CAR-Treg-based therapy 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 the benefit and priority of U.S. Provisional Application No. 62 / 648,684, filed Mar. 27, 2018, the content of which is incorporated herein by reference in its entirety.

[0002] Field of the Invention The present invention provides CAR-Treg compositions that specifically modulate immune responses and inflammation associated with various neurodegenerative diseases (e.g., progressive supranuclear palsy and Parkinson's disease), and methods of using the same.

Background Art

[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. Treatments that focus on curing these diseases, as well as managing and delaying the progression of symptoms, are unknown.

[0004] One such disease, PSP, mimics Parkinson's disease (PD) and is an idiopathic degenerative disease not uncommon in the elderly. Its clinical picture includes the tetrad of supranuclear gaze palsy, axial rigidity, dementia, and pseudobulbar palsy. It is associated with bradykinesia, severe postural impairment, and frequent falls. The pathology is associated with cell loss and Tau neurofibrillary changes mainly in the brainstem, globus pallidus, subthalamic nucleus, and dentate nucleus. PSP has a prevalence of 5-6 per 100,000 and 5,000-25,000 patients occur in the United States per year. The average age of onset of the above diseases is 63 years, and the normal prognosis ranges from 5 to 10 years from diagnosis to death. There are no available disease-modifying treatments.

[0005] Parkinson's disease is another neurodegenerative disease with no known cure. Parkinson's disease 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 of the brain, along with astrocyte death in the substantia nigra and an increase in microglia that results in dopamine deficiency in that area. Inclusion bodies called Lewy bodies occur in damaged cells prior to cell death. Although mechanisms underlying the cell death in the brain in Parkinson's disease are hypothesized to drive it, they remain poorly understood, and current treatments focus on managing the symptoms of the disease. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEMS

[0006] Abstract The compositions and methods of the present invention use immunosuppressive proteins expressed by regulatory T lymphocytes (Tregs) or Treg cells to modulate neurodegenerative immune responses targeting glial cells in the central nervous system (CNS). By linking the Treg or immunosuppressive protein to either a chimeric antigen receptor (CAR) or a single-chain variable fragment (scFv) that specifically recognizes and binds to a glial cell marker, the immunosuppressive Treg or protein reduces inflammation and induces glial cells of the CNS to defend the CNS from autoimmune attack.

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

[0008] ​The present invention directs immunosuppressive molecules (Tregs or immunosuppressive proteins) to oligodendrocyte (ODC) glial cells. The resulting compounds and methods of use thereof recruit the body's own immune system to counter 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 nerve function but do not depend on any specific biochemical cause of the underlying disease (i.e., autoimmune attack on the central nervous system). Thus, the compounds and methods of the present invention can provide a therapeutic effect across a number of neurodegenerative diseases.

[0009] Aspects of the present invention are 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) that express 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 can be human.

[0010] The glial cell marker can be myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (OM4), nerve / 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 can be myelin oligodendrocyte glycoprotein (MOG).

[0011] Neurodegenerative diseases 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 regulatory T cells (Tregs) engineered to a therapeutically effective amount for treating a neurodegenerative disease that is not 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), nerve / glial antigen 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), or myelin oligodendrocyte specific protein (MOSP).

[0013] The composition can 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 glial cell-specific binding proteins linked to molecules expressed by regulatory T cells (Tregs). The molecules expressed by Tregs can be extracellular immunosuppressive enzymes. In certain embodiments, the molecules expressed by Tregs can be CD73, CD39, indoleamine 2,3-dioxygenase (IDO), or glutamate oxaloacetate 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 molecule expressed by the Treg can bind to myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (OM4), nerve / glia 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 molecules expressed by Tregs can be extracellular immunosuppressive enzymes such as CD73, CD39, indoleamine 2,3-dioxygenase (IDO), or glutamate oxaloacetate transaminase 1 (GOT1). The glial cell-specific binding protein bound to the mimicked molecule can bind to myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (OM4), nerve / glia 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) A method for treating a neurodegenerative disease in a subject, said method comprising administering to said subject a therapeutically effective amount of regulatory T cells (Tregs) each expressing a chimeric antigen receptor (CAR) that specifically binds to a glial cell marker, for protecting neural tissue and reducing inflammation in said neural tissue, thereby treating said neurodegenerative disease, provided that said neurodegenerative disease is not multiple sclerosis. (Item 2) The method according to item 1, wherein said subject is a human. (Item 3) The method according to item 1, wherein said glial cell marker is selected from the group consisting of myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (OM4), nerve / glial marker 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and myelin oligodendrocyte specific protein (MOSP). (Item 4) The method according to item 3, wherein said glial cell marker is myelin oligodendrocyte glycoprotein (MOG). (Item 5) The method according to item 1, wherein said 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) The method according to item 5, wherein said neurodegenerative disease is progressive supranuclear palsy (PSP). (Item 7) The method according to item 5, wherein said neurodegenerative disease is Alzheimer's disease (AD). (Item 8) The method according to item 5, wherein said neurodegenerative disease is Parkinson's disease (PD). (Item 9) A composition comprising a plurality of engineered regulatory T cells (Tregs) in a therapeutically effective amount for treating a neurodegenerative disease other than 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) The composition according to 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), nerve / glial antigen 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and myelin oligodendrocyte specific protein (MOSP). (Item 11) The composition according to item 9, wherein the glial cell marker is myelin oligodendrocyte glycoprotein (MOG). (Item 12) The composition according to 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 14) The composition according to item 12, wherein the neurodegenerative disease is Alzheimer's disease (AD). (Item 15) The composition according to 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 a regulatory T cell (Treg). (Item 17) The engineered protein according to item 16, wherein the molecule expressed by the Treg is an extracellular immunosuppressive enzyme. (Item 18) The molecule expressed by Treg is the engineered protein according to item 17, selected from the group consisting of CD73, CD39, indoleamine 2,3-dioxygenase (IDO), and glutamate oxaloacetate transaminase 1 (GOT1). (Item 19) The glia cell-specific binding protein is the engineered protein according to item 16, which is a tetrameric single-chain variable fragment (scFv) of an antibody molecule. (Item 20) The glia cell-specific binding protein is the engineered protein according to item 16, which binds to a marker selected from the group consisting of myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (OM4), neuron / glia marker 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and myelin oligodendrocyte-specific protein (MOSP). (Item 21) The glia cell-specific binding protein is the engineered protein according to item 20, which binds to myelin oligodendrocyte glycoprotein (MOG). (Item 22) The glia cell-specific binding protein is the engineered protein according to item 21, which is a single-chain variable fragment (scFv) of an antibody molecule containing an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 6, and 12. (Item 23) An engineered protein comprising a glia cell-specific binding protein linked to a molecule that mimics the activity of a molecule expressed by regulatory T cells (Treg). (Item 24) The molecule expressed by Treg is the engineered protein according to item 23, which is an extracellular immunosuppressive enzyme. (Item 25) The molecule expressed by Treg is the engineered protein according to item 24, selected from the group consisting of CD73, CD39, indoleamine 2,3-dioxygenase (IDO), and glutamate oxaloacetate transaminase 1 (GOT1). (Item 26) The glia cell-specific binding protein is the engineered protein according to item 23, which is a tetrameric single-chain variable fragment (scFv) of an antibody molecule. (Item 27) The glia cell-specific binding protein is the engineered protein according to item 23, which binds to a marker selected from the group consisting of myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (OM4), neuron / glia marker 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and myelin oligodendrocyte-specific protein (MOSP). (Item 28) The glia cell-specific binding protein is the engineered protein according to item 27, which binds to myelin oligodendrocyte glycoprotein (MOG). (Item 29) The glia cell-specific binding protein is the engineered protein according to item 28, which is a single-chain variable fragment (scFv) of an antibody molecule containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, and 12.

Brief Description of the Drawings

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[0018] Detailed Description The present invention relates to compositions for modulating the autoimmune components 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) into the CNS to prevent autoimmune attacks contributing 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 disease.

[0019] The blood-brain barrier can act as an obstacle to the treatment of brain or CNS disorders. This is because this barrier can block access of therapeutic compounds to diseased cells. Importantly, Tregs can cross the blood-brain barrier and can be localized to CNS neurons by glial cells bound to Tregs, thereby potentially enabling the compounds of the present invention to effectively treat neurodegenerative disorders of the CNS.

[0020] The compounds and methods of the present invention do not rely on any disease-specific biochemical mechanism. Instead, they avoid the immune responses that many neurodegenerative diseases affect mental and physical deterioration. Thus, the compounds and methods can provide a therapeutic effect against many neurodegenerative diseases.

[0021] For example, PSP is associated with tau protein accumulation and neurofibrillary changes, leading to damage and loss of neurons and glial cells, and ultimately death, as well as physical and mental deterioration. Parkinson's disease is accompanied by neuron loss in the basal ganglia, along with astrocyte death and microglial enlargement in the substantia nigra. Inclusion bodies called Lewy bodies occur in damaged cells prior to cell death. ALS is characterized by the death of motor neurons in the motor cortex after the generation of protein-rich inclusion bodies in the cell bodies and axons of motor neurons.

[0022] The present invention recognizes that 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 immune components that contribute to inflammation and CNS deterioration, despite their underlying causes and disease mechanisms being different. 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 Perkinson’s disease, Lancet Neurology, 16(10):769-71" (the contents of each of which are incorporated herein by reference). Thus, the compounds and methods of the present invention, which focus on suppressing immune responses in the CNS and addressing chronic inflammation that causes many neurodegenerative diseases, may be therapeutically effective in treating many of those 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 regulatory T cells (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 nervous system (CNS) and peripheral nervous system of various animals, including humans. Glial cells include oligodendrocytes, astrocytes, ependymal cells, and microglia. As a result of their functions in maintaining CNS neurons, glial cells migrate to CNS neurons and can thus be used to localize therapeutic compounds there. For example, oligodendrocyte (ODC) glial cells traffic to the CNS and maintain axonal insulation by making myelin sheaths. The compounds and methods of the present invention involve linking immunosuppressive molecules to glial cells such as ODCs, such that as the glial cells perform their functions, the immunosuppressive molecules are brought into the immediate vicinity of CNS neurons, as shown in FIGS. 1 and 2. The presence of the immunosuppressive molecules modulates any ongoing immune responses and chronic inflammation that may be present in the CNS and contribute to neurodegenerative disease symptoms in diseases such as PD, PSP, etc.

[0024] Glia cell-specific targets include proteins and other markers expressed by various glia cells, such as myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (OM4), neuron / glia 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, a CAR, scFv, or antibody can be bound to an immunosuppressive molecule and used to target glia cells. A CAR is an engineered receptor 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, each of which is incorporated herein by reference. Using a similar principle, the compounds and methods of the present invention involve engineering a CAR specific for a marker found on glia cells, such as ODC, but instead of grafting a glia cell-specific CAR onto cytotoxic T cells, they are grafted onto engineered immunosuppressive Tregs.

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

[0027] An scFv is a fusion protein containing the variable region of the heavy chain (VH) and the variable region of the light chain (VL) of an immunoglobulin. An scFv can be produced by cloning the VH gene and the VL gene from a mouse or other animal immunized with a desired target molecule (e.g., MOG). The VH gene and the VL gene can then be expressed in a number of 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. An scFv or antibody specific for a glial cell marker discussed above can be linked to an immunosuppressive protein discussed below to form a fusion protein that can provide the CNS localization immunosuppressive therapy shown in Figure 2 and discussed below.

[0028] Antibodies targeting glial cell markers can be generated by methods known in the art, including services commercially available for generating custom antibodies from, for example, 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 and preferably can be 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 nucleic acids encoding engineered chimeric antigen receptors. CAR-Treg cells can then be experimentally verified before being introduced into the patient's system for treatment.

[0030] Regulatory T cells, or Tregs, regulate the immune system and generally down-regulate the induction and proliferation of effector T cells. Tregs prevent autoimmune responses and help the immune system distinguish self from non-self. Regulatory T cells produce inhibitory cytokines, including transforming growth factor β, 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 direct interactions with dendritic cells and reverse signaling via induction of immunosuppressive indoleamine 2,3-dioxygenase. Tregs can also down-regulate the immune response through the production of immunosuppressive adenosine and through the ectoenzymes CD39 and CD73. Tregs also suppress the immune response through direct interactions with dendritic cells by LAG3 and by TIGIT. Another control mechanism is through the IL-2 feedback loop. Another mechanism of immunosuppression by Tregs is through the action of the CTLA-4 molecule, through CD28 on effector T cells, via prevention of co-stimulation.

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

[0032] Figure 2 shows the glial cell-targeted immunosuppressive protein (GTIP) of the present invention that suppresses immune attack on neurons. GTIP can include enzymes such as extracellular enzymes that scavenge immunosuppressive proteins or immunostimulatory metabolites (e.g., ATP, AMP, tryptophan, and glutamate) present in Treg cells. Such extracellular enzymes can include CD73, CD39, indoleamine 2,3-dioxygenase (IDO), and glutamate oxaloacetate transaminase 1 (GOT1). In Figure 2, glial cells expressing MOG are bound by GTIP consisting of an anti-MOG scFV linked to an immunosuppressive enzyme (IE). The glial cells, in performing their neuron-related functions, 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 may regulate or halt the immune response, thereby maintaining neurons and reducing the symptoms of underlying neurodegenerative diseases. 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 GTIP of the present invention can include 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 can be linked by any known means to form the GTIP of the present invention (e.g., including fusion proteins or biotin-streptavidin linkage).

[0034] The adoptive cell transfer technology 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 content of which is incorporated herein by reference.

[0035] The CAR-Tregs or glial cell-targeted immunosuppressive proteins of the present invention can be incorporated into a carrier system comprising one or more of the therapeutic compounds described herein. In certain embodiments, the carrier system can be nanoparticles comprising crosslinked polyethyleneimine (CLPEI) and lipids. The lipids can be bile acids (e.g., cholic acid, deoxycholic acid, and lithocholic acid). Such carrier systems are 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 content of which is incorporated herein by reference in its entirety.

[0036] As used herein, the terms "parenteral administration" and "administered parenterally" mean a mode of administration other than enteral administration and topical administration (usually by injection), including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subdural, intraspinal, and intrasternal injections and infusions.

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

[0038] When the compounds of the present invention are administered to humans and animals as medicaments, they can be administered by themselves or in combination with a pharmaceutically acceptable carrier, for example, as a pharmaceutical composition containing 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 agent can be readily determined by one of ordinary skill in the art, taking into account typical factors such as the age, weight, gender, and clinical history of the patient. Generally, the appropriate daily dosage of the compounds of the present invention is the amount of the compound that is the lowest dosage effective to produce a therapeutic effect. Such effective dosages generally depend on the factors described above.

[0040] If desired, the effective daily dosage of the above active compound can be administered in unit dosage form, as two, three, four, five, six, or more sub-dosages, administered separately at appropriate intervals throughout the day, as necessary.

[0041] The pharmaceutical composition of the present invention comprises a "therapeutically effective amount" or "prophylactically effective amount" of one or more of the compounds of the present invention or their functional derivatives. The "effective amount" is, as defined in the Definitions section herein, the amount necessary to achieve a desired therapeutic result, e.g., a reduction or prevention of effects associated with neuropathic and / or inflammatory pain, in an amount and over a period of time effective for this purpose. The therapeutically effective amount of the compound of the present invention or its functional derivative can vary according to factors such as the disease state, age, sex, and weight of the subject, as well as the ability of the therapeutic compound to elicit the desired response in the subject. The therapeutically effective amount is also an amount where any toxic or detrimental effects of the therapeutic agent are outweighed by the therapeutically beneficial effects.

[0042] The "prophylactically effective amount" refers to the amount effective in an amount and over a period of time necessary to achieve the desired prophylactic result. Typically, since prophylactic dosages are used in a subject prior to or at an earlier stage of a disease, the prophylactically effective amount may be less than the therapeutically effective amount. The prophylactically or therapeutically effective amount is also an amount where any toxic or detrimental effects of the compound are outweighed by the beneficial effects.

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

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

[0045] In some embodiments, a therapeutically effective amount can first be predicted in cell culture assays, or in animal models, typically in any of mice, rabbits, dogs, or pigs. Such animal models are also used to achieve the desired concentration ranges and routes of administration. Such information can then be used to determine useful dosages 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 one of ordinary skill in the art using conventional considerations (e.g., by appropriate conventional pharmacological protocols). A physician can, for example, initially prescribe at a relatively low dosage and then increase the dosage until an appropriate response is obtained. The dosage administered to a patient is sufficient to provide a beneficial therapeutic response over time in the patient, or to reduce symptoms, or other appropriate activities, for example, depending on the application. The dosage is determined by the effectiveness of the particular formulation, and the activity, stability, or serum half-life of the compound of the invention or its functional derivative, as well as the condition of the patient, and the weight or body surface area of the patient being treated. The size of the dosage is also determined by the presence, nature, and extent of any adverse side effects associated with the administration of a particular vector, formulation, etc. in a particular subject. A therapeutic composition comprising one or more compounds of the invention or its functional derivative is tested, if necessary, in one or more appropriate in vitro models and / or in vivo disease animal models (e.g., models of neuropathic and / or inflammatory pain) according to methods well known in the art to confirm effectiveness, tissue metabolism, and estimate dosage. In particular, the dosage can first be determined in a relevant assay by activity, stability, or other appropriate measure of treatment relative to non-treatment (e.g., comparison of treated cells or animal models to untreated cells or animal models).The formulation is administered, for example, when applied to the patient's weight and overall health status, at a rate determined by observation of any side effects at various concentrations of the LD50 of the relevant formulation and / or the compound of the present invention or its functional derivatives. Administration can be achieved via a single dose or divided doses.

[0046] Administration typically involves administering a pharmaceutically acceptable dosage form, which means a dosage form of the compounds described herein, for example, tablets, dragees, powders, elixirs, syrups, liquid preparations (including suspensions), sprays, inhalants tablets, lozenges, emulsions, solutions, granules, capsules, and suppositories, as well as liquid preparations for injection (including liposomal preparations). Techniques and formulations can generally be found in Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latest edition (which is incorporated by reference in its entirety). Administration can be oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal. The compound may be administered alone or together with a suitable pharmaceutical carrier and may be in solid or liquid form (e.g., tablets, capsules, powders, solutions, suspensions, or emulsions).

[0047] A pharmaceutical composition containing an active ingredient may be in a form suitable for oral use, such as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents selected from sweetening agents, flavoring agents, coloring agents, and preservatives to provide a pharmaceutically elegant and palatable preparation. Tablets contain the active ingredient mixed with non-toxic pharmaceutically acceptable excipients appropriate for the manufacture of tablets. These excipients may be, for example, inert diluents (such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate), granulating and disintegrating agents, such as corn starch, or alginic acid; binders (such as starch, gelatin or acacia gum), and lubricants (such as magnesium stearate, stearic acid or talc). The tablets may or may not be coated and may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, time delay materials such as glyceryl monostearate or glyceryl distearate may be used. They may be coated by the techniques described in U.S. Patent Nos. 4,256,108, 4,166,452 and 4,265,874, the contents of each of which are hereby incorporated by reference in their entirety, to form therapeutic osmotic tablets for controlled release.

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

[0049] The formulation may also contain a complex of the parent (non-ionized) compound and a derivative of β-cyclodextrin, in particular, hydroxypropyl-β-cyclodextrin.

[0050] Alternative oral formulations can be achieved using a controlled-release formulation in which the above compound is encapsulated in an enteric coating.

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

[0052] The oily suspension can be formulated by suspending the active ingredient in a vegetable oil (e.g., peanut oil, olive oil, sesame oil or coconut oil) or in a mineral oil (e.g., liquid paraffin). The said oily suspension may contain a thickening agent (e.g., beeswax, solid paraffin or cetyl alcohol). Sweetening agents (e.g., those mentioned above) and flavoring and odor-masking agents may be added to provide an acceptable oral preparation. These compositions can be preserved by the addition of an antioxidant (e.g., ascorbic acid).

[0053] Powders and granules with suitable dispersibility for the preparation of an aqueous suspension by the addition of water provide the active ingredient in a state of being mixed 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, sweetening agents, flavoring and odor-masking agents 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 a vegetable oil (e.g., olive oil or peanut oil), or a mineral oil (e.g., liquid paraffin) or a mixture thereof. Suitable emulsifying agents can be natural gums (e.g., acacia gum or tragacanth gum), natural phosphatides (e.g., soy lecithin), and esters or partial esters derived from fatty acids and hexitol anhydrides (e.g., sorbitan monooleate), and condensation products of the said partial esters and ethylene oxide (e.g., polyoxyethylene sorbitan monooleate). The said emulsion may also contain sweetening agents and flavoring and odor-masking agents.

[0055] Syrups and elixirs can be formulated using sweeteners (e.g., glycerol, propylene glycol, sorbitol, or sucrose). Such formulations can also include demulcents, preservatives, as well as flavoring and coloring agents. The pharmaceutical composition can be in the form of a sterile aqueous or oily suspension for injection. This suspension can be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents mentioned above. The sterile injectable preparation can also be in the form of a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, and can exist, for example, as a solution in 1,3 - butanediol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile non - volatile oils are conventionally used as solvents or suspending media. For this purpose, any non - irritating non - volatile oil can be used (such as synthetic monoglycerides or diglycerides). Furthermore, fatty acids such as oleic acid are found to be used in the preparation of injectables.

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

[0057] For topical use, creams, ointments, jellies, solutions or suspensions are suitable. Topical applications include the use of mouse washes and gargles.

[0058] The term "pharmaceutical composition" means a composition comprising a compound as described herein, as well as a pharmaceutically acceptable carrier, diluent, adjuvant, excipient, or vehicle (e.g., at least one component including preservatives, fillers, disintegrants, wetting agents, emulsifying agents, suspending agents, sweetening agents, 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" is used to mean any carrier, diluent, adjuvant, excipient, or vehicle as described herein. 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, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc. Prolonged absorption of the above injectable pharmaceutical forms can be brought about by using agents that delay absorption (e.g., aluminum monostearate and gelatin). Examples of suitable carriers, diluents, solvents, or vehicles include water, ethanol, polyols, suitable mixtures of these, 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 disintegrants 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 free of excessive toxicity, irritation, allergic response, etc., and is suitable for use in contact with cells of humans and lower animals within the scope of sound medical judgment, and is commensurate with a reasonable benefit / risk ratio.

[0060] Incorporation by reference References and citations to other documents such as patents, patent applications, patent gazettes, academic journals, books, papers, and web content are made throughout this disclosure. All such documents are hereby incorporated by reference in their entirety for all purposes.

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

Examples

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

[0063] The main objective is to evaluate the safety and feasibility of intravenous infusion of autologous CNS-specific CAR-Tregs selected, expanded, and transduced ex vivo in at least 5 patients with PSP.

[0064] The temporary outcome measures are as follows: 1. Adverse events 2. Abnormalities in examinations 3. Injection reactions 4. Complications related to infection 5. Potential negative impact on the course of PSP

[0065] The secondary objective is to evaluate the effect of CNS-specific CAR-Tregs on PSP and to obtain indications regarding potential applications in other neurodegenerative diseases.

[0066] The endpoints are as follows: 1. Evaluate the effect of CNS-specific CAR-Tregs on clinical parameters, neuropsychological parameters, radiological parameters, and biochemical parameters in PSP patients 2. Obtain indications regarding potential therapeutic uses of CNS-specific CAR-Tregs in other neurodegenerative diseases, including Alzheimer's disease (AD) 3. Obtain indications in a potential Phase II placebo-controlled randomized double-blind trial that could provide valuable insights into the potential efficacy of CAR-Tregs for neurodegenerative disorders.

[0067] Patient evaluation Clinical and neuropsychological evaluations: A detailed description of the inclusion and exclusion criteria, as well as clinical evaluations (motor and neuropsychological) and neuroimaging evaluations, are performed as previously reported (Giordano et al., J. Transl. Med. 2014; Canesi et al., J. Transl. Med. 2016, incorporated herein by reference). Patients undergo neurological examinations and their motor function is evaluated using the following scales: Unified Parkinson's Disease Rating Scale (UPDRS Part III, motor score), Hoehn and Yahr staging (H&Y), PSP Rating Scale (PSP-RS) (Goetz et al., Mov. Disord. 2004; Golbe et al., Brain 2007 (the content of each of these is incorporated herein by reference). The Mini-Mental State Examination (MMSE) is also performed as previously described (Folstein et al. J. Psychiatr. Res. 1975 (incorporated herein by reference)). All of these tests are 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 did not decrease by more than 30% compared to baseline, and if the H&Y severity classification did not change at the defined time point, the clinical status is classified as "stable" (Canesi et al., J. Transl. Med. 2016 (incorporated herein by reference)).

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

[0069] Regarding SPECT, intravenous administration of 110 - 140 MBq of [123I]FP-CIT (Datscan, GE-Health, Amersham, UK) is performed in all patients 30 - 40 minutes after thyroid block (oral administration of 10 - 15 mg of Lugol's solution). Analysis is performed as previously described (Isaias et al., NeuroReport 2007 (incorporated herein by reference)). A volumetric measurement template of gray matter anatomy is generated from the Montreal Neurological Institute MRI single-subject brain atlas by applying macroscopic anatomy (automated anatomical labeling), and reoriented and reformatted to obtain reference cross-sections 2.64 cm thick. A template of 8 irregular regions of interest (ROIs) is hand-drawn on this cross-section to evaluate the anatomical extent of the striatum and occipital lobe structures with specific and non-specific uptake of [123I]FP-CIT, respectively. The above ROI template is also placed on the reference SPECT cross-section and adjusted for both the striatum and occipital cortex. The striatal ROI is also divided into its anterior (caudate nucleus) and posterior (putamen) parts.

[0070] The specific striatal dopamine uptake transporter (DAT) binding of [123I]FP-CIT is calculated in the whole striatum, putamen, and caudate nucleus using the formula: [(mean in specific ROI)-(mean in occipital ROI)] / (mean in occipital ROI). The putamen / caudate nucleus ratio for each subject is also calculated.

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

[0072] Biomechanical evaluation: Biomechanical evaluation is performed at baseline, and 6 and 12 months after CAR-Treg cell administration. Two specific sets of parameters (one for standing and one for gait initiation) are automatically extracted by an ad hoc algorithm (Carpinella et al, IEEE Trans Neural Syst Rehabil Eng. 2007 (incorporated herein by reference)). For standing, the center of pressure (CoP) mean velocity and spatial displacement are measured (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., the unstable and unloaded phases), and the following parameters are measured: (1) the duration of both phases, (2) the anterior-posterior (AP) and medial-lateral (ML) shifts and velocities of the CoP, (3) the CoP mean length and velocity, (4) the length of the first step, and (5) the velocity. Spatial parameters are normalized based on body height (%BH).

[0073] Preparation and Administration of CAR-Treg Cells Treg Isolation and Expansion: PolyTregs were selected and expanded from 5 individuals with PSP based on three cell surface markers (CD4, CD25, and CD127) to purify FOXP3+ Tregs present in peripheral blood 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 pack unit containing citrate phosphate dextrose and processed within 24 hours for isolation of PBMCs via Ficoll density gradient. Tregs were isolated using a high-speed cell sorter with the following GMP-grade lyophilized antibodies: CD4-PerCP (peridinin chlorophyll protein) (clone L200), CD127-PE (phycoerythrin) (clone 40131), and CD25-APC (allophycocyanin) (clone 2A3). Sorted CD4+CD127lo / −CD25+ cells were collected into 3 ml of X-VIVO 15 medium (Lonza, catalog number 04-418Q) containing 10% heat-inactivated pooled human AB serum (Valley Biomedical). Tregs were analyzed for purity after sorting. The predicted purity of CD4+CD127lo / −CD25+ cells is greater than 96% (Bluestone et al., Sci. Transl. Med., 2015, incorporated herein by reference).

[0075] The purified Tregs are cultured with clinically graded 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). Blood units are expected to yield between 4.2×106 and 11.8×106 purified CD4+CD127lo / −CD25+ Tregs (Bluestone et al., Sci. Transl. Med., 2015, incorporated herein by reference). The expanded Treg preparation is expected to be approximately 90% FOXP3+. Check the Treg preparation for viability, percentage of CD4+, and CD8+ cell contamination (Bluestone et al., Sci. Transl. Med., 2015, incorporated herein by reference).

[0076] Phenotypic and TCR analysis of expanded poly Tregs: Check CD4 and CD127, important cell surface markers used to isolate Tregs, after expansion.

[0077] Previous data have shown that naïve CD45RA+ Tregs preferentially expand in these cultures, and that CD45RA+RO− cells downregulate CD45RA and upregulate CD45RO over the expansion period (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 the expanded Tregs is also analyzed to determine the polyclonality of the expanded Tregs compared to freshly isolated populations. The expanded cells are predicted to show polyclonality indistinguishable from pre-expansion cultures and that the Tregs 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: After Treg expansion, the following assays are performed (Bluestone et al., Sci. Transl. Med., 2015, incorporated herein by reference): - DNA methylation status of the enhancer region of the FOXP3 locus to assess the overall purity and stability of the expanded Tregs. - Cytokine production (IFNγ, IL-4, IL-5, and IL-17) to evaluate lymphocyte phenotype. - In vitro suppressive activity to determine the functional potential of the expanded cells.

[0079] Generation and functional analysis of CAR-Tregs: CAR RNA is transfected onto Tregs after electroporation of human Tregs and using published protocols (Zhao, Y et al., 2010 Cancer Res and Beatty, GL et al., 2014 Cancer Immunol Res; Singh, Optimize for anti-MOG CAR expression on murine Tregs after adoptive transfer into the PSP mouse model according to Net al., 2014 Oncoimmunol (the content of each of these is incorporated herein by reference). Delivery of anti-MOG CAR using a second-generation lentiviral vector and a standard protocol (Levine, B.L. et al., 2017 Mol Ther Methods & Clin Dev (incorporated herein by reference)) is also optimized. Human Tregs are transduced either under GMP conditions (about 1 mg / 3×10 6 Tregs) using electroporation of RNA or by lentiviral transduction (1×10 6 pfu / 3×10 6 Tregs). Generate clinical-grade RNA for anti-MOG CAR. About 0.9 mg RNA / patient is required for 2.6×10 9 Treg cells; 4.5 mg is required for 5 patients. Generate clinical-grade lentivirus. About 8.7×10 9 pfu / patient is required for 2.6×10 9 Treg cells; 4.3×10 10 pfu is required for 5 patients. Perform functional analysis of MOG-specific CAR-Tregs as described above in 3.3.

[0080] Cell Administration Perform a single administration of MOG-specific CAR-Tregs for each patient (2.6×10 9 CAR-Treg / Patients). Administer the cells to at least 5 PSP patients. The patients receive pre-medication with acetaminophen and diphenhydramine. Infuse the CAR-Treg over 10 - 30 minutes via a peripheral venous line. Measure vital signs 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. Repeat the chemical properties with various blood cell counts and the complete blood count the day after discharge from the clinical research unit. Examine the patients for follow-up evaluations on the 4th day after infusion, then once a week for 4 weeks, then once every 13 weeks for 1 year, and once every 26 weeks for 2 years. Conduct telephone monitoring for adverse events every 6 months for 5 years, and then perform the final clinic visit.

[0081] Patient Evaluation after CAR-Treg Cell Infusion Evaluate the effects of CNS-specific CAR-Treg on clinical parameters, neuropsychological parameters, radiological parameters, and biochemical parameters in PSP patients as described above. All tests are performed at each follow-up point: 1 month, 3 months, 6 months, and 12 months after cell administration.

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

[0083] For relapsing-remitting MS (RRMS — 85% of diagnosed MS), there are 11 FDA-approved drugs. There are several orally available and antibody-based drugs that are currently approved or in 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). Ocrelizumab provides a 25% reduction in symptoms and is currently the only immunomodulator for PPMS in the USA. Secondary progressive MS (SPMS) always develops in patients with RRMS and there are also limited disease-modifying options for it.

[0084] Generation of Biologics Anti-MOG hybridomas are generated by immunizing mice with recombinant human MOG through a CRO. The VH gene and VL gene are cloned to produce an anti-scFv molecule. The orientation of VH and VL, as well as the linker (between scFvs or within each scFv), can greatly affect the stability, expression level, and binding ability of the GTIP. In some cases, only one of these forms generates a functional molecule. Therefore, several orientations of VH-VL are expressed on a small scale and tested before scale-up production. A linker is connected, then a central linker, and then an expression construct that encodes four anti-MOG scFvs and is connected to a Treg-related enzyme or mimetic is generated.

[0085] Verification of GTIP Protein Product in Mouse MS Model GTIP is tested in acute and chronic EAE models of mouse MS. The levels of Th1, Th17, CTL (blood and CNS) specific to myelin basic protein (MBP), bone marrow inflammatory cells (macrophages and neutrophils), and anti-MBP antibodies are measured. The immunological response correlates with disease progression. The administration is varied to gain insights into potential use in late-stage MS. The product is administered in normal mice to gain insights into any potential off-target effects.

[0086] Clinical evaluation Conduct clinical trials to test the efficacy of GTIP as a disease-modifying agent in MS. The product is first tested in RRMS patients who do not respond to first use drugs. Safety and tolerability are measured using a dosing regimen similar to that for antibody therapy (e.g., three i.v. doses every two weeks for the first two weeks, then every four weeks over 20 weeks). In the Phase 2 trial, the primary measures are reduced disease relapse frequency and brain lesions. The secondary measures are reduced inflammatory cytokines, Th1 / Th17 cells, and other white blood cell levels in the blood. Side effects may include increased susceptibility to infection. These trials enable the inventors to benchmark the efficacy of the above compounds against other second use drugs that show a reduction of up to 49% in relapse frequency. If the above products show an acceptable efficacy level, they enter a longer-term Phase 2 clinical trial in PPMS patients. The primary measures are delayed decline in motor function and reduced brain lesions, and the secondary measures are reduced inflammatory cytokines, Th1 / Th17 cells, and other white blood cell levels in the blood.

[0087] Example 3 Using the tetramer binding assay, as shown in Figure 3, the binding affinity of GITP to target cells is compared with that of a known tetramer for T cells (Ober, B et al, 2000 Int Immunol (incorporated herein by reference)). The relative binding affinity of the H-Y 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 and half-life (t 1 / 2) It is. MOG target cells are generated by gene transfection of non-adherent target cells (e.g., RMA or Jurkat cells). Antibody staining and flow cytometry (FCM) are used to confirm the surface expression of MOG. Antibody staining and FCM are used to identify transfectants having the same level of MOG as the B6.2.16 TCR on CTLs. The maximum staining and half-life of the staining of MOG target cells with the labeled GITP protein are measured and compared with those of CTLs and pMHC tetramers as shown in FIGS. 4 and 5. The purpose is to generate a GITP having a binding force for cell interaction comparable to or better than those of CTLs and pMHC tetramers. If the tetramer anti-MOG scFv in the GITP molecule is below this bar, the valence binding valence of the scFv can be increased. If an even higher binding valence is required, a nanoparticle scaffold can be used to achieve the required binding force for target cell binding.

[0088] Example 4 The ability of GTIP bound to MOG target cells to suppress the proliferation of T effector (Teff) cells is tested. GTIP composed of a tetramer of a given scavenger IE (see Table 1 below) is bound to MOG target cells, washed, and then incubated with proliferating human Teff (e.g., generated using standard procedures 3 days after anti-CD3 / CD28 and IL-2 stimulation) (the middle column in FIG. 6).

Table 1

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

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

[0091] Determine the relative binding affinity of the pMHC target to the relevant CTL clone (B6.2.16) (left column of Figure 7). Label the target with the vital dye PKH26 (red), label the CTL with CFSE (green), co-incubate for 4 hours, then subject to standard shear forces and examine by FCM. The conjugates are detected as double-stained doublets and are dependent on the presence of the H-Y peptide antigen. The relative binding affinity of the pMHC target:B6.2.16 CTL interaction is measured by two parameters (the maximum level of conjugate formation (about 80% of the total input cells) and the half-life of conjugate dissociation). Incubate MOG target cells 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). Measure the half-life of the conjugates between the labeled cells by FCM (right column of Figure 7). A conjugate half-life comparable to that of CTL:pMHC / target cells indicates the physiological binding affinity of the anti-MOG scFv / CAR for T cells against MOG-positive target cells.

[0092] Example 6 Human scFv antibodies specific for 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 the purity of the target. To reduce non-specific binding factors, pre-counter selection was first performed on the phage library using polystyrene flat-bottom plates and blocking buffer prior to targeted screening.

[0093] After three rounds of biopanning, positive enrichment was observed. Twenty clones were randomly picked from the third round and QC monoclonal phage ELISA was performed. Eighteen clones were found to bind to the target compared to the control. All 18 positive clones were sequenced.

[0094] Twenty other clones were picked up starting from the third time and subjected to QC monoclonal phage ELISA. All 20 of the second clone set were found to bind to the target as compared to the control. All of them were sequenced.

[0095] After analysis of 38 positive clones, 7 positive clones (clone 1, 3, 6, 10, 13, 17, 21) with unique sequences were identified. The sequences of the 7 scFv proteins and the DNA sequences encoding them are listed below:

[0096]

Chem.

Chem.

Chem.

Chem.

Chem.

[0097] Expression vectors were constructed for each of the above 7 scFv proteins. Then, the above cell lysates were coated for ELISA. Next, soluble ELISA was performed using cell lysates from both 30 °C and 37 °C. Differences were easily observed in all 7 clones as compared to the control. Among the above 7 positive clones, clones 1, 6 and 13 were much stronger than the others.

[0098] ELISA involving quantification was performed on the soluble scFvs generated from the above 7 positive clones to rank their ability to bind to MOG. The above 7 scFvs were subcloned into pET-26b and constructed in the scFv-myc-6×His format. The purity of the above 7 scFvs induced at 16°C was >85%, while the purity when induced at 37°C was lower. Therefore, 16°C was determined to be a more appropriate condition for production. QC ELISA was performed to analyze the binding ability to the target MOG for each of the above 7 scFvs. When compared with the control, differences were easily found in each of the above 7 positive clones (clone 1, 3, 6, 10, 13, 17, 21). Among the above 7 positive clones, 3 clones (clone 3, 6 and 17) showed a stronger binding ability to the target.

[0099] Quantification by QC ELISA was performed for each of the above 7 clones induced at 16°C. Seven different concentrations of the above 7 clones were used for quantification by ELISA. The results showed that all 7 clones could specifically bind to the target MOG. Among the above 7 clones, clone 3, 6 and 17 still showed a stronger binding affinity to the above target. The results are shown in Figure 8. Figure 8 illustrates that the anti-hMOG1 scFv of clone 17 showed the strongest binding, followed by the binding of clone 6, and then the binding of clone 3. The remaining 4 clones showed significantly weaker binding than those of clone 17, 6 and 3.

Claims

1. 1. A composition comprising a plurality of engineered regulatory T cells (Tregs), the engineered Tregs of the plurality of engineered Tregs are CD4+CD127-CD25+FOXP3+ cells; wherein each engineered Treg of the plurality of engineered Tregs expresses 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: 12; composition.

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

12.

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

4. 4. The composition of claim 3, wherein the CAR is capable of directing the engineered Tregs to glial target cells that express MOG.

5. 1. A composition comprising a plurality of engineered regulatory T cells (Tregs), the engineered Tregs of the plurality of engineered Tregs are CD4+CD127-CD25+FOXP3+ cells; wherein each engineered Treg of the plurality of engineered Tregs expresses 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; composition.

6. The composition of claim 5 , wherein the scFv has at least 99% sequence identity to SEQ ID NO:

6.

7. The composition of claim 6, wherein the scFv is capable of specifically binding to the glial cell marker myelin oligodendrocyte glycoprotein (MOG).

8. 8. The composition of claim 7, wherein the CAR is capable of directing the engineered Tregs to glial target cells that express MOG.

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