oligonucleotides synthesized and tested. In some embodiments, the antisense molecule targets the IgV domain of Siglec15. In other embodiments, the antisense molecule targets a region within MAG or LRRC4C where interaction with Siglec15 occurs. In some embodiments, the antisense molecule targets a region spanning residue 143 of Siglec15.

[0177] Antisense nucleic acid molecules of the invention are generally administered to a subject (e.g., by direct injection at a tissue site) or generated in situ, such that they hybridize or bind to cellular mRNA and / or genomic DNA encoding the target protein, thereby inhibiting protein expression, e.g., by inhibiting transcription and / or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For systemic administration, antisense molecules are engineered to specifically bind to receptors or antigens expressed on the surface of selected cells. For example, the antisense nucleic acid molecule can be modified by linking a peptide or antibody that binds to a cell surface receptor or antigen to the antisense nucleic acid molecule. Antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of antisense molecules, vector constructs can be used in which the antisense nucleic acid molecule is under the control of a strong pol II or pol III promoter.

[0178] CRISPR technology: Expression of a target polynucleotide (e.g., DNA or RNA of Siglec15 and / or its binding ligands, such as MAG and LRRC4C) can be modified by binding a CRISPR complex to a polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within the target polynucleotide, the guide sequence linked to a tracr mate sequence, and the tracr mate sequence further hybridizes to the tracr sequence. In some embodiments, binding of the CRISPR complex to the target polynucleotide results in increased expression of the target polynucleotide. In other embodiments, binding of the CRISPR complex to the target polynucleotide results in decreased expression of the target polynucleotide (e.g., DNA or RNA of Siglec15 and / or its binding ligands, such as MAG and LRRC4C).

[0179] Clustered, regularly interspaced, short palindromic repeat (CRISPR) technology is included in the present invention as an RNA-guided nuclease-generating method with customizable specificity for targeted genome editing. Genome editing mediated by these nucleases has been used to rapidly, easily, and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that are traditionally difficult to genetically manipulate.

[0180] In general, the term "CRISPR system" collectively refers to transcripts and other elements involved in the expression or directing activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (e.g., tracr RNA or active partial tracr RNA), tracr mate sequences (including "direct repeats" and partial direct repeats processed by tracr RNA in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), or other sequences and transcripts derived from a CRISPR locus. In some embodiments of the present invention, the terms guide sequence and guide RNA are used interchangeably. In some embodiments, one or more elements of a CRISPR system are derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system are derived from a particular organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes. Generally, CRISPR system is characterized by an element (also referred to as protospacer in endogenous CRISPR system) that promotes the formation of CRISPR complex at the site of target sequence.In the formation of CRISPR complex, "target sequence" refers to the sequence that guide sequence is designed to have complementarity with, and the hybridization between target sequence and guide sequence promotes the formation of CRISPR complex.Target sequence can comprise any polynucleotide, such as DNA or RNA polynucleotide (for example, DNA or RNA of Siglec15 and / or its binding ligand, for example, MAG and LRRC4C).In some embodiments, target sequence is located in the nucleus or cytoplasm of cell.

[0181] In a preferred embodiment of the present invention, the CRISPR / Cas system is a type II CRISPR system. The Cas enzyme is Cas9, which catalyzes DNA cleavage. Enzymatic action by Cas9 from Streptococcus pyogenes or any closely related Cas9 results in a double-stranded break at the target site sequence, which hybridizes to 20 nucleotides of the guide sequence and has a protospacer adjacent motif (PAM) sequence NGG after 20 nucleotides of the target sequence. CRISPR activity through Cas9 for site-specific DNA recognition and cleavage is defined by the guide sequence, a tracr sequence that hybridizes in part to the guide sequence, and the PAM sequence. Further aspects of the CRISPR system are described in Karginov and Hannon, "The CRISPR system: small RNA-guided Defense in bacteria and archae, Mol. Cell, 2010, 37(1):7.

[0182] The type II CRISPR locus from Streptococcus pyogenes SF370 contains a cluster of four genes, Cas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA elements, the tracr RNA and a unique array of direct repeats spaced by short stretches of non-repetitive sequences (spacers, each approximately 30 bp long). In this system, targeted DNA double-strand breaks (DSBs) occur in four sequential steps. First, two non-coding RNAs, the pre-crRNA array and the tracr RNA, are transcribed from the CRISPR locus. Second, the tracr RNA hybridizes with the direct repeats of the pre-crRNA, which are then processed into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracr RNA complex guides Cas9 to the DNA target consisting of the protospacer and the corresponding PAM via heteroduplex formation between the spacer region of the crRNA and the protospacer DNA. Finally, Cas9 mediates cleavage of the target DNA upstream of the PAM, generating a DSB within the protospacer. Several aspects of the CRISPR system can be further improved to increase the efficiency and versatility of CRISPR targeting. Optimal Cas9 activity is achieved by the addition of free Mg 2+ This may depend on the availability of NGG motifs at levels higher than those present in mammalian nuclei (see, e.g., Jinek et al., 2012, Science 337:816), and the preference for NGG motifs immediately downstream of the protospacer limits the ability to target every 12 bp on average in the human genome.

[0183] The inhibitory nucleic acid strategies described above can also be used to reduce the level of sialyl-Tn. For example, RNA interference, siRNA molecules, antisense nucleic acids, and / or CRISPR technology can be used to modulate the expression level of enzymes involved in the formation of sialyl-Tn. As described herein, sialyl-Tn is a truncated O-glycan containing sialic acid α-2,6-linked to GalNAc α-O-Ser / Thr. Exemplary enzymes involved in the synthesis of sialyl-Tn include sialyltransferase ST6GalNAc1 and COSMC (core 1 β3-Gal-T specific molecular chaperone). For example, modulation of enzymes involved in sialyl-Tn production, such as sialyltransferase ST6GalNAc1 or COSMC, using inhibitory nucleic acids can be used to reduce the level of sialyl-Tn in vivo or in vitro.

[0184] ii. Antagonist antibodies The present invention further contemplates methods and compositions comprising antagonist antibodies that inhibit Siglec15 and / or its binding ligands, such as MAG, LRRC4C, and / or sialyl-Tn, thereby reducing the expression and / or activity of Siglec15 and promoting an immune response against cancer.

[0185] In some embodiments, Siglec15 and / or its binding ligand, e.g. MAG, LRRC4C, and / or sialyl-Tn antagonist antibodies or antigen-binding portions thereof reduce the expression and / or activity of Siglec15 and / or its binding ligands, such as MAG, LRRC4C, and / or sialyl-Tn.

[0186] In some embodiments, the antagonist antibody of Siglec15 and / or the antagonist antibody of Siglec15 ligand, e.g., MAG, LRRC4C, and / or sialyl-Tn, or antigen-binding portion thereof, blocks the interaction between Siglec15 and its binding ligand, e.g., MAG, LRRC4C, and / or sialyl-Tn. In some embodiments, the antagonist antibody of Siglec15 and / or its binding ligand, e.g., MAG, LRRC4C, and / or sialyl-Tn, or antigen-binding portion thereof, binds to the IgV domain of Siglec15. In other embodiments, the antagonist antibody of Siglec15 and / or its binding ligand, e.g., MAG, LRRC4C, and / or sialyl-Tn, or antigen-binding portion thereof, binds to residue 143 of Siglec15. In some embodiments, an antagonist antibody or antigen-binding portion thereof for Siglec15 and / or its binding ligand, e.g., MAG, LRRC4C, and / or sialyl-Tn, binds to a region within Siglec15's ligand, e.g., MAG, LRRC4C, and / or sialyl-Tn, where interaction between Siglec15 and its ligand occurs.

[0187] In one embodiment, the antagonist antibody or antigen-binding portion thereof binds to an extracellular epitope of Siglec15. For example, the antagonist antibody or antigen-binding portion thereof may bind to Siglec15 at an extracellular epitope involved in the interaction between Siglec15 and Siglec15 ligands, such as MAG, LRRC4C, and / or sialyl-Tn. Such an antibody may partially or completely block the interaction between Siglec15 and Siglec15 ligands, such as MAG, LRRC4C, and / or sialyl-Tn. In an exemplary embodiment, the anti-Siglec15 antagonist antibody or antigen-binding portion thereof binds to Siglec15 at an epitope comprising residue 143. In one embodiment, the antagonist anti-Siglec15 antibody does not transduce signals through Siglec15. In one embodiment, the antagonist anti-Siglec15 antibody induces endocytosis of Siglec15. In another embodiment, the antagonist anti-Siglec15 antibody does not induce endocytosis of Siglec15.

[0188] Anti-Siglec15 antibodies can be screened for their ability to antagonize the function of Siglec15 using standard methods. Exemplary anti-Siglec15 antagonist antibodies include S15m03 and S15m02 described herein. Antibodies or antigen-binding fragments thereof derived from S15m03 or S15m02 can also be useful as Siglec15 antagonists. For example, derivatives of S15m03 or S15m02 can be chimeric, humanized, or human variants of S15m03 or S15m02. Exemplary derivatives of S15m03 or S15m02 include one, two, three, four, five, or six complementarity-determining regions (CDRs) of S15m03 or S15m02. For example, a derivative of S15m03 or S15m02 may contain the heavy and / or light chain CDR3 of S15m03 or S15m02. In another example, a derivative of S15m03 or S15m02 may contain the heavy and / or light chain CDR2 of S15m03 or S15m02. In another example, a derivative of S15m03 or S15m02 may contain the heavy and / or light chain CDR1 of S15m03 or S15m02. In another example, a derivative of S15m03 or S15m02 may contain the heavy and / or light chain CDR1, CDR2, and CDR3 of S15m03 or S15m02. In a preferred embodiment, a derivative of S15m03 or S15m02 maintains the antigen binding specificity of S15m03 or S15m02 for Siglec15.

[0189] In one embodiment, the anti-Siglec15 antibody is an antibody described in U.S. Provisional Patent Application No. 62 / 397,794, filed September 21, 2016, the entire contents of which are incorporated herein by reference.

[0190] Other exemplary anti-Siglec15 antibodies that may be useful as Siglec15 antagonists include, but are not limited to, mAb A9E8 (described in US Pat. No. 9,447,192), mAb DS-1501 (Daiichi Sankyo), mAb 32A1 (described in US Pat. No. 8,575,316), mAb AB-25E9 (Alethia Biotherapeutics), mAb 25B8, mAb 25E6, and mAb 25E9 (described in US Pat. No. 9,388,242). The entire contents of US Pat. Nos. 9,447,192, 8,575,316, and 9,388,242 are incorporated herein by reference. Other exemplary anti-Siglec15 antibodies include antibodies that compete for binding with the aforementioned anti-Siglec15 antibodies and / or antibodies that bind to the same or overlapping epitopes of Siglec15 as the aforementioned anti-Siglec15 antibodies. In one embodiment, the Siglec15 antagonist is not mAb A9E8 or an antigen-binding fragment thereof. In another embodiment, the Siglec15 antagonist is not mAb DS-1501 or an antigen-binding fragment thereof. In another embodiment, the Siglec15 antagonist is not mAb AB-25E9 or an antigen-binding fragment thereof.

[0191] In one embodiment, the antagonist antibody or antigen-binding portion thereof binds to an extracellular epitope of MAG. For example, the antagonist antibody or antigen-binding portion thereof may bind to MAG at an extracellular epitope involved in the interaction between MAG and Siglec15. Such an antibody may partially or completely block the interaction between MAG and Siglec15. In an exemplary embodiment, the anti-MAG antagonist antibody or antigen-binding portion thereof binds to MAG at an epitope that interacts with residue 143 of Siglec15. In one embodiment, the antagonist anti-MAG antibody does not transduce signals through MAG.

[0192] In one embodiment, the antagonist antibody or antigen-binding portion thereof binds to an extracellular epitope of LRRC4C. For example, the antagonist antibody or antigen-binding portion thereof may bind to LRRC4C at an extracellular epitope involved in the interaction between LRRC4C and Siglec15. Such an antibody may partially or completely block the interaction between LRRC4C and Siglec15. In an exemplary embodiment, the anti-LRRC4C antagonist antibody or antigen-binding portion thereof binds to LRRC4C at an epitope that interacts with residue 143 of Siglec15. In one embodiment, the antagonist anti-LRRC4C antibody does not transduce signals through LRRC4C.

[0193] In one embodiment, the antagonist antibody or antigen-binding portion thereof binds to sialyl-Tn. Such an antibody may partially or completely block the interaction between sialyl-Tn and Siglec15. In an exemplary embodiment, the anti-sialyl-Tn antagonist antibody or antigen-binding portion thereof binds to sialyl-Tn at an epitope that interacts with Siglec15. In one embodiment, the antagonist anti-sialyl-Tn antibody does not transduce signals through polypeptides containing sialyl-Tn.

[0194] Antagonist antibodies suitable for use in the methods of the invention can be identified using a variety of assays known in the art (e.g., Antibodies: A Laboratory Manual, 2nd Edition, Greenfield, ed., 2010). Antibodies can be identified by various techniques (see, for example, ELISA, Western blot, or surface plasmon resonance) and screened (e.g., using phage display) or characterized for their physical / chemical properties and / or biological activity. The binding specificity of an antibody to its antigen can be tested by methods known in the art, such as ELISA, Western blot, or surface plasmon resonance.

[0195] Antibodies can be produced using recombinant methods and compositions known in the art, for example, as described in U.S. Patent No. 4,816,567, incorporated herein by reference. For example, an isolated nucleic acid encoding an anti-Siglec15 antibody is used to transform a host cell for expression. Such a nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In further embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In further embodiments, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., has been transformed with) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL and an amino acid sequence comprising the VH of the antibody, or a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, or Sp20 cell).

[0196] For recombinant production of an anti-Siglec15 antibody, nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the antibody heavy and light chains).

[0197] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein.For example, antibody can be produced in bacteria, especially when glycosylation and Fc effector function are not required.For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523.After expression, antibody can be isolated from bacterial cell paste in soluble fraction and further purified.

[0198] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast, including fungal and yeast strains in which the glycosylation pathway has been "humanized" resulting in the production of antibodies with partially or fully human glycosylation patterns, are suitable cloning or expression hosts for antibody-encoding vectors. Gerngross, Nat. Biotech., 22:140 9-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0199] Suitable host cells for the expression of glycosylated antibodies can also be obtained from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant cells and insect cells.A number of baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for the transfection of Spodoptera frugiperda cells.

[0200] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to growth in suspension can be useful. Other examples of useful mammalian host cell lines include SV40 the monkey kidney CV1 strain (COS-7) transformed by 3 or 293 cells); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod., 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TR1 cells described in Mather et al., Annals NY Acad. Sci., 383:44-68 (1982)); MRC 5 Other useful mammalian host cell lines include DHFR cells; and FS4 cells. - Examples of suitable host cell lines for antibody production include Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, 248 (ed. BKC Lo, Humana Press, Totowa, NJ), 255-268 (2003).

[0201] iii. Small molecule inhibitors Other inhibitory modulators that can be used to specifically inhibit the activity of Siglec15 protein are chemical compounds that directly inhibit Siglec15 activity or that inhibit the interaction between Siglec15 and its binding ligands, such as MAG, LRRC4C, and / or sialyl-Tn. Such compounds may be natural products or members of combinatorial chemistry libraries and can be identified using screening assays described in detail below.

[0202] In some embodiments, the small molecule inhibitors of the present invention can block the interaction between Siglec15 and its binding ligand. For example, the small molecule inhibitors bind to the IgV domain of Siglec15. In other embodiments, the small molecule inhibitors of the present invention can bind to a region of MAG, LRRC4C, or sialyl-Tn that interacts with the IgV domain of Siglec15, thereby blocking the interaction between Siglec15 and its ligand, such as MAG, LRRC4C, or sialyl-Tn.

[0203] In some embodiments, small molecule inhibitors are selected to bind to domains that share homology with Siglec15. For example, small molecules of the present invention may target domains that are at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% or 99% identical to the IgV domain of Siglec15. Such small molecules are likely to bind to protein domains within Siglec15 that are functionally similar to, for example, the IgV domain of Siglec15.

[0204] The small molecule inhibitors of the present invention can also bind to specific motifs or consensus sequences derived from the IgV domain of Siglec15, thereby enabling the small molecule inhibitors to specifically bind to domains shared among members of the Siglec family. In another embodiment, the small molecules of the present invention bind to protein motifs or consensus sequences that represent the three-dimensional structure of a protein. Such motifs or consensus sequences are not contiguous stretches of amino acids, but represent non-linear amino acid arrangements (i.e., structural motifs) resulting from the three-dimensional folding of Siglec15. An example of such a motif is a motif designed based on the IgV domain of Siglec15. Such motifs and consensus sequences can be designed according to any method known in the art.

[0205] In some embodiments, the small molecule binds to a specific sequence of the Siglec15 protein, for example, residue 143 of Siglec15.

[0206] iv. Fusion or mutant proteins Variants of Siglec15 proteins or variants of Siglec15 binding ligands (e.g., MAG and LRRC4C) that function as antagonists and inhibit Siglec15 activity can be used in the methods of the present invention. For example, Siglec15 Fc fusion proteins, which function as blocking proteins similar to anti-Siglec15 antagonist antibodies, can be used as inhibitory modulators in the present invention. In an exemplary embodiment, the Siglec15-Fc fusion protein antagonist is a soluble Siglec15-Fc fusion protein. Alternatively, Siglec15 mutants with reduced binding activity to their ligands can also function as inhibitory modulators for use in the methods of the present invention. For example, Siglec15 mutants with a single substitution mutation at residue 143 or Siglec15 mutants lacking the IgV domain can be used as inhibitory modulators in the present invention.

[0207] In some embodiments, variants of Siglec15 binding ligands (e.g., MAG and LRRC4C) block the interaction between Siglec15 and its binding ligand. In some embodiments, Fc fusion proteins of MAG or LRRC4C, which function as blocking proteins similar to anti-Siglec15 antagonist antibodies, can be used as inhibitory modulators in the present invention. In another example, proteins containing sialyl-Tn can function as blocking agents by binding to and sequestering Siglec15. Alternatively, MAG or LRRC4C mutants with reduced binding activity to Siglec15 can also function as inhibitory modulators for use in the methods of the present invention. For example, MAG or LRRC4C mutants with mutations that abolish interaction with the IgV domain of Siglec15 can be used as inhibitory modulators in the present invention.

[0208] Recombinant fusion proteins of Siglec15 and / or its binding ligands, such as MAG or LRRC4C, for use in the methods of the present invention can be produced from recombinant vectors according to methods known in the art. The recombinant vector may contain a nucleic acid encoding a Siglec15 protein in a form suitable for expression of the nucleic acid in a host cell. In some embodiments, a nucleic acid sequence encoding an IgG Fc domain can be operably linked to a nucleic acid molecule encoding a protein of interest, such as Siglec15 and / or its binding ligands, such as MAG and LRRC4C, or a functional segment thereof, in an expression vector. The resulting fusion protein can then be easily purified from cells using art-recognized methods, such as using an anti-Fc antibody.

[0209] In some embodiments, a recombinant vector may contain one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed, selected based on the host cell to be used for expression (i.e., a recombinant expression vector). Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest and the regulatory sequence(s) are linked in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Methods in Enzymology: Gene Expression Technology, Vol. 185. , Academic Press, San Diego, CA (1991). Regulatory sequences are These include those that direct constitutive expression of a nucleotide sequence in many types of host cell, and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will appreciate that the design of the expression vector can depend on factors such as, for example, the choice of the host cell to be transformed, the desired expression level of protein, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by the nucleic acids described herein.

[0210] The recombinant expression vectors of the invention can be designed for expression of a polypeptide or functional fragment thereof in prokaryotic cells (e.g., E. coli) or eukaryotic cells (e.g., insect cells, yeast cells, or mammalian cells using baculovirus expression vectors). Suitable host cells can include, but are not limited to, E. coli cells, Bacillus cells, Saccharomyces cells, Pochia cells, NSO cells, COS cells, Chinese hamster ovary (CHO) cells, myeloma cells, or cells described herein.

[0211] Another aspect of the present invention relates to a host cell into which a recombinant vector of the present invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but still fall within the scope of the term as used herein.

[0212] Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" refer to various art-recognized techniques for introducing foreign nucleic acid into host cells, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.

[0213] v.Siglec15-derived peptide Inhibitory modulators for use in the methods of the present invention are peptide compounds derived from the amino acid sequence of Siglec15 and / or its binding ligand (e.g., the sequences disclosed herein as SEQ ID NOS: 2, 4, and 6). In particular, inhibitory compounds include a portion of Siglec15 (or a mimetic thereof) that mediates the interaction between Siglec15 and its binding ligand, such as MAG, LRRC4C, or sialyl-Tn. Thus, contact of Siglec15 with the peptide compound competitively inhibits the interaction between Siglec15 and its binding ligand, such as MAG, LRRC4C, or sialyl-Tn. For example, a Siglec15-derived peptide containing an amino acid sequence within the IgV domain can function as an inhibitory modulator. Alternatively, a Siglec15-derived peptide containing an amino acid sequence surrounding residue 143 of Siglec15 can function as an inhibitory modulator. In other embodiments, a MAG- or LRRC4C-derived peptide containing an amino acid sequence within the region where interaction with Siglec15 occurs can function as an inhibitory modulator.

[0214] The peptide compounds of the present invention can be produced intracellularly in a cell by introducing into the cell an expression vector encoding the peptide. Such expression vectors can be produced by standard techniques. The peptide can be expressed intracellularly as a fusion with another protein or peptide (e.g., a GST fusion). As an alternative to recombinantly synthesizing the peptide in a cell, the peptide can be expressed intracellularly using standard peptide synthesis techniques. The synthesized peptide can then be introduced into cells by various means known in the art for introducing peptides into cells (e.g., liposomes, etc.).

[0215] B. Irritant modulators In some embodiments, the modulator for use in the methods of the invention is a stimulatory modulator that increases the expression and / or activity of Siglec15 and / or its binding ligand, e.g., MAG, LRRC4C, or sialyl-Tn, in a subject in need thereof, thereby reducing inflammation. Examples of such stimulatory modulators include proteins, nucleic acid molecules, e.g., expression vectors comprising nucleic acid molecules, and small molecules that stimulate the expression and / or activity of Siglec15 and / or its binding ligand, e.g., MAG, LRRC4C, or sialyl-Tn, in cells.

[0216] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of DNA or RNA produced using nucleotide analogs. Nucleic acid molecules may be single-stranded or double-stranded, but are preferably double-stranded DNA. Nucleic acid molecules used in the methods of the present invention can be isolated using standard molecular biology techniques.

[0217] In some embodiments, the stimulatory regulator suitable for use in the methods of the present invention is a Siglec15 protein. For example, the stimulatory regulator is a full-length Siglec15 protein, a functional fragment of Siglec15, or the IgV domain of Siglec15. The stimulatory regulator promotes the interaction between Siglec15 and its binding ligand, such as MAG, LRRC4C, or sialyl-Tn. In other embodiments, the stimulatory regulator suitable for use in the methods of the present invention is a nucleic acid molecule encoding a Siglec15 protein. For example, cDNA (full-length or partial cDNA sequence) is cloned into a recombinant expression vector, and the vector is transfected into cells using standard molecular biology techniques. The cDNA can be obtained, for example, by amplifying using polymerase chain reaction (PCR) or by screening an appropriate cDNA library.

[0218] In some embodiments, the stimulatory regulator suitable for use in the methods of the present invention is a binding ligand of Siglec15 protein, such as MAG, LRRC4C, or sialyl-Tn. The stimulatory regulator promotes the interaction between Siglec15 and its binding ligand. In other embodiments, the stimulatory regulator suitable for use in the methods of the present invention is a nucleic acid molecule encoding a binding ligand of Siglec15 protein, such as MAG or LRRC4C.

[0219] In some embodiments, stimulatory modulators for use in the methods of the invention are intracellular binding molecules that act to specifically activate the expression, stability, and / or activity of Siglec15 and / or its binding ligands, such as MAG, LRRC4C, or sialyl-Tn. As used herein, the term "intracellular binding molecule" is intended to include molecules that act intracellularly to increase protein expression or activity by binding to the protein or a nucleic acid (e.g., an mRNA molecule) encoding the protein.

[0220] In other embodiments, the stimulatory modulator is an agonist antibody or antigen-binding antibody against Siglec15 and / or its binding ligand, e.g., MAG or LRRC4C. Such agonistic antibodies against Siglec15 and / or its binding ligands, such as MAG or LRRC4C, can be identified and generated using the methods described herein.

[0221] In one embodiment, the stimulatory regulator is a Siglec15 fusion protein. In an exemplary embodiment, the stimulatory Siglec15 fusion protein is immobilized, for example, on a cell or a solid support, such as a bead. For example, a membrane-anchored Siglec15-Fc fusion protein can increase the activity of Siglec15 or a Siglec15-binding ligand and function as a Siglec15 agonist. In another embodiment, the agonist is a Siglec-Fc fusion protein that contains a mutation in FcR binding.

[0222] In some embodiments, stimulatory nucleic acids can be used to activate the expression and / or activity of Siglec15 and / or its binding ligands, such as MAG and LRRC4C, based on the CRISPR technology described herein.

[0223] Other stimulatory modulators that can be used to specifically activate the activity of Siglec15 protein and / or its binding ligand, e.g., MAG, LRRC4C, or sialyl-Tn, are chemical compounds that directly activate Siglec15 activity or that promote the interaction between Siglec15 and / or its binding ligand, e.g., MAG, LRRC4C, or sialyl-Tn. Such compounds can be identified using screening assays that select for such compounds, as described in detail below.

[0224] V. Screening Assays Modulators that affect Siglec15 activity can be known (e.g., antibodies that interfere with Siglec15 activity, or Siglec15 mutant proteins) or can be identified using the methods described herein. The present invention provides methods (also referred to herein as "screening assays") for identifying other modulators, i.e., candidate or test compounds or modulators (e.g., peptides, small molecules, or other drugs) that modulate Siglec15 expression and / or activity, and for testing or optimizing the activity of other modulators.

[0225] In some embodiments, molecules that bind to Siglec15 and / or its binding ligands, such as MAG, LRRC4C, or sialyl-Tn, or that have a stimulatory or inhibitory effect on the expression and / or activity of Siglec15 or its binding ligands, such as MAG, LRRC4C, or sialyl-Tn, can be identified.

[0226] In one embodiment, the ability of a compound to directly modulate the expression, post-translational modification, or activity of Siglec15 or its binding ligand, such as MAG, LRRC4C, or sialyl-Tn, is measured as an index using a screening assay of the present invention.

[0227] Modulators identified by the methods of the present invention that can inhibit the expression, stability, and / or activity of Siglec15 or its binding ligands, such as MAG, LRRC4C, or sialyl-Tn, are useful as candidate compounds for treating cancer in a subject in need thereof, for reducing tumor size or prolonging survival in a subject in need thereof, or for increasing an immune response against a tumor in a subject in need thereof.

[0228] Modulators identified by the methods of the present invention that are capable of increasing the expression, stability, and / or activity of Siglec15 or its binding ligands, such as MAG, LRRC4C, or sialyl-Tn, are useful as candidate compounds useful for treating an autoimmune disease or reducing an inflammatory response in a subject in need thereof.

[0229] For example, in one aspect, the present invention provides a method for identifying a compound useful for treating an autoimmune disease or cancer in a subject. The method includes providing a test compound (or a plurality of test compounds), determining the effect of the test compound on Siglec15 expression and / or activity, and selecting a compound that modulates Siglec15 expression and / or activity, thereby identifying a compound useful for treating an autoimmune disease or cancer in the subject. In some embodiments, an increase in Siglec15 expression and / or activity indicates that the compound is useful for treating an autoimmune disease. In other embodiments, a decrease in Siglec15 expression and / or activity indicates that the compound is useful for treating cancer.

[0230] In another aspect, the present invention provides a method for identifying a compound useful for increasing an immune response against a tumor in a subject in need thereof, the method comprising the steps of providing a test compound (or a plurality of test compounds), determining the effect of the test compound on Siglec15 expression and / or activity, and selecting a compound that reduces Siglec15 expression and / or activity, thereby identifying a compound useful for increasing an immune response against a tumor in the subject.

[0231] In yet another aspect, the present invention provides a method for identifying a compound useful for reducing a brain inflammatory response in a subject in need thereof, the method comprising the steps of providing a test compound (or a plurality of test compounds), determining the effect of the test compound on Siglec15 expression and / or activity, and selecting a compound that increases Siglec15 expression and / or activity, thereby identifying a compound useful for reducing a brain inflammatory response in the subject.

[0232] Examples of modulators, candidate compounds, or test compounds include, but are not limited to, nucleic acids (e.g., DNA and RNA), carbohydrates, lipids, proteins, peptides, peptidomimetics, small molecules, and other drugs. Modulators can be obtained using any of a number of techniques in combinatorial library methods known in the art, including biological libraries; spatially addressable parallel solid-phase or solution-phase libraries; synthetic library methods requiring deconvolution; "one bead one compound" library methods; and synthetic library methods using affinity chromatography selection. While the biological library technique is limited to peptide libraries, the other four techniques are applicable to peptide, non-peptide oligomer, or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145; U.S. Pat. No. 5,738,996; and U.S. Pat. No. 5,807,683, the entire contents of each of the foregoing references being incorporated herein by reference).

[0233] Examples of methods for synthesizing molecular libraries can be found in the art, for example, in DeWitt et al. (1993), Proc. Natl. Acad. Sci. USA, 90:6909; Erb et al. (1993), Proc. Natl. Acad. Sci. USA, 90:6909; 994), Proc. Natl. Acad. Sci. USA, 91:11422; Zuckermann et al. (1994), J. Med. Chem., 37:2678; Cho et al. (1993), Science , 261:1303; Carrell et al. (1994), Angew. Chem. Int. Ed. Engl., 33:2059; Carell et al. (1994), Angew. Chem. Int. Ed. Engl., 33:2061; and Gallop et al. (1994), J. Med. Chem., 37:12 33 (the entire contents of each of the foregoing references are incorporated herein by reference). Libraries of compounds can be displayed, for example, in solution (e.g., Houghten (1992) Bio / Techniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), on chips (Fodor (1993) Nature 364:555-556), on bacteria (U.S. Pat. No. 5,223,409), on spores (Patent Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869), or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382; and Felici (1991) J. Mol. Biol. 222:301-310). The entire contents of each of the foregoing references are incorporated herein by reference.

[0234] The test compound can be contacted with cells that express the Siglec15 protein or a molecule with which Siglec15 directly interacts, such as MAG or LRRC4C. For example, the test compound can be contacted with cells that naturally express the protein(s) or that have been engineered to express the protein(s) by introducing an expression vector encoding the protein into the cell.

[0235] Alternatively, the test compound can be provided to a cell-free composition containing the protein(s) (eg, a cell extract or composition containing, eg, purified native or recombinant protein).

[0236] Compounds that modulate the expression and / or activity of Siglec15 or a binding ligand of Siglec15, such as MAG, LRRC4C, or sialyl-Tn, can be identified using a variety of "readouts."

[0237] For example, cells can be transfected with an expression vector and incubated in the presence or absence of a test compound to determine the effect of the compound on Siglec15 expression or on biological responses regulated by Siglec15. The biological activity of Siglec15 includes activities determined in vivo or in vitro according to standard techniques. The activity can be a direct activity, such as association with a binding ligand, for example, MAG, LRRC4C, or sialyl-Tn. Alternatively, the activity can be an indirect activity, such as an increase in brain inflammatory response.

[0238] To determine whether test compound modulates Siglec15 protein expression, in vitro transcription assay can be carried out.To determine whether test compound modulates Siglec15 mRNA expression, various methodologies can be carried out, such as quantitative PCR or real-time PCR.

[0239] A variety of reporter genes are known in the art and are suitable for use in the screening assays of the present invention. Examples of suitable reporter genes include genes encoding chloramphenicol acetyltransferase, beta-galactosidase, alkaline phosphatase, green fluorescent protein, or luciferase. Standard methods for measuring the activity of these gene products are known in the art.

[0240] A variety of cell types are suitable for use as indicator cells in screening assays. These cells express low levels of endogenous Siglec15, and are suitable for use as indicator cells in screening assays. It is preferred to use cell lines engineered to do so. Cells for use in the subject assays include eukaryotic cells. For example, in one embodiment, the cell is a fungal cell, such as a yeast cell. In another embodiment, the cell is a plant cell. In yet another embodiment, the cell is a vertebrate cell, such as an avian cell or a mammalian cell (e.g., a mouse cell or a human cell).

[0241] For example, recombinant expression vectors that can be used to express Siglec15 are known in the art. For example, first, cDNA is introduced into a recombinant expression vector using standard molecular biology techniques. cDNA can be obtained, for example, by amplifying using polymerase chain reaction (PCR) or by screening an appropriate cDNA library. The nucleotide sequences or molecules of cDNA in the involved signal transduction pathways (e.g., human, mouse, and yeast) are known in the art and can be used to design PCR primers that allow cDNA amplification by standard PCR methods, or to design hybridization probes that can be used to screen cDNA libraries using standard hybridization methods.

[0242] In another embodiment, the test compound can be administered to a cell-free composition containing the protein(s) (e.g., a cell extract or composition containing, for example, purified native or recombinant protein). Siglec15 expressed by recombinant methods in host cells or culture medium can be isolated from the host cells or cell culture medium using standard methods for purifying proteins. For example, ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification using antibodies can be used to produce purified or semi-purified proteins that can be used in cell-free compositions. Alternatively, a lysate or extract of cells expressing the protein of interest can be prepared for use as a cell-free composition.

[0243] In one embodiment, compounds that specifically modulate Siglec15 activity or the activity of its binding ligand in a signal transduction pathway involving Siglec15 are identified based on their ability to modulate the interaction between Siglec15 and its binding ligand. The binding ligand can be an mRNA molecule or a protein molecule, such as MAG or LRRC4C. Suitable assays that allow for the detection of protein-protein interactions (e.g., immunoprecipitation, two-hybrid assay, etc.) or the detection of the interaction between Siglec15 and mRNA (e.g., electrophoretic mobility shift assay, DNAse I footprinting assay, etc.) are known in the art. By performing such assays in the presence and absence of test compounds, these assays can be used to identify compounds that modulate (e.g., inhibit or enhance) the activity of Siglec15 with its binding ligand.

[0244] Compounds identified in the subject screening assays can be used in methods of modulating one or more biological responses regulated by Siglec15. It will be understood that it may be desirable to formulate such compound(s) as pharmaceutical compositions described herein and then contact them with cells.

[0245] Once a test compound that directly or indirectly modulates, for example, Siglec15 expression or activity has been identified by one of the various methods described above, the selected test compound (or "compound of interest") can then be examined for its effect on cells, for example, by combining the compound of interest with the cells in vivo (e.g., by administering the compound of interest to an organism). ) or ex vivo (e.g., by isolating the cells from an organism and contacting the isolated cells with the compound of interest, or alternatively, by contacting the compound of interest with a cell line), and further evaluating the effect of the compound of interest on the cells relative to a suitable control (e.g., untreated cells, or cells treated with a control compound or carrier that does not modulate the biological response).

[0246] In another aspect, the invention relates to a combination of two or more of the assays described herein. For example, a cell-based or cell-free assay can be used to identify a modulator, and the ability of the modulator to increase or decrease the activity of Siglec15 or a protein with which Siglec15 interacts can be confirmed in vivo, for example, in an animal model, such as, for example, a glioblastoma tumor model.

[0247] Furthermore, modulators (e.g., antisense nucleic acid molecules, or specific antibodies, or small molecules) of Siglec15 or molecules in a signaling pathway involving Siglec15 identified as described herein can be used in animal models to determine the efficacy, toxicity, or side effects of treatment with such modulators. Alternatively, modulators identified as described herein can be used in animal models to determine the mechanism of action of such modulators.

[0248] In another embodiment, it will be appreciated that similar screening assays can be used to identify compounds that indirectly modulate Siglec15 activity and / or expression, for example, by performing a screening assay such as the screening assays described above using a molecule with which Siglec15 interacts, such as MAG, LRRC4C, or sialyl-Tn, or any molecule that acts either upstream or downstream of Siglec15 in the pathway.

[0249] Compounds identified by the screening assays of the present invention are considered candidate therapeutic compounds useful for treating the diseases described herein, such as cancer or autoimmune diseases. Accordingly, the present invention also encompasses compounds identified in the screening assays, as well as methods of their administration and use in treating, preventing, or delaying the onset or progression of the diseases described herein.

[0250] It is to be understood that the present invention is not limited to the particular assay methods or test agents and experimental conditions described, as such methods and agents may 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.

[0251] The present invention is further illustrated by the following examples, which are not intended to be limiting in any way. The entire contents of all references, patents and published patent applications, and figures cited throughout this application are hereby incorporated by reference. [Example]

[0252] Example 1 Identification of Siglec15 by genome-scale T cell activity array technology We developed the genome-scale T cell avidity array (GS-TCAA) to conduct a human genome-wide search for targets for immunotherapy. To construct the GS-TCAA, we prepared, quantified, and diluted 6402 human membrane cDNAs, covering 90% of the human membrane genome, using a Qiagen miniprep kit.

[0253] A set of four 1536-well plates containing human receptor arrays was centrifuged (600 g, 2 min) and then reverse transfection was performed. Briefly, 2 μl of optiMEM containing Lipofectamine 3000 (7 μl lipo / ml) was dispensed per well into the 1536-well array plates using a robotic dispenser (Multidrop Combi, Thermo Scientific) and immediately shaken on an ultra-high speed orbital shaker for 1 min. All plates were stored at room temperature for 20 min, after which 293T.2A.m. anti-CD3 cells were added (2000 cells in 4 μl per well) using Multidrop for T cell stimulation. The plates were then further centrifuged (1000 g, 4 min) to remove air bubbles inside each well and then incubated at 37°C. T cell reporter cells or primary T cells (4000 cells), such as engineered Jurkat cell lines carrying different GFP reporters, were loaded onto the array plate 24 hours after transfection. After 12 hours of co-culture of T cells with 293T-based T cell stimulators, plate imaging was performed using an InCell image analyzer (GE). After optimal imaging analysis using Cellprofiler software, gene candidates with potential regulatory functions were identified. For example, if a molecule is ineffective in modulating T cell activity, the GFP signal remains similar. If the molecule acts as a stimulatory signal for T cell activity, a relatively stronger GFP signal is detected. Alternatively, if the molecule is inhibitory, a much weaker GFP signal is observed. Based on the genome-scale T cell activity array, Siglec15 was identified as an immunomodulator, suggesting that Siglec15 may have immunological functions.

[0254] Example 2 Brain- and bone marrow-associated Siglec15 expression Siglec15 expression was assessed in various mouse tissues using RT-PCR with a mouse cDNA library (Clontech Laboratories). Siglec15 plasmid (Origene) was used as a positive control. The results are shown in Figure 1A. Microarray analysis of Siglec15 RNA expression in human tissues (BioGPS.org) is shown in Figure 1B. The data indicate that Siglec15 is expressed in brain- and bone marrow-associated cells in both humans and mice under normal conditions. Microarray data from BioGPS are shown in Figure 1C, demonstrating that Siglec15 is expressed in monocytes, macrophages, dendritic cells, B cells, and osteoclasts. Therefore, Siglec15 may have physiological functions in brain-associated diseases and / or regulating immune responses.

[0255] Example 3 Siglec15 expression in cancer A meta-analysis of Siglec15 expression in human cancers was performed using the TCGA cancer microarray database. Siglec15 mRNA expression in a number of human cancers was compared with normal tissue counterparts (Figure 2). The original dataset was collected from UCSC Cancer Normalization was performed using Genomics Browser software (https: / / genome-cancer.ucsc.edu) and analyzed using the program R. The data indicate that Siglec15 is overexpressed in many human cancers and may play a role in cancer development and progression.

[0256] Siglec15 expression was also assessed in several human cancer cell lines. Siglec15 expression in human cancer cell lines was determined by NCI60 microarray (BioGPS) (Figure 3A, expression values ​​are shown in arbitrary units). Several human cancer cell lines were stained with the anti-Siglec15 antibody m03. Antibody m03 was developed by immunizing NZB / W F1 mice with a mouse Siglec15 ectodomain fusion protein and detecting the expression of human Siglec15. The stained cells were analyzed for Siglec15 expression using FACS (Figure 3B). This data indicates that Siglec15 is also upregulated in human tumor cell lines.

[0257] Example 4 Identification of MAG or LRRC4C as a ligand for Siglec15 by receptor array technology To identify ligands for Siglec15, Siglec15-Fc fusion proteins were generated by fusing the extracellular domain of each molecule with a mouse or human Fc tag (Dong H et al., Nat Med., 1999;5(12):1365-9) and screened on a newly established genome-scale human receptor array (Yao, S et al., Immunity, 2011;34(5):729-40). Briefly, 6,200 A complete human membrane gene library containing genes from multiple species was collected, maintained, and diluted in OPTI-MEM medium and then individually distributed at 40 ng / well into four 1,536-well plates using a robotic system. Lipofectamine 2000 was added to each well and mixed with the plasmid for 30 minutes. 2,000 HEK293T cells were then added to each well for transient transfection. Eight hours after transfection, 10 ng of human Siglec15-Fc and an anti-Fc FMAT blue secondary antibody were added to each well. Twenty-four hours after transfection, the plates were read using the Applied Biosystems 8200 Cell Detection System and analyzed using CDS 8200 software. Human Fc receptor genes served as internal positive controls for the assay.

[0258] Two positive hits were identified for Siglec15 ligands: myelin-associated glycoprotein (MAG) and leucine-rich repeat-containing 4C (LRRC4C) (Figure 4A). MAG, also known as Siglec4, is found primarily in the brain and is involved in neuronal myelination. MAG is also present in myeloid cells, particularly microglia. MAG knockout (KO) mice exhibit phagocytosis problems. LRRC4C is an LRR family molecule associated with neuronal growth, dendrite formation, and axon elongation. LRRC4C is primarily localized at the postsynaptic side of excitatory synapses and interacts with its presynaptic ligand, netrin-G1, to regulate excitatory synapse formation. MAG expression has been shown to be enriched in the brain or brain-related tumors (Figure 5), and LRRC4C mRNA levels were also upregulated in several types of cancer, including brain cancer, breast cancer, ovarian cancer, renal cell carcinoma, and Ewing's sarcoma (Figure 6). It has previously been reported that Siglec15 interacts with sialyl-Tn. To confirm this finding, we measured the binding of the sialyl-Tn antigen Neu5Acα2-6GalNAc to a Siglec15 fusion protein (Siglec15-mIg) using an Octet streptavidin biosensor (ForteBio). The Octet streptavidin biosensor was preloaded with NeuAa-2-6 GalNAc-biotin (Glycotech, 50 μg / ml), and the response to either Siglec15-mIg or control mIg (two-fold serial dilutions starting at 100 μg / ml) was determined over time (Figure 4B). Sialyl-Tn expression has been demonstrated in many human and mouse cancers. Therefore, sialyl-Tn may be an additional binding partner that serves as a ligand / receptor for Siglec15.

[0259] The specificity of the interaction between Siglec15 and MAG or LRRC4C was further verified by flow cytometry analysis. All antibodies for flow cytometry staining were purchased from BD Bioscience (San Jose, CA) or eBioscience (San Diego, CA). Siglec15 fusion proteins strongly bound to HEK293T cells transfected with MAG or LRRC4C, but not to MAG or LRRC4C. , but not to control cells, and this interaction was completely blocked by the inclusion of an anti-Siglec15 mAb. Indeed, the interaction between Siglec15 and MAG or LRRC4C is well conserved between mice and humans. As demonstrated in Figure 4A, human Siglec15 can bind to both human and mouse MAG, and mouse Siglec15 can also recognize both mouse and human LRRC4, suggesting a cross-species interaction.

[0260] Example 5 Identification of the binding domains of Siglec15 for MAG and LRRC4C To determine the binding domain of Siglec15 to MAG or LRRC4C, we constructed and purified Siglec15 mutants with domain deletions. Siglec15 contains an intracellular domain, a transmembrane domain, an IgC domain, and an IgV domain. Human or mouse Siglec15 was generated by PCR using a method similar to that previously described (Sedy JR et al., Nat Immunol., 2005;6(1):90-8). Siglec15 point mutations were selected and generated using PCR according to a previous publication (Cheung TC et al., Proc Natl Acad Sci U S A., 2005;102(37) :13218-23; Compaan DM et al., J Biol Chem., 2005; 280(47 No. 39553-61). As demonstrated in Figure 7, deletion of the IgV domain from Siglec15 completely abolished the interaction between Siglec15 and MAG and LRRC4C. Furthermore, a point mutation at residue 143 in the IgV domain (R143A mutation) also eliminated the interaction between Siglec15 and MAG or LRRC4C, suggesting that the IgV domain of Siglec15 is required for the interaction with MAG or LRRC4C, more specifically, for the interaction involving the R143 residue in the IgV domain.

[0261] Example 6 Siglec15 directly inhibits T cell activity To determine the effect of Siglec15 on human T cell activity, human PBMCs were stimulated for 72 hours with immobilized anti-CD3 antibody (ranging from 0.03 μg / ml to 1 μg / ml) and either immobilized human Siglec15-mIgG fusion protein (S15-mIg) or control mouse IgG (mIg) (5 μg / ml). After 16 hours, the effect of Siglec15 on expanded T cells was assessed. 3 H-thymidine incorporation was analyzed (Fig. 8A). 3 The level of 3H thymidine incorporation was significantly reduced, indicating that exposure to Siglec15 as a ligand reduced T cell proliferation.

[0262] A membrane-bound OKT3 expression construct was generated by fusing nucleic acids encoding the scFv fragment of the anti-human CD3 antibody OKT3 with nucleic acids encoding CD14. This construct was transfected into 293T cells to generate 293T.m.OKT3 cells expressing OKT3 scFv on the cell surface. Jurkat NF-AT luciferase reporter cells were co-cultured for 12 hours with 293T.m.OKT3 cells overexpressing mock plasmid, full-length FASLG (Fas ligand), LRRC4C, Siglec15, or a gene encoding the Siglec15 ectodomain with the B7-H6 transmembrane domain (Siglec15 ATM). Luciferase activity was monitored 4 hours after co-culture (Figure 8B). In this system, overexpression of FASLG nearly eliminated NF-AT signaling compared to mock plasmid. Furthermore, it was observed that expression of either full-length Siglec15 or Siglec15 ATM could similarly significantly inhibit NF-AT luciferase signaling.

[0263] The above experiments showed that human Siglec15, which acts as a ligand, inhibits T cell activity. It is shown that it can be obtained.

[0264] To confirm that mouse Siglec15 similarly inhibits mouse T cell activity, we performed a cytotoxic assay in mouse splenocytes after exposure to mouse Siglec15. 3 H-thymidine incorporation was analyzed. Mouse splenocytes were stimulated with immobilized anti-CD3 (ranging from 0 μg / ml to 2 μg / ml) and exposed to immobilized or soluble mouse Siglec15-mIgG fusion protein (S15-mIg) or control mouse IgG (mIg) (5 μg / ml) for 72 hours. Thymidine incorporation in expanded T cells was analyzed 16 hours later. 3 H-thymidine incorporation is shown in Figure 9A. 3 H thymidine incorporation is shown in Figure 9B. In cells exposed to mouse S15-mIg,3 The level of 3H thymidine incorporation was reduced, indicating that contact with Siglec15 ligand reduced proliferation of mouse splenocytes.

[0265] 293T cells were transfected with a plasmid encoding H2-Kb fused to the OVA peptide (SIINFEKL) to generate 293T-Kb-OVA cells. The cells were then infected with a lentiviral vector encoding full-length Siglec15 to generate 293T-Kb-OVA-S15 cells. Several cell lines with different Siglec15 expression were isolated by FACS screening. OT-1 mice transgenic for an OVA-specific TCR recognizing the H-2kb OVA SIINFEKL peptide were obtained from The Jackson Laboratory. Splenocytes from OT-1 transgenic mice were preactivated with SIINFEKL peptide plus IL-2 for 3 days. The activated cells were then cocultured with 293T-KbOVA cells overexpressing full-length mouse Siglec15 (KbOVA-S15) or a mock-transfected control (KbOVA-control) for 3 days. The expanded T cells 3 H-thymidine incorporation was analyzed 16 hours later (Fig. 9C). Splenocytes exposed to Siglec15 in the context of 293T-KbOVA cells showed 3 The level of H-thymidine incorporation was reduced, indicating that cell-based contact with Siglec15 reduced proliferation of mouse splenocytes.

[0266] As shown by FACS analysis followed by staining with the anti-Siglec15 antibody m03, different levels of mouse Siglec15 expression were identified in three 293T-KbOVA cell lines overexpressing mouse Siglec15 (Figure 9D). Splenocytes from OT-1 transgenic mice were preactivated with SIINFEKL peptide and IL-2 as described above. The activated splenocytes were then cocultured for 3 days with 293T-KbOVA cell lines expressing elevated levels of Siglec15, as shown in Figure 9D. IFN-γ (Figure 9E) and TNF-α (Figure 9F) levels in the supernatants were measured using a Cytometric Bead Array (CBA) (BD Pharmingen). IFN-γ and TNF-α production decreased with increasing levels of Siglec15 expression, indicating that Siglec15 inhibits T cell function in a dose-dependent manner.

[0267] Example 7 Cell-associated Siglec15 reduces T cell cytotoxicity An adhesion-based cytotoxicity assay was used to investigate the effect of Siglec15 on T cell cytotoxicity and its association with tumor killing.

[0268] 293T-KbOVA target cells were plated onto 384 E-plates (ACEA Biosciences) at 10 cells per well. 4 Cells were plated at a density of 1 / 3 and allowed to grow overnight. Cells that adhere to the plate generate an increasing electrical signal. If left undisturbed, the electrical signal The signal increases over time and then declines due to cell overgrowth. To test the effect of antigen-specific T cells in this system, 293T-KbOVA target cells were cocultured with different ratios of preactivated OT-1 T cells (ranging from 0:1 to 2:1). The adhesion signal of growing target cells over time is shown in Figure 10A. A dose-response relationship was observed regarding the level of killing of 293T-KbOVA target cells by OT-1 T cells. The higher the ratio of OT-1 T cells:293T-KbOVA target cells present in the assay, the smaller the signal detected, indicating that fewer live 293T-KbOVA target cells were detected as the amount of OT-1 T cells increased.

[0269] In this system, T cell killing of target cells overexpressing Siglec15 (293T-KbOVA-S15) was compared with T cell killing of Siglec15-negative target cells (293T-KbOVA-control) in the presence of various ratios of OT-1 T cells. In the absence of T cells (0:1 ratio), the signal of 293T-KbOVA cells overexpressing Siglec15 was weaker than that of control cells. However, in the presence of T cells (1:1 or 2:1 ratio), a higher signal was generated in the Siglec15-overexpressing cells, indicating resistance to T cell killing (Figure 10B). These data indicate that cell-associated Siglec15 (e.g., against tumor cells expressing Siglec15) can render cells resistant to T cell cytotoxicity.

[0270] Example 8 Siglec15 deficiency elicits enhanced T cell responses Wild-type (WT) or Siglec15 whole-body knockout (KO) mice received splenocytes from OT-1 / RagKO mice intravenously (iv) according to the schedule in Figure 11A. Briefly, mice received 2 x 10 6Splenocytes were received on day -1 and boosted intraperitoneally (ip) with 100 μg of OVA peptide plus 100 μg of poly i:c on day 0. The percentage of OT-1 cells relative to the total CD8 T cell population was assessed in the blood on day 4 and in the spleen on day 5 (Figure 11B). The percentage of OT-1 cells was determined by FACS staining using OT-1 tetramer.

[0271] The percentage of antigen-specific CD8+ T cells in the blood and spleen was increased in Siglec15 KO mice compared with WT mice, suggesting that removing the inhibitory effect of Siglec15 allows T cells to respond robustly to antigen priming.

[0272] As shown in Figure 11A, OT-1 T cells were injected into WT or S15KO mice on day -1, followed by peptide stimulation on day 0. Mice received 5-ethynyl-2'-deoxyuridine (EdU) (0.8 mg / ml) in their drinking water, which was changed every 2 days starting on day 0. On day 5, the proliferation of blood OT-1 cells was analyzed by anti-EdU staining and calculated as the percentage of EdU-positive OT-1 cells / total OT-1-positive cells (Figure 11C). Splenocytes were also isolated, cultured overnight without stimulation, and stained for Annexin V. Apoptosis was calculated as Annexin V-positive OT-1 cells / total OT-1-positive cells (Figure 11D).

[0273] Example 9 Bone marrow-derived Siglec15 is critically involved in Siglec15 function in vivo To identify the cell population involved in the Siglec15 response, we developed macrophage-specific Siglec15 knockout mice (LysM-Cre KO), which allowed us to analyze which cells express Siglec15 and inhibit T cells. Siglec15 conditional knockout mice (Strain 1) were purchased from Netherland. B6.Cg-Siglec15 tm1.1Cfg / Mmucd; reference number MMRRC:032723-UCD) were backcrossed with LysM-Cre mice (Jax Labs) to generate LysM-Cre Siglec15 knockout mice. These mice were subsequently backcrossed with C57 / BL6 mice for six generations before experiments were performed.

[0274] Using the OT-1 T cell transfer system described in Example 8, we compared the expansion of OT-1 T cells in Siglec15 whole-body knockout (KO) mice with that in macrophage-specific Siglec15 knockout mice (LysM-Cre KO). Blood was collected at different time points, and the percentage of OT-1 cells in the total CD8 T cell population was determined (Figure 12A). OT-1 T cell expansion peaked on day 3 in WT mice and later in the two types of KO mice. The contraction phase, in which T cells contract after initial expansion, remained high in both KO models, suggesting that T cells are maintained at higher numbers in the absence of Siglec15 inhibition. Siglec15 whole-body KO and LysM-Cre KO mice behaved similarly, suggesting that macrophages may be significantly involved in the inhibitory effects of Siglec15 molecules. IL-10 levels in the plasma of whole-body KO and LysM-Cre KO mice were also assessed during the OT-1 T cell response and compared with wild-type mice (Figure 12B). Lower levels of IL-10 were identified in the plasma of whole-body KO and LysM-Cre KO mice compared with wild-type mice.

[0275] Example 10 Siglec15 deficiency has only a minor effect on the endogenous immune cell pool To determine whether the lack of Siglec15 in knockout mice affects the composition of the endogenous pool of immune cells, the percentages of myeloid, CD8, or CD4 populations in the blood of wild-type (WT) and Siglec15 knockout (S15KO) mice were determined at 11 months of age (Figure 13).

[0276] These data demonstrate that Siglec15 KO mice have significantly enhanced antigen-specific T cell responses, but do not have a distinct genetic immune phenotype (either myeloid immune cells or T cells) under normal conditions, indicating that Siglec15 may operate with induced expression patterns and / or functionality.

[0277] Example 11 Cell-based Siglec15 inhibits macrophage responses Mouse peritoneal macrophages were cocultured with 293T cells overexpressing mock plasmid (control), full-length LRRC4C, or Siglec15 in the presence of various doses of LPS for 24 hours. Cytokine levels in the supernatant were measured using a Cytometric Bead Array (CBA) (BD Pharmingen). IL-6, TNF-α, and TGF-β1 production by macrophages was reduced when the cells were cocultured with cells overexpressing Siglec15 (Figure 14). This data indicates that cell-based Siglec15 directly inhibits myeloid cell responses. Thus, Siglec15 may act as a ligand to impose inhibitory activity on the immune system through both myeloid cells and T cells.

[0278] Example 12 Effects of blocking the interaction between Siglec15 and MAG or LRRC4C in EAE mice To determine the role of the interaction between Siglec15 and MAG or LRRC4C in regulating brain inflammatory diseases, we used a mouse model of experimental autoimmune encephalomyelitis (EAE). EAE is an inflammatory model of encephalitis and demyelination that affects the spinal cord and brain, causing paralysis. The EAE model is widely used as a model for multiple sclerosis (MS), a human inflammatory demyelinating disease. The degree of paralysis can be quantified by the EAE score.

[0279] Briefly, female C57BL / 6 mice (6–10 weeks old) were purchased from the National Cancer Institute, NIH (Frederick, MD). Eight to 12-week-old C57BL / 6 mice were subcutaneously immunized with 100 μg of MOG peptide (35–55) emulsified in complete Freund's adjuvant (CFA) (Difco) on day 0 to induce EAE. Two injections of 400 ng of pertussis toxin (Sigma) in 200 μl of PBS were administered on days 0 and 2. Each mouse was intraperitoneally injected with 200 μg of Siglec15 mAb (S15m02, S15m03), control antibody, or the indicated fusion protein on days 7 and 10. Mouse anti-mouse Siglec15 mAb was generated by immunizing NZB / W F1 mice with mouse Siglec15-Ig fusion protein. All fusion proteins were generated by fusing the extracellular domain of each molecule with a mouse or human Fc tag (Dong H et al., Nat Med., 1999;5(12):1365-9).

[0280] Disease severity was scored based on the following scale as previously described: 0, no disease; 1, tail paralysis; 2, paraparesis; 3, paraplegia; 4, paraplegia or paralysis with forelimb weakness; and 5, moribund or dead (Stromnes IM et al., Nature protocols., 2006;1(4):1810-9). Figure 15 illustrates that the Siglec15 antibody clone S15m02 acted as a blocking antibody, preventing Siglec15 from binding to MAG or LRRC4C. As a result, mice receiving the S15m02 antibody developed more severe disease symptoms than their counterparts injected with control mAb or S15m03 antibody (Figure 16), suggesting that the interaction between Siglec15 and MAG or LRRC4C is essential for regulating inflammatory responses in the brain. The results were further confirmed by treating EAE mice with the Siglec15-Fc fusion protein Siglec15-mIg. EAE was accelerated after mice received Siglec15-Fc fusion protein (FIG. 17).

[0281] Example 13 The role of Siglec15 in suppressing brain inflammatory responses To confirm that Siglec15 plays a role in regulating brain inflammation, a Siglec15 knockout (KO) mouse model was purchased from MMRRC (UC Davis) (Tao et al., J Immunol., 2008;180(10):6649-55). Both wild-type (WT) and Siglec15 knockout mice were immunized with MOG(33-35) peptide to induce experimental autoimmune encephalomyelitis (EAE), and then clinical scores for EAE disease were measured. As demonstrated in Figure 18, Siglec15 KO mice showed more severe EAE disease symptoms than WT mice, indicating an inhibitory role for Siglec15 in regulating brain inflammatory responses.

[0282] Example 14 Siglec15 deficiency enhances autoimmunity Wild-type (WT) and Siglec15 knockout (Siglec15 KO) mice were immunized with the MOG peptide to induce EAE. All mice were boosted with pertussis toxin on days 0 and 1. Clinical scores for EAE were determined for WT and Siglec15 KO mice, and it was confirmed that Siglec15 KO mice exhibited more severe EAE disease symptoms than WT mice.

[0283] Separate cohorts of wild-type (WT) mice were immunized with MOG peptide and boosted with pertussis toxin on days 0 and 1. Subsequently, treatment with Siglec15-mIg fusion protein (Siglec15-mIg) or control mIg, or Siglec15-hIg fusion protein (Siglec15-hIg) or control hIg (100 μg) was initiated on day 6 and administered twice weekly for a total of four doses. EAE clinical scores were assessed over time (Figure 19A). On day 12, splenocytes from mice treated with control mIg were restimulated with MOG peptide (60 μg / ml) in the presence of 5 μg / ml Siglec15-mIg (S15-mIg) or control mIg (mIg) for 3 days. After an additional 16 h of incubation, EAE in both groups was significantly elevated. 3 H-thymidine incorporation was measured (Figure 19B). This data indicates that soluble Siglec15-mIg behaves as an antagonist and can stimulate antigen-specific T cell responses in vivo. This mechanistic study advances our understanding of the functional role of Siglec15 fusion proteins in the brain inflammation model (EAE).

[0284] Example 15 The role of Siglec15 in suppressing immune responses in brain cancer A brain tumor model was used to test the impact of Siglec15 in regulating immune responses in cancer. C57BL / 6 mice were intracranially injected with GL261 tumor cells containing a luciferase reporter used to quantify tumor size. Four days after tumor injection, the mice underwent low-dose whole-brain radiation therapy and then treated with Siglec15-Fc or control Ig on days 5 and 10. Tumor size in both groups was monitored intensively. As demonstrated in Figures 20 and 21, blocking Siglec15 with Siglec15-Fc treatment significantly reduced tumor size (Figure 20) and prolonged mouse survival (Figure 21). Mice treated with Siglec15-Fc also showed synergistic effects when treated with an anti-PD-L1 antibody, significantly enhancing the efficacy of anti-PD-L1 in tumor reduction and survival benefit (Figure 22). These results suggest that blocking or otherwise inhibiting the activity of Siglec15 could enhance the immune response against cancer.

[0285] Example 16 Significant infiltration of tumor-associated CD8 T cells in tumor-bearing mice As demonstrated above, Siglec15 is expressed in the brain. We investigated the number and activity of T cells in wild-type (WT) and Siglec15 knockout (Siglec15 KO) brain tumor models. WT or Siglec15 KO mice were intracranially inoculated with GL261 glioblastoma cells on day 0. Tumor burden in the brain was monitored by measuring luciferase activity at different time points (Figure 23A). Luciferase activity in the brains of WT and KO mice was imaged on days 13 and 18 after inoculation with GL261 cells (Figure 23B). Survival curves of GL261-inoculated WT and KO mice demonstrate that Siglec15 KO mice survive longer than WT mice (Figure 23C). On day 14, some mice from both groups were sacrificed. The numbers and percentages of CD8 T cells (Figure 24A), CD4 T cells (Figure 24B), or myeloid cell populations (Figure 24C) in the brain or spleen were monitored. Furthermore, brain lymphocytes from tumor-bearing WT or KO mice were restimulated overnight with GL-261 tumor cells, and the percentages and total numbers of IFN-γ-positive CD8 or CD4 T cells were determined (Figure 24D).

[0286] Significant infiltration of functional CD8 T cells was identified at the tumor site of Siglec15 KO mice but not at the tumor site of WT mice. Since there was no difference in the spleen, this effect is tumor-related. Furthermore, significant differences were observed in brain dendritic cell / macrophage populations. These data indicate that Siglec15 can influence immune cell responses at the tumor site. In particular, Siglec15 in the tumor / brain microenvironment. Expression can inhibit T cells and / or myeloid cells, shutting down the immune response against tumors.

[0287] Example 17 Growth of tumor cells expressing Siglec15 MC38 colon adenocarcinoma cells were infected with lentiviral expression constructs encoding Siglec15 (S15+) or control (S15-). After lentiviral infection, MC38-S15- and MC38-S15+ cell populations were sorted using FACS. Siglec15 expression in MC38-S15- and MC38-S15+ cells was confirmed by FACS staining with a monoclonal antibody (Figure 25A). B6 mice were subcutaneously inoculated with MC38-S15- and MC38-S15+ cells (0.4M cells per mouse), and tumor growth was monitored (Figure 25B). Tumors derived from MC38 cells expressing Siglec15 were larger than tumors derived from MC38 cells lacking Siglec15 expression.

[0288] Example 18 Siglec15 antagonist inhibits the growth of Siglec15-expressing tumor cells The MC38-S15+ stable cell line was subcutaneously inoculated into B6 mice (0.4M cells per mouse). Mice were treated with control antibody, anti-Siglec15 antibody m01, or S15-mIg (ip, 200 μg / mouse) on day 6, and then every 4 days for a total of 4 doses. The average tumor size in each group is shown in Figure 26. Mice treated with Siglec15 antagonist m01 or S15-mIg showed significant tumor size reduction.

[0289] equivalent 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 which equivalents are intended to be encompassed by the following claims.

Claims

[Claim 1] The invention as described in the drawings.

Images