Car-treg-based therapies for treating neurodegenerative diseases

Abstract

To provide CAR-Treg-based therapies for treating neurodegenerative diseases.SOLUTION: The invention provides compositions and methods for suppressing autoimmune components of neurodegenerative diseases, thereby providing therapeutic effects to patients suffering from such diseases. Compositions and methods include immunosuppressive moieties such as regulatory T cells (Tregs) and proteins expressed by Tregs coupled to a chimeric antigen receptor or protein that specifically binds one or more glial cell markers. Therapeutically effective doses of the compounds for treating neurodegenerative diseases including progressive supranuclear palsy (PSP), Parkinson's disease (PD), Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy (CTE), and prion diseases are disclosed.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 648,684, filed March 27, 2018, the contents of which are incorporated by reference in their entirety.

[0002] FIELD OF THE INVENTION The present invention provides CAR-Treg compositions and methods of use that specifically regulate immune responses and inflammation associated with various neurodegenerative diseases (e.g., progressive supranuclear palsy and Parkinson's disease). [Background technology]

[0003] background Neurodegenerative diseases such as Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease, amyotrophic lateral sclerosis (ALS), and progressive supranuclear palsy (PSP) affect a significant number of people and often result in rapid physical and / or mental deterioration and death. There are no known cures for these diseases, and no known treatments that focus on managing symptoms and slowing deterioration.

[0004] One such disorder, PSP, mimics Parkinson's disease (PD) and is a not-rare idiopathic degenerative disorder in elderly individuals. Its clinical manifestations include the tetralogy of supranuclear gaze palsy, axial rigidity, dementia, and pseudobulbar palsy. It is associated with bradykinesia, severe postural disturbances, and frequent falls. Pathology is associated with cell loss and tau neurofibrillary tangles, primarily in the brainstem, globus pallidus, subthalamic nucleus, and dentate nucleus. PSP has a prevalence of 5–6 cases per 100,000, with an annual incidence of 5,000–25,000 cases in the United States. The mean age at onset of the disease is 63 years, and the usual prognosis ranges from 5–10 years from diagnosis to death. No disease-modifying treatments are available.

[0005] Parkinson's disease is another neurodegenerative disorder with no known cure. It has a prevalence of approximately 1 to 2 cases per 1,000 people. Parkinson's disease is characterized by cell death in the basal ganglia, along with astrocyte death in the substantia nigra and an increase in microglia, resulting in dopamine deficiency in that region. Inclusions called Lewy bodies develop in damaged cells prior to cell death. While the underlying mechanisms driving brain cell death in Parkinson's disease are speculated to drive this, they remain poorly understood, and treatments currently focus on managing disease symptoms. Summary of the Invention [Means for solving the problem]

[0006] Abstract The compositions and methods of the present invention use regulatory T lymphocytes (Tregs) or immunosuppressive proteins expressed by Treg cells to regulate neurodegenerative immune responses that target glial cells in the central nervous system (CNS). By linking Tregs or immunosuppressive proteins to either chimeric antigen receptors (CARs) or single-chain variable fragments (scFvs) that specifically recognize and bind to glial cell markers, the immunosuppressive Tregs or proteins induce CNS glial cells to reduce inflammation and protect the CNS from autoimmune attack.

[0007] The present invention recognizes the lack of effective treatment options for most neurodegenerative diseases, as well as the presence of autoimmune and / or inflammatory components to some such diseases, and designs compositions to specifically inhibit those disease components. The compounds and methods of the present invention inhibit glial cells from transforming into type 1 helper cells (Th1), T helper 17 cells (Th17), and cytotoxic T cells. It allows for the regulation of damaging immune cells such as cytotoxic T cells (CTLs), M1 macrophages, and polymorphonuclear neutrophils (PMNs).

[0008] The present invention targets immunosuppressive molecules (Tregs or immunosuppressive proteins) to oligodendrocyte (ODC) glial cells. The resulting compounds and methods of use mobilize the body's own immune system to combat the effects of neurodegenerative diseases such as Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease, amyotrophic lateral sclerosis (ALS), and progressive supranuclear palsy (PSP). The present invention addresses a mechanism by which several neurodegenerative diseases disrupt neuronal function that is independent of any specific biochemical cause of the underlying disease (i.e., autoimmune attack of the central nervous system). Thus, the compounds and methods of the present invention may provide therapeutic benefits across a range of neurodegenerative diseases.

[0009] Aspects of the present invention include methods for treating a neurodegenerative disease in a subject, the method comprising administering to the subject a therapeutically effective amount of regulatory T cells (Tregs) expressing a chimeric antigen receptor (CAR) that specifically binds to a glial cell marker, wherein the neurodegenerative disease is not multiple sclerosis (MS). The CAR-Tregs then protect neural tissue and reduce inflammation in the neural tissue, thereby treating the neurodegenerative disease. In various embodiments, the subject may be human.

[0010] The glial cell marker may be oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (OM4), neural / glial marker 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), or myelin oligodendrocyte-specific protein (MOSP). In some embodiments, the glial cell marker may be myelin oligodendrocyte glycoprotein (MOG).

[0011] The neurodegenerative disease that can be treated can be progressive supranuclear palsy (PSP), Alzheimer's disease (AD), Huntington's disease, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy (CTE), or prion disease. In some embodiments, the neurodegenerative disease is progressive supranuclear palsy (PSP). In other embodiments, the neurodegenerative disease is Alzheimer's disease (AD). In still other embodiments, the neurodegenerative disease is Parkinson's disease (PD).

[0012] In certain aspects, the present invention provides a composition comprising a therapeutically effective amount of engineered regulatory T cells (Tregs) for treating a neurodegenerative disease other than multiple sclerosis, wherein the engineered Tregs express a chimeric antigen receptor (CAR) that specifically binds to a glial cell marker. The glial cell marker in the composition can be myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (OM4), neural / glial marker 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), or myelin oligodendrocyte-specific protein (MOSP).

[0013] The compositions may be therapeutically effective for treating progressive supranuclear palsy (PSP), Parkinson's disease (PD), Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy (CTE), or prion disease.

[0014] Various aspects of the present invention include engineered proteins comprising a glial cell-specific binding protein linked to a molecule expressed by regulatory T cells (Tregs). The molecule expressed by Tregs can be an extracellular immunosuppressive enzyme. In certain embodiments, the molecule expressed by Tregs can be CD73, CD39, indoleamine 2,3-dioxygenase (IDO), or glutamic oxaloacetic transaminase 1 (GOT1). The glial cell-specific binding protein can be a tetrameric single-chain variable fragment (scFv) of an antibody molecule.

[0015] In certain embodiments, the glial cell-specific binding protein bound to the Treg-expressed molecule may bind to myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (OM4), neural / glial marker 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), or myelin oligodendrocyte-specific protein (MOSP).

[0016] In some aspects, the present invention provides engineered proteins comprising glial cell-specific binding proteins linked to molecules that mimic the activity of molecules expressed by regulatory T cells (Tregs). The mimicked molecule expressed by Tregs can be an extracellular immunosuppressive enzyme such as CD73, CD39, indoleamine 2,3-dioxygenase (IDO), or glutamic oxaloacetic transaminase 1 (GOT1). The glial cell-specific binding protein linked to the mimicked molecule can bind to myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (OM4), neural / glial marker 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), or myelin oligodendrocyte-specific protein (MOSP). In certain embodiments, for example, the following are provided: (Item 1) 1. A method for treating a neurodegenerative disease in a subject, the method comprising administering to the subject regulatory T cells (Tregs), each expressing a chimeric antigen receptor (CAR) that specifically binds to a glial cell marker, in a therapeutically effective amount to protect neural tissue and reduce inflammation in the neural tissue, thereby treating the neurodegenerative disease, wherein the neurodegenerative disease is not multiple sclerosis. (Item 2) Item 10. The method of item 1, wherein the subject is a human. (Item 3) 2. The method of claim 1, wherein the glial cell marker is selected from the group consisting of oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (OM4), neural / glial marker 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and myelin oligodendrocyte-specific protein (MOSP). (Item 4) 4. The method of claim 3, wherein the glial cell marker is myelin oligodendrocyte glycoprotein (MOG). (Item 5) Item 1. The method of item 1, wherein the neurodegenerative disease is selected from the group consisting of progressive supranuclear palsy (PSP), Alzheimer's disease (AD), Huntington's disease, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy (CTE), and prion disease. (Item 6) Item 6. The method of item 5, wherein the neurodegenerative disease is progressive supranuclear palsy (PSP). (Item 7) 6. The method of claim 5, wherein the neurodegenerative disease is Alzheimer's disease (AD). (Item 8) 6. The method of claim 5, wherein the neurodegenerative disease is Parkinson's disease (PD). (Item 9) 1. A composition comprising a plurality of engineered regulatory T cells (Tregs) in a therapeutically effective amount for treating a neurodegenerative disease that is not multiple sclerosis, wherein each of the plurality of engineered Tregs expresses a chimeric antigen receptor (CAR) that specifically binds to a glial cell marker. (Item 10) 10. The composition of item 9, wherein the glial cell marker is selected from the group consisting of myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (OM4), neural / glial marker 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and myelin oligodendrocyte-specific protein (MOSP). (Item 11) 10. The composition of item 9, wherein the glial cell marker is myelin oligodendrocyte glycoprotein (MOG). (Item 12) Item 10. The composition of item 9, wherein the neurodegenerative disease is selected from the group consisting of progressive supranuclear palsy (PSP), Parkinson's disease (PD), Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy (CTE), and prion disease. (Item 13) The composition according to Item 12, wherein the neurodegenerative disease is progressive supranuclear palsy (PSP). Item 13. The composition of item 12, wherein the neurodegenerative disease is Alzheimer's disease (AD). (Item 15) Item 13. The composition of item 12, wherein the neurodegenerative disease is Parkinson's disease (PD). (Item 16) An engineered protein comprising a glial cell-specific binding protein linked to a molecule expressed by regulatory T cells (Tregs). (Item 17) 17. The engineered protein of claim 16, wherein the molecule expressed by Tregs is an extracellular immunosuppressive enzyme. (Item 18) 18. The engineered protein of claim 17, wherein the molecule expressed by Tregs is selected from the group consisting of CD73, CD39, indoleamine 2,3-dioxygenase (IDO), and glutamic oxaloacetic transaminase 1 (GOT1). (Item 19) 17. The engineered protein of claim 16, wherein the glial cell-specific binding protein is a tetrameric single-chain variable fragment (scFv) of an antibody molecule. (Item 20) 17. The engineered protein of paragraph 16, wherein the glial cell-specific binding protein binds to a marker selected from the group consisting of myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (OM4), neural / glial marker 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and myelin oligodendrocyte-specific protein (MOSP). (Item 21) 21. The engineered protein of paragraph 20, wherein the glial cell-specific binding protein binds to myelin oligodendrocyte glycoprotein (MOG). (Item 22) 22. The engineered protein of claim 21, wherein the glial cell-specific binding protein is a single-chain variable fragment (scFv) of an antibody molecule comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, and 12. (Item 23) An engineered protein comprising a glial cell-specific binding protein linked to a molecule that mimics the activity of a molecule expressed by regulatory T cells (Tregs). (Item 24) 24. The engineered protein of claim 23, wherein the molecule expressed by Tregs is an extracellular immunosuppressive enzyme. (Item 25) 25. The engineered protein of claim 24, wherein the molecule expressed by Tregs is selected from the group consisting of CD73, CD39, indoleamine 2,3-dioxygenase (IDO), and glutamic oxaloacetic transaminase 1 (GOT1). (Item 26) 24. The engineered protein of claim 23, wherein the glial cell-specific binding protein is a tetrameric single-chain variable fragment (scFv) of an antibody molecule. (Item 27) 24. The engineered protein of paragraph 23, wherein the glial cell-specific binding protein binds to a marker selected from the group consisting of myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (OM4), neural / glial marker 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and myelin oligodendrocyte-specific protein (MOSP). (Item 28) 28. The engineered protein of paragraph 27, wherein the glial cell-specific binding protein binds to myelin oligodendrocyte glycoprotein (MOG). (Item 29) 29. The engineered protein of claim 28, wherein the glial cell-specific binding protein is a single-chain variable fragment (scFv) of an antibody molecule comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, and 12. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 illustrates glial cell-specific CAR-Tregs and their immunosuppressive function. [Figure 2] FIG. 2 illustrates glial cell-targeted immunosuppressive proteins and their immunosuppressive functions. [Figure 3] FIG. 3 illustrates the binding of pMHC-tetramers to cytotoxic T cells and the binding of GITP of the present invention to the target MOG protein. [Figure 4]FIG. 4 illustrates the maximal staining of MOG target cells with labeled GITP protein compared to that of CTL and pMHC. [Figure 5] FIG. 5 illustrates the half-life of staining of MOG target cells with labeled GITP protein compared to that of CTL and pMHC. [Figure 6] FIG. 6 illustrates a comparison of GTIP-bound MOG target cells suppressing T effector cell proliferation compared to negative and positive controls. [Figure 7] FIG. 7 illustrates the relative avidity of pMHC of the corresponding cytotoxic T cell clones compared to the relative avidity of CAR molecules expressing scFv specific for MOG on MOG-target cells. [Figure 8] FIG. 8 illustrates the relative immunoreactivity of seven different scFv proteins against human MOG-1. DETAILED DESCRIPTION OF THE INVENTION

[0018] Detailed Description The present invention relates to compositions for regulating the autoimmune component of various neurodegenerative diseases. The compositions and methods provided herein target glial cell-specific markers to induce immunosuppressive molecules (e.g., Tregs or immunosuppressive proteins expressed by Tregs) in the CNS, thereby disrupting the autoimmune attacks that contribute to the neurodegenerative effects of diseases such as progressive supranuclear palsy (PSP), Alzheimer's disease (AD), Huntington's disease, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy (CTE), or prion diseases.

[0019] The blood-brain barrier can act as an obstacle to the treatment of brain or CNS disorders, because this barrier can block therapeutic compounds from accessing affected cells.Importantly, Tregs can cross the blood-brain barrier and be localized in CNS neurons by Treg-associated glial cells, thereby allowing the compounds of the present invention to effectively treat CNS neurodegenerative disorders.

[0020] The compounds and methods of the present invention do not rely on any disease-specific biochemical mechanisms, but instead avoid the immune response that contributes to the mental and physical deterioration of many neurodegenerative diseases, and thus may provide therapeutic benefits for many neurodegenerative diseases.

[0021] For example, PSP is accompanied by tau protein accumulation and neurofibrillary tangles, resulting in the damage and loss of neurons and glial cells, and ultimately death, associated with physical and mental deterioration.Parkinson's disease is accompanied by astrocyte death and microglia proliferation in the substantia nigra, as well as neuronal loss in the basal ganglia.Inclusions called Lewy bodies occur in damaged cells before cell death.ALS is characterized by the death of motor neurons in the motor cortex, followed by the development of protein-rich inclusions in the cell bodies and axons of motor neurons.

[0022] The present invention recognizes that, despite differing underlying causes and disease mechanisms, PSP, Parkinson's disease, and ALS, along with neurodegenerative diseases including Alzheimer's disease (AD), Huntington's disease, chronic traumatic encephalopathy (CTE), and prion diseases, likely involve an immune component that contributes to inflammation and CNS deterioration. Malaspina, et al., 2015, Disease origin and progression See, in amyotrophic lateral sclerosis: an immunology perspective, International Immunology, 27(3): 117-129; Mosley R, Gendelman H, 2017, T cells and Parkinson's disease, Lancet Neurology, 16(10):769-71, the contents of each of which are incorporated herein by reference. Thus, compounds and methods of the present invention that focus on suppressing immune responses in the CNS and addressing the chronic inflammation that drives many neurodegenerative diseases may be therapeutically effective in treating many of these diseases.

[0023] The compounds and methods of the present invention use chimeric antigen receptors (CARs), antibodies, or single-chain variable fragments (scFvs) that specifically bind to glial cell markers. The glial cell-binding molecules are linked to Tregs, immunosuppressive proteins expressed by Tregs, or molecules configured to mimic immunosuppressive proteins expressed by Tregs. Glial cells are non-neuronal cells that perform many functions in supporting neurons in the central and peripheral nervous systems of various animals, including humans. Glial cells include oligodendrocytes, astrocytes, ependymal cells, and microglia. As a result of their function in maintaining CNS neurons, glial cells migrate to CNS neurons and can be used to localize therapeutic compounds there. For example, oligodendrocyte (ODC) glial cells traffic to the CNS and maintain axonal insulation by producing myelin sheaths. The compounds and methods of the present invention involve linking immunosuppressive molecules to glial cells, such as ODCs, so that as the glial cells perform their function, the immunosuppressive molecules are brought into close proximity to neurons in the CNS, as shown in Figures 1 and 2. The presence of such immunosuppressive molecules modulates any ongoing immune response and chronic inflammation that may be present in the CNS and contributes to neurodegenerative disease symptoms in PD, PSP, etc.

[0024] Glial cell-specific targets include proteins and other markers expressed by various glial cells, such as myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (OM4), neural / glial marker 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), or myelin oligodendrocyte-specific protein (MOSP).

[0025] In various embodiments, CARs, scFvs, or antibodies can be linked to immunosuppressive molecules and used to target glial cells. CARs are engineered receptors that can provide specificity to immune effector cells (T cells). CARs have been used to confer tumor cell specificity to cytotoxic T lymphocytes for use in cancer immunotherapy. See Couzin-Frankel, 2013, Cancer immunotherapy, Science, 342(6165): 1432-33; Smith, et al., 2016, Chimeric antigen receptor (CAR) T cell therapy for malignant cancers: Summary and perspective, Journal of Cellular Immunotherapy, 2(2):59-68 (the contents of each of which are incorporated herein by reference). Using a similar principle, the compounds and methods of the present invention involve engineering CARs specific for markers found on glial cells, such as ODCs, but instead of grafting glial cell-specific CARs onto cytotoxic T cells, they are grafted onto engineered immunosuppressive Tregs.

[0026] The CAR-Tregs of the present invention may express multiple chimeric antigen receptors that target the same or two or more different glial cell markers.

[0027] An scFv is a fusion protein containing the variable region of an immunoglobulin heavy chain (VH) and the variable region of a light chain (VL). An scFv can be generated by cloning the VH and VL genes of a mouse or other animal immunized with a desired target molecule (e.g., MOG). The VH and VL genes can then be expressed in multiple orientations and with various linkers to form various scFvs that can be experimentally verified to provide the desired stability, expression level, and binding affinity for glial cells or their specific markers. The scFvs or antibodies specific for the glial cell markers discussed above can be linked to the immunosuppressant proteins discussed below to form fusion proteins that can provide CNS-localized immunosuppressive therapy, as shown in Figure 2 and discussed below.

[0028] Antibodies targeting glial cell markers can be generated by methods known in the art, including, for example, commercially available services for generating custom antibodies from Pacific Immunology (San Diego, CA) or ABclonal (Woburn, MA).

[0029] CAR-Treg can be engineered by known methods for preparing CAR-T cells. Treg cells can be isolated from a subject, preferably autologous Treg cells derived from the patient to be treated. The genes of the Treg cells can then be modified through known techniques, such as electroporation, viral vectors, or other forms of transfection with a nucleic acid encoding a selected engineered chimeric antigen receptor. The CAR-Treg cells can then be experimentally validated before being introduced into the patient's system for treatment.

[0030] Regulatory T cells (Tregs) regulate the immune system, generally downregulating the induction and proliferation of effector T cells. Tregs prevent autoimmune responses and help the immune system distinguish between self and nonself. Regulatory T cells produce inhibitory cytokines, including transforming growth factor beta, interleukin-35, and interleukin-10, and can induce other cell types to express interleukin-10. Tregs can also produce granzyme B, which can subsequently induce apoptosis of effector cells. Tregs also function through reverse signaling via direct interaction with dendritic cells and induction of immunosuppressive indoleamine 2,3-dioxygenase. Tregs can also downregulate immune responses through the ectozymes CD39 and CD73, along with the production of immunosuppressive adenosine. Tregs also suppress immune responses through direct interaction with dendritic cells via LAG3 and TIGIT. Another control mechanism is via the IL-2 feedback loop. Another mechanism of immune suppression by Tregs is via the prevention of costimulation through CD28 on effector T cells by the action of the CTLA-4 molecule.

[0031] Figure 1 illustrates glial cell-targeting CAR-Treg and its therapeutic mechanism. The CAR-Treg cells express a CAR that specifically binds to a marker on glial cells. The CAR-Treg cells are then bound to glial cells, transported across the blood-brain barrier, and localized to neurons in the CNS through natural glial cell function. The bound Treg cells then perform their natural regulatory function by suppressing local immune attack of neurons.

[0032] Figure 2 shows a glial cell-targeted immunosuppressive protein (GTIP) of the present invention, which suppresses immune attack of neurons. GTIP may include enzymes, such as extracellular enzymes, that scavenge immune-activating metabolites (e.g., ATP, AMP, tryptophan, and glutamate) present in Treg cells or immune-activating proteins. Examples of such extracellular enzymes include CD73, CD39, indoleamine 2,3-dioxygenase (IDO), and glutamic oxaloacetic transaminase 1 (GOT1). In Figure 2, MOG-expressing glial cells are bound by GTIP, which consists of an anti-MOG scFV linked to an immunosuppressive enzyme (IE). In performing their neuron-related functions, the glial cells localize the bound IE to neurons under immune attack by various immune cells (Th17 cells, Th1 cells, CTL cells, M1 cells, and PMN cells) and modulate or terminate the immune response, thereby preserving neurons and reducing the symptoms of the underlying neurodegenerative disease. GTIP may be useful in treating neurodegenerative diseases such as progressive supranuclear palsy (PSP), Alzheimer's disease (AD), Huntington's disease, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy (CTE), multiple sclerosis (MS), and prion diseases.

[0033] The GTIPs of the invention may comprise one or more immunosuppressive proteins (including two or more different proteins) linked to one or more scFVs or antibodies that target the same or two or more different glial cell markers. The proteins may be linked by any known means (including, for example, fusion proteins or biotin-streptavidin linkages) to form the GTIPs of the invention.

[0034] Adoptive cell transfer techniques, as used in cancer immunotherapy techniques, including those involving cytotoxic T lymphocytes, can be used to prepare autologous CAR-Tregs for use in the compounds and methods of the present invention. See Rosenberg, et al., 2008, Adoptive cell transfer: a clinical path to effective cancer immunotherapy, Nat Rev Cancer, 8(4):299-308, the contents of which are incorporated herein by reference.

[0035] The CAR-Treg or glial cell-targeting immunosuppressive protein of the present invention can be incorporated into a carrier system comprising one or more of the therapeutic compounds described herein. In some embodiments, the carrier system can be a nanoparticle comprising disulfide-crosslinked polyethyleneimine (CLPEI) and a lipid. The lipid can be a bile acid (e.g., cholic acid, deoxycholic acid, and lithocholic acid). Such a carrier system is further described in the following examples. Other exemplary carrier systems are described, for example, in Wittrup et al. (Nature Reviews / Genetics, 16:543-552, 2015), the contents of which are incorporated herein by reference in their entirety.

[0036] The phrases "parenteral administration" and "administered parenterally," as used herein, mean modes of administration other than enteral and topical administration (usually by injection), including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0037] The phrases "systemic administration," "administered systematically," "peripheral administration," and "administered peripherally," as used herein, refer to the administration of a compound, drug, or other substance other than directly to the central nervous system (e.g., subcutaneous administration) so that it enters the patient's system and is therefore subject to metabolism and other similar processes.

[0038] When the compounds of the present invention are administered to humans and animals as pharmaceuticals, they may be given as they are, or may be given in combination with a pharmaceutically acceptable carrier as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of the active ingredient, i.e., at least one therapeutic compound of the present invention and / or its derivative.

[0039] The effective dosage of each drug can be easily determined by those skilled in the art, taking into account typical factors such as patient's age, weight, sex and clinical history.Generally, the suitable daily dose of the compound of the present invention is the amount of compound that is the minimum effective dose to produce therapeutic effect.This effective dosage generally depends on the factors described above.

[0040] If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

[0041] The pharmaceutical compositions of the present invention comprise a "therapeutically effective amount" or a "prophylactically effective amount" of one or more compounds of the present invention, or functional derivatives thereof. An "effective amount," as defined in the definitions section herein, refers to an amount effective at dosages and for periods of time necessary to achieve the desired therapeutic result, e.g., reduction or prevention of the effects associated with neuropathic and / or inflammatory pain. A therapeutically effective amount of a compound of the present invention or its functional derivative may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the therapeutic compound to elicit a desired response in the subject. A therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic agent are outweighed by the therapeutically beneficial effects.

[0042] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so the prophylactically effective amount may be less than the therapeutically effective amount. A prophylactically or therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the beneficial effects.

[0043] The dosage regimen can be adjusted to provide the optimum desired response (e.g., therapeutic or prophylactic response). For example, a single bolus can be administered, or several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of active ingredient effective to achieve the desired therapeutic response, composition, and mode of administration for a particular subject without being toxic to the patient.

[0044] The term "dosage unit," as used herein, refers to a physically discrete unit suitable as a unitary dosage for the mammalian subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect, together with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are dictated by or directly dependent upon (a) the unique properties of the compound, and (b) the limitations inherent in the art of formulating such active compounds for the treatment of sensitivities in individuals.

[0045] In some embodiments, a therapeutically effective amount can be initially predicted using cell culture assays or animal models, usually mice, rabbits, dogs, or pigs. The animal models are also used to achieve a desired concentration range and route of administration. Such information can then be used to determine useful doses and routes of administration in other subjects. Generally, the therapeutically effective amount is sufficient to reduce or inhibit neuropathic and / or inflammatory pain in a subject. In some embodiments, the therapeutically effective amount is sufficient to eliminate neuropathic and / or inflammatory pain in a subject. Dosages for a particular patient can be determined by those skilled in the art using conventional considerations (e.g., by appropriate conventional pharmacological protocols). A physician may, for example, prescribe a relatively low dose initially, subsequently increasing the dose until an appropriate response is obtained. The dose administered to a patient is sufficient to produce a beneficial therapeutic response in the patient over time, or to reduce symptoms or other appropriate activity, for example, depending on the application. The dosage is determined by the effectiveness of a particular formulation, the activity, stability, or serum half-life of the compound of the present invention or its functional derivative, as well as the patient's condition and the patient's body weight or body surface area to be treated. The size of the dosage is also determined by the existence, nature, and extent of any adverse side effects associated with the administration of a particular vector, formulation, etc. in a particular subject. Therapeutic compositions containing one or more compounds of the present invention or their functional derivatives are optionally tested in one or more appropriate in vitro and / or in vivo animal models of disease (e.g., models of neuropathic and / or inflammatory pain) to confirm efficacy, tissue metabolism, and estimate dosage, according to methods well known in the art. In particular, dosage can be initially determined by activity, stability, or other appropriate measures of treatment versus non-treatment in relevant assays (e.g., comparing treated cells or animal models to untreated cells or animal models).The formulation is administered at a rate determined, for example, by the LD50 of the relevant formulation and / or by observing any side effects at various concentrations of the compound of the present invention or its functional derivative, as applied to the patient's weight and overall health. Administration can be achieved via a single dose or divided doses.

[0046] Administration typically involves administering a pharmaceutically acceptable dosage form, which refers to dosage forms of the compounds described herein, including, for example, tablets, dragees, powders, elixirs, syrups, liquid preparations (including suspensions), sprays, inhalant tablets, lozenges, emulsions, solutions, granules, capsules, and suppositories, as well as liquid preparations for injection (including liposomal preparations). Techniques and formulations are generally described in Remington's Pharmaceuticals, Inc. Sciences, Mack Publishing Co., Easton, Pa., latest edition, which is incorporated by reference in its entirety. Administration can be orally, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, or intranasally. The compound can be administered alone or with a suitable pharmaceutical carrier, and can be in solid or liquid form (e.g., tablet, capsule, powder, solution, suspension, or emulsion).

[0047] The pharmaceutical composition comprising active ingredient can be in the form suitable for oral use, for example, tablet, troche, lozenge, aqueous or oily suspension, dispersible powder or granule, emulsion, hard or soft capsule, or syrup or elixir.The composition intended for oral use can be prepared according to any method known in the art for the preparation of pharmaceutical compositions, and such composition can comprise one or more agents selected from sweeteners, flavoring agents, coloring agents and preservatives to provide pharmacologic and palatable preparations.Tablet comprises active ingredient mixed with non-toxic pharmaceutically acceptable excipients suitable for the manufacture of tablets. These excipients can be, for example, inert diluents (e.g., calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate), granulating and disintegrating agents, such as cornstarch or alginic acid; binders (e.g., starch, gelatin, or gum acacia); and lubricants (e.g., magnesium stearate, stearic acid, or talc). The tablets can be uncoated, or they can be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over a longer period. For example, a time delay material, such as glyceryl monostearate or glyceryl distearate, can be used. They can be coated by the techniques described in U.S. Pat. Nos. 4,256,108, 4,166,452, and 4,265,874 (the contents of each of which are incorporated herein by reference in their entireties) to form therapeutic osmotic tablets for controlled release.

[0048] Formulations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., calcium carbonate, calcium phosphate, or kaolin), or as soft gelatin capsules in which the active ingredient is mixed with water or an oil medium (e.g., peanut oil, liquid paraffin, or olive oil).

[0049] Formulations may also include complexes of the parent (non-ionized) compound with derivatives of β-cyclodextrin, particularly hydroxypropyl-β-cyclodextrin.

[0050] An alternative oral formulation may be achieved using a controlled release formulation in which the compound is encapsulated in an enteric coating.

[0051] Aqueous suspensions contain the active substance in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia); dispersing or wetting agents (e.g., naturally occurring phosphatides, such as lecithin) or condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), or condensation products of ethylene oxide with long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), or condensation products of ethylene oxide with fatty acids and hexitol partial esters (e.g., partial esters derived from polyoxyethylene with fatty acids and hexitol anhydrides), such as polyoxyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example, ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0052] Oily suspensions can be prepared by suspending the active ingredient in vegetable oil (e.g., peanut oil, olive oil, sesame oil or coconut oil) or mineral oil (e.g., liquid paraffin).The oily suspensions can contain thickening agents (e.g., beeswax, hard paraffin or cetyl alcohol).Sweeteners (e.g., those mentioned above) and flavoring agents can be added to provide a palatable oral preparation.These compositions can be preserved by adding antioxidants (e.g., ascorbic acid).

[0053] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified, and for example, sweeteners, flavorings, and coloring agents may also be present.

[0054] The pharmaceutical composition of the present invention can also be in the form of an oil-in-water emulsion. The oil phase can be vegetable oil (e.g., olive oil or peanut oil), mineral oil (e.g., liquid paraffin), or a mixture thereof. Suitable emulsifiers can be naturally occurring gums (e.g., acacia gum or tragacanth gum), naturally occurring phosphatides (e.g., soybean lecithin), and esters or partial esters derived from fatty acids and hexitol anhydrides (e.g., sorbitan monooleate), and condensation products of the partial esters with ethylene oxide (e.g., polyoxyethylene sorbitan monooleate). The emulsion can also contain sweeteners and flavoring agents.

[0055] Syrups and elixirs may be formulated with sweetening agents (e.g., glycerol, propylene glycol, sorbitol, or sucrose). Such formulations may also contain demulcents, preservatives, and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents mentioned above. The sterile injectable preparation may also be in a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as solvents or suspending media. For this purpose, any bland, fixed oil may be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[0056] Each active agent can also be administered in the form of a suppository for rectal administration. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient. This non-irritating excipient is solid at normal temperatures but liquid at rectal temperature, and therefore melts in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycol.

[0057] For topical use, creams, ointments, jellies, solutions or suspensions are suitable. Topical application includes the use of mouthwashes and gargles.

[0058] The term "pharmaceutical composition" means a composition comprising a compound as described herein and at least one component including a pharmaceutically acceptable carrier, diluent, adjuvant, excipient, or vehicle (e.g., preservatives, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweeteners, flavoring agents, perfuming agents, antibacterial agents, antifungal agents, lubricants, and dispersing agents), depending on the mode of administration and the nature of the dosage form. The term "pharmaceutically acceptable carrier" means a composition comprising a compound as described herein and at least one component including a pharmaceutically acceptable carrier, diluent, adjuvant, excipient, or vehicle (e.g., preservatives, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweeteners, flavoring agents, perfuming agents, antibacterial agents, antifungal agents, lubricants, and dispersing agents), depending on the mode of administration and the nature of the dosage form. The term "carrier" is used herein to mean any carrier, diluent, adjuvant, excipient, or vehicle. Examples of suspending agents include ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, and tragacanth gum, or mixtures of these substances. Prevention of microbial activity can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic agents It may also be desirable to include an absorbent or dispersant (e.g., an absorbent or dispersant agent), such as sugar, sodium chloride, or the like. Prolonged absorption of the injectable pharmaceutical forms can be brought about by the use of agents delaying absorption, such as aluminum monostearate and gelatin. Examples of suitable carriers, diluents, solvents, or vehicles include water, ethanol, polyols, suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Examples of excipients include lactose, milk sugar, sodium citrate, calcium carbonate, and dicalcium phosphate. Examples of disintegrating agents include starch, alginic acid, and certain complex silicates. Examples of lubricants include magnesium stearate, sodium lauryl sulfate, talc, and high molecular weight polyethylene glycol.

[0059] The term "pharmaceutically acceptable" means that it is suitable for use in contact with the cells of humans and lower animals without undue toxicity, irritation, allergic response, etc., within the scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio.

[0060] Incorporation by Reference References and citations to other documents, such as patents, patent applications, patent publications, journals, books, articles, web content, etc. are made throughout this disclosure, and all such documents are incorporated herein by reference in their entirety for all purposes.

[0061] equivalent Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the entire contents of this document, including reference to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of the invention in its various embodiments and equivalents thereof. [Example]

[0062] Example 1 Study design Participants with progressive supranuclear palsy (PSP) will receive a single infusion of ex vivo expanded autologous CD4+CD127lo / -CD25+ CAR-regulatory T cells (Tregs). CAR-Tregs are designed to specifically recognize myelin-oligodendrocyte protein (MOG), a glycoprotein specifically expressed in the central nervous system (CNS), and induce immune tolerance and anti-inflammatory effects in the brain.

[0063] The primary objective is to evaluate the safety and feasibility of intravenous infusion of ex vivo selected, expanded, and transduced autologous CNS-specific CAR-Tregs in at least five patients with PSP.

[0064] The temporal outcome measures were: 1. Adverse events 2. Abnormalities in the examination 3. Infusion Reactions 4. Infection-related complications 5. Potential negative impact on the course of PSP

[0065] Secondary objectives are to evaluate the effect of CNS-specific CAR-Tregs on PSP and to gain indications regarding their potential application in other neurodegenerative diseases.

[0066] The endpoints are: 1. To evaluate the effects of CNS-specific CAR-Tregs on clinical, neuropsychological, radiological, and biochemical parameters in patients with PSP. 2. Gaining indications regarding the potential therapeutic use of CNS-specific CAR-Tregs in other neurodegenerative diseases, including Alzheimer's disease (AD). 3. Obtaining indications in a potential placebo-controlled, phase II, randomized, double-blind study that may provide valuable insight into the potential efficacy of CAR-Tregs for neurodegenerative disorders.

[0067] Patient evaluation Clinical and neuropsychological assessments: A detailed description of the inclusion and exclusion criteria, as well as clinical (motor and neuropsychological) and neuroimaging assessments will be performed as previously reported (Giordano et al., J. Transl. Med. 2014; Canesi et al., J. Transl. Med. 2016, incorporated herein by reference). Patients will undergo a neurological examination and motor function will be assessed using the following scales: Unified Parkinson's Disease Rating Scale (UPDRS Part III, Motor Score), Hoehn and Yahr staging (H&Y), and PSP Rating Scale (PSP-RS) (Goetz et al. al., Mov. Disord. 2004; Golbe et al., Brain 2007 (each of which is incorporated herein by reference). Mini-Mental State Examination (MMSE) will also be performed as previously described (Folstein et al. J. Psychiatr. Res. 1975 (incorporated herein by reference)). All these tests will be evaluated at baseline and at each follow-up point (1 month, 3 months, 6 months, and 12 months after cell administration). If the UPDRS and PSP-RS scores do not decrease by more than 30% compared to baseline, and if the H&Y severity classification does not change at the specified time points, the clinical condition will be classified as "stable" (Canesi et al., J. Transl. Med. 2016 (incorporated herein by reference)).

[0068] Neuroimaging: All patients will undergo longitudinal neuroimaging evaluation using brain magnetic resonance imaging (MRI) (at baseline, 24 hours after cell administration, and 1 year), striatal dopamine transporter single-photon emission computed tomography (SPECT) and positron emission tomography (PET) (both at baseline and 12 months). Tropanic tracers labeled with iodine-123 (FP-CIT) and 18F-fluoro-2-deoxyglucose (Beta-CIT) will be used for SPECT and PET / TC imaging, respectively.

[0069] For SPECT, intravenous administration of 110–140 MBq of [123I]FP-CIT (Datscan, GE-Health, Amersham, UK) was performed in all patients 30–40 minutes after thyroid block (10–15 mg of Lugol's solution orally). Analysis was performed as previously described (Isaias et al., NeuroReport 2007, incorporated herein by reference). A volumetric template of gray matter anatomical distribution was generated from the Montreal Neurological Institute MRI single-participant brain atlas by applying macroscopic dissection (automated anatomical labeling), reoriented, and reformatted to obtain a 2.64 cm thick reference section. Eight irregular region-of-interest (ROI) templates were manually delineated on this section to assess the anatomical extent of striatal and occipital lobe structures with specific and nonspecific uptake of [123I]FP-CIT, respectively. The ROI template is also placed on the reference SPECT slice and adjusted for both the striatum and occipital cortex. The striatum ROI is also divided into its anterior (caudate) and posterior (putamen) portions.

[0070] Specific striatal dopamine uptake transporter (DAT) binding of [I]FP-CIT is calculated in the whole striatum, putamen, and caudate nucleus using the formula: [(average number in a specific ROI)-(average number in occipital ROI)] / (average number in occipital ROI). The putamen / caudate ratio for each subject is also calculated.

[0071] All patients also undergo F-fluoro-2-deoxyglucose positron emission tomography (FDGPET) scanning at rest after intravenous injection of 170 MBq. Each acquisition includes a computed tomography (CT) transmission scan of the head (50 mA for 16 seconds), followed by a 15-minute three-dimensional (3D) static emission scan using a Biograph Truepoint 64 PET / CT scanner (Siemens). PET sections are reconstructed using an iterative algorithm (OS-EM) and corrected for scatter and attenuation using density coefficients derived from a low-dose CT scan of the head obtained on the same scanner. Images are reconstructed in the form of 128-pixel longitudinal transverse images with a 128 Å-2 mm resolution using the iterative algorithm Ordered Subset Expectation Maximization (OSEM). The resolution of the PET system is 4-5 mm FWHM.

[0072] Biomechanical assessment: Biomechanical assessment is assessed at baseline and 6 and 12 months after CAR-Treg cell administration.Two specific sets of parameters (one for standing and one for walking initiation) are automatically extracted by ad-hoc algorithm (Carpinella et al., IEEE Trans Neural Syst Rehabil Eng. 2007 (incorporated herein by reference)).For standing, measure center of pressure (CoP) mean velocity and spatial displacement (Canesi et al., J. Transl. Med. 2016 (incorporated herein by reference)). To examine gait initiation, anticipatory postural adjustments are analyzed (Canesi et al., J. Transl. Med. 2016, incorporated herein by reference) (i.e., unstable and unweight-bearing phases), and the following parameters are measured: (1) duration of both phases, (2) CoP anterior-posterior (AP) and mediolateral (ML) shifts and velocities, (3) CoP mean length and velocity, (4) first step length, and (5) velocity. Spatial parameters are normalized based on body height (%BH).

[0073] Preparation and administration of CAR-Treg cells Treg isolation and expansion: Poly-Tregs were selected and expanded from five individuals with PSP based on three cell surface markers (CD4, CD25, and CD127) to purify peripheral blood-resident FOXP3+ Tregs as previously described (Putnam et al., Diabetes 2009; Bluestone et al., Sci. Transl. Med., 2015, incorporated herein by reference).

[0074] 400 ml of fresh peripheral blood was collected into a blood-packed unit containing citrate phosphate dextrose and processed within 24 hours for PBMC isolation via a Ficoll density gradient. Tregs were isolated using a high-speed cell sorter using the following GMP-grade lyophilized antibodies: CD4-PerCP (peridinin chlorophyll protein) (L200 clone), CD127-PE (phycoerythrin) (40131 clone), and CD25-APC (allophycocyanin) (2A3 clone). Sorted CD4+CD127lo / -CD25+ cells were collected in 3 ml of X-VIVO 15 medium (Lonza, catalog number 04-418Q) containing 10% human heat-inactivated pooled AB serum (Valley Biomedical). Tregs were analyzed for purity after sorting. The expected purity of CD4+CD127lo / -CD25+ cells is greater than 96% (Bluestone et al., Sci. Transl. Med., 2015, incorporated herein by reference).

[0075] Purified Tregs are cultured with clinical-grade Dynabeads coated with anti-CD3 and anti-CD28 recombinant IL-2 as previously described (Bluestone et al., Sci. Transl. Med., 2015, incorporated herein by reference). A blood unit is expected to yield between 4.2 x 10 and 11.8 x 10 purified CD4+CD127lo / -CD25+ Tregs (Bluestone et al., Sci. Transl. Med., 2015, incorporated herein by reference). Expanded Treg preparations are expected to be approximately 90% FOXP3+. Treg preparations are checked for viability, CD4+ percentage, and CD8+ cell contamination (Bluestone et al., Sci. Transl. Med., 2015, incorporated herein by reference).

[0076] Phenotype and TCR analysis of expanded poly-Tregs: CD4 and CD127, key cell surface markers used to isolate Tregs, will be checked after expansion.

[0077] Previous data have shown that natural CD45RA+ Tregs are preferentially expanded in these cultures, and CD45RA+RO- cells downregulate CD45RA and upregulate CD45RO throughout the expansion phase (Bluestone et al., Sci. Transl. Med., 2015, incorporated herein by reference). CCR7 (a Treg trafficking receptor), CD38 (a multifunctional extracellular enzyme associated with enhanced Treg function), and CD45RO are determined before and after expansion. The TCRβ repertoire of expanded Tregs is also analyzed to determine their polyclonality compared to freshly isolated populations. It is expected that expanded cells will exhibit polyclonality indistinguishable from pre-expansion cultures, and that Tregs will remain a highly diverse population after expansion (Bluestone et al., Sci. Transl. Med., 2015, incorporated herein by reference).

[0078] Functional analysis of expanded poly-Tregs: The following assays (Bluestone et al., Sci. Transl. Med., 2015, incorporated herein by reference) are performed after Treg expansion: - DNA methylation status of the enhancer region of the FOXP3 locus to assess the overall purity and stability of expanded Tregs. - Cytokine production (IFNγ, IL-4, IL-5 and IL-17) to assess lymphocyte phenotype. - In vitro inhibitory activity to determine the functional potential of expanded cells.

[0079] Generation and functional analysis of CAR-Treg cells: CAR RNA was transfected onto Tregs after electroporation of human Tregs and following published protocols (Zhao, Y et al., 2010 Cancer Res and Beatty, GL et al., 2014 Cancer Immunol Res; Singh, Anti-MOG CAR expression on mouse Tregs after adoptive transfer into a PSP mouse model will be optimized according to N et al., 2014 Oncoimmunol (the contents of each of which are incorporated herein by reference). Delivery of the anti-MOG CAR using second-generation lentiviral vectors and standard protocols (Levine, BL et al., 2017 Mol Ther Methods & Clin Dev (incorporated herein by reference)) will also be optimized. Human Tregs will be transfected under GMP conditions (approximately 1 mg / 3 x 10 cells) using RNA electroporation. 6 Tregs) or under lentiviral transduction (1 × 10 6 pfu / 3×10 6 Transduce either 2.6 x 10 Tregs or 2.6 x 10 Tregs to generate clinical-grade RNA of anti-MOG CAR. 9 Approximately 0.9 mg RNA / patient for Treg cells; 4.5 mg per 5 patients is required. Produce clinical grade lentivirus. 2.6 x 10 9 Approximately 8.7 × 10 for Treg cells 9 pfu / patient: 4.3 × 10 for 5 patients 10 pfu is required. Functional analysis of MOG-specific CAR-Tregs is performed as described above in 3.3.

[0080] Cell administration A single administration of MOG-specific CAR-Tregs will be administered to each patient (2.6 × 10 9CAR-Tregs / patient). Cells will be administered to at least five PSP patients. Patients will be premedicated with acetaminophen and diphenhydramine. CAR-Tregs will be infused over 10-30 minutes via a peripheral intravenous line. Vital signs will be measured before and after the infusion, then every 15 minutes for at least 1 hour, then every hour for the first 4 hours, and every 4 hours for 20 hours. Chemistry and complete blood counts with various blood counts will be repeated the next day before discharge from the clinical research unit. Patients will be seen for follow-up assessments on day 4 after the infusion, then weekly for 4 weeks, then every 13 weeks for 1 year, and every 26 weeks for 2 years. Telephone monitoring for adverse events will continue every 6 months for 5 years, followed by a final clinic visit.

[0081] Patient evaluation after CAR-Treg cell infusion The effects of CNS-specific CAR-Tregs on clinical, neuropsychological, radiological, and biochemical parameters in PSP patients will be evaluated as described above. All studies will be performed at follow-up points: 1 month, 3 months, 6 months, and 12 months after cell administration.

[0082] Example 2 Develop and test multiple sclerosis (MS) drugs that harness components of the immunosuppressive regulatory T lymphocytes (Tregs) that halt the damaging immune response that causes disease. The global MS market is approximately $21.5 billion, yet approved drugs for the most common forms of MS offer only modest disease modification with significant side effects. For more severe forms of MS, treatment options are limited to just one recently approved drug.

[0083] There are 11 FDA-approved medications for relapsing-remitting MS (RRMS—85% of diagnosed MS cases). There are several orally available and antibody-based medications currently approved or under clinical evaluation for RRMS. In March 2017, the FDA approved the use of ocrelizumab (an anti-CD20 antibody, Roche) for primary progressive MS (PPMS—approximately 10% of diagnosed MS cases). Ocrelizumab provides a 25% reduction in symptoms and is currently the only immunomodulatory agent for PPMS in the USA. Secondary progressive MS (SPMS) always develops in patients with RRMS, and for it, limited disease-modifying options exist.

[0084] Biologics Production Anti-MOG hybridomas are generated through a CRO by immunizing mice with recombinant human MOG. The VH and VL genes are cloned and the anti-scFv molecules are generated. The orientation of the VH and VL, as well as the linker (between the scFvs or within each scFv), can significantly affect the stability, expression level, and binding capacity of GTIP. In some cases, only one of these configurations produces a functional molecule. Therefore, several VH-VL orientations are expressed on a small scale and tested before scale-up production. Expression constructs encoding the four anti-MOG scFvs, along with the connecting linker, central linker, and then linked to a Treg-associated enzyme or mimic, are generated.

[0085] Validation of GTIP protein products in mouse MS models GTIP will be tested in acute and chronic EAE models of MS in mice. Levels of Th1, Th17, and CTL (blood and CNS) specific for myelin basic protein (MBP), bone marrow inflammatory cells (macrophages and neutrophils), and anti-MBP antibodies will be measured. Immunological responses correlate with disease progression. Dosing will be varied to gain insight into potential use in late-stage MS. The product will be administered in normal mice to gain insight into any potential off-target effects.

[0086] Clinical evaluation Clinical trials will be conducted to test the efficacy of GTIP as a disease-modifying agent in MS. The product will be initially tested in RRMS patients who do not respond to first-use drugs. Safety and tolerability will be measured using a dosing regimen similar to that for antibody therapy (e.g., three iv doses every two weeks initially, then every four weeks for 20 weeks). In Phase 2 trials, the primary endpoints are reduced disease relapse frequency and brain lesions. Secondary endpoints are reduced levels of inflammatory cytokines, Th1 / Th17 cells, and other white blood cells in the blood. Side effects may include increased susceptibility to infection. These trials will allow us to benchmark the efficacy of the compound against other second-use drugs, which have shown up to a 49% reduction in relapse frequency. If the product demonstrates acceptable efficacy levels, it will enter a longer-term Phase 2 clinical trial in PPMS patients. The primary measures are slowed decline in motor function and reduced brain lesions, and the secondary measures are reduced levels of inflammatory cytokines, Th1 / Th17 cells and other white blood cells in the blood.

[0087] Example 3 Using a tetramer binding assay, the avidity of GITP binding to target cells is compared to that of a known tetramer for T cells (Ober, B et al., 2000 Int Immunol, incorporated herein by reference) as shown in Figure 3. The relative avidity of the HY peptide / MHC H-2Db (pMHC) tetramer for the TCR on B6.2.16 CTL is measured by determining two parameters using cell staining and flow cytometry (FCM). These are the concentration required to give maximal staining of the tetramer staining (after cell washing) and the half-life (t 1 / 2) MOG target cells are generated by genetic transfection of nonadherent target cells (e.g., RMA or Jurkat cells). Antibody staining and flow cytometry (FCM) confirm surface expression of MOG. Antibody staining and FCM identify transfectants with MOG levels equivalent to the B6.2.16 TCR on CTLs. The maximum staining and half-life of MOG target cells stained with labeled GITP protein are measured and compared with those of CTLs and pMHC tetramers, as shown in Figures 4 and 5. The goal is to generate GITP with avidity for cell interaction comparable to or better than that of CTLs and pMHC tetramers. If the tetrameric anti-MOG scFv in the GITP molecule falls below this bar, the balance valency of the scFv can be increased. If even higher valency is required, nanoparticle scaffolds can be used to achieve the necessary avidity for target cell binding.

[0088] Example 4 The ability of GTIP bound to MOG target cells to suppress T effector (Teff) cell proliferation is tested. GTIP, composed of tetramers of predetermined scavenger IEs (see Table 1 below), is bound to MOG target cells, washed, and then incubated with proliferating human Teffs (e.g., generated using standard procedures after 3 days of anti-CD3 / CD28 and IL-2 stimulation) (Figure 6, center column). [Table 1]

[0089] Cells are cultured in medium supplemented with a related mitogenic metabolite (M) (see table above), which is a substrate for IEs in GTIP. Over time, the concentration of M and the number of Teffs are measured, as shown in Figure 6. The number of GTIP-modified MOG-target cells is titrated against the number of Teffs after a certain time period to provide an index of suppressive activity. In negative control experiments (left column of Figure 6), tetrameric scFv binding to MOG-target cells results in a longer M half-life, a higher number of Teffs after a certain time period, and no suppressive activity against Teff cell accumulation. As a positive control (right column of Figure 6), human Tregs (generated under standard conditions, e.g., after 9 days of CD3 / CD28 and TGF-β stimulation) are cocultured with Teff cells, and the suppressive activity is compared to that of GTIP-modified MOG-target cells. The cell-to-cell efficacy of GTIP-modified MOG target cells comparable to that of human Treg cells serves as a positive validation of the GTIP molecule in a particular IE. The assay identifies the GTIP composed of the most effective IE molecules. Efficacy can be increased by adding more than one type of IE molecule to the GTIP molecule and / or by increasing the valency of the IE molecule.

[0090] Example 5 The assay is used to measure the ability of CAR molecules expressing MOG-specific scFv to allow Treg cells to bind to MOG-expressing target cells with a relative avidity that approximates the physiologically relevant T cell:target cell interaction. The physiologically relevant T cell:target cell interaction used for comparison is the CTL:peptide / MHC (pMHC) / target cell interaction. This is performed using a flow cytometry (FCM)-based assay for cell-cell binding (Opferman, JT et al., 2001 Int Immunol. (incorporated herein by reference)).

[0091] The relative avidity of pMHC targets to a related CTL clone (B6.2.16) is determined (left column in Figure 7). Targets are labeled with the vital dye PKH26 (red), and CTLs are labeled with CFSE (green), co-incubated for 4 hours, then subjected to standard shear force and examined by FCM. Binders are detected as double-stained doublets and depend on the presence of the HY peptide antigen. The relative avidity of pMHC target:B6.2.16 CTL interactions is measured by two parameters: the maximum level of conjugate formation (approximately 80% of all input cells) and the half-life of conjugate dissociation. MOG target cells are incubated with CAR-anti-MOG scFv-expressing human T cells generated under standard conditions (e.g., lentiviral transduction of anti-CD3 / CD28 IL-2-stimulated T cells). The half-life of the conjugates between the labeled cells is measured by FCM (right column in Figure 7). The conjugate half-life comparable to that of CTL:pMHC / target cells indicates the physiological avidity of anti-MOG scFv / CAR to T cells for MOG-positive target cells.

[0092] Example 6 Specific scFv antibodies against human MOG were generated by affinity panning of a human phage display scFv library. QC SDS-PAGE was performed prior to library screening to assess target purity. To reduce nonspecific binders, pre-counter selection was first performed on the phage library prior to target screening using polystyrene flat-bottom plates and blocking buffer.

[0093] Positive enrichment was observed after three rounds of biopanning. 20 clones were randomly picked from the third round and subjected to QC monoclonal phage ELISA. 18 clones were found to bind to the target compared to the control. All 18 positive clones were sequenced.

[0094] Another 20 clones were picked from the third round and subjected to QC monoclonal phage ELISA. All 20 of the second set of clones were found to bind to the target compared to the control. All of them were sequenced.

[0095] After analysis of 38 positive clones, seven positive clones with unique sequences were identified (clones 1, 3, 6, 10, 13, 17, 21). The sequences of the seven scFv proteins and the DNA sequences encoding them are listed below:

[0096] [ka] [ka] [ka] [ka] [ka]

[0097] Expression vectors were constructed for each of the seven scFv proteins. The cell lysates were then coated for ELISA. Soluble ELISA was then performed using cell lysates from both 30°C and 37°C. Compared with the control, differences were readily observed in all seven clones. Among the seven positive clones, clones 1, 6, and 13 were much stronger than the others.

[0098] ELISA with quantitative determination was performed on the soluble scFvs produced from the seven positive clones to rank their MOG-binding ability. The seven scFvs were subcloned into pET-26b to construct the scFv-myc-6xHis format. The purity of the seven scFvs induced at 16°C was >85%, whereas the purity was lower when induced at 37°C. Therefore, 16°C was determined to be a more suitable condition for production. QC ELISA was performed to analyze the binding ability of each of the seven scFvs to the target MOG. Compared with the control, differences were readily detected among the seven positive clones (clones 1, 3, 6, 10, 13, 17, and 21). Among the seven positive clones, three clones (clones 3, 6, and 17) showed stronger binding ability to the target.

[0099] QC ELISA was performed on each of the seven clones induced at 16°C. Seven different concentrations of the seven clones were used for ELISA quantification. The results showed that all seven clones could specifically bind to the target MOG. Among the seven clones, clones 3, 6, and 17 still exhibited stronger binding affinity to the target. The results are shown in Figure 8. Figure 8 illustrates that the anti-hMOG1 scFv of clone 17 exhibited the strongest binding, followed by the binding of clone 6, and then the binding of clone 3. The remaining four clones exhibited significantly weaker binding than those of clones 17, 6, and 3.

Claims

1. 1. A pharmaceutical composition for treating a neurodegenerative disease in a subject, said pharmaceutical composition comprising an effective amount of engineered regulatory T cells (Tregs), wherein The engineered Tregs are CD4 ＋ CD127 - CD25 ＋ FOXP3 ＋ cells, the engineered Tregs express a chimeric antigen receptor (CAR) that specifically binds to a glial cell marker; The CAR comprises a single chain variable fragment (scFv) comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain present in SEQ ID NO: 6 or SEQ ID NO:

12. Pharmaceutical compositions.

2. 2. The pharmaceutical composition of claim 1, wherein the neurodegenerative disease is selected from the group consisting of progressive supranuclear palsy (PSP), Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and chronic traumatic encephalopathy (CTE).

3. 3. The pharmaceutical composition of claim 2, wherein the neurodegenerative disease is progressive supranuclear palsy (PSP).

4. 3. The pharmaceutical composition of claim 2, wherein the neurodegenerative disease is Parkinson's disease (PD).

5. 3. The pharmaceutical composition of claim 2, wherein the neurodegenerative disease is Alzheimer's disease (AD).

6. 3. The pharmaceutical composition of claim 2, wherein the neurodegenerative disease is Huntington's disease (HD).

7. 3. The pharmaceutical composition of claim 2, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).

8. 3. The pharmaceutical composition of claim 2, wherein the neurodegenerative disease is chronic traumatic encephalopathy (CTE).

9. 2. The pharmaceutical composition of claim 1, wherein the scFv has at least 99% sequence identity with SEQ ID NO:

12.

10. The pharmaceutical composition of claim 1, wherein the scFv has at least 99% sequence identity to SEQ ID NO:

6.

11. 2. The pharmaceutical composition of claim 1, wherein the scFv is capable of specifically binding to the glial cell marker, myelin oligodendrocyte glycoprotein (MOG).

12. 12. The pharmaceutical composition of claim 11, wherein the CAR is capable of directing the engineered Tregs to glial target cells that express MOG.

13. 10. The pharmaceutical composition of claim 1, wherein the engineered Tregs regulate a neurodegenerative immune response that targets glial cells.

14. 15. The pharmaceutical composition of claim 14, wherein the engineered Tregs reduce the neurodegenerative immune response that targets the glial cells.

15. 15. The pharmaceutical composition of claim 14, wherein the glial cells are oligodendrocytes, astrocytes, ependymal cells, or microglia.

16. 10. The pharmaceutical composition of claim 1, wherein the engineered Tregs reduce inflammation in neural tissue of the subject.

17. 10. The pharmaceutical composition of claim 1, wherein the engineered Tregs are administered to the subject by intravenous administration.

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