CAR-TREG-based therapy for treating neurodegenerative diseases

Abstract

The present invention provides compositions and methods for inhibiting the autoimmune component of neurodegenerative diseases and thereby providing a therapeutic effect to patients suffering from such diseases. The compositions and methods comprise immunosuppressive moieties, such as regulatory T cells (Tregs) and proteins expressed by Tregs coupled to a chimeric antigen receptor or that specifically bind to 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.

Description

[0001] This application is a divisional application of Chinese patent application No. 201980035934.X, filed on March 21, 2019, entitled "CAR-TREG-based therapy for treating neurodegenerative diseases".

[0002] Related applications This application claims priority and benefit to U.S. Provisional Application No. 62 / 648,684, filed March 27, 2018, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention provides CAR-Treg compositions and methods of use thereof, which specifically modulate immune responses and inflammation associated with various neurodegenerative diseases, such as progressive supranuclear palsy and Parkinson's disease. Background Technology

[0004] 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 many people and often lead to rapid physical and / or mental decline and death. There is no known cure for these diseases, and treatment focuses on managing symptoms and slowing the decline.

[0005] One such disease, PSP, is an idiopathic degenerative disorder that is not uncommon in older adults and mimics Parkinson's disease (PD). Clinical manifestations include a tetrad of symptoms—supranuclear gaze palsy, axial stiffness, dementia, and pseudobulbar palsy. It is associated with bradykinesia, severe postural disturbances, and frequent falls. Pathology involves cell loss and Tau neurofibrillary tangles, primarily in the brainstem, globus pallidus, hypothalamic nucleus, and dentate nucleus. The prevalence of PSP is 5–6 per 100,000 people, resulting in 5,000–25,000 new cases annually in the United States. The average age of onset is 63 years, the general prognosis from diagnosis to death ranges from 5 to 10 years, and there are no disease-modifying treatments available.

[0006] Parkinson's disease is another neurodegenerative disease for which there is currently no known cure. The prevalence of Parkinson's disease is approximately 1-2 per 1,000 people. Parkinson's disease is characterized by cell death in the basal ganglia and astrocytes, as well as an increase in microglia in the substantia nigra, leading to a lack of dopamine in these areas. Inclusions called Lewy bodies are produced in damaged cells prior to cell death. There are speculations about the potential mechanisms driving brain cell death in Parkinson's disease, but little is known about these mechanisms, and current treatments focus on managing disease symptoms. Summary of the Invention

[0007] The compositions and methods of the present invention utilize regulatory T lymphocytes (Tregs) or immunosuppressive proteins expressed by Treg cells to modulate neurodegenerative immune responses targeting glial cells in the central nervous system (CNS). By coupling Tregs or immunosuppressive proteins to chimeric antigen receptors (CARs) or single-chain variable fragments (scFvs) that specifically recognize and bind to glial cell markers, immunosuppressive Tregs or proteins are inhaled into glial cells of the CNS to reduce inflammation and protect the CNS from autoimmune attack.

[0008] This invention recognizes that most neurodegenerative diseases lack effective treatment options and that several such diseases possess autoimmune and / or inflammatory components, and engineeres compositions that specifically inhibit those disease components. The compounds and methods of this invention allow glial cells to modulate impaired immune cells, such as type 1 helper cells (Th1), T helper 17 cells (Th17), cytotoxic T cells (CTLs), M1 macrophages, and polymorphonuclear neutrophils (PMNs).

[0009] This invention directs immunosuppressive molecules (Treg or immunosuppressive proteins) to oligodendrocytes (ODCs) glial cells. The resulting compounds and their methods of use recruit the body's own immune system to counteract 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). This invention addresses the mechanism by which several neurodegenerative diseases disrupt nerve function, independent of any specific biochemical cause of the underlying disease. Therefore, the compounds and methods of this invention can provide therapeutic effects against several neurodegenerative diseases.

[0010] Aspects of the present invention include methods for treating a subject with a neurodegenerative disease, the methods comprising administering to the subject a therapeutically effective amount of a regulatory T cell (Treg) expressing a chimeric antigen receptor (CAR), the chimeric antigen receptor specifically binding to a glial cell marker, wherein the neurodegenerative disease is not multiple sclerosis (MS). The CAR-Treg then protects neural tissue and reduces inflammation in the neural tissue, thereby treating the neurodegenerative disease. In various embodiments, the subject may be a human being.

[0011] The glial cell marker may be oligodendrocyte glycoprotein (MOG), oligodendrocyte marker O1 (OM1), oligodendrocyte marker O4 (OM4), glial marker 2 (NG2), A2B5, galactosylceramidinase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), or myelin oligodendrocyte-specific protein (MOSP). In some embodiments, the glial cell marker is myelin oligodendrocyte glycoprotein (MOG).

[0012] The treated neurodegenerative disease 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 prions. 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).

[0013] In some aspects, the present invention provides a composition comprising a therapeutically effective amount of engineered regulatory T cells (Tregs) for treating a neurodegenerative disease, 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 may be myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker O1 (OM1), oligodendrocyte marker O4 (OM4), neuroglia / glial marker 2 (NG2), A2B5, galactosylceramidinase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), or myelin oligodendrocyte-specific protein (MOSP).

[0014] This composition can effectively treat progressive supranuclear palsy (PSP), Parkinson's disease (PD), Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy (CTE), or prions.

[0015] Various aspects of the present invention include an engineered protein comprising a glial cell-specific binding protein coupled to a molecule expressed by a regulatory T cell (Treg). The molecule expressed by the Treg may be an extracellular immunosuppressive enzyme. In some embodiments, the molecule expressed by the Treg may be CD73, CD39, indoleamine 2,3-dioxygenase (IDO), or glutamate-oxaloacetate transaminase 1 (GOT1). The glial cell-specific binding protein may be a tetrameric single-chain variable fragment (scFv) of an antibody molecule.

[0016] In some embodiments, the glial cell-specific binding proteins expressed by Treg may bind to myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker O1 (OM1), oligodendrocyte marker O4 (OM4), neuroglia / glial marker 2 (NG2), A2B5, galactosylceramidinase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), or myelin oligodendrocyte-specific protein (MOSP).

[0017] In some aspects, the present invention provides an engineered protein comprising a glial cell-specific binding protein coupled to a molecule that mimics the activity of a molecule expressed by regulatory T cells (Tregs). The mimic molecule expressed by Tregs may be an extracellular immunosuppressive enzyme, such as CD73, CD39, indoleamine 2,3-dioxygenase (IDO), or glutamate-oxaloacetate transaminase 1 (GOT1). The glial cell-specific binding protein to which the mimic molecule binds may bind to myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker O1 (OM1), oligodendrocyte marker O4 (OM4), neuroglia / glial marker 2 (NG2), A2B5, galactosylceramidinase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), or myelin oligodendrocyte-specific protein (MOSP). Attached Figure Description

[0018] Figure 1 This study demonstrated glial cell-specific CAR-Tregs and their immunosuppressive function.

[0019] Figure 2 This study demonstrates the targeting of glial cells to immunosuppressive proteins and their immunosuppressive functions.

[0020] Figure 3 This demonstrates the binding of pMHC-tetramer to cytotoxic T cells and the binding of the GITP of this invention to the target MOG protein.

[0021] Figure 4 This study demonstrates the maximum staining effect of labeled GITP protein on MOG target cells compared to CTL and pMHC.

[0022] Figure 5 The half-life of staining MOG target cells with labeled GITP protein was shown compared to CTL and pMHC.

[0023] Figure 6 This study demonstrates a comparison of the inhibition of T effector cell proliferation by MOG target cells bound to GTIP compared to negative and positive controls.

[0024] Figure 7 The relative affinity of pMHC for the corresponding cytotoxic T cell clones was demonstrated compared to the relative affinity of CAR molecules expressing scFv, which are specific to MOG target cells.

[0025] Figure 8 The relative immunoreactivity of seven different scFv proteins against human MOG-1 was demonstrated. Detailed Implementation

[0026] This 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 attract immunosuppressive molecules (e.g., Tregs or immunosuppressive proteins expressed by Tregs) to the CNS and disrupt the autoimmune attack that contributes 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 prions.

[0027] The blood-brain barrier can serve as a barrier to the treatment of brain or CNS conditions because it can prevent therapeutic compounds from entering the affected cells. Importantly, Tregs can cross the blood-brain barrier and can be localized to neurons in the CNS via glial cells that bind to Tregs, thereby enabling the compounds of the present invention to effectively treat neurodegenerative conditions of the CNS.

[0028] The compounds and methods of this invention do not rely on any disease-specific biochemical mechanisms, but rather short-circuit the immune response through which many neurodegenerative diseases affect mental and physical decline. Therefore, the same compounds and methods can provide therapeutic effects for many neurodegenerative diseases.

[0029] For example, PSP involves tau protein buildup and neurofibrillary tangles, leading to damage and loss of neurons and glial cells, associated physical and mental decline, and ultimately death. Parkinson's disease involves neuronal loss in the basal ganglia, astrocyte death, and an increase in microglia in the substantia nigra. Inclusions known as Lewy bodies are produced in damaged cells prior to cell death. ALS is characterized by the death of motor neurons in the motor cortex following the production of protein-rich inclusions in their cell bodies and axons.

[0030] This invention recognizes that, despite differences in underlying causes and disease mechanisms, PSP, Parkinson's disease, and ALS, as well as neurodegenerative diseases (including Alzheimer's disease (AD), Huntington's disease, chronic traumatic encephalopathy (CTE), and prions), may contain immune components that promote inflammation and CNS degeneration. See Malaspina et al., 2015, “Disease origin and progression 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 these references are incorporated herein by reference. Therefore, the compounds and methods of the present invention, which focus on inhibiting the immune response in the CNS and resolving the chronic inflammation that drives many neurodegenerative diseases, may be therapeutically effective in treating many of those diseases.

[0031] The compounds and methods of this invention utilize chimeric antigen receptors (CARs), antibodies, or single-chain variable fragments (scFvs) that specifically bind to glial cell markers. Glial cell-binding molecules are coupled 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 numerous 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. Because glial cells maintain the function of neurons in the CNS, they migrate to neurons in the CNS and can therefore be used to localize therapeutic compounds therein. For example, oligodendrocytes (ODCs) glial cells circulate into the CNS by producing myelin sheaths to maintain axonal insulation. The compounds and methods of this invention involve coupling immunosuppressive molecules to glial cells such as ODCs such that the immunosuppressive molecules are in close proximity to the cells as the glial cells perform their functions. Figure 1 and 2 The diagram shows neurons in the CNS. The presence of immunosuppressive molecules regulates any ongoing immune responses and chronic inflammation that may be present in the CNS, and promotes symptoms of neurodegenerative diseases such as PD and PSP.

[0032] Glial cell-specific targets include proteins and other markers expressed by various glial cells, such as myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker O1 (OM1), oligodendrocyte marker O4 (OM4), neuroglia / glial marker 2 (NG2), A2B5, galactosylceramidinase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), or myelin oligodendrocyte-specific protein (MOSP).

[0033] In various embodiments, CARs, scFvs, or antibodies can bind to immunosuppressive molecules and be 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 specificity on tumor cells to cytotoxic T lymphocytes for cancer immunotherapy. See Cousin-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 these references are incorporated herein by reference. Using a similar principle, the compounds and methods of the present invention involve engineering a CAR (which is specific for markers found on glial cells such as ODCs), but instead of transplanting the glial cell-specific CAR into cytotoxic T cells, the glial cell-specific CAR is transplanted into an engineered immunosuppressive Treg.

[0034] The CAR-Treg of the present invention can express multiple chimeric antigen receptors that target the same or two or more different glial cell markers.

[0035] ScFvs are fusion proteins containing variable regions of the heavy chain (VH) and light chain (VL) of immunoglobulins. ScFvs can be generated by cloning the VH and VL genes of mice or other animals immunized with a desired target molecule (e.g., MOG). The VH and VL genes can then be expressed in various orientations and with various linkers to form a variety of scFvs, which can then be experimentally validated to provide the desired stability, expression levels, and binding affinity to glial cells or their specific markers. ScFvs or antibodies specific to glial cell markers discussed above can be conjugated with immunosuppressive proteins discussed below to form proteins capable of providing, for example, specificity to glial cell markers. Figure 2 The fusion protein shown and discussed below is a CNS-localized immunosuppressive therapy.

[0036] Antibodies targeting glial cell markers can be generated using methods known in the art, which include commercially available services for generating custom antibodies from companies such as Pacific Immunology (San Diego, California) or ABclonal (Woburn, Massachusetts).

[0037] CAR-Treg cells can be engineered using known methods for preparing CAR-T cells. Treg cells can be isolated from a subject, preferably from an autologous Treg cell from a patient to be treated. The genes of the Treg cells can then be modified using known techniques such as electroporation, viral vectors, or other forms of transfection with nucleic acids encoding a selected engineered chimeric antigen receptor. The CAR-Treg cells can then be experimentally validated before being introduced into a patient system for treatment.

[0038] Regulatory T cells, or Tregs, modulate the immune system and typically downregulate the induction and proliferation of effector T cells. Tregs prevent autoimmune responses and help the immune system distinguish between self and non-self. Regulatory T cells produce repressive 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 in turn can induce apoptosis in effector cells. Tregs also function via reverse signaling through direct interaction with dendritic cells and the induction of immunosuppressive indoleamine 2,3-dioxygenase. Tregs can also downregulate immune responses through extracellular enzymes CD39 and CD73, which produce immunosuppressive adenosine. Tregs can also suppress immune responses through direct interaction with dendritic cells via LAG3 and TIGIT. Another control mechanism is through the IL-2 feedback loop. Another mechanism of Treg immunosuppression is through the action of the molecule CTLA-4 via CD28 on effector T cells to prevent co-stimulation.

[0039] Figure 1 This study demonstrates CAR-Treg cells targeting glial cells and their therapeutic mechanism. CAR-Treg cells express CARs that specifically bind to markers on glial cells. Thus, CAR-Treg cells bind to glial cells and are carried across the blood-brain barrier, localizing to neurons in the CNS via the natural functions of glial cells. The bound Treg cells then perform their natural regulatory functions by suppressing the immune attack on local neurons.

[0040] Figure 2This invention illustrates a glial cell-targeted immunosuppressive protein (GTIP) that inhibits the immune attack of neurons. GTIPs may comprise immunosuppressive proteins or enzymes present in Treg cells, such as extracellular enzymes that clear immune-activating metabolites (e.g., ATP, AMP, tryptophan, and glutamate). Such extracellular enzymes may comprise CD73, CD39, indoleamine 2,3-dioxygenase (IDO), and glutamate-oxaloacetate transaminase 1 (GOT1). Figure 2 In this process, glial cells expressing MOG bind to GTIP, which consists of anti-MOG scFV linked to an immunosuppressive enzyme (IE). When performing their neuronal-related functions, glial cells localize the bound IE to neurons subjected to immune attacks by various immune cells (Th17 cells, Th1 cells, CTL cells, M1 cells, and PMN cells) and modulate or shut down the immune response, thereby preserving neurons and alleviating symptoms of underlying neurodegenerative diseases. GTIP can be used to treat 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 prions.

[0041] The GTIP of the present invention may comprise one or more immunosuppressive proteins (comprising two or more different proteins) linked to one or more scFVs or antibodies targeting the same or two or more different glial cell markers. The proteins may be conjugated in any known manner to form the GTIP of the present invention, comprising, for example, fusion proteins or biotin-streptavidin linkages.

[0042] For example, adoptive cell transfer techniques used in cancer immunotherapy (including those involving cytotoxic T lymphocytes) can be used to prepare autologous CAR-Tregs used in the compounds and methods of this 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.

[0043] The CAR-Treg or glial cell-targeting immunosuppressive proteins of the present invention can be incorporated into a carrier system containing one or more therapeutic compounds described herein. In some embodiments, the carrier system may be nanoparticles comprising disulfide-crosslinked polyethyleneimine (CLPEI) and lipids. The lipids may be bile acids, such as cholic acid, deoxycholic acid, and lithocholic acid. Such carrier systems are further described in the examples below. 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.

[0044] As used herein, the phrase “parenteral administration and administered parenterally” refers to administration methods that are normally administered by injection, other than enteral and local administration, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-occipital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions.

[0045] As used in this article, the phrases “systemic administration” and “administered systematically” and “peripheral administration” refer to the administration of compounds, drugs or other materials, other than for direct use in the central nervous system, to the patient’s system and thus to undergo metabolism and other similar processes, such as subcutaneous administration.

[0046] When the compounds of the present invention are administered as medicine to humans and mammals, they may be given alone or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably 0.5 to 90%) of an active ingredient, i.e., at least one therapeutic compound of the present invention and / or its derivatives in combination with a pharmaceutically acceptable carrier.

[0047] Taking into account each typical factor, such as the patient's age, weight, sex, and clinical history, the effective dose of each agent can be easily determined by a technician. Generally, the appropriate daily dose of the compounds of the present invention will be the amount of the lowest dose of compound that effectively produces a therapeutic effect. This effective dose typically depends on the factors mentioned above.

[0048] If necessary, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-dose administered individually at appropriate intervals throughout the day, optionally in unit dosage form.

[0049] The pharmaceutical compositions of the present invention comprise one or more of the compounds of the present invention or their functional derivatives thereof in a “therapeutic effective amount” or “preventative effective amount.” An “effective amount” is an amount as defined in the definition section herein and refers to an amount that is effective at the necessary dose and time period for achieving the desired therapeutic outcome, such as reducing or preventing effects associated with neurogenic and / or inflammatory pain. The therapeutically effective amount of the compounds of the present invention or their functional derivatives thereof can vary depending on factors such as the subject’s disease state, age, sex, weight, and the ability of the therapeutic compound to elicit the desired response in the subject. A therapeutically effective amount is also the amount in which the beneficial therapeutic effect of the therapeutic agent outweighs any of its toxic or harmful effects.

[0050] "Prophylactic effective dose" refers to the amount that is effective in achieving the desired preventive outcome at the necessary dosage and time period. Typically, because the preventive dose is administered to the subject before or early in the course of the disease, the preventive effective dose can be less than the therapeutic effective dose. The preventive or therapeutic effective dose is also the amount at which the beneficial effects of a compound outweigh any of its toxic or harmful effects.

[0051] Dosing regimens can be adjusted to provide the optimal desired response (e.g., therapeutic or preventative response). For example, a single large pill can be administered, several fractionated doses can be administered over time, or the dose can be reduced or increased proportionally as indicated by the urgency of the treatment situation. It is particularly advantageous to prepare parenteral compositions in dose units for ease of administration and dosage uniformity. The actual dose level of the active ingredient in the pharmaceutical compositions of the present invention can be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response for a particular subject, composition, and administration mode, while being non-toxic to the patient.

[0052] As used herein, the term "dosage unit" refers to a physically discrete unit suitable as a single dose to a mammalian subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the desired drug carrier. Specifications of the dosage unit form of the present invention are subject to and directly depend on: (a) the unique characteristics of the compound and (b) the inherent limitations in the art posed by such active compounds for the treatment of individual sensitivities.

[0053] In some embodiments, the therapeutically effective dose may be initially estimated in cell culture assays or in animal models (typically mice, rabbits, dogs, or pigs). Animal models may also be used to determine the desired concentration range and route of administration. This information can then be used to determine the dose and route of administration that would be useful for administration in other subjects. Typically, the therapeutically effective dose is sufficient to reduce or suppress neurogenic and / or inflammatory pain in the subject. In some embodiments, the therapeutically effective dose is sufficient to eliminate neurogenic and / or inflammatory pain in the subject. Those skilled in the art can determine the dose for a particular patient using conventional considerations, such as with the aid of appropriate conventional pharmacology protocols. A physician may, for example, prescribe a relatively low dose initially and then increase the dose until an appropriate response is obtained. Depending on the application, the dose administered to the patient is sufficient to produce a beneficial therapeutic response or, for example, relief of symptoms or other appropriate activity in the patient over time. The dose is determined by the efficacy of the particular formulation, the activity, stability, or serum half-life of the compound of the present invention or its functional derivatives, and the patient’s condition and the weight or surface area of ​​the patient to be treated. The magnitude of the dose is also determined by the presence, nature, and extent of any adverse side effects accompanying the administration of the particular carrier, formulation, etc., in a particular subject. A therapeutic composition comprising one or more compounds of the present invention or functional derivatives thereof may optionally be tested in in vitro and / or in vivo animal models of one or more suitable diseases (such as models of neurological and / or inflammatory pain) to confirm efficacy, tissue metabolism, and estimate dosage, according to methods well known in the art. Specifically, the dosage may be initially determined in relevant assays by other suitable measures of activity, stability, or therapeutic versus non-therapeutic (e.g., comparison of treated versus untreated cell or animal models). The formulation is administered at a rate determined by the LD50 of the relevant formulation and / or by observing any side effects (e.g., on the patient's quality and overall health) at various concentrations of the compound or functional derivative thereof. Administration may be achieved via a single or multiple doses.

[0054] Administration typically involves administering a pharmaceutically acceptable dosage form, which means the dosage form of the compound described herein, and includes, for example, tablets, sugar-coated pills, powders, elixirs, syrups, liquid formulations including suspensions, sprays, inhalers, tablets, lozenges, emulsions, solutions, granules, capsules, and suppositories, as well as liquid formulations for injection (including liposomal formulations). Techniques and formulations are commonly found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference in its entirety. Administration may be oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal. The compound may be administered alone or with a suitable drug carrier and may be in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, or emulsions.

[0055] Pharmaceutical compositions containing an active ingredient can be in forms suitable for oral administration, such as tablets, lozenges, tablets, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard capsules or soft capsules, or syrups or elixirs. Compositions intended for oral administration can be prepared according to any method known in the art for preparing pharmaceutical compositions, and such compositions may contain one or more agents selected from sweeteners, flavoring agents, coloring agents, and preservatives to provide a pharmaceutically palatable formulation. Tablets contain the active ingredient mixed with non-toxic, pharmaceutically acceptable excipients suitable for preparing tablets. For example, these excipients may be inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrants such as corn starch or alginate; binding agents such as starch, gelatin, or gum arabic; and lubricants such as magnesium stearate, stearic acid, or talc. Tablets may be uncoated, or they may be coated using known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a prolonged effect over a longer period of time. For example, time-delaying materials such as glyceryl monostearate or glyceryl distearate can be used. They can also be coated using the techniques described in U.S. Patents 4,256,108, 4,166,452, and 4,265,874 (the contents of each of these U.S. Patents are incorporated herein by reference in their entirety) to form permeable therapeutic tablets for controlled release.

[0056] Oral formulations 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 an aqueous or oily medium (e.g., peanut oil, liquid paraffin, or olive oil).

[0057] The formulation may also contain complexes of parent (unionized) compounds with P-cyclodextrin, particularly derivatives of hydroxypropyl-β-cyclodextrin.

[0058] Alternative oral formulations can be achieved using controlled-release formulations, in which the compound is encapsulated in an enteric coating.

[0059] Aqueous suspensions contain active materials mixed with excipients suitable for preparing aqueous suspensions. Such excipients are suspending agents, such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and gum arabic; dispersants or wetting agents, such as naturally occurring phospholipids (e.g., lecithin), or condensation products of olefin oxides and fatty acids (e.g., polyoxyethylene stearate), or condensation products of ethylene oxide and long-chain fatty alcohols (e.g., heptadecetylacetyl cetyl alcohol), or condensation products of ethylene oxide and esters derived from fatty acids and hexitols (e.g., polyoxyethylene sorbitan monooleate with esters derived from fatty acids and hexitols). Aqueous suspensions may also contain one or more preservatives (e.g., ethylparaben or n-propylparaben), one or more colorants, one or more flavoring agents, and one or more sweeteners (e.g., sucrose or saccharin).

[0060] Oily suspensions can be formulated by suspending the active ingredient in vegetable oils (e.g., peanut oil, olive oil, sesame oil, or coconut oil) or mineral oils (e.g., liquid paraffin). Oily suspensions may contain thickeners such as beeswax, hard paraffin, or cetyl alcohol. Sweeteners (as described above) and flavoring agents may be added to provide palatable oral formulations. These compositions may be preserved by adding antioxidants such as ascorbic acid.

[0061] By adding water, dispersible powders and granules suitable for preparing aqueous suspensions provide active ingredients that can be mixed with dispersants or wetting agents, suspending agents, and one or more preservatives. Examples of suitable dispersants or wetting agents and suspending agents may also include, for example, sweeteners, flavoring agents, and coloring agents.

[0062] These pharmaceutical compositions 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 of these oils. Suitable emulsifiers can be naturally occurring gums (e.g., gum arabic or tragacanth), naturally occurring phospholipids (e.g., soybean, lecithin), and esters or metaesters derived from fatty acids and hexitan anhydrides (e.g., sorbitan monooleate) and condensation products of such metaesters with ethylene oxide (e.g., polyoxyethylene sorbitan monooleate). The emulsion may also contain sweeteners and flavorings.

[0063] Syrups and elixirs can be formulated with sweeteners (e.g., glycerol, propylene glycol, sorbitol, or sucrose). Such formulations may also contain modifiers, preservatives, flavoring agents, and coloring agents. Pharmaceutical compositions can be in the form of sterile injectable aqueous or oily suspensions. Such suspensions can be formulated using suitable dispersants or wetting agents and suspending agents as known in the art. Sterile injectable formulations can also be sterile injectable solutions or suspensions in non-toxic, parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable mediators and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile, non-volatile oils are commonly used as solvents or suspension media. For this purpose, any non-irritating, non-volatile oil, including synthetic monoglycerides or diglycerides, can be used. Furthermore, fatty acids, such as oleic acid, can be used in the preparation of injectable formulations.

[0064] Each active agent can also be administered in suppository form for rectal drug administration. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and thus melts in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycol.

[0065] For topical application, creams, ointments, gels, solutions, or suspensions are suitable. Topical applications include the use of mouthwash and rinsing solutions.

[0066] The term "pharmaceutical composition" means a composition comprising a compound as described herein and at least one component, which includes pharmaceutically acceptable carriers, diluents, adjuvants, excipients, or mediators such as preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, aroma agents, antibacterial agents, antifungal agents, lubricants, and dispersants, depending on the method of administration and the nature of the dosage form. The term "pharmaceuticalally acceptable carrier" is used to mean any carrier, diluent, adjuvant, excipient, or mediator as described herein. Examples of suspending agents include ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum hydroxide, bentonite, agar, and astragalus gum, or mixtures of these substances. Antimicrobial activity can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It is also desirable to include isotonic agents, such as sugars, sodium chloride, etc. Prolonged absorption of injectable drug forms can be achieved by using agents with delayed absorption (e.g., aluminum monosterate and gelatin). Examples of suitable carriers, diluents, solvents, or mediators include water, ethanol, polyols, suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Examples of excipients include lactose, milk sugar, sodium citrate, calcium carbonate, and dicalcium phosphate. Examples of disintegrants include starch, alginate, and certain complex silicates. Examples of lubricants include magnesium stearate, sodium lauryl sulfate, talc, and high molecular weight polyethylene glycol.

[0067] The term "pharmaceutically acceptable salt" refers to salts that, within reasonable medical judgment, are suitable for contact with cells in humans and lower animals without causing excessive toxicity, irritation, allergic reactions, etc., and that are commensurate with a reasonable benefit / risk ratio.

[0068] References Throughout this disclosure, references and citations have been made to other sources, such as patents, patent applications, patent publications, magazines, books, papers, and web content. All of these sources are incorporated herein by reference in their full text for all purposes.

[0069] equivalent Based on the entire contents of this document, including references to scientific and patent literature cited herein, various modifications to the invention and many other embodiments thereof will become apparent to those skilled in the art, in addition to those shown and described herein. The subject matter of this document contains important information, illustrations, and guidance suitable for practicing the invention in different embodiments and equivalents thereof.

[0070] Example Example 1 Research Design Participants with progressive supranuclear palsy (PSP) will receive a single infusion of ex vivo expanded autologous CD4+CD1271o / -CD25+ CAR-T regulatory cells (Tregs). The CAR-Tregs will be engineered to specifically recognize myelin oligodendrocyte protein (MOG), a glycoprotein specifically expressed in the central nervous system (CNS), and to induce immune tolerance and anti-inflammatory effects in the brain.

[0071] The primary objective will be 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.

[0072] The primary outcome measure will be: 1. Adverse events 2. Laboratory abnormalities 3. Infusion reaction 4. Infection-related complications 5. Potential negative impacts on the PSP process A secondary objective will be to evaluate the effects of CNS-specific CAR-Treg on PSP and to obtain indications of its potential application in other neurodegenerative diseases.

[0073] The destination will be: 1. To evaluate the effects of CNS-specific CAR-Treg on clinical, neuropsychological, radiological, and biomechanical parameters in PSP patients.

[0074] 2. To obtain indications of potential therapeutic use of CNS-specific CAR-Tregs in other neurodegenerative diseases, including Alzheimer's disease (AD).

[0075] 3. To obtain indications of potential phase II randomized, double-blind, placebo-controlled trials that may provide valuable insights into the potential efficacy of CAR-Treg for neurodegenerative diseases.

[0076] Patient assessment Clinical and neuropsychological assessments: As previously reported, inclusion and exclusion criteria, as well as clinical (motor and neuropsychological) and neuroimaging assessments, will be described in detail (Giordano, *J. Transl. Med.* 2014; Canesi et al., *J. Transl. Med.* 2016, these references are incorporated herein by reference). Patients will undergo neurological examination to assess motor function 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., *Mov. Disord.* 2004; Golbe et al., *Brain* 2007; the contents of each of these references are incorporated herein by reference). As previously described, the Mini-Mental State Examination (MMSE) will also be performed (Folstein et al., *Journal of Psychiatric Research*, 1975, which is incorporated herein by reference). All of these tests will be assessed at baseline and at each follow-up point (1, 3, 6, and 12 months after cell administration). If the UPDRS and PSP-RS scores decrease by no more than 30% from baseline and the H&Y stage remains unchanged at the defined time points, the clinical condition will be classified as “stable” (Canesi et al., *Journal of Translational Medicine*, 2016, which is incorporated herein by reference).

[0077] Neuroimaging: All patients will undergo longitudinal neuroimaging assessment using brain magnetic resonance imaging (MRI) (baseline, 24 hours after cell administration, and 1 year later), 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 (β-CIT) will be used for SPECT and PET / TC imaging, respectively.

[0078] For SPECT, in all patients, 110–140 MBq [123I] FP-CIT (Datscan, GE-Health, Amersham, UK) will be administered intravenously 30–40 minutes after thyroid blockade (10–15 mg oral Lugol solution). The analysis will be performed as previously described (Isaias et al., NeuroReport 2007, which is incorporated herein by reference). A volumetric template of the gray matter anatomical distribution will be generated from a single-participant brain atlas from MRI at the Montreal Neurological Institute using a macro-anatomical approach (automated anatomical markers) and will be reoriented and reformatted to obtain a 2.64 cm thick reference portion. Templates of eight irregular regions of interest (ROIs) will be manually drawn on this portion to assess the anatomical extent of the striatum and occipital structures with specific and nonspecific uptake of [123I] FP-CIT, respectively. The ROI template will also be located on the reference SPECT portion and adjusted on both the striatal cortex and the occipital cortex. The striatal ROI will also be divided into its anterior (caudate nucleus) and posterior (putamen) portions.

[0079] The specific striatal dopamine uptake transporter (DAT) combination in the entire striatum, putamen, and caudate nucleus of FP-CIT will be calculated using the following formula [123 I]: [(mean count in specific ROI) - (mean count in occipital ROI)] / (mean count in occipital ROI). The putamen / caudate nucleus ratio for each subject will also be calculated.

[0080] Following an intravenous injection of 170 MBq, all patients underwent F-fluoro-2-deoxyglucose positron emission tomography (FDGPET) at rest. Each acquisition included a computed tomography (CT) transmission scan of the head (50 mA, 16 seconds), followed by a 15-minute three-dimensional (3D) static emission scan using a Biograph Truepoint 64 PET / CT scanner (Siemens). The PET portion was reconstructed using an iterative algorithm (OS-EM), corrected for scattering and attenuation using density coefficients derived from low-dose CT scans of the head obtained with the same scanner. Images were reconstructed as 2 mm, 128 Å–128 pixel transaxial images using an iterative algorithm, ordered subset expectation maximization (OSEM). The PET system had a resolution of 4–5 mm FWHM.

[0081] Biomechanical evaluation: Biomechanical evaluation will be assessed at baseline and at 6 and 12 months after CAR-Treg cell administration. Two specific sets of parameters will be automatically extracted using an ad hoc algorithm (Carpinella et al., IEEE TransNeural Syst Rehabil Eng. 2007, which is incorporated herein by reference). One set will be for standing and the other for gait initiation. For standing, mean velocity and spatial displacement of the center of pressure (CoP) will be measured (Canesi et al., Translational Medicine Journal 2016, which is incorporated herein by reference). To examine gait initiation, anticipated postural adjustments (Canesi et al., Translational Medicine Journal 2016, which is incorporated herein by reference) (i.e., the imbalance and unloading phases) will be analyzed and the following parameters will be measured: (1) the duration of both phases, (2) the anterior-posterior (AP) and medial-lateral (ML) displacement and velocity of the CoP, and (3) the mean length and velocity of the CoP. The (4) length and (5) velocity of the first step will also be measured. Spatial parameters will be normalized based on height (BH%).

[0082] Preparation and administration of CAR-Treg cells Treg isolation and expansion: PolyTregs were selected from five individuals with PSP based on three cell surface markers—CD4, CD25, and CD127—and expanded to purify FOXP3+ Tregs present in peripheral blood as previously described (Putnam et al., Diabetes 2009; Bluestone et al., Sci. Transl. Med., 2015, these references are incorporated herein by reference).

[0083] 400 ml of fresh peripheral blood was collected into a blood packaging unit containing citrate-phosphate dextran and processed within 24 hours to separate PBMCs via a Ficoll density gradient. Tregs were separated on a high-speed cell sorter using the following GMP-grade lyophilized antibodies: CD4-PerCP (polydinophyte chlorophyll protein) (L200 clone), CD127-PE (phycoerythrin) (40131 clone), and CD25-APC (allophycocyanin) (2A3 clone). The sorted CD4+CD1271o / -CD25+ cells were collected in 3 ml of X-VIVO 15 medium (Lonza, catalog number 04-418Q) containing 10% human heat-inactivated combined AB serum (Valley Biomedical). The purity of the Tregs was analyzed after sorting. The expected purity of CD4+CD1271o / -CD25+ cells is greater than 96% (Bluestone et al., Science Translational Medicine, 2015, which is incorporated herein by reference).

[0084] As previously described, purified Tregs will be cultured with clinical-grade Dynabeads coated with anti-CD3 and anti-CD28, as well as recombinant IL-2 (Bluestone et al., Science Translational Medicine, 2015, which is incorporated herein by reference). The expected yield per unit blood volume is between 4.2 × 10⁶ and 11.8 × 10⁶ purified CD4+CD1271o / -CD25+ Tregs (Bluestone et al., Science Translational Medicine, 2015, which is incorporated herein by reference). The expanded Treg formulation is expected to achieve approximately 90% FOXP3+. The viability, CD4+ percentage, and CD8+ cell contamination of the Treg formulation will be examined (Bluestone et al., Science Translational Medicine, 2015, which is incorporated herein by reference).

[0085] Phenotypic and TCR analyses of polyTregs were performed after expansion: Following the expansion, key cell surface markers CD4 and CD127, used to isolate Tregs, were examined.

[0086] Previous data have shown that untreated CD45RA+ Tregs preferentially expand in these cultures, and that CD45RA+RO- cells downregulate CD45RA and upregulate CD45RO during the expansion period (Bluestone et al., Science Translational Medicine, 2015, which is incorporated herein by reference). CCR7 (the Treg transport receptor), CD38 (a multifunctional exonuclease associated with enhanced Treg function), and CD45RO will be measured before and after expansion. The TCRP library of expanded Tregs will also be analyzed and compared with a freshly isolated population to determine the polyclonal nature of the expanded Tregs. It is expected that expanded cells will exhibit indistinguishable polyclonal nature from the unexpanded culture, and that the expanded Treg population will maintain a highly diverse population (Bluestone et al., Science Translational Medicine, 2015, which is incorporated herein by reference).

[0087] Functional analysis following the expansion of polyTreg: After Treg expansion, the following assays were performed (Bluestone et al., Science Translational Medicine, 2015, which is incorporated herein by reference): DNA methylation status of the enhancer region of the FOXP3 locus was analyzed to assess the overall purity and stability of the expanded Treg.

[0088] - Cytokine production (IFNγ, IL-4, IL-5, and IL-17) to assess lymphocyte phenotype - In vitro inhibition of activity to determine the functional potential of expanded cells.

[0089] CAR-Treg cell generation and functional analysis: Following published protocols, CAR RNA was optimized for anti-MOG CAR expression in Tregs after electroporation of human Tregs and in mouse Tregs after adoptive transfer to PSP mouse models (Zhao, Y et al., 2010 *Cancer Res*; Beatty, GL et al., 2014 *Cancer Immunol Res*; Singh, N et al., 2014 *Oncoimmunol*; the contents of each of these publications are incorporated herein by reference). Delivery of anti-MOG CARs using second-generation lentiviral vectors and standard protocols (Levine, BL et al., 2017 *Molecular Ther Methods & Clin Dev*, which is incorporated herein by reference) was also optimized. Human Tregs were electroporated with RNA under GMP conditions (approximately 1 μg / 3 × 10⁻⁶). 6Treg) or via lentivirus transduction (1 × 10 6 pfu / 3 × 10 6 Transduction using Treg cells will produce clinical-grade RNA targeting anti-MOGCAR. This is achieved against 2.6 × 10⁻⁶ Treg cells. 9 Each patient will require approximately 0.9 mg of RNA per Treg cell; 5 patients will require 4.5 mg. This will produce clinical-grade lentivirus. Targeting 2.6 × 10⁻⁶ Treg cells. 9 Each patient will require approximately 8.7 × 10 Treg cells. 9 PFU; 5 patients will require 4.3 × 10⁻⁶ pfu. 10 The functional analysis of MOG-specific CAR-Tregs will be performed as described in section 3.3 above.

[0090] Cellular administration Each patient will receive a single dose of MOG-specific CAR-Treg (2.6 × 10⁻⁶ doses per patient). 9 CAR-Treg cells will be administered to at least 5 PSP patients. Patients will receive a pre-operative medication regimen of acetaminophen and diphenhydramine. CAR-Treg will be infused via a peripheral intravenous catheter over 10 to 30 minutes. Vital signs will be taken before and after infusion, then every 15 minutes for at least 1 hour, then every hour for the first 4 hours, and then every 4 hours for 20 hours. A repeat chemistry test and complete blood count will be performed the day before discharge from the clinical study unit. Patients will be followed up on day 4 post-infusion, then weekly for 4 weeks, then every 13 weeks for 1 year, and then every 26 weeks for 2 years. Adverse events will continue to be monitored by telephone every 6 months post-infusion for 5 years, followed by a final clinical visit.

[0091] Patient assessment after CAR-Treg cell infusion The effects of CNS-specific CAR-Treg on clinical, neuropsychological, radiological, and biomechanical parameters in PSP patients will be evaluated as described above. All tests will be performed at each follow-up point: 1 month, 3 months, 6 months, and 12 months after cell administration.

[0092] Example 2 Multiple sclerosis (MS) drugs that utilize components of immunosuppressive T regulatory lymphocytes (Tregs) to shut down the impaired immune response that causes the disease will be developed and tested. The global MS market is worth approximately $21.5 billion, but drugs approved for the most common forms of MS produce only modest disease modifications and have significant side effects. For more severe forms of MS, treatment options are limited to just one recently approved drug.

[0093] There are 11 FDA-approved medications for relapsing-remitting MS (RRMS – accounting for 85% of diagnosed MS). Several oral and antibody-based medications are currently approved for RRMS or are undergoing clinical evaluation for RRMS. In March 2017, the FDA approved olizumab (an anti-CD20 antibody, Roche) for primary progressive MS (PPMS – accounting for approximately 10% of diagnosed MS). Ocrelizumab reduced symptoms by 25% and is currently the only immunomodulatory agent for PPMS in the United States. Secondary progressive MS (SPMS) always develops in patients with RRMS, and disease-modifying options for it are limited.

[0094] Biological production Anti-MOG hybridomas will be generated by immunizing mice with recombinant human MOG via a CRO. The VH and VL genes, as well as the anti-scFv molecule, will be cloned. The orientation of VH and VL, and the linkers (between or within each scFv), can significantly influence the stability, expression level, and binding ability of GTIP. In some cases, only one of these forms will yield a functional molecule. Therefore, several orientations of VH-VL will be expressed on a small scale and tested before large-scale production. An expression construct will be generated that encodes four anti-MOG scFvs with connecting linkers and a central linker, and then ligated to a Treg-associated enzyme or mimicry.

[0095] Validation of GTIP protein products in a mouse MS model GTIP will be tested in mouse models of acute and chronic EAE of MS. Levels of Th1, Th17, and CTLs (blood and CNS) specific for myelin basic protein (MBP), myelitis inflammatory cells (macrophages and neutrophils), and anti-MBP antibodies will be measured. Immunological responses will be correlated with disease progression. Dosage will be varied to explore potential use in advanced MS. The product will be administered to normal mice to explore any potential off-target effects.

[0096] Clinical evaluation Clinical studies will be conducted to test the efficacy of GTIP as a disease modifier in MS. These products will initially be tested in RRMS patients who are unresponsive to first-line drug use. Safety and tolerability will be measured using a dosing regimen similar to that used for antibody therapy (e.g., three intravenous doses every two weeks initially, followed by four-week dosing over 20 weeks). In Phase 2 studies, the primary measures will be the frequency of disease relapses and the reduction in encephalopathy. Secondary measures will be the reduction in blood levels of inflammatory cytokines, Th1 / Th17 cells, and other white blood cells. Side effects may include increased susceptibility to infection. These studies will benchmark the efficacy of the compounds relative to other second-line drugs that reduce the frequency of relapses by up to 49%. If the products demonstrate acceptable levels of efficacy, they will proceed to a long-term Phase 2 clinical study in PPMS patients. The primary measures will be the decline in delayed motor function and the reduction in encephalopathy, and secondary measures will be the reduction in blood levels of inflammatory cytokines, Th1 / Th17 cells, and other white blood cells.

[0097] Example 3 like Figure 3 As shown, the tetramer binding assay will be used to compare the affinity of GITP binding to target cells with that of known tetramers targeting T cells (Ober, B et al., 2000, *International Journal of Immunology*). Two parameters will be determined using cell staining and flow cytometry (FCM) to measure the relative affinity of the HY peptide / MHC H-2Db (pMHC) tetramer for the TCR on B6.2.16 CTLs. These will be parameters that allow the tetramer staining (after cell washing) to reach maximum staining and half-life (t). 1 / 2 The required concentration of MOGs will be determined. MOG target cells will be generated through gene transfection of non-adhesive target cells (e.g., RMA or Jurkat cells). Antibody staining and flow cytometry (FCM) will confirm MOG surface expression. Antibody staining and FCM will identify transfectants with the same MOG levels as B6.2.16 TCRs on CTLs. Figure 4 and 5 As shown, the maximum staining and half-life of MOG target cells with labeled GITP protein will be measured and compared with those of CTL and pMHC tetramers. The goal is to generate GITP with cell interaction affinity comparable to or better than that of CTL and pMHC tetramers. If the tetramer in the GITP molecule does not meet this standard for anti-MOG scFv, the valence of the scFv can be increased. If even higher valence is required, a nanoparticle scaffold can be used to achieve the desired affinity for binding to target cells.

[0098] Example 4 The ability of GTIP bound to MOG target cells to inhibit the proliferation of T effector (T eff) cells will be tested. GTIP, consisting of a tetramer of a given scavenger IE (see Table 1 below), will be bound to MOG target cells, washed, and then incubated with proliferating human Teff (e.g., generated using a standard procedure 3 days after stimulation with anti-CD3 / CD28 and IL-2). Figure 6 (the middle column).

[0099] Table 1

[0100] Cells will be cultured in a medium containing a relevant mitotic-promoting metabolite (M), which is a substrate of IE in GTIP (see table above). Over time, as... Figure 6 As shown, the concentration of M and the number of Teff will be measured. After a fixed time period, the number of GTIP-modified MOG target cells will be titrated relative to the number of Teff to provide an indicator of inhibitory activity. Negative control experiment ( Figure 6 The left column (of which only tetramer scFv binds to MOG target cells) will produce a longer M half-life, a larger amount of Teff after a fixed time, and no inhibitory activity against Teff cell accumulation. As a positive control ( Figure 6 Human Tregs (e.g., those generated under standard conditions 9 days after stimulation with CD3 / CD28 and TGF-β) will be co-cultured with Teff cells and exhibit inhibitory activity compared to GTIP-modified MOG target cells. Cell-to-cell comparable efficacy on a GTIP-modified MOG target cell basis compared to human Treg cells will serve as a positive validation of GTIP molecules with specific IEs. These assays will identify GTIPs composed of the most potent IE molecules. Efficacy can be increased by adding more than one type of IE molecule to the GTIP molecule and / or increasing the valence of the IE molecule.

[0101] Example 5 An assay will be used to measure the ability of CAR molecules expressing scFv specific for MOG to bind Treg cells to MOG-expressing target cells, where the relative affinity approximates the affinity of physiologically significant T cell:target cell interactions. The physiologically significant T cell:target cell interaction used for comparison is the CTL peptide / MHC (pMHC) / target cell interaction. This will be performed using a flow cytometry (FCM)-based assay for cell-cell conjugates (Opferman, JT et al., 2001 *International Journal of Immunology*, which is incorporated herein by reference).

[0102] The relative affinity of the pMHC target of the relevant CTL clone (B6.2.16) will be determined. Figure 7 (Left column). Targets will be labeled with the live dye PKH26 (red), and CTLs with CFSE (green), co-incubated for 4 hours, then subjected to standard shear stress and examined by FCM. Conjugates will be detected as double-staining bimodal and will depend on the presence of the HY peptide antigen. pMHC target: B6.2.16 The relative affinity of CTL interactions will be measured by two parameters—the maximum level of conjugate formation (approximately 80% of total input cells) and the half-life of conjugate dissociation. MOG target cells will be co-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 labeled cells will be measured by FCM. Figure 7 (Right column). The half-life of the conjugate compared to CTL: pMHC / target cells will indicate the physiological affinity of anti-MOG scFv / CAR on T cells for MOG-positive target cells.

[0103] Example 6 scFv antibodies specific to human MOG were generated through affinity panning of human phage-displayed scFv libraries. QC SDS-PAGE was performed prior to library screening to assess target purity. To minimize non-specific binding agents, pre-counter selection was performed using polystyrene flat-bottom plates and blocking buffer specific to the phage library before target screening.

[0104] After three rounds of biological panning, positive enrichment was observed. Twenty clones were randomly selected from the third round and subjected to QC monoclonal phage ELISA. Compared to the control, 18 clones were found to bind to the target. All 18 positive clones were sequenced.

[0105] From the third round, another 20 clones were selected and subjected to a QC monoclonal phage ELISA. Compared to the control, all 20 clones in the second group were found to bind to the target. All of them were then sequenced.

[0106] After analyzing 38 positive clones, seven positive clones with unique sequences were identified (clones 1, 3, 6, 10, 13, 17, and 21). The sequences of the seven scFv proteins and their encoding DNA sequences are listed below: Clone 1 DNA (SEQ ID NO.1):

[0107] Cloned 1 protein (SEQ ID NO.2):

[0108] Cloned DNA 3 (SEQ ID NO.3):

[0109] Cloned 3 protein (SEQ ID NO.4):

[0110] Cloned DNA 6 (SEQ ID NO.5):

[0111] Cloned 6 protein (SEQ ID NO. 6):

[0112] Cloned DNA 10 (SEQ ID NO.7):

[0113] Cloned 10 protein (SEQ ID NO. 8):

[0114] Cloned DNA 13 (SEQ ID NO. 9):

[0115]

[0116] Cloned protein 13 (SEQ ID NO. 10):

[0117] Cloned DNA 17 (SEQ ID NO. 11):

[0118]

[0119] Cloned 17 protein (SEQ ID NO.12):

[0120] Cloned DNA 21 (SEQ ID NO.13):

[0121] Cloned 21 protein (SEQ ID NO.14):

[0122] Expression vectors were constructed for each of the seven scFv proteins. Cell lysates were then coated for ELISA. Soluble ELISA was then performed using the cell lysates at both 30°C and 37°C. Differences were readily observed in all seven clones compared to the control. Of the seven positive clones, clones 1, 6, and 13 were significantly stronger than the others.

[0123] Soluble scFvs generated from seven positive clones were titrated with an ELISA to rank their MOG-binding ability. The seven scFvs were subcloned into pET-26b to construct the scFv-myc-6×His form. The purity of the seven scFvs induced at 16°C was >85%, while the purity induced at 37°C was lower. Therefore, 16°C was determined to be a more suitable production condition. A QC ELISA was performed to analyze the binding ability of each of the seven scFvs to the target MOG. Differences were readily apparent in each of the seven positive clones (clones 1, 3, 6, 10, 13, 17, and 21) compared to the control. Among the seven positive clones, three clones (clones 3, 6, and 17) indicated stronger binding affinity to the target.

[0124] QC ELISA titration was performed on each of the seven clones induced at 16 °C. Seven clones at seven different concentrations were used for ELISA titration. The results indicated that all seven clones could specifically bind to the target MOG. Among the seven clones, clones 3, 6, and 17 still indicated stronger binding affinity to the target. Figure 8 The results are shown in the figure. Figure 8 The study showed that clone 17 exhibited the strongest binding to hMOG1 scFv, followed by clone 6 and then clone 3. The remaining four clones showed significantly weaker binding than clones 17, 6, and 3.

Claims

1. A method for treating a subject with a neurodegenerative disease, the method comprising administering to the subject a therapeutically effective amount of regulatory T cells (Tregs), each regulatory T cell expressing a chimeric antigen receptor (CAR) that specifically binds to a glial cell marker to protect neural tissue and reduce inflammation in the neural tissue, thereby treating the neurodegenerative disease, provided that the neurodegenerative disease is not multiple sclerosis.

2. The method of claim 1, wherein the subject is a human being.

3. The method according to claim 1, wherein the glial cell marker is selected from the group consisting of: oligodendrocyte glycoprotein (MOG), oligodendrocyte marker O1 (OM1), oligodendrocyte marker O4 (OM4), neuroglia / glial marker 2 (NG2), A2B5, galactosylceramidinase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and myelin oligodendrocyte-specific protein (MOSP).

4. The method according to claim 3, wherein the glial cell marker is myelin oligodendrocyte glycoprotein (MOG).

5. The method of claim 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 prions.

6. The method of claim 5, wherein the neurodegenerative disease is progressive supranuclear palsy (PSP).

7. The method of claim 5, wherein the neurodegenerative disease is Alzheimer's disease (AD).

8. The method of claim 5, wherein the neurodegenerative disease is Parkinson's disease (PD).

9. A composition comprising a therapeutically effective amount of a plurality of engineered regulatory T cells (Tregs) for treating a neurodegenerative disease, wherein the neurodegenerative disease is not multiple sclerosis, and each of the plurality of engineered Tregs expresses a chimeric antigen receptor (CAR) that specifically binds to a glial cell marker.

10. The composition of claim 9, wherein the glial cell marker is selected from the group consisting of: myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker O1 (OM1), oligodendrocyte marker O4 (OM4), neuroglia / glial marker 2 (NG2), A2B5, galactosylceramidinase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and myelin oligodendrocyte-specific protein (MOSP).

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