CAR-TREG-BASED THERAPY TARGETING MYELINAL OLIGOdendroglia GLYCOPROTEIN (MOG) FOR TREATING NEURODEGENERATIVE

Abstract

The present invention provides compositions and methods for suppressing autoimmune components of neurodegenerative diseases and 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 the Tregs coupled to chimeric antigen receptors or proteins that specifically bind to one or more glial cell markers. Disclosed are therapeutically effective doses of the compounds for the treatment of neurodegenerative diseases, including progressive supranuclear paralysis (PSP), Parkinson's disease (PD), Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy (CTE), multiple sclerosis, and prion diseases.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 202180059182.8, invention name “CAR-TREG-based therapy targeting myelin oligodendrocyte glycoprotein (MOG) for the treatment of neurodegenerative diseases” and application date May 28, 2021 (PCT application number PCT / US2021 / 034968).

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 031,261, filed May 28, 2020, which is incorporated by reference in its entirety.

[0004] Sequence Listing

[0005] This application contains a sequence listing with XX sequences, which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy created on XX, 2021 is named 48219WO_CRF_sequencelisting.txt and is XXX bytes in size. Technical Field

[0006] The present invention provides CAR-Treg compositions and methods of using the same that specifically regulate immune responses and inflammation associated with various neurodegenerative diseases such as progressive supranuclear palsy and Parkinson's disease. Background Art

[0007] 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, often leading to rapid physical and / or mental decline and death. There is no known cure for those diseases, and treatment focuses on controlling symptoms and slowing decline.

[0008] One such disease, PSP, is an idiopathic degenerative disease not uncommon in the elderly, similar to Parkinson's disease (PD). Clinical manifestations include supranuclear gaze palsy, axial rigidity, dementia, and tetralogy of pseudobulbar palsy. It is associated with bradykinesia, severe postural disorders, and frequent falls. Pathology is associated with cell loss and Tau neurofibrillary tangles, primarily in the brainstem, globus pallidus, subthalamic nucleus, and dentate nucleus. The prevalence of PSP is 5-6 per 100,000 people, resulting in 5000-25000 patients per year in the U.S. The average age of onset of the disease is 63 years old, and the usual prognosis from diagnosis to death is in the range of 5 to 10 years, and there is no available disease-modifying treatment.

[0009] Parkinson's disease is another neurodegenerative disorder with 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 astrocyte death and increased microglia in the substantia nigra, resulting in dopamine deficiency in those areas. Prior to cell death, inclusions known as Lewy bodies form in damaged cells. There has been speculation about the underlying mechanisms driving brain cell death in Parkinson's disease, but the underlying mechanisms remain poorly understood and treatment currently focuses on controlling disease symptoms. Summary of the invention

[0010] The compositions and methods of the present invention use T regulatory lymphocytes (Treg) or immunosuppressive proteins expressed by Treg cells to regulate neurodegenerative immune responses targeting glial cells in the central nervous system (CNS). By coupling Treg or immunosuppressive proteins with chimeric antigen receptors (CAR) or single-chain variable fragments (scFv) that specifically recognize and bind to glial cell markers, immunosuppressive Treg or proteins are attracted to glial cells in the CNS to reduce inflammation and protect the CNS from autoimmune attacks.

[0011] The present invention recognizes the lack of effective treatment options for most neurodegenerative diseases and the presence of autoimmune and / or inflammatory components of several such diseases, and engineers compositions to specifically curb those disease components. The compounds and methods of the present invention allow glial cells to modulate destructive immune cells, such as type 1 helper cells (Th1), T helper 17 cells (Th17), cytotoxic T cells (CTL), M1 macrophages, and polymorphonuclear neutrophils (PMN).

[0012] The present invention guides immunosuppressive molecules (Treg or immunosuppressive proteins) to dendrites (ODC) glial cells. The resulting compounds and methods of use thereof recruit the body's autoimmune 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). The present invention addresses a mechanism (i.e., autoimmune attacks of the central nervous system) by which several neurodegenerative diseases destroy neural function, but do not rely on any specific biochemical causes of the underlying disease. Therefore, the compounds and methods of the present invention can provide therapeutic effects across a variety of neurodegenerative diseases.

[0013] Aspects of the present invention include methods for treating a neurodegenerative disease in a subject, the method comprising administering to the subject a therapeutically effective amount of regulatory T cells (Treg), the regulatory T cells expressing a chimeric antigen receptor (CAR) specifically bound to a glial cell marker, wherein the neurodegenerative disease is multiple sclerosis (MS). CAR-Treg then protects neural tissue and reduces inflammation in neural tissue, thereby treating neurodegenerative diseases. In various embodiments, the subject may be a person.

[0014] The glial cell marker can be oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (OM4), neural / glial marker 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acid protein (GFAP) or myelin oligodendrocyte specific protein (MOSP). In some embodiments, the glial cell marker is myelin oligodendrocyte glycoprotein (MOG).

[0015] The neurodegenerative disease 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), multiple sclerosis 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).

[0016] In some aspects, the present invention provides a composition comprising a therapeutically effective amount of engineered regulatory T cells (Treg) to treat non-multiple sclerosis neurodegenerative diseases, and the engineered Treg expresses a chimeric antigen receptor (CAR) that specifically binds to a glial cell marker. The glial cell marker in the composition can be myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (OM4), neural / glial marker 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP) or myelin oligodendrocyte-specific protein (MOSP).

[0017] The composition can be therapeutically effective to treat progressive supranuclear palsy (PSP), Parkinson's disease (PD), Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy (CTE), multiple sclerosis, or a prion disease.

[0018] 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 Treg can be an extracellular immunosuppressive enzyme. In certain embodiments, the molecule expressed by Treg 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.

[0019] 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), neural / glial marker 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP) or myelin oligodendrocyte-specific protein (MOSP).

[0020] In some aspects, the present invention provides an engineered protein comprising a glial cell-specific binding protein coupled to a molecule simulating the activity of a molecule expressed by a regulatory T cell (Treg). The simulated molecule expressed by Treg can 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 bound by the simulated molecule can bind to myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (OM4), neural / glial marker 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP) or myelin oligodendrocyte-specific protein (MOSP). BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Glial cell-specific CAR-Treg and its immunosuppressive function are shown.

[0022] Figure 2 Glial cells are shown to target immunosuppressive proteins and their immunosuppressive functions.

[0023] Figure 3 The binding of pMHC-tetramers to cytotoxic T cells and the binding of GITP of the present invention to the target MOG protein are shown.

[0024] Figure 4 Shown is maximal staining of MOG target cells with labeled GITP protein compared to CTL and pMHC.

[0025] Figure 5 Shown is the half-life of staining of MOG target cells with labeled GITP protein compared to CTL and pMHC.

[0026] Figure 6 Shown is a comparison of the inhibition of T effector cell proliferation by GTIP-bound MOG-target cells compared to negative and positive controls.

[0027] Figure 7 Shown are the relative avidity of pMHC for the corresponding cytotoxic T cell clones compared to the relative avidity of CAR molecules expressing scFv specific for MOG for MOG-target cells.

[0028] Figure 8 Shown are the relative immunoreactivity of seven different scFv proteins against human MOG-1.

[0029] Fig. 9 scFvs 3, 4, 6 and 17 were shown to bind cell surface MOG-1 protein.

[0030] Fig.10 FACS validation of the binding of scFvs (PMC 669 (clone 17H-L), 670 (clone 17L-H), 696 (clone 3H-L), 697 (clone 3L-H), 698 (clone 6H-L), and 699 (clone 6L-H)) to MOG-1 is shown.

[0031] Fig.11 Shown are cells transduced with PMC671 lentivirus and selected with 2 μg / ml puromycin.

[0032] Fig.12 Depicted constructs PMC691 and PMC692 can successfully express myelin oligodendrocyte glycoprotein both intracellularly and extracellularly.

[0033] Fig.13 CD69 binding assay is shown.

[0034] Fig.14 Depicted are the results of a CD69 binding assay of scFv clone 3 (PMC 696).

[0035] Fig.15 CAR-Treg suppression assay is shown.

[0036] Fig.16 This is a vector map of PMC 669: [clone 17H-LscFv]-CD8-CD28-CD3z with a FLAG tag.

[0037] Fig.17 This is a vector map of PMC 670: [clone 17L-HscFv]-CD8-CD28-CD3z with a FLAG tag.

[0038] Fig.18 This is the vector map of PMC 691.

[0039] Fig.19 This is the vector map of PMC 692.

[0040] Fig. 20 This is a vector map of PMC 696: [clone 3H-LscFv]-CD8-CD28-CD3z with a FLAG tag.

[0041] Fig.21 This is a vector map of PMC 697: [clone 3L-HscFv]-CD8-CD28-CD3z with a FLAG tag.

[0042] Fig. 22 This is a vector map of PMC 698: [clone 6H-LscFv]-CD8-CD28-CD3z with a FLAG tag.

[0043] Fig.23 This is a vector map of PMC 699: [clone 6L-HscFv]-CD8-CD28-CD3z with a FLAG tag.

[0044] Fig.24 This is the vector map of the scFv4[8-18C5]CAR sequence.

[0045] Fig.25 This is the vector map of the scFv4[18C5-8]CAR sequence.

[0046] Fig.26A -D shows the alignment of the first eight scFV amino acid sequences and cassettes. DETAILED DESCRIPTION

[0047] The present invention relates to compositions for regulating 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 prion diseases.

[0048] The blood-brain barrier can serve as an obstacle to treating brain or CNS disorders because the barrier can prevent therapeutic compounds from accessing affected cells. Importantly, Tregs are able to cross the blood-brain barrier and can localize to neurons of the CNS via Treg-bound glial cells, allowing the compounds of the invention to effectively treat neurodegenerative disorders of the CNS.

[0049] The compounds and methods of the present invention do not rely on any disease-specific biochemical mechanism, but rather short-circuit the immune response that affects the mental and physical decline of many neurodegenerative diseases. Therefore, the same compounds and methods can provide therapeutic benefits for multiple neurodegenerative diseases.

[0050] For example, PSP involves accumulation of tau protein and neurofibrillary tangles, and leads to neuronal and glial cell damage and loss, associated physical and mental decline and eventual death. Parkinson's disease involves neuronal loss in the basal ganglia and astrocyte death and microglia increase in the substantia nigra. Before cell death, inclusions known as Lewy bodies form in damaged cells. ALS is characterized by the death of motor neurons in the motor cortex after the formation of protein-rich inclusions in their cell bodies and axons.

[0051] The present invention recognizes that, despite the different 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 prion diseases, may include immune components that contribute to 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 which are incorporated herein by reference. Therefore, the compounds and methods of the present invention, which focus on curbing immune responses in the CNS and addressing chronic inflammation that drives many neurodegenerative diseases, can be therapeutically effective in treating many of those diseases.

[0052] The compounds and methods of the present invention use chimeric antigen receptors (CARs), antibodies or single-chain variable fragments (scFv) 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 a variety of functions to support neurons in the central and peripheral nervous systems of various animals including humans. Glial cells include oligodendrocytes, astrocytes, ependymal cells, and microglia. Due to their function in maintaining neurons in the CNS, glial cells migrate to neurons in the CNS and can therefore be used to locate therapeutic compounds there. For example, oligodendrocytes (ODC) glial cells are transported to the CNS to maintain axon insulation by forming myelin sheaths. The compounds and methods of the present invention include coupling immunosuppressive molecules to glial cells such as ODCs, so that when glial cells perform their functions, the immunosuppressive molecules are in close proximity to neurons in the CNS, such as Figure 1 and 2 The presence of immunosuppressive molecules regulates any ongoing immune responses and chronic inflammation that may be present in the CNS and contribute to neurodegenerative disease symptoms in PD, PSP, etc.

[0053] Glial cell-specific targets include proteins expressed by various glial cells and other markers such as myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (OM4), neural / glial marker 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), or myelin oligodendrocyte-specific protein (MOSP).

[0054] In various embodiments, CAR, scFv or antibody can be bound to immunosuppressive molecules and used to target glial cells. CAR is an engineered receptor that can provide specificity for immune effector cells (T cells). CAR has been used to confer tumor cell specificity to cytotoxic T lymphocytes used in cancer immunotherapy. See, Couzin-Frankel, 2013, Cancer immunotherapy, Science, 342 (6165): 1432-33; Smith et al., 2016, Chimericantigen receptor (CAR) T cell therapy for mal ignant cancers: Summary and perspective, Journal of Cellular Immunotherapy, 2 (2): 59-68; The contents of each are incorporated herein by reference. Using similar principles, the compounds and methods of the present invention include engineering CARs that are specific for markers found on glial cells such as ODCs, but instead of transplanting glial cell-specific CARs to cytotoxic T cells, the glial cell-specific CARs are transplanted to engineered immunosuppressive Tregs.

[0055] The CAR-Treg of the present invention may express multiple chimeric antigen receptors targeting the same or two or more different glial cell markers.

[0056] ScFv is a fusion protein comprising the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin. ScFv can be produced by cloning the VH and VL genes of mice or other animals immunized with the desired target molecule (e.g., MOG). The VH and VL genes can then be expressed in multiple orientations and formed with various linkers to form various scFvs, which can then be experimentally validated to provide the desired stability, expression level, and binding affinity to glial cells or their specific markers. The ScFv or antibodies specific for glial cell markers discussed above can be linked to the immunosuppressive proteins discussed below to form fusion proteins capable of providing CNS-localized immunosuppressive therapy, such as Figure 2 As shown and discussed below.

[0057] Antibodies targeted to glial markers can be generated by methods known in the art, including commercially available services for generating custom antibodies from, for example, Pacific Immunology (San Diego, CA) or ABclonal (Woburn, MA).

[0058] CAR-Treg can be engineered by known methods for preparing CAR-T cells. Treg cells can be separated from subjects, preferably from autologous Treg cells of patients to be treated. Then the genes of Treg cells can be modified by known techniques such as electroporation, viral vectors or other forms of nucleic acid transfection encoding selected engineered chimeric antigen receptors. Allogeneic cells (i.e., cells that are not HLA matched or only partially matched with the subject) can also be used for the methods and treatments described herein. The source of such allogeneic cells includes peripheral blood mononuclear cells (PBMC). PBMC can be separated by Ficol l-Hypaque density gradient centrifugation of samples obtained from discarded, de-identified leukocyte reduction filters (American Red Cross) or blood donations from informed consent healthy volunteers. Cell populations, sources and descriptions of methods for selecting or enriching desired cell types can be found, for example, in U.S. Patent No. 9,347,044. CAR-Treg cells can then be verified by experiments before the system of the patient is introduced for treatment.

[0059] Regulatory T cells, or Tregs, regulate the immune system and often 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 inhibitory cytokines, including transforming growth factor beta, interleukin 35, and interleukin 10, and can induce other cell types to express interleukin 10. Tregs can also produce granzyme B, which in turn can induce apoptosis of effector cells. Tregs also act by reverse signaling and induction of the immunosuppressive indoleamine 2,3-dioxygenase through direct interactions with dendritic cells. Tregs can also downregulate immune responses through the production of the exoenzymes CD39 and CD73 and immunosuppressive adenosine. Tregs also suppress immune responses through direct interactions with dendritic cells through LAG3 and TIGIT. Another control mechanism is through the IL-2 feedback loop. Another immunosuppressive mechanism of Tregs is through preventing CD28 co-stimulation of effector T cells through the action of the molecule CTLA-4.

[0060] Figure 1CAR-Treg targeting glial cells and its therapeutic mechanism are shown. CAR-Treg cells express CARs that specifically bind to markers on glial cells. CAR-Treg cells thus bind to glial cells and cross the blood-brain barrier and are located in neurons of the CNS through the natural functions of glial cells. The bound Treg cells then perform their natural regulatory functions by curbing immune attacks on local neurons.

[0061] Figure 2 The glial cell targeted immunosuppressive protein (GTIP) of the present invention is shown to suppress the immune attack of neurons. GTIP may include immunosuppressive proteins or enzymes present in Treg cells, such as extracellular enzymes that clear immune activation metabolites (e.g., ATP, AMP, tryptophan and glutamate). Such extracellular enzymes may include CD73, CD39, indoleamine 2,3-dioxygenase (IDO) and glutamate-oxaloacetate aminotransferase 1 (GOT1). In Figure 2 In the present invention, MOG-expressing glial cells are bound by GTIP, which consists of an anti-MOG scFV linked to an immunosuppressive enzyme (IE). Glial cells localize the bound IE to neurons that are under immune attack by various immune cells (Th17 cells, Th1 cells, CTL cells, M1 cells, and PMN cells) while performing their neuron-related functions, and regulate or shut down the immune response, thereby protecting neurons and reducing the 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 prion diseases.

[0062] The GTIP of the present invention may comprise one or more immunosuppressive proteins (including two or more different proteins) connected to one or more scFv or antibodies targeting the same or two or more different glial cell markers. Proteins may be connected by any known means to form the GTIP of the present invention, including, for example, fusion proteins or biotin-streptavidin linkages.

[0063] Adoptive cell transfer techniques such as those used in cancer immunotherapy techniques (including those involving cytotoxic T lymphocytes) can be used to prepare autologous CAR-Tregs for use in the compounds and methods of the present invention. See, Rosenberg et al., 2008, Adoptive cell transfer: a clinical path to effective cancer immunotherapy, Nat Rev Cancer, 8(4): 299-308, the contents of which are incorporated herein by reference.

[0064] The CAR-Treg or glial cell targeted immunosuppressive protein of the present invention can be incorporated into a carrier system containing one or more therapeutic compounds described herein. In certain embodiments, the carrier system can be a nanoparticle comprising disulfide cross-linked polyethyleneimine (CLPEI) and lipids. The lipid can be a bile acid, 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.

[0065] The phrases "parenteral administration" and "administered parenterally" as used herein mean modes of administration other than enteral administration and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0066] As used herein, the phrases "systemic administration," "administered systemically," "peripheral administration," and "peripherally administered" mean administration of a compound, drug, or other material other than directly into the central nervous system so that it enters the patient's system and thereby undergoes metabolism and other similar processes, such as subcutaneous administration.

[0067] When the compounds of the present invention are administered to humans and mammals as medicines, they can be administered per se or as a pharmaceutical composition containing, for example, 0.1% to 99.5% (more preferably 0.5% to 90%) of active ingredient (i.e., at least one therapeutic compound of the present invention and / or its derivatives) in combination with a pharmaceutically acceptable carrier.

[0068] The effective dose of each agent can be easily determined by a skilled technician taking into account typical factors such as the patient's age, weight, sex and clinical history. In general, a suitable daily dose of a compound of the invention will be the amount of the lowest dose that effectively produces a therapeutic effect of the compound. This effective dose will generally depend on the above factors.

[0069] If desired, the effective daily dose of active compound may optionally be administered in unit dosage form as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day.

[0070] The pharmaceutical compositions of the present invention comprise a "therapeutically effective amount" or a "prophylactically effective amount" of one or more compounds of the present invention or their functional derivatives. "Effective amount" is an amount as defined herein in the definition section, and refers to an amount that is effective to achieve the desired therapeutic outcome (e.g., to reduce or prevent the effects associated with neuropathic and / or inflammatory pain) within the necessary dosage and time period. The therapeutically effective amount of a compound of the present invention or its functional derivative may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the therapeutic compound to elicit the desired response in the subject. A therapeutically effective amount is also an amount in which the therapeutic beneficial effects outweigh any toxic or deleterious effects of the therapeutic agent.

[0071] A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result at the necessary dosage and time period. Typically, because a prophylactic dose is used in a subject before disease or at an early stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount. A prophylactically or therapeutically effective amount is also an amount in which the beneficial effects outweigh any toxic or deleterious effects of the compound.

[0072] An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired result.

[0073] The dosage regimen may be adjusted to provide the optimal 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 dose may be proportionally reduced or increased as indicated by the urgency of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention may be varied in order to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration without being toxic to the patient.

[0074] The term "dosage unit" as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the compound, and (b) the limitations inherent in the art of compounding such an active compound for the sensitivity of the individual to be treated.

[0075] In some embodiments, the therapeutically effective amount can be initially estimated in a cell culture assay or in an animal model (typically a mouse, rabbit, dog or pig). Animal models are also used to achieve desirable concentration ranges and routes of administration. This information can then be used to determine useful doses and routes for administration in other subjects. Typically, 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.

[0076] The dosage for a particular patient can be determined by a person of ordinary skill in the art using conventional considerations (e.g., with the aid of an appropriate conventional pharmacology regimen). A physician may, for example, first formulate a relatively low dose, then increase the dose until an appropriate response is obtained. Depending on the application, the dose applied to the patient is sufficient to achieve a beneficial therapeutic response in the patient over time, or, for example, to reduce symptoms, or other appropriate activity. The dosage is determined by the efficacy of a particular preparation and the activity, stability or serum half-life of the compound of the present invention or its functional derivative and the patient's condition and the weight or surface area of ​​the patient to be 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 carrier, formulation, etc. in a particular subject. The therapeutic composition comprising one or more compounds of the present invention or its functional derivative is optionally tested in one or more appropriate in vitro and / or in vivo animal models of the disease, such as models of neuropathic and / or inflammatory pain to confirm efficacy, tissue metabolism, and estimate dosage according to methods well known in the art. In particular, the dosage can initially be determined by activity, stability, or other appropriate measures (e.g., comparison of treatment to untreated cells or animal models) of treatment versus non-treatment in a relevant assay. The formulation is administered at a rate determined by the LD50 of the relevant formulation and / or the observation of any adverse effects of various concentrations of the compound of the invention or its functional derivative, such as on the patient's quality and overall health when used. Administration may be accomplished via a single dose or divided doses.

[0077] Administration generally involves administration of pharmaceutically acceptable dosage forms, which means dosage forms of the compounds described herein, and includes, for example, tablets, lozenges, powders, elixirs, syrups, liquid preparations, including suspensions, sprays, inhalants, tablets, lozenges, emulsions, solutions, granules, capsules and suppositories, and liquid preparations for injection, including liposomal preparations. Techniques and formulations can generally be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., the latest version, which is hereby incorporated by reference in its entirety. Administration can be performed orally, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously or intranasally. The compound can be administered alone or with a suitable pharmaceutical carrier, and can be in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions or emulsions.

[0078] Pharmaceutical compositions containing active ingredients may be in a form suitable for oral use, for example in the form of 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 of pharmaceutical composition manufacture, and such compositions may contain one or more agents selected from sweeteners, flavoring agents, colorants and preservatives, so as to provide pharmaceutically excellent and palatable preparations. Tablets contain active ingredients mixed with non-toxic pharmaceutically acceptable excipients suitable for making tablets. These excipients may be, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating agents and disintegrants such as corn starch or alginic acid; binders 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 by known techniques to delay disintegration and absorption in the gastrointestinal tract, and thereby provide sustained action over a longer period of time. For example, a time-delay material such as glyceryl monostearate or glyceryl distearate may be used. They may also be coated by the techniques described in U.S. Pat. Nos. 4,256,108, 4,166,452 and 4,265,874 (each of which is incorporated herein by reference in its entirety) to form an osmotic therapeutic tablet for controlled release.

[0079] Formulations 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 peanut oil, liquid paraffin or olive oil.

[0080] Formulations may also include complexes of the parent (non-ionized) compound with a β-cyclodextrin derivative, especially hydroxypropyl-β-cyclodextrin.

[0081] An alternative oral formulation may be achieved using a controlled release formulation in which the compound is encapsulated in an enteric coating.

[0082] Aqueous suspensions contain active materials mixed with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum arabic; dispersants or wetting agents, such as naturally occurring phospholipids (e.g., lecithin), or condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), or condensation products of ethylene oxide with long-chain fatty alcohols (e.g., heptadecaethyleneoxyhexadecanoic acid), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol, such as polyoxyethylene with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). Aqueous suspensions may also contain one or more preservatives (e.g., ethyl or n-propyl p-hydroxybenzoate), one or more coloring agents, one or more flavoring agents, and one or more sweeteners (e.g., sucrose or saccharin).

[0083] Oily suspensions can be prepared by suspending the active ingredient in a vegetable oil (e.g., peanut oil, olive oil, sesame oil, or coconut oil) or a mineral oil (e.g., liquid paraffin). Oily suspensions may contain thickeners, such as beeswax, hard paraffin, or cetyl alcohol. Sweeteners (e.g., those described above) and flavorings may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

[0084] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified, for example, sweetening, flavoring and coloring agents may also be present.

[0085] 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 of these. Suitable emulsifiers can be naturally occurring gums, such as gum arabic or tragacanth; naturally occurring phospholipids, such as soybeans, lecithin, and esters or partial esters (e.g., sorbitan monooleate) derived from fatty acids and hexitol anhydrides; and condensation products of the partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion can also contain sweeteners and flavorings.

[0086] Syrups and elixirs can be formulated with sweeteners, such as glycerol, propylene glycol, sorbitol or sucrose. Such preparations may also contain demulcents, preservatives and flavoring agents and coloring agents. Pharmaceutical compositions may be in the form of sterile injectable aqueous or oily suspensions. Such suspensions may be prepared according to known techniques using suitable dispersants or wetting agents and suspending agents as mentioned above. Sterile injectable preparations may also be in the form of sterile injectable solutions or suspensions in non-toxic parenteral acceptable diluents or solvents, for example as solutions in 1,3-butanediol. Acceptable vehicles and solvents that may be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvents or suspension media. For this purpose, any mild fixed oil may be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid may be used to prepare injectables.

[0087] The term "pharmaceutical composition" means a composition comprising a compound as described herein and at least one component including a pharmaceutically acceptable carrier, diluent, adjuvant, excipient or vehicle, such as a preservative, filler, disintegrant, wetting agent, emulsifier, suspending agent, sweetener, flavoring agent, fragrance, antibacterial agent, antifungal agent, lubricant and dispersant, depending on the mode of administration and the nature of the dosage form.

[0088] The term "pharmaceutically acceptable carrier" is used to mean any carrier, diluent, adjuvant, excipient or vehicle as described herein. Examples of suspensions include ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth or mixtures of these substances. Prevention of microbial action 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. Extended absorption of injectable drug forms can be caused by using agents that delay absorption (such as aluminum monostearate and gelatin). Examples of suitable carriers, diluents, solvents or vehicles 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, alginic acid and certain complex silicates.Examples of lubricants include magnesium stearate, sodium lauryl sulfate, talc and high molecular weight polyethylene glycols.

[0089] The term "pharmaceutically acceptable" means that it is suitable, within the scope of sound medical judgment, for use in contact with cells of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and is commensurate with a reasonable benefit / risk ratio.

[0090] Example

[0091] Example 1

[0092] The tetramer binding assay will be used to compare the affinity of GITP binding to target cells with the affinity of known tetramers for T cells (Ober, B et al., 2000 Int Immunol, incorporated herein by reference), as Figure 3 The relative affinity of the HY peptide / MHCH-2Db (pMHC) tetramer for the TCR on B6.2.16CTL will be measured by determining two parameters using cell staining and flow cytometry (FCM). These will be the half-life (t) to obtain maximum staining and tetramer staining (after cell washing). 1 / 2 ) at the desired concentration. MOG target cells will be generated by gene transfection of non-adherent target cells (e.g., RMA or Jurkat cells). Antibody staining and flow cytometry (FCM) will confirm surface expression of MOG. Antibody staining and FCM will identify transfectants with the same MOG levels as the B6.2.16 TCR on CTL. Maximum staining and staining half-life of MOG target cells with labeled GITP protein will be measured and compared to CTL and pMHC tetramers, as Figure 4 and 5 The goal would be to generate GITP with comparable or better cell interaction avidity than CTL and pMHC tetramers. If the tetrameric anti-MOG scFv in the GITP molecule does not meet this standard, the valency of the scFv can be increased. If even higher valency is desired, the nanoparticle scaffold can be used to achieve the desired avidity for target cell binding.

[0093] Example 2

[0094] The ability of GTIPs bound to MOG target cells to suppress T effector (T eff) cell proliferation will be tested. GTIPs consisting of tetramers 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 standard procedures after 3 days of anti-CD3 / CD28 and IL-2 stimulation) ( Figure 6 middle column).

[0095] Table 1

[0096]

[0097] Cells will be cultured in medium spiked with relevant mitogenic metabolites (M) that are substrates for IE in GTIP (see table above). Over time, the concentration of M and the amount of Teff will be measured, as Figure 6 After a fixed time, the number of GTIP-modified MOG target cells will be titrated against the number of Teff to give an index of inhibitory activity. Negative control experiment ( Figure 6 ), where only tetrameric scFv binding to MOG target cells will result in a longer M half-life, a larger amount of Teff after a fixed time, and no inhibitory activity on Teff cell accumulation. As a positive control ( Figure 6 The right column of the table shows that human Tregs (e.g., generated under standard conditions after 9 days of CD3 / CD28 and TGF-β stimulation) will be co-cultured with Teff cells and have suppressive activity compared to GTIP-modified MOG target cells. Comparable efficacy of GTIP-modified MOG target cells and human Treg cells on a cell-to-cell basis will serve as a positive validation of GTIP molecules with specific IEs. The assay will identify GTIPs composed of the most effective IE molecules. Efficacy can be improved by adding more than one type of IE molecule to the GTIP molecule and / or increasing the valence of the IE molecule.

[0098] Example 3

[0099] A kind of assay is used to measure the CAR molecule expressing scFv specific to MOG to make Treg cells bind to the ability of target cells expressing MOG with a relative affinity close to the relative affinity of the relative affinity of the physiologically significant T cell: target cell interaction. Physiologically significant T cells for comparison: target cell interaction cells are CTL: peptide / MHC (pMHC) / target cell interactions. This is done using a flow cytometry (FCM) assay based on cell-cell conjugates (Opferman, JT et al., 2001 Int Immunol., incorporated herein by reference).

[0100] Determine the relative affinity of the pMHC target for the relevant CTL clone (B6.2.16) Figure 7 Left column). The targets were labeled with the vital dye PKH26 (red) and the CTLs were labeled with CFSE (green), co-incubated for 4 hours, then subjected to standard shear forces and examined by FCM. The conjugates were detected as dual-stained doublets and were dependent on the presence of the HY peptide antigen. The relative affinity of the pMHC target: B6.2.16 CTL interaction was measured by two parameters - the maximum level of conjugate formation (approximately 80% of the total input cells) and the half-life of conjugate dissociation. MOG target cells were incubated with human T cells expressing CAR-anti-MOG scFv generated under standard conditions (e.g., lentiviral transduction of anti-CD3 / CD28IL-2 stimulated T cells). The half-life of the conjugate between labeled cells will be measured by FCM ( Figure 7 Comparable conjugate half-life and CTL:pMHC / target cell half-life would indicate the physiological avidity of the anti-MOG scFv / CAR T cells for MOG-positive target cells.

[0101] Example 4

[0102] ScFv antibodies specific for human MOG were generated by affinity panning of a human phage-displayed scFv library. QC SDS-PAGE was performed prior to library screening to assess the purity of the target. To reduce nonspecific binders, pre-counter selection was first performed using polystyrene flat-bottom plates and blocking buffer against the phage library prior to targeted screening.

[0103] After three rounds of bio-panning, positive enrichment was observed. 20 clones were randomly picked from the third round and subjected to QC monoclonal phage ELISA. 18 clones were found to bind to the target compared to the control. All 18 positive clones were sequenced.

[0104] Another 20 clones were picked from round 3 and subjected to QC monoclonal phage ELISA. All 20 of the second set of clones were found to bind to the target compared to the control. All of these were sequenced.

[0105] After analysis of 38 positive clones, 8 positive clones with unique sequences were identified (clones 1, 3, 4, 6, 10, 13, 17, 21). The sequences of the 8 scFv proteins and the DNA sequences encoding them are listed below.

[0106] Table 2

[0107]

[0108]

[0109] Table 3

[0110]

[0111]

[0112] Clone 1 DNA (SEQ ID NO: 1):

[0113] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTTCTGATTATGGTAATACTACAGCTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAATGCTAATTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACTTCTACTTATCCTGGTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0114] Clone 1 scFv Protein (SEQ ID NO:2):

[0115] MEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISDYGNTTAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNANYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTSTYPGTFGQGTKVEIK

[0116] Clone 3 DNA (SEQ ID NO: 3):

[0117] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTCTTCTTATGGTTCTTATACAGGTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAATGGTTATGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCGCTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAATGATGCTTCTCCTAATACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0118] Clone 3 scFv Protein (SEQ ID NO:4):

[0119] MEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISSYGSYTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNGYAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNDASPNTFGQGTKVEIK

[0120] Clone 6 DNA (SEQ ID NO: 5):

[0121] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTTCTACTTATGGTGATTATACAACTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGTAGTTATACTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCTGCATCCTATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTCTAATGCTACTCCTTCTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0122] Clone 6 scFv Protein (SEQ ID NO:6):

[0123] MEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISTYGDYTTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSYTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYSASYLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNATPSTFGQGTKVEIK

[0124] Clone 10 DNA (SEQ ID NO: 7):

[0125] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTACTAATTATGGTTATACTACATATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATCTTCTTATTCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCGCTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTCTGCTTATTATCCTGATACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0126] clone scFv 10 Protein (SEQ ID NO: 8):

[0127] MEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGITNYGYTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSYSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSAYYPDTFGQGTKVEIK

[0128] Clone 13 DNA (SEQ ID NO: 9):

[0129] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAATTCTGCTGGTGGTTCTACATATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTATATTACTGTGCGAAAAATTCTGCTTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACTGATACTTATCCTACTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0130] Clone 13 scFv Protein (SEQ ID NO: 10):

[0131] MEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSINSAGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNSAYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTDTYPTTFGQGTKVEIK

[0132] Clone 17 DNA (SEQ ID NO: 11):

[0133] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTTCTACTTCTGGTAGTTATACAGCTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGTGGTTATACTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTGATGGTAATCCTACTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0134] Clone 17 scFv Protein (SEQ ID NO: 12):

[0135] MEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISTSGSYTAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGYTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYSASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSDGNPTTFGQGTKVEIK

[0136] Clone 21 DNA (SEQ ID NO: 13):

[0137] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCCTCTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGCGCAGCCTCTGGATTCCACCTTTAGCAGCTATGCCATGACCGGGTCCCCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTTCGAATCGGGGTAAGTAGACAATTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAACATAATGCGCATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGGCATCCAGGTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTGCGATGGTGCCTCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0138] Clone 21 scFv Protein (SEQ ID NO: 14):

[0139] MEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISYSGAYTAYADSVNGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSGTDFDYWGQGTLVTVSSGGG GSGGGGSGGGGSTDIQMTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNYDPSTFGQGTKVEIK

[0140] An expression vector was constructed for each of the eight scFv proteins. Afterwards, the cell lysate was packaged for ELISA. Soluble ELISA was then performed using cell lysates from 30°C and 37°C. Differences were easily observed in all 7 clones compared to the control. Among the 7 positive clones, clones 1, 6, and 13 were much stronger than the other clones.

[0141] Soluble scFvs produced from seven positive clones were subjected to ELISA and titration to rank their ability to bind MOG. The seven scFvs were subcloned into pET-26b to be constructed as scFv-myc-6×His formats. The expression cassettes of the seven scFv clones are shown below, with each corresponding scFv polynucleotide or amino acid sequence underlined:

[0142] Clone 1 cassette DNA (SEQ ID NO: 15):

[0143] ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCC ATG GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGG ATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTT CTGATTATGGTAATACTACAGCTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAAC ACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAATGCTAATTATTT TGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTG GCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACT TGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGAT CTATGATGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCA CCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACTTCTACTTATCCTGGTACGTTCGGC CAAGGGACCAAGGTGGAAATCAAAGAATTC GAGCAGAAACTCATCTCAGAAAGAGGATCTGCTCGAGCACCACCACCACCACCACTGA

[0144] Clone 1 cassette protein (SEQ ID NO:16):

[0145] MKYLLPTAAAGLLLLAAQPAMA MEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE WVSSISDYGNTTAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNANYFDYWGQGTLVTVSSGGGGSGG GGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTSTYPGTFGQGTKVEIKEF EQKLISEEDLLEHHHHHH

[0146] Clone 3 cassette DNA (SEQ ID NO:17):

[0147] ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCC ATG GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGG ATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTT CTTCTTATGGTTCTTATACAGGTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAAC ACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAATGGTTATGCTTT TGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTG GCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACT TGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGAT CTATGGTGCATCCGCTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCA CCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAATGATGCTTCTCCTAATACGTTCGGC CAAGGGACCAAGGTGGAAATCAAAGAATTC GAGCAGAAACTCATCTCAGAAGAGGATCTGCTCGAGCACCACCACCACCACCACTGA

[0148] Clone 3 cassette protein (SEQ ID NO:18):

[0149] MKYLLPTAAAGLLLLAAQPAMA MEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE WVSSISSYGSYTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNGYAFDYWGQGTLVTVSSGGGGSGG GGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASALQSGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQNDASPNTFGQGTKVEIKEF EQKLISEEDLLEHHHHHH

[0150] Clone 6 cassette DNA (SEQ ID NO:19):

[0151] ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCC ATG GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGG ATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTT CTACTTATGGTGATTATACAACTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAAC ACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGTAGTTATACTTT TGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTG GCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACT TGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGAT CTATTCTGCATCCTATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCA CCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTCTAATGCTACTCCTTCTACGTTCGGC CAAGGGACCAAGGTGGAAATCAAAGAATTC GAGCAGAAACTCATCTCAGAAGAGGATCTGCTCGAGCACCACCACCACCACCACTGA

[0152] Clone 6 heavy chain protein (SEQ ID NO:20):

[0153] MKYLLPTAAAGLLLLAAQPAMA MEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE WVSTISTYGDYTTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSYTFDYWGQGTLVTVSSGGGGSGG GGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYSASYLQSGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQSNATPSTFGQGTKVEIKEFEQKLISEEDLLEHHHHHH

[0154] Clone 10 DNA (SEQ ID NO:21):

[0155] ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCC ATG GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGG ATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTA CTAATTATGGTTATACTACATATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAAC ACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATCTTCTTATTCTTT TGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTG GCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACT TGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGAT CTATGCTGCATCCGCTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCA CCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTCTGCTTATTATCCTGATACGTTCGGC CAAGGGACCAAGGTGGAAATCAAAGAATTC GAGCAGAAACTCATCTCAGAAGAGGATCTGCTCGAGCACCACCACCACCACCACTGA

[0156] Clone 10 Box Protein (SEQ ID NO:22):

[0157] MKYLLPTAAAGLLLLAAQPAMA MEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE WVSGITNYGYTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSSYSFDYWGQGTLVTVSSGGGGSGG GGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASALQSGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQSAYYPDTFGQGTKVEIKEF EQKLISEEDLLEHHHHHH

[0158] Clone 13 Box DNA (SEQ ID NO:23):

[0159] ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCC ATG GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGG ATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTA ATTCTGCTGGTGGTTCTACATATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAAC ACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTATATTACTGTGCGAAAAATTCTGCTTATTT TGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTG GCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACT TGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGAT CTATGATGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCA CCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACTGATACTTATCCTACTACGTTCGGC CAAGGGACCAAGGTGGAAATCAAAGAATTC GAGCAGAAACTCATCTCAGAAGAGGATCTGCTCGAGCACCACCACCACCACCACTGA

[0160] Clone 13 cassette protein (SEQ ID NO:24):

[0161] MKYLLPTAAAGLLLLAAQPAMA MEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE WVSSINSAGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNSAYFDYWGQGTLVTVSSGGGGSGG GGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASNLQSGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQTDTYPTTFGQGTKVEIKEF EQKLISEEDLLEHHHHHH

[0162] Clone 17 cassette DNA (SEQ ID NO:25):

[0163] ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCC ATG GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGG ATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTT CTACTTCTGGTAGTTATACAGCTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAAC ACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGTGGTTATACTTT TGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTG GCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACT TGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGAT CTATTCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCA CCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTGATGGTAATCCTACTACGTTCGGC CAAGGGACCAAGGTGGAAATCAAAGAATTC GAGCAGAAACTCATCTCAGAAGAGGATCTGCTCGAGCACCACCACCACCACCACTGA

[0164] Clone 17 cassette protein (SEQ ID NO:26):

[0165] MKYLLPTAAAGLLLLAAQPAMA MEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE WVSSISTSGSYTAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGYTFDYWGQGTLVTVSSGGGGSGG GGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYSASTLQSGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQSDGNPTTFGQGTKVEIKEF EQKLISEEDLLEHHHHHH

[0166] Clone 21 cassette DNA (SEQ ID NO:27):

[0167] ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCC ATG GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGG ATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCGTCTATTT CTTATTCTGGTGCTTATACAGCTTACGCAGACTCCGTGAACGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAAC ACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATCTGGTACTGATTT TGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTG GCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACT TGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGAT CTATGGTGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCA CCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTCTAATTATGATCCTAGTACGTTCGGC CAAGGGACCAAGGTGGAAATCAAAGAATTC GAGCAGAAACTCATCTCAGAAGAGGATCTGCTCGAGCACCACCACCACCACCACTGA

[0168] Clone 21 box protein (SEQ ID NO:28):

[0169] MKYLLPTAAAGLLLLAAQPAMA MEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE WVSSISYSGAYTAYADSVNGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSGTDFDYWGQGTLVTVSSGGGGSGG GGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASNLQSGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQSNYDPSTFGQGTKVEIKEF EQKLISEEDLLEHHHHHH

[0170] The purity of the 7 scFvs induced at 16°C was>85%, while the purity was lower when induced at 37°C. Therefore, 16°C was determined to be a more suitable production condition. QC ELISA was performed to analyze the binding ability of each of the seven scFvs to the target MOG. Compared with the control, differences were easily found in each of the 7 positive clones (clones 1, 3, 6, 10, 13, 17, 21). Among the 7 positive clones, three clones (clones 3, 6 and 17) indicated stronger binding ability to the target.

[0171] QC ELISA titration was performed on each of the 7 clones induced at 16°C. Seven different concentrations of the 7 clones were used for ELISA titration. The results indicated that all 7 clones could specifically bind to the target MOG. Among the 7 clones, clones 3, 6, and 17 still indicated stronger binding ability to the target. The results are shown in Figure 8 , it is shown that the anti-hMOG1 scFv of clone 17 exhibited the strongest binding, followed by clone 6 and then clone 3.

[0172] Example 5

[0173] ScFv production

[0174] E. coli BL21 (DE3) strain was transfected with pET26b-scFv expression vector in 2YT-K medium. When the strain was growing logarithmically, 1mM and 0.2mM IPTG were added to the medium to induce the expression of scFv for 16 hours at 26°C. The expression and solubility of the antibody were tested by SDS-PAGE. scFv was highly expressed in E. coli BL21 strain, but mainly in inclusion bodies.

[0175] To obtain soluble scFv antibodies, expression conditions were optimized. When E. coli BL21 were transfected with pET26b-scFv, they grew logarithmically, and 0.1 mM IPTG was added to the medium to induce expression of scFv overnight at 22°C. Purification was performed by affinity chromatography.

[0176] ScFv was also purified by inclusion body refolding. The inclusion bodies were washed once with PBS and then twice with 1M urea. 8M urea was added to dissolve the inclusion bodies. The scFv was then purified by dialysis bags (8KDa, Biotics, F132579) and Millipore concentrators. A total of 1.17 mg of scFv antibody fragments (0.9 mg / mL, 1.3 mL) were finally obtained.

[0177] Example 6

[0178] ScFv labeled with FITC

[0179] The scFv was diluted to 1 mg / mL with sodium carbonate buffer (pH 9.0). It was mixed with FITC solution (Sigma, F7250) and incubated overnight in the dark at 4 ° C. The labeled scFv was concentrated by Millipore concentrator. A total of 0.83 mg of FITC-labeled scFv antibodies were finally obtained. The labeling efficiency was measured, and the result showed that 2.52 FITC molecules were labeled for each scFv antibody fragment.

[0180] DNA (pelB-VH-linker-VL-6His) (SEQ ID NO:29):

[0181] ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCATGGCCGAAGTGAAACTGCATGAAAGCGGCGCGGGCCTGGTGAAACCGGGCGCGAGCGTGGAAATTAGCTGCAAAGCGACCGGCTATACCTTTAGCAGCTTTTGGATTGAATGGGTGAAACAGCGCCCGGGCCATGGCCTGGAATGGATTGGCGAAATTCTGCCGGGCCGCGGCCGCACCAACTATAACGAAAAATTTAAAGGCAAAGCGACCTTTACCGCGGAAACGAGCAGCAACACCGCGTATATGCAGCTGAGCAGCCTGACGAGCGAAGATAGCGCTGTATATTACTGTGCGACGGGCAACACCATGGTGAACATGCCGTATTGGGGCCAAGGCACCACCGTGACCGTGAGCTCGGGTGGAGGCGGTTCAGGCGGAGGTGGTTCTGGCGGTGGCGGATCGGACATTGAACTGACGCAGAGCCCGAGCAGCCTGGCGGTGAGCGCGGGCGAAAAAGTGACCATGAGCTGCAAAAGCAGTCAGAGCCTGCTGAACAGCGGCAATCAGAAAAACTATCTGGCGTGGTATCAGCAGAAACCGGGCCTGCCGCCGAAACTGCTGATTTATGGCGCGAGCACCCGCGAAAGCGGCGTGCCGGATCGCTTTACCGGCAGCGGCAGCGGGACCGATTTTACCCTGACCATTAGCAGCGTGCAAGCGGAAGATCTGGCGGTGTATTACTGTCAGAATGATCATAGCTATCCGCTGACCTTTGGCGCGGGCACCAAACTGGAAATTAAACGCCTCGAGCACCACCACCACCACCACTGA

[0182] Protein (pelB-VH-linker-VL-6His) (SEQ ID NO:30): scFv 4 sequence underlined

[0183] MKYLLPTAAAGLLLLAAQPAMA MAEVKLHESGAGLVKPGASVEISCKATGYTFSSFWIEWVKQRPGHG LEWIGEILPGRGRTNYNEKFKGKATFTAETSSNTAYMQLSSLTSEDSAVYYCATGNTMVNMPYWGQGTTVTVSSGG GGSGGGGSGGGGSDIELTQSPSSLAVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGLPPKLLIYGASTRESG VPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHSYPLTFGAGTKLEIKRLE HHHHHH*

[0184] SEQ ID NO:41: Amino acid sequence of scFv 4:

[0185] MAEVKLHESGAGLVKPGASVEISCKATGYTFSSFWIEWVKQRPGHGLEWIGEILPGRGRTNYNEKFKGKATFTAETSSNTAYMQLSSLTSEDSAVYYCATGNTMVNMPYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPSSLAVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGLPPKLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHSYPLTFGAGTKLEIKRLE

[0186] Protein 1PKQ_1|Chain A, F|(8-18C5) Chimeric Fab, Light Chain|Mus musculus (10090) (SEQ ID NO:31):

[0187] MKQSTIALALLPLLFTPVTKADIELTQSPSSLAVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGLPPKLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHSYPLTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0188] Protein 1PKQ_2|Chain B, G|(8-18C5) Chimeric Fab, Heavy Chain|Mus musculus (10090) (SEQ ID NO:32):

[0189] MKKTAIAIAVALAGFATVAQAEVKLHESGAGLVKPGASVEISCKATGYTFSSFWIEWVKQRPGHGLEWIGEILPGRGRTNYNEKFKGKATFTAETSSNTAYMQLSSLTSEDSAVYYCATGNTMVNMPYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCSAWSHPQFEK

[0190] Example 7

[0191] scFv CAR antigen recognition

[0192] HEK293 expressing MOG-1: HEK293 cells expressing MOG-1 were generated by lentiviral transduction of the parental HEK293 epithelial cell line with lentivirus encoding MOG-1 and a puromycin resistance gene. Cells were maintained in DMEM medium containing 10% FCS and 2ug / mL puromycin to ensure retention of MOG-1 expression. Cells were regularly FACS purified for MOG-1 expression using a FITC-labeled anti-MOG 1 antibody.

[0193] scFv anti-MOG-1 binding activity: 10^4 MOG-1 expressing or parental HEK293 cells were seeded in 96-well plates at 10^5 / mL and incubated overnight at 37°C in 5% CO2. The cells were then stained with FITC-labeled scFv in DMEM medium containing 10% FCS for 20 minutes at 37°C (at the indicated concentrations). The cells were then fixed and permeabilized according to the manufacturer's protocol (100uL fixation permeabilization 30 minutes Cytofix-Cytoperm Biolegend at room temperature). The cells were then washed and stained with Cell Mask Red (2mg / mL) in the permeabilization wash solution for 30 minutes to visualize the plasma membrane, and then stained with DAPI (500nM) for 5 minutes to visualize the nucleus. The cells were then washed and stored in PBS until imaging on a Leica EVOSM7000 fluorescence microscope. Binding activity was measured by calculating the ratio of the scFv-FITC signal and the Cell Mask Red signal. Cel Mask Red and scFv-FITC minimum thresholds were set to 3x background. scFv binding values ​​were limited to 8x background to eliminate nonspecific spot staining patterns. Results were then plotted as binding activity versus scFv concentration. Fig. 9 .

[0194] T cells were transduced with anti-MOG-1CAR constructs encoding DNA, and their ability to recognize MOG-1 on the cell surface was measured. In brief, total T cells (Stem cel technologies) were activated with anti-CD3, CD28 antibodies at 2.5e10^4 / mL for 48 hours at 37°C. Cells were then transduced with anti-MOG-1CAR lentivirus (MOI=5). After 7 days of culture, T cells were harvested and counted, and cryopreserved or downstream for CAR recognition assays. 10^4 MOG-1 expression or parent HEK293 cells were inoculated in 96-well plates at 10^5 / mL and incubated overnight at 37°C in 5% CO2. CAR T cells were then incubated with cells at a ratio of 100:1, 50:1 or 25:1Treg to HEK cells at 37°C for 4 hours. After stimulation, surface staining was performed to identify the activation marker CD69 (Biolegend 310930) and upregulation of T-reg phenotypic markers CD4, CD25, and CD127 to identify T-regs, in addition to intracellular staining for FOXP3 and FLAG tags to identify transduced T-regs. Cells were read on a FACS Aria at Tufts University FlowCore and data were analyzed using FlowJo, with analysis performed in GraphPadPrism. CD69 MFI was measured from the CD4+CD25hi, CD127lo, FLAG+ gates and compared between MOG-HEK and HEK-parental groups. (See Figure 11-13 ).

[0195] One vial of virus was used to transduce 5E5 donor PBMCs in a 24-well plate. Following the amplification protocol, cells were harvested and Flag expression was determined by flow cytometry. Anti-Flag antibody (mouse anti-DDDDK, ProMab, catalog number 20201) was used. Viral transduction was performed on two different days A and B (see Fig.10 ).

[0196] DNA PMC 669: [Clone 17H-LscFv]-CD8-CD28-CD3z with FLAG tag (SEQ ID NO: 33)：

[0197]

[0198] DNA PMC 670: [Clone 17L-HscFv]-CD8-CD28-CD3z with FLAG tag (SEQ ID NO: 34)：

[0199]

[0200] DNA PMC 696: [Clone 3H-L scFv]-CD8-CD28-CD3z with FLAG tag (SEQ ID NO: 35)：

[0201]

[0202] DNA PMC 697: [Clone 3L-H scFv]-CD8-CD28-CD3z with FLAG tag (SEQ ID NO: 36)：

[0203]

[0204] DNA PMC 698: [Clone 6H-L scFv]-CD8-CD28-CD3z with FLAG tag (SEQ ID NO: 37)：

[0205]

[0206] DNA PMC 699: [Clone 6L-H scFv]-CD8-CD28-CD3z with FLAG tag (SEQ ID NO: 38)：

[0207]

[0208] DNA scFv4[8-18C5]CAR sequence (SEQ ID NO:39):

[0209]

[0210] DNA scFv4[18C5-8]CAR sequence (SEQ ID NO:40):

[0211]

[0212] Two different CAR constructs of each scFv were tested. One construct encodes the variable domains in order VH-VL, and the other construct encodes the variable domains in order VL-VH. Testing two different CAR variants based on the core scFv sequence allows selection of CARs with optimal surface expression, stability, antigen recognition, and signal transduction capabilities.

[0213] Example 8

[0214] Study Design

[0215] Participants with progressive supranuclear palsy (PSP) will receive a single infusion of ex vivo expanded autologous CD4+CD127lo / -CD25+CAR-T regulatory cells (Tregs). CAR-Tregs will be designed to specifically recognize myelin oligodendrocyte protein (MOG), a glycoprotein specifically expressed in the central nervous system (CNS), and induce immune tolerance and anti-inflammatory effects in the brain.

[0216] 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 5 PSP patients.

[0217] The main outcome measures will be:

[0218] 1. Adverse events

[0219] 2. Laboratory abnormalities

[0220] 3. Infusion reaction

[0221] 4. Complications related to infection

[0222] 5. Potential negative impact on the PSP process

[0223] Secondary objectives will be to evaluate the effects of CNS-specific CAR-Tregs on PSP and to gain indications for potential applications in other neurodegenerative diseases.

[0224] The end point will be:

[0225] 1. To evaluate the effects of CNS-specific CAR-Treg on clinical, neuropsychological, radiological, and biomechanical parameters in patients with PSP.

[0226] 2. Obtain indications for the potential therapeutic use of CNS-specific CAR-Tregs in other neurodegenerative diseases, including Alzheimer's disease (AD).

[0227] 3. Obtain instructions for a potential Phase II randomized, double-blind, placebo-controlled trial that could provide valuable insights into the potential efficacy of CAR-Treg for neurodegenerative disorders.

[0228] Patient evaluation

[0229] Clinical and neuropsychological assessments: A detailed description of the inclusion and exclusion criteria and clinical (motor and neuropsychological) and neuroimaging assessments will be performed as previously reported (Giordano et al., J. Trans l. Med. 2014; Canes i et al., J. Trans l. Med. 2016, incorporated herein by reference). Patients will undergo neurological examinations 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), PSP rating scale (PSP-RS) (Goetz et al., Mov. Disord. 2004; Golbe et al., Brain 2007; the contents of each of which are incorporated herein by reference). A Mini-Mental State Examination (MMSE) will also be performed as previously described (Folstein et al. J. Psychiatr. Res. 1975, 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). The clinical status will be classified as “stable” if the UPDRS and PSP-RS scores do not decrease by more than 30% compared to baseline and the H&Y stage does not change at the defined time point (Canesi et al., J. Transl. Med. 2016, incorporated herein by reference).

[0230] Neuroimaging: All patients will undergo longitudinal neuroimaging assessments 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) (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 imaging and PET / TC imaging, respectively.

[0231] For SPECT, intravenous administration of 110–140 MBq of [123I] FP-CIT (Datscan, GE-Health, Amersham, UK) will be performed in all patients 30–40 minutes after thyroid blockade (oral administration of 10–15 mg of Lugol's solution). Analysis will be performed as previously described (Isaias et al., NeuroReport 2007, incorporated herein by reference). Volume templates for gray matter anatomical distribution will be generated from the Montreal Neurological Institute MRI single participant brain atlas by applying a macroscopic anatomical method (automatic anatomical labeling), and will be redirected and reformatted to obtain a 2.64-cm thick reference section. Templates of eight irregular target regions (ROIs) will be manually drawn on this section to assess the anatomical extent of striatal and occipital structures with specific and nonspecific uptake of [123I] FP-CIT, respectively. The ROI template will also be positioned on the reference SPECT section and adjusted on the striatum and occipital cortex. The striatal ROIs will also be segmented into their anterior (caudate) and posterior (putamen) portions. [123I] Specific striatal dopamine uptake transporter (DAT) binding of FP-CIT will be calculated throughout the striatum, putamen, and caudate using the following formula: [(mean counts in the specific ROI) - (mean counts in the occipital ROI)] / (mean counts in the occipital ROI). The putamen and caudate ratios will also be calculated for each subject.

[0232] All patients will also undergo a F-fluoro-2-deoxyglucose positron emission tomography scan (FDGPET) at rest after an intravenous injection of 170 MBq. Each acquisition will consist of a computed tomography (CT) transmission scan of the head (50 mAs for 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 will be reconstructed using an iterative algorithm (OS-EM) and corrected for scatter and attenuation using density coefficients derived from low-dose CT scans of the head acquired with the same scanner. The images will be reconstructed as 128-pixel axial images with a resolution of 2 mm. The resolution of the PET system will be 4–5 mm FWHM.

[0233] Biomechanical evaluation: Biomechanical evaluation will be evaluated at baseline and 6 and 12 months after CAR-Treg cell administration. Two groups of specific parameters (one group for standing and one group for gait initiation) will be automatically extracted by a special algorithm (Carpinell la et al., IEEE Trans Neural Syst Rehabi l Eng. 2007, incorporated herein by reference). For standing, the average velocity and spatial displacement of the center of pressure (CoP) will be measured (Canes i et al., J. Transl. Med. 2016, incorporated herein by reference). In order to check gait initiation, the expected posture adjustment (Canes i et al., J. Trans l. Med. 2016, incorporated herein by reference) (i.e., unbalanced and unloading phases) will be analyzed and the following parameters will be measured: (1) the duration of the two phases, (2) the front-back (AP) and medial-lateral (ML) offsets and speeds of CoP, (3) the average length and speed of CoP. The (4) length and (5) speed of the first step will also be measured. The spatial parameters will be normalized according to body height (BH%).

[0234] Preparation and administration of CAR-Treg cells

[0235] Treg isolation and expansion: PolyTregs were selected and expanded from five 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. Trans l. Med., 2015, incorporated herein by reference).

[0236] 400 ml of fresh peripheral blood will be collected into blood pack units containing citrate phosphate dextrose and processed within 24 hours to separate PBMCs by Ficol density gradient. Tregs will be isolated on a high-speed cell sorter using the following GMP-grade lyophilized antibodies: CD4-PerCP (peridin chlorophyll protein) (L200 clone), CD127-PE (phycoerythrin) (40131 clone) and CD25-APC (allophycocyanin) (2A3 clone). The sorted CD4+CD127lo / -CD25+ cells will be collected into 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 Tregs will be analyzed after sorting. The expected purity of CD4+CD127lo / -CD25+ cells is over 96% (Bluestone et al., Sci. Transl. Med., 2015, incorporated herein by reference).

[0237] Purified Tregs will be cultured with clinical-grade Dynabeads coated with anti-CD3 and anti-CD28 plus recombinant IL-2, as previously described (Bluestone et al., Sci. Trans l. Med., 2015, incorporated herein by reference). One unit of blood is expected to produce between 4.2 x 106 and 11.8 x 106 purified CD4+CD127lo / -CD25+Treg, (Bluestone et al., Sci. Trans l. Med., 2015, incorporated herein by reference). The expanded Treg preparation is expected to be around 90% FOXP3+. The viability, CD4+ percentage and CD8+ cell contamination of the Treg preparation will be checked (Bluestone et al., Sci. Trans l. Med., 2015, incorporated herein by reference).

[0238] Phenotypic and TCR Analysis of Expanded PolyTregs: The key cell surface markers CD4 and CD127 for isolating Tregs will be examined after expansion.

[0239] Previous data indicate that initial CD45RA+Tregs preferentially expand in these cultures, and that CD45RA+RO- cells downregulate CD45RA and upregulate CD45RO during the expansion period (Bluestone et al., Sci. Trans l. Med., 2015, incorporated herein by reference). CCR7 (a Treg trafficking receptor), CD38 (a multifunctional extracellular enzyme associated with enhanced Treg function), and CD45 RO will be measured before and after expansion. The TCRβ repertoire of the expanded Tregs will also be analyzed and compared with the freshly isolated population to determine the polyclonality of the expanded Tregs. The expanded cells are expected to exhibit polyclonality indistinguishable from the pre-expansion culture, and Tregs remain a highly diverse population after expansion (Bluestone et al., Sci. Trans l. Med., 2015, incorporated herein by reference).

[0240] Functional analysis of expanded polyTregs: The following assays will be performed after Treg expansion (Bluestone et al., Sci. Transl. Med., 2015, incorporated herein by reference):

[0241] -DNA methylation status of the enhancer region of the FOXP3 locus to assess the overall purity and stability of the expanded Tregs.

[0242] - Cytokine production (IFNγ, IL-4, IL-5, and IL-17) to assess lymphocyte phenotype

[0243] - In vitro suppressive activity to determine the functional potential of the expanded cells.

[0244] Generation and functional analysis of CAR-Treg cells: CAR RNA will be optimized for anti-MOG CAR expression on Treg after electroporation of human Tregs and on mouse Tregs after adoptive transfer to the PSP mouse model according to published protocols (Zhao, Y et al., 2010 Cancer Res and Beatty; GL et al., 2014 Cancer Immunol Res; Singh, N et al., 2014 Oncoimmunol; the contents of each of which are incorporated herein by reference). The delivery of anti-MOG CAR using second-generation lentiviral vectors and standard protocols will also be optimized (Levine, BL et al., 2017 Mol Ther Methods & Clin Dev, incorporated herein by reference). Electroporation of RNA (approximately 1 μg / 3 x 10 6 Treg) or by lentiviral transduction (1x 10 6 pfu / 3x 10 6 Clinical-grade RNA for anti-MOG CAR will be generated. For 2.6x 10 9 Treg cells per patient approximately 0.9 mg RNA; for 5 patients 4.5 mg will be required. Clinical grade lentivirus will be produced. For 2.6x10 9 Treg cells, about 8.7 x 10 per patient 9 pfu; for 5 patients would require 4.3x10 10 pfu. Functional analysis of MOG-specific CAR-Treg will be performed as described above in 3.3.

[0245] Cell administration

[0246] Each patient will receive a single administration of MOG-specific CAR-Treg (2.6 x 10 9CAR-Treg). The cells will be administered to at least 5 PSP patients. Patients will receive premedication with acetaminophen and diphenhydramine. CAR-Treg will be infused over 10 to 30 minutes via a peripheral intravenous line. Vital signs will be obtained before and after the infusion, then every 15 minutes for at least 1 hour, then every hour for the first 4 hours, and every 4 hours for 20 hours. Chemistry and complete blood counts and blood cell differential counts will be repeated the day before discharge from the clinical research unit. Patients will receive follow-up assessments on the 4th day after infusion, then once a week for 4 weeks, then every 13 weeks for 1 year, and then every 26 weeks for 2 years. Telephone monitoring of adverse events will continue every 6 months for 5 years after infusion, followed by the last outpatient visit.

[0247] Patient evaluation after CAR-Treg cell infusion

[0248] 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, 3, 6 and 12 months after cell administration.

[0249] Example 9

[0250] Drugs that employ components of immunosuppressive T regulatory lymphocytes (Tregs) to shut down the destructive immune response that causes the disease will be developed and tested for multiple sclerosis (MS). The global MS market is approximately $21.5 billion, but approved drugs for the most common form of MS yield only modest disease improvements with significant side effects. For more severe forms of MS, treatment options are limited to 1 recently approved drug.

[0251] There are 11 FDA-approved drugs for the relapsing-remitting form of MS (RRMS - 85% of diagnosed MS). There are several orally available and antibody-based drugs currently approved or in clinical evaluation for RRMS. In March 2017, the FDA approved ocrelizumab (anti-CD20 antibody, Roche) for primary progressive multiple sclerosis (PPMS - approximately 10% of diagnosed MS). Ocrelizumab reduces symptoms by 25% and is currently the only immunomodulator for PPMS in the United States. Secondary progressive multiple sclerosis (SPMS) invariably occurs in patients with RRMS, for whom disease treatment options are also limited.

[0252] Production of biologics

[0253] Anti-MOG hybridomas will be produced by CRO with recombinant human MOG immunized mice. VH and VL genes and anti-scFv molecules will be cloned. The orientation of VH and VL and the joint (between scFv or inside each scFv) may greatly affect the stability, expression level and binding capacity of GTIP. In some cases, only one of these forms will produce functional molecules. Therefore, several orientations of VH-VL will be expressed on a small scale and tested before expanding production. An expression construct encoding 4 anti-MOG scFvs will be produced, which has a connecting joint, a central joint, and then connected to a Treg-related enzyme or a mimic.

[0254] Validation of GTIP protein products in mouse MS models

[0255] GTIP will be tested in mice, acute and chronic EAE models of MS. Levels of Th1, Th17, CTLs (blood and CNS), bone marrow inflammatory cells (macrophages and neutrophils), and anti-MBP antibodies specific for myelin basic protein (MBP) will be measured. Immune responses will be correlated with disease progression. Doses will be varied to gain insight into potential use in advanced MS. The product will be dosed in normal mice to gain insight into any potential off-target effects.

[0256] Clinical evaluation

[0257] Clinical studies will be conducted to test the efficacy of GTIP as a disease modifier in MS. These products will first be tested in RRMS patients who are unresponsive to the first use of drugs. Safety and tolerability will be measured using a dosing regimen similar to antibody therapy (e.g., the first 3 intravenous doses are once every 2 weeks, and then once every 4 weeks for 20 weeks). In Phase 2 studies, the main measurement will be the reduced disease relapse frequency and brain damage. The secondary measurement will be the inflammatory cytokines, Th1 / Th17 cells and other leukocyte levels reduced in the blood. Side effects may include increased susceptibility to infection. These studies will enable us to compare the effectiveness of the compound with other secondary use drugs, which can reduce the relapse frequency by 49%. If the product shows an acceptable level of efficacy, they will enter a long-term clinical Phase 2 study in PPMS patients. The main measurement will be a delayed decline in motor function and a reduction in brain damage, and the secondary measurement will be the inflammatory cytokines, Th1 / Th17 cells and other leukocyte levels reduced in the blood.

[0258] 1. EQUIVALENTS AND INCORPORATION BY REFERENCE

[0259] All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent specifically and individually indicated as being incorporated by reference in its entirety for all purposes. Such statements of incorporation by reference are contemplated by applicant under 37 CFR § 1.57 (b) (1) to relate to each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent, each of which is expressly identified under 37 CFR § 1.57 (b) (2), even if such reference is not immediately adjacent to a specific statement of incorporation by reference. Inclusion of a specific statement of incorporation by reference in the specification, if any, does not in any way impair such general statement of incorporation by reference. Citation of references herein does not constitute an admission that the references are relevant prior art, nor does it constitute any admission of the contents or dates of these publications or documents.

[0260] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

Claims

1. An engineered regulatory T cell (Treg), The engineered Tregs are CD4+CD127–CD25+FOXP3+ cells. wherein the engineered Treg expresses a chimeric antigen receptor (CAR) comprising a single chain variable fragment (scFv), The scFv comprises the VH region and VL region of SEQ ID NO:

6.

2. The engineered Treg of claim 1, wherein the CAR is encoded by SEQ ID NO:

38.

3. The engineered Treg of claim 1, wherein the CAR is capable of specifically binding to a glial cell marker.

4. The engineered Treg of claim 3, wherein the glial cell marker is myelin oligodendrocyte glycoprotein (MOG).

5. The engineered Treg of claim 4, wherein the CAR is capable of directing the engineered Treg to glial target cells expressing MOG. 6 . The engineered Treg according to claim 1 , wherein the order of the VH region and the VL region in the scFv from the N-terminus to the C-terminus is VL region-VH region.

7. The engineered Treg according to claim 1, wherein the order of the VH region and the VL region in the scFv from the N-terminus to the C-terminus is VH region-VL region.

8. The engineered Treg of claim 1, wherein the VH region and the VL region of the scFv are connected by a linker.

9. The engineered Treg of claim 8, wherein the linker comprises one or more amino acids. 10 . A pharmaceutical composition comprising a plurality of engineered Tregs according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

11. Use of the pharmaceutical composition of claim 10 in the preparation of a medicament for treating a neurodegenerative disease in a subject.

12. The use according to claim 11, wherein the neurodegenerative disease is selected from the group consisting of progressive supranuclear palsy (PSP), Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS) and chronic traumatic encephalopathy (CTE).

13. The use according to claim 12, wherein the neurodegenerative disease is progressive supranuclear palsy (PSP).

14. The use according to claim 12, wherein the neurodegenerative disease is Alzheimer's disease (AD).

15. The use according to claim 12, wherein the neurodegenerative disease is Parkinson's disease (PD).

16. The use according to claim 12, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).

17. The use according to claim 12, wherein the neurodegenerative disease is Huntington's disease (HD).

18. The use according to claim 12, wherein the neurodegenerative disease is multiple sclerosis (MS).

19. The use according to claim 12, wherein the neurodegenerative disease is chronic traumatic encephalopathy (CTE).

20. The use of claim 11, wherein use of the pharmaceutical composition reduces inflammation in the subject's neural tissue.

21. The use of claim 11, wherein the subject is a human.

Images