Patent Application
20230210899
The instant application contains a Sequence Listing with XX sequences, which has been submitted via EFS-Web and is hereby incorporated herein by reference in its entirety. Said ASCII copy, created on Month XX, 2021, is named 48219WO_CRF_sequencelisting.txt, and is XXX bytes in size.
The invention provides CAR-Treg compositions and methods of use thereof that specifically regulate immune response and inflammation related to various neurodegenerative diseases such as progressive supranuclear palsy and Parkinson's disease.
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 resulting in rapid physical and/or mental deterioration and death. There are no known cures for those diseases and treatments focus on managing symptoms and delaying deterioration.
One such disease, PSP, is an idiopathic degenerative disease, not uncommon in the elderly, which mimics Parkinson's disease (PD). The clinical presentation includes the tetrad of supranuclear gaze paralysis, axial rigidity, dementia, and pseudobulbar palsy. It is associated with bradykinesia, severe postural disorder and frequent falls. Pathology is associated with cell loss and Tau neurofibrillary tangles, mainly in the brain stem, globus pallidus, subthalamic nucleus, and dentates nucleus. PSP has a prevalence of 5-6 per 100,000, resulting in 5000-25000 patients per year in the USA. The mean age of onset of the disease is 63 years, with a usual prognosis ranging from 5 to 10 years from diagnosis to death and there are no disease modifying treatments available.
Parkinson's disease is another neurodegenerative disease with no known cure. Parkinson's has a prevalence of about 1-2 per 1,000. Parkinson's is characterized by cell death in the basal ganglia along with astrocyte death and an increase in microglia in the substantia nigra resulting in a dopamine deficiency in those areas. Inclusions called Lewy bodies develop in the damaged cells before cell death. There is speculation regarding the underling mechanisms driving brain cell death in Parkinson's but they remain poorly understood and treatments are current focused on managing the disease symptoms.
Compositions and methods of the invention use T regulatory lymphocytes (Tregs) or immunosuppressive proteins expressed by Treg cells to modulate neurodegenerative immune responses targeting glial cells in the central nervous system (CNS). By coupling either Tregs or immunosuppressive proteins to a chimeric antigen receptors (CAR) or a single-chain variable fragment (scFv) that specifically recognizes and binds glial cell markers, the immunosuppressive Tregs or proteins are drawn to glial cells of the CNS to reduce inflammation and protect the CNS from autoimmune attack.
The present invention recognizes the lack of effective treatment options for most neurodegenerative diseases and the presence of an autoimmune and/or inflammation component to several such diseases and engineers compositions to specifically suppress those disease components. Compounds and methods of the invention allow glial cells to modulate damaging immune cells such as Type 1 helper cells (Th1), T helper 17 cells (Th17), cytotoxic T cells (CTL), M1 macrophages, and polymorphonuclear neutrophils (PMN).
The present invention directs immunosuppressive molecules (Tregs or immunosuppressive proteins) to oligodendrocyte (ODC) glial cells. The resulting compounds and methods of use thereof recruit the body's own immune system to counter the effects of neurodegenerative diseases such as Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease, amyotrophic lateral sclerosis (ALS), and progressive supranuclear palsy (PSP). The invention addresses a mechanism (i.e., autoimmune attack of the central nervous system) by which several neurodegenerative diseases disrupt neural function but does not depend on any particular biochemical causes of the underlying disease. Accordingly, the compounds and methods of the invention can provide therapeutic effects across several neurodegenerative diseases.
Aspects of the invention include methods for treating a neurodegenerative disease in a subject including steps of administering to said subject a therapeutically effective amount of regulatory T cells (Treg) expressing a chimeric antigen receptor (CAR) that specifically binds to a glial cell marker, wherein the neurodegenerative disease is Multiple Sclerosis (MS). The CAR-Treg then protects neural tissue and reduces inflammation in the neural tissue, thereby treating the neurodegenerative disease. In various embodiments, the subject may be a human.
The glial cell marker may be oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (0M4), neural/glial marker 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), or myelin oligodendrocyte specific protein (MOSP). In some embodiments, the glial cell marker is myelin oligodendrocyte glycoprotein (MOG).
The neurodegenerative disease treated may 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 a 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).
In certain aspects, the invention provides a composition comprising an engineered regulatory T cell (Treg) in a therapeutically effective amount to treat a neurodegenerative disease that is not multiple sclerosis, the engineered Treg expressing a chimeric antigen receptor (CAR) that specifically binds to a glial cell marker. The glial cell marker in the composition may be myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (0M4), neural/glial marker 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), or myelin oligodendrocyte specific protein (MOSP).
The composition may be therapeutically effective to treat progressive supranuclear palsy (PSP), Parkinson's disease (PD), Alzheimer's, Huntington's disease, amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy (CTE),_multiple sclerosis, or a prion disease.
Various aspects of the invention include an engineered protein comprising a glial cell-specific binding protein coupled to a molecule expressed by a regulatory T cell (Treg). The molecule expressed by the Treg may be an extracellular immune-suppressive enzyme. In certain embodiments, the molecule expressed by a 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.
In certain embodiments the Treg-expressed-molecule-bound glial cell-specific binding protein may bind myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (0M4), neural/glial marker 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), or myelin oligodendrocyte specific protein (MOSP).
In some aspects, the invention provides an engineered protein comprising a glial cell-specific binding protein coupled to a molecule that mimics the activity of a molecule expressed by a regulatory T cell (Treg). The mimicked molecule expressed by a Treg can be an extracellular immune-suppressive enzyme such as CD73, CD39, indoleamine 2,3-dioxygenase (IDO), or glutamate-oxaloacetate transaminase 1 (GOT1). The mimicked-molecule-bound glial cell-specific binding protein may bind myelin oligodendrocyte glycoprotein (MOG), oligodendrocyte marker 01 (OM1), oligodendrocyte marker 04 (0M4), neural/glial marker 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), or myelin oligodendrocyte specific protein (MOSP).
The invention relates to compositions for regulating autoimmune components of various neurodegenerative diseases. Compositions and methods provided herein target glial-cell-specific markers to draw immunosuppressive molecules (e.g., Tregs or immunosuppressive proteins expressed by Tregs) to the CNS and disrupt autoimmune attacks that contribute to the neurodegenerative effects of diseases such as progressive supranuclear palsy (PSP), Alzheimer's disease (AD), Huntington's disease, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy (CTE), or a prion disease.
The blood-brain barrier can serve as an impediment to treating disorders of the brain or CNS as the barrier can block therapeutic compounds from accessing the affected cells. Importantly, Tregs are able to cross the blood-brain barrier and can be localized to neurons of the CNS by Treg-bound glial cells, thereby allowing compounds of the invention to effectively treat neurodegenerative disorders of the CNS.
Compounds and methods of the invention do not rely on any disease-specific biochemical mechanisms and instead short circuit the immune response by which many neurodegenerative diseases affect mental and physical deterioration. Accordingly, the same compounds and methods may provide therapeutic effects for a number of neurodegenerative diseases.
For example, PSP involves tau protein buildup and neurofibrillary tangles and results in neuron and glial cell damage and loss, associated physical and mental deterioration, and eventual death. Parkinson's disease involves neuron loss in the basal ganglia along with astrocyte death and an increase in microglia in the substantia nigra. Inclusions called Lewy bodies develop in the damaged cells before cell death. ALS is marked by the death of motor neurons in the motor cortex after developing protein-rich inclusions in their cell bodies and axons.
The present invention recognizes that, despite differing underlying causes and disease mechanisms, PSP, Parkinson's, and ALS, along with neurodegenerative diseases including Alzheimer's disease (AD), Huntington's disease, chronic traumatic encephalopathy (CTE), and prion diseases likely include an immune component that contributes to inflammation and CNS degradation. 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. Accordingly, compounds and methods of the invention, focused on suppressing immune response in the CNS and addressing the chronic inflammation driving many neurodegenerative disease, may be therapeutically effective in treating many those diseases.
Compounds and methods of the invention use chimeric antigen receptors (CAR), antibodies, or single-chain variable fragments (scFv) that specifically bind glial cell markers. The glial cell binding molecules are coupled to a Treg, an immunosuppressive protein expressed by Tregs, or a molecule configured to mimic the immunosuppressive proteins expressed by Tregs. Glial cells are non-neuronal cells that perform a number of functions in supporting neurons in the central and peripheral nervous systems various animals including humans. Glial cells include oligodendrocytes, astrocytes, ependymal cells and microglia. As a result of their functions in maintaining neurons of the CNS, glial cells migrate to neurons of the CNS and can therefore be used to localize therapeutic compounds there. For example, oligodendrocyte (ODC) glial cells traffic to the CNS to maintain axon insulation by creating the myelin sheath. Compounds and methods of the invention include coupling immunosuppressive molecules to glial cells such as ODCs such that, as the glial cells perform their functions, the immunosuppressive molecules are brought into close proximity to the neurons of the CNS as shown in
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 (0M4), neural/glial marker 2 (NG2), A2B5, galactosylceramidase (GALC), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), or myelin oligodendrocyte specific protein (MOSP).
In various embodiments, CARs, scFvs, or antibodies can be bound to immunosuppressive molecules and used to target glial cells. CARs are engineered receptors that can provide specificity to immune effector cells (T cells). CARs have been used to confer tumor cell specificity to cytotoxic T lymphocytes for use in cancer immunotherapies. See, Couzin-Frankel, 2013, Cancer immunotherapy, Science, 342(6165):1432-33; Smith, et al., 2016, Chimeric antigen receptor (CAR) T cell therapy for malignant cancers: Summary and perspective, Journal of Cellular Immunotherapy, 2(2):59-68; the contents of each of which are incorporated herein by reference. Using similar principles, compounds and methods of the invention include engineering CARs that are specific to markers found on glial cells such as ODCs but, instead of grafting the glial-cell-specific CARs to cytotoxic T cells, they are grafted onto engineered immunosuppressive Tregs.
CAR-Tregs of the invention may express multiple chimeric antigen receptors targeting the same or two or more different glial cell markers.
ScFvs are fusion proteins including variable regions of the heavy (VH) and light chains (VL) of immunoglobulins. ScFvs may be created by cloning 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 with various linkers to form a variety of scFvs which may then be experimentally verified to provide desired stability, expression levels, and binding affinity for glial cells or specific markers thereof. ScFvs or antibodies specific to glial cell markers discussed above can be joined to the immunosuppressive proteins discussed below to form fusion proteins capable of providing CNS-localized immunosuppression therapy as shown in
Antibodies targeting glial cell markers can be produced by methods known in the art including commercially available services for producing custom antibodies from, for example, Pacific Immunology (San Diego, Calif.) or ABclonal (Woburn, Mass.).
CAR-Tregs may be engineered by known methods for preparing CAR-T cells. Treg cells may be isolated from a subject, preferably autologous Treg cells from the patient to be treated. The genes of the Treg cells can then be modified through known techniques such as electroporation, viral vectors, or other forms of transfection with nucleic acids encoding the engineered chimeric antigen receptor of choice. Allogeneic cells (i.e. those that are not HLA-matched, or are only partially matched to the subject) can also be utilized in the methods and treatments described herein. A source of such allogenic cells includes peripheral blood mononuclear cells (PBMCs). PBMCs can be isolated by Ficoll-Hypaque density gradient centrifugation of samples obtained from discarded, de-identified leukocyte reduction filters (American Red Cross), or blood donations from healthy volunteers with informed consent. Descriptions of cell populations, sources and methods for selecting or enriching for desired cell types can be found, for example in: U.S. Pat. No. 9,347,044. CAR-Treg cells can then be experimentally verified before introduction into the patient's system for treatment.
Regulatory T cells or Tregs modulate the immune system and generally downregulate the induction and proliferation of effector T cells. Tregs prevent auto-immune responses and aid in the discrimination of self and non-self by the immune system. 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 function through reverse signaling through direct interaction with dendritic cells and the induction of immunosuppressive indoleamine 2,3-dioxygenase. Tregs can also downregulate immune response through the ectoenzymes CD39 and CD73 with the production of immunosuppressive adenosine. Tregs also suppress immune response through direct interactions with dendritic cells by LAG3 and by TIGIT. Another control mechanism is through the IL-2 feedback loop. Another mechanism of immune suppression by Tregs is through the prevention of co-stimulation through CD28 on effector T cells by the action of the molecule CTLA-4.
GTIPs of the invention may include one or more immunosuppressive proteins (including two or more different proteins) linked to one or more scFvs or antibodies targeting the same or two or more different glial cell markers. Proteins can be joined by any known means to form GTIPs of the invention including, for example, fusion proteins or biotin-streptavidin linkage.
Adoptive cell transfer techniques as used in cancer immunotherapy techniques including those involving cytotoxic T lymphocytes may be used to prepare autologous CAR-Tregs for use in compounds and methods of the 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.
A CAR-Treg or glial-cell-targeted immunosuppressive protein of the invention may be incorporated into carrier systems containing one or more of the therapeutic compounds described herein. In certain embodiments, the carrier system can be a nanoparticle that includes disulfide-crosslinked polyethyleneimine (CLPEI) and a lipid. The lipid may be a bile acid, such as cholic acid, deoxycholic acid, and lithocholic acid. Such carrier systems are described further in the Examples below. Other exemplary carrier systems are described for example in Wittrup et al. (Nature Reviews/Genetics, 16:543-552, 2015), the content of which is incorporated by reference herein in its entirety.
The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
The phrases “systemic administration,” “administered systematically,” “peripheral administration” and “administered peripherally” as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
When the compounds of the present invention are administered as pharmaceuticals, to humans and mammals, they can be given 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 a therapeutic compound of the invention and/or derivative thereof, in combination with a pharmaceutically acceptable carrier.
The effective dosage of each agent can readily be determined by the skilled person, having regard to typical factors each as the age, weight, sex and clinical history of the patient. In general, a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
The pharmaceutical compositions of the invention include a “therapeutically effective amount” or a “prophylactically effective amount” of one or more of the compounds of the present invention, or functional derivatives thereof. An “effective amount” is the amount as defined herein in the definition section and refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, e.g., a diminishment or prevention of effects associated with neuropathic and/or inflammatory pain. A therapeutically effective amount of a compound of the present invention or functional derivatives thereof may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the therapeutic compound to elicit a desired response in the subject. A therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic agent are outweighed by the therapeutically beneficial effects.
A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to, or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount. A prophylactically or therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the beneficial effects.
An “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired result.
Dosage regimens may be adjusted to provide the optimum desired response (e.g. a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigency of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the patient.
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 containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification 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 treatment of sensitivity in individuals.
In some embodiments, therapeutically effective amount can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in other subjects. Generally, the therapeutically effective amount is sufficient to reduce or inhibit neuropathic and/or inflammatory pain in a subject. In some embodiments, the therapeutically effective amount is sufficient to eliminate neuropathic and/or inflammatory pain in a subject.
Dosages for a particular patient can be determined by one of ordinary skill in the art using conventional considerations, (e.g. by means of an appropriate, conventional pharmacological protocol). A physician may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. The dose administered to a patient is sufficient to effect a beneficial therapeutic response in the patient over time, or, e.g., to reduce symptoms, or other appropriate activity, depending on the application. The dose is determined by the efficacy of the particular formulation, and the activity, stability or serum half-life of the compounds of the invention or functional derivatives thereof, and the condition of the patient, as well as the body weight or surface area of the patient to be treated. The size of the dose is also determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular vector, formulation, or the like in a particular subject. Therapeutic compositions comprising one or more compounds of the invention or functional derivatives thereof are optionally tested in one or more appropriate in vitro and/or in vivo animal models of disease, such as models of neuropathic and/or inflammatory pain, to confirm efficacy, tissue metabolism, and to estimate dosages, according to methods well known in the art. In particular, dosages can be initially determined by activity, stability or other suitable measures of treatment vs. non-treatment (e.g., comparison of treated vs. untreated cells or animal models), in a relevant assay. Formulations are administered at a rate determined by the LD50 of the relevant formulation, and/or observation of any side-effects of compounds of the invention or functional derivatives thereof at various concentrations, e.g., as applied to the mass and overall health of the patient. Administration can be accomplished via single or divided doses.
Administering typically involves administering pharmaceutically acceptable dosage forms, which means dosage forms of compounds described herein, and includes, for example, tablets, dragees, powders, elixirs, syrups, liquid preparations, including suspensions, sprays, inhalants tablets, lozenges, emulsions, solutions, granules, capsules, and suppositories, as well as liquid preparations for injections, including liposome preparations. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latest edition, which is hereby incorporated by reference in its entirety. Administering may be carried out orally, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, or intranasally. Compounds may be administered alone or with suitable pharmaceutical carriers, and can be in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, or emulsions.
A pharmaceutical composition containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the techniques described in U.S. Pat. Nos. 4,256,108, 4,166,452 and 4,265,874 (the content of each of which is incorporated by reference herein in its entirety), to form osmotic therapeutic tablets for control release.
Formulations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
Formulations may also include complexes of the parent (unionized) compounds with derivatives of β-cyclodextrin, especially hydroxypropyl-β-cyclodextrin.
An alternative oral formulation can be achieved using a controlled-release formulation, where the compound is encapsulated in an enteric coating.
Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as a naturally occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such a polyoxyethylene with partial esters derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
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.
The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally occurring phosphatides, for example soya bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.
Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be in a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
The term “pharmaceutical composition” means a composition comprising a compound as described herein and at least one component comprising pharmaceutically acceptable carriers, diluents, adjuvants, excipients, or vehicles, such as preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispensing agents, depending on the nature of the mode of administration and dosage forms.
The term “pharmaceutically acceptable carrier” is used to mean any carrier, diluent, adjuvant, excipient, or vehicle, as described herein. Examples of suspending agents include ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monosterate 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 disintegrating agents include starch, alginic acids, and certain complex silicates. Examples of lubricants include magnesium stearate, sodium lauryl sulphate, talc, as well as high molecular weight polyethylene glycols.
The term “pharmaceutically acceptable” means it is, within the scope of sound medical judgment, suitable for use in contact with the cells of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio.
Tetramer binding assays will be used to compare the avidity of GITP binding to target cells with that of known tetramers against T cells (Ober, B et al., 2000 Int Immunol, incorporated herein by reference) as shown in
The ability of GTIP bound to MOG-target cells to suppress the proliferation of T effector (T eff) cells will be tested. GTIP composed of a tetramer of a given scavenger IE (see table 1 below) will be bound to MOG-target cells, washed then incubated with proliferating human Teff (generated using standard procedures e.g. 3 days after anti-CD3/CD28 and IL-2 stimulation) (middle column of
Cells will be cultured in medium spiked with the relevant mitogenic metabolite (M) that is the substrate of the IE in the GTIP (see table above). Over time the concentration of M and the number of Teff will be measured as shown in
An assay is used to measure the ability of CAR molecules expressing scFv specific for MOG to bind Treg cells to MOG-expressing target cells with a relative avidity approximating that of a physiologically meaningful T cell:target cell interaction. The physiologically meaningful T cell:target cell interaction used for comparison is a CTL: peptide/MHC(pMHC)/target cell interaction. This is done using a flow cytometry (FCM)-based assay for cell-cell conjugates (Opferman, J T et al., 2001 Int Immunol., incorporated herein by reference).
The relative avidity of pMHC target for the relevant CTL clone (B6.2.16) is determined (left column of
ScFv antibodies were generated specific to human MOG by affinity panning of a human phage display scFv library. QC SDS-PAGE was conducted before library screening to assess purity of the target. In order to reduce non-specific binders, pre-counter selections were performed using polystyrene flat bottom plates and a blocking buffer against the phage library first before targeting screening.
After three rounds of biopanning, positive enriching was observed. 20 clones were randomly picked from the third round and QC monoclonal phage ELISA was conducted. 18 clones were found to bind to the target compared to the control. All 18 positive clones were sequenced.
Another 20 clones were picked from the third round and subjected to QC monoclonal phage ELISA. All 20 of the second set of clones were found to bind to the target compared to the control. All of them were sequenced.
After analysis of the 38 positive clones, 8 positive clones (clone 1, 3, 4, 6, 10, 13, 17, 21) with unique sequence were identified. The sequences of the 8 scFv proteins and the DNA sequences encoding them are listed below.
Expression vectors were constructed for each of the eight scFv proteins. After that, the cell lysates were coated for ELISA. Soluble ELISA was then conducted using the cell lysates from both 30° C. and 37° C. Compared to the control, differences were readily observed in all 7 clones. Among the 7 positive clones, clones 1, 6 and 13 were much stronger than the others.
ELISA with titration was conducted on soluble scFv produced from the seven positive clones to rank their ability to bind MOG. The 7 scFvs were subcloned into pET-26b to be constructed as scFv-myc-6×His format. Expression cassettes for the 7 scFv clones are shown below with each respective scFv polynucleotide or amino acid sequence underlined:
CTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTA
TGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAG
TATTTCTGATTATGGTAATACTACAGCTTACGCAGACTCCGTGAAGGGCCGGTTC
ACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTG
AGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAATGCTAATTATTTTGACT
ACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCG
GAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCAT
CCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCA
GAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAA
GCTCCTGATCTATGATGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGT
GGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAG
ATTTTGCAACTTACTACTGTCAACAGACTTCTACTTATCCTGGTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAAGAATTCGAGCAGAAACTCATCTCAGAAGAGGA
MSWVRQAPGKGLEWVSSISDYGNTTAYADSVKGRFTISRDNSKNTLYLQMNSLRAE
DTAVYYCAKNANYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLS
ASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTD
FTLTISSLQPEDFATYYCQQTSTYPGTFGQGTKVEIKEFEQKLISEEDLLEHHHHHH
CTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTA
TGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATC
TATTTCTTCTTATGGTTCTTATACAGGTTACGCAGACTCCGTGAAGGGCCGGTTCA
CCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGA
GAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAATGGTTATGCTTTTGACTA
CTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGG
AGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATC
CTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAG
AGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAG
CTCCTGATCTATGGTGCATCCGCTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTG
GCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGA
TTTTGCAACTTACTACTGTCAACAGAATGATGCTTCTCCTAATACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAAGAATTCGAGCAGAAACTCATCTCAGAAGAGGA
MSWVRQAPGKGLEWVSSISSYGSYTGYADSVKGRFTISRDNSKNTLYLQMNSLRAE
DTAVYYCAKNGYAFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLS
ASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASALQSGVPSRFSGSGSGTD
FTLTISSLQPEDFATYYCQQNDASPNTFGQGTKVEIKEFEQKLISEEDLLEHHHHHH
CTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTA
TGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAC
TATTTCTACTTATGGTGATTATACAACTTACGCAGACTCCGTGAAGGGCCGGTTC
ACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTG
AGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGTAGTTATACTTTTGACT
ACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCG
GAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCAT
CCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCA
GAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAA
GCTCCTGATCTATTCTGCATCCTATTTGCAAAGTGGGGTCCCATCAAGGTTCAGT
GGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAG
ATTTTGCAACTTACTACTGTCAACAGTCTAATGCTACTCCTTCTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAAGAATTCGAGCAGAAACTCATCTCAGAAGAGGA
MSWVRQAPGKGLEWVSTISTYGDYTTYADSVKGRFTISRDNSKNTLYLQMNSLRAE
DTAVYYCAKGSYTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSA
SVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYSASYLQSGVPSRFSGSGSGTDF
TLTISSLQPEDFATYYCQQSNATPSTFGQGTKVEIKEFEQKLISEEDLLEHHHHHH
CTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTA
TGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGG
TATTACTAATTATGGTTATACTACATATTACGCAGACTCCGTGAAGGGCCGGTTC
ACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTG
AGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATCTTCTTATTCTTTTGACT
ACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCG
GAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCAT
CCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCA
GAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAA
GCTCCTGATCTATGCTGCATCCGCTTTGCAAAGTGGGGTCCCATCAAGGTTCAGT
GGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAG
ATTTTGCAACTTACTACTGTCAACAGTCTGCTTATTATCCTGATACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAAGAATTCGAGCAGAAACTCATCTCAGAAGAGGA
MSWVRQAPGKGLEWVSGITNYGYTTYYADSVKGRFTISRDNSKNTLYLQMNSLRA
EDTAVYYCAKSSYSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLS
ASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASALQSGVPSRFSGSGSGTD
FTLTISSLQPEDFATYYCQQSAYYPDTFGQGTKVEIKEFEQKLISEEDLLEHHHHHH
CTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTA
TGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATC
TATTAATTCTGCTGGTGGTTCTACATATTACGCAGACTCCGTGAAGGGCCGGTTC
ACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTG
AGAGCCGAGGACACGGCGGTATATTACTGTGCGAAAAATTCTGCTTATTTTGACT
ACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCG
GAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCAT
CCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCA
GAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAA
GCTCCTGATCTATGATGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGT
GGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAG
ATTTTGCAACTTACTACTGTCAACAGACTGATACTTATCCTACTACGTTCGGCCA
AGGGACCAAGGTGGAAATCAAAGAATTCGAGCAGAAACTCATCTCAGAAGAGG
MSWVRQAPGKGLEWVSSINSAGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAE
DTAVYYCAKNSAYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLS
ASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYDASNLQSGVPSRFSGSGSGTD
FTLTISSLQPEDFATYYCQQTDTYPTTFGQGTKVEIKEFEQKLISEEDLLEHHHHHH
CTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTA
TGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAG
TATTTCTACTTCTGGTAGTTATACAGCTTACGCAGACTCCGTGAAGGGCCGGTTC
ACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTG
AGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGTGGTTATACTTTTGACT
ACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCG
GAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCAT
CCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCA
GAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAA
GCTCCTGATCTATTCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGT
GGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAG
ATTTTGCAACTTACTACTGTCAACAGAGTGATGGTAATCCTACTACGTTCGGCCA
AGGGACCAAGGTGGAAATCAAAGAATTCGAGCAGAAACTCATCTCAGAAGAGG
MSWVRQAPGKGLEWVSSISTSGSYTAYADSVKGRFTISRDNSKNTLYLQMNSLRAE
DTAVYYCAKGGYTFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLS
ASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYSASTLQSGVPSRFSGSGSGTD
FTLTISSLQPEDFATYYCQQSDGNPTTFGQGTKVEIKEFEQKLISEEDLLEHHHHHH
CTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTA
TGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCGTC
TATTTCTTATTCTGGTGCTTATACAGCTTACGCAGACTCCGTGAACGGCCGGTTCA
CCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGA
GAGCCGAGGACACGGCCGTATATTACTGTGCGAAATCTGGTACTGATTTTGACTA
CTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGG
AGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATC
CTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAG
AGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAG
CTCCTGATCTATGGTGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTG
GCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGA
TTTTGCAACTTACTACTGTCAACAGTCTAATTATGATCCTAGTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAAGAATTCGAGCAGAAACTCATCTCAGAAGAGGA
MSWVRQAPGKGLEWVSSISYSGAYTAYADSVNGRFTISRDNSKNTLYLQMNSLRAE
DTAVYYCAKSGTDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSA
SVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASNLQSGVPSRFSGSGSGTDF
TLTISSLQPEDFATYYCQQSNYDPSTFGQGTKVEIKEFEQKLISEEDLLEHHHHHH
The purity of the 7 scFvs induced at 16° C. was >85% while the purity when induced at 37° C. was lower. Accordingly, 16° C. was determined to be a more suitable condition for production. QC ELISA was conducted to analyze the binding ability to the target MOG for each of the seven scFvs. Compared to the control, differences were readily found in each of the 7 positive clones (clone 1, 3, 6, 10, 13, 17, 21). Among the 7 positive clones, three clones (clone 3, 6 and 17) indicated stronger binding ability to the target.
QC ELISA titration for each of the 7 clones induced at 16° C. was conducted. Seven different concentrations of the 7 clones were employed for ELISA titration. The results indicated that all the 7 clones can specifically bind to the target MOG. Among the 7 clones, clone 3, 6 and 17 still indicated stronger binding ability to the target. The results are shown in
E. coli BL21 (DE3) strains were transfected with pET26b-scFv expression vector in 2YT-K medium. When the strains grew logarithmically, 1 mM and 0.2 mM IPTG were added respectively into the medium to induce the expression of scFv at 26° C. for 16 h. The expression and solubility of the antibodies were tested by SDS-PAGE. The scFv were highly expressed in E. coli BL21 strain, but mainly existed in inclusion bodies.
In order to obtain soluble scFv antibodies, the expression conditions were optimized. When the E. coli BL21 were transfected with pET26b-scFv, they grew logarithmically, and 0.1 mM IPTG were added into medium to induce the expression of scFv at 22° C. overnight. Purification was preformed through affinity chromatography.
The scFv were also purified by inclusion body renaturation. The inclusion bodies were washed once with PBS and then washed twice with 1 M urea. 8 M urea was added to dissolve the inclusion body. Then the scFv was purified through a dialysis bag (8 KDa, Biotics, F132579) and a Millipore concentrator. A total of 1.17 mg of scFv antibody fragments (0.9 mg/mL, 1.3 mL) were obtained finally.
ScFv Labeled with FITC
scFvs were diluted with sodium Carbonate buffer, pH 9.0 to 1 mg/mL. This was mixed with FITC solution (Sigma, F7250), and incubated in the dark at 4° C. overnight. The labeled scFv was concentrated by a Millipore concentrator. A total of 0.83 mg FITC-labeled scFv antibody was finally obtained. The labeling efficiency was determined, and the results showed that 2.52 FITC molecules were labeled for each scFv antibody fragment.
WIEWVKQRPGHGLEWIGEILPGRGRTNYNEKFKGKATFTAETSSNTAYMQLSSLTSE
DSAVYYCATGNTMVNMPYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPSSL
AVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGLPPKLLIYGASTRESGVPDR
FTGSGSGTDFTLTISSVQAEDLAVYYCQNDHSYPLTFGAGTKLEIKRLEHHHHHH*
scFv CAR Antigen Recognition
MOG-1 Expressing HEK293: MOG-1 expressing HEK293 cells 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 media containing 10% FCS and 2 ug/mL puromycin to ensure retention of MOG-1 expression. Cells were periodically FACS purified for MOG-1 expression using FITC-labeled anti-MOG 1 antibody scFv anti-MOG-1 binding activity: 10 ∧4 MOG-1 expressing or parental HEK293 cells were plated at 10 ∧5/mL in a 96 well plate and incubated at 37 C overnight in 5% CO2. Cells were then stained with FITC labeled scFv for 20 minutes (at the concentrations indicated) in DMEM medium with 10% FCS at 37 C. Cells were then fixed and permeabilized according to manufacturer's protocol (100 uL fix perm at room temp for 30 minutes Cytofix-Cytoperm Biolegend). Cells were then washed and stained in perm wash with Cell Mask Red (2 mg/mL) for 30 min to visualize the plasma membrane and subsequently with DAPI (500 nM) for 5 min to visualize the nucleus. Cells were then washed and stored in PBS until imaging on the Leica EVOS M7000 fluorescence microscope. Binding activity was measured by calculating the ratio scFv-FITC signal and the Cell Mask Red signal. Cell Mask Red and scFv-FITC minimum threshold values were set at 3×background. scFv binding values were limited it to 8×background to eliminate nonspecific puncta staining patterns. Results were then plotted as Binding activity vs scFv concentration. See
T cells were transduced with DNA encoding anti-MOG-1 CAR constructs and their ability to recognize MOG-1 on the surface of cells was measured. Briefly, total T cells (Stem cell technologies) were activated with anti-CD3, CD28 antibodies at 2.5e 10 ∧4/mL for 48 h at 37 C. Then the cells were transduced with anti-MOG-1 CAR lentivirus (MOI=5). After 7 days of culture, T cells were harvested and counted and either cryopreserved or used downstream for the CAR recognition assay. 10 ∧4 MOG-1 expressing or parental HEK293 cells were plated at 10 ∧5/mL in a 96 well plate and incubated at 37 C overnight in 5% CO2. CAR T-cells were then incubated with the cells at a ratio of 100:1, 50:1, or 25:1 Treg to HEK cell for 4 h at 37 C. After stimulation a surface stain was performed to identify upregulation of the activation marker CD69 (Biolegend 310930) and the T-reg phenotypic markers CD4, CD25, and CD127 to identify T-regs in addition to an intracellular stain for FOXP3 and FLAG Tag to identify transduced T-regs. The cells were read on a FACS aria at the tufts university flow core and data was analyzed using flowjo with analysis being performed in graphpad prism. CD69MFI was measured from the CD4+CD25hi, CD127lo, FLAG+ gate with the MOG-HEK and HEK-Parental groups being compared. (See
One vial of virus was used to transduce 5 E5 donor PBMCs in a 24 well plate. Following expansion protocol, cells were harvested and Flag expression was determined by flow. Flag tag was detected using anti-Flag antibody (mouse anti-DDDDK, ProMab, Cat. #20201). Viral transduction was performed on 2 separate dates, A and B. (See
Two different CAR constructs for each scFv were tested. One construct encodes the variable domains in the order VH-VL and the other in the order VL-VH. Testing two different CAR variants based on a core scFv sequence allows for selection of a CAR with the best surface expression, stability, antigen recognition and signaling capabilities.
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.
The primary objective will be to assess the safety and feasibility of intravenous infusion of ex vivo—selected, expanded and transduced autologous CNS-specific CAR-Tregs in at least 5 patients with PSP.
Primary outcome measures will be:
1. adverse events
2. laboratory abnormalities
3. infusion reactions
4. complications related to infection
5. potential negative impact on the course of PSP
The secondary objective will be to assess the effect of CNS-specific CAR-Tregs on PSP and obtain indication on the potential application in other neurodegenerative diseases. The end points will be:
1. Assess the effect of CNS-specific CAR-Tregs on clinical, neuropsychological, radiological and biomechanical parameters in PSP patients.
2. Obtain indication on the potential therapeutic usage of CNS-specific CAR-Tregs in other neurodegenerative diseases, including Alzheimer's disease (AD).
3. Obtain indication on a potential phase-II randomized, double-blind, placebo-controlled trial that may provide valuable insights into the potential efficacy of CAR-Tregs for neurodegenerative disorders.
Clinical and neuropsychological assessment: 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. Transl. Med. 2014; Canesi et al., J. Transl. Med. 2016, incorporated herein by reference). The 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). Mini mental state evaluation (MMSE) will be also performed as previously described (Folstein et al. J. Psychiatr. Res. 1975, incorporated herein by reference). All these tests will be assessed at baseline and at each follow-up point (1, 3, 6 and 12 months after cell administration). The clinical conditions will be classified as “stable” if the UPDRS and PSP-RS scores did not diminished by more than 30% compared to baseline and the H&Y staging did not change at the defined time point (Canesi et al., J. Transl. Med. 2016, incorporated herein by reference).
Neuroimaging: All patients will undergo longitudinal neuroimaging assessments, using brain magnetic resonance imaging (Mill) (baseline, 24 h after cell administration and after 1 year), striatal dopamine transporter single photon emission computed tomography (SPECT) and positron emission tomography (PET) (both at baseline and after 12 months). Tropanic tracers labeled with Iodine-123 (FP-CIT) and 18F-Fluoro-2-deoxyglucose (Beta-CIT) will be used for SPECT imaging and for PET/TC imaging, respectively.
For SPECT, intravenous administration of 110-140 MBq of [1231] FP-CIT (Datscan, GE-Health, Amersham, UK) will be performed 30-40 min after thyroid blockade (10-15 mg of Lugol solution per os) in all patients. The analysis will be performed as previously described (Isaias et al., NeuroReport 2007, incorporated herein by reference). A volumetric template of grey matter anatomic distribution will be generated from the Montreal Neurological Institute MRI single participant brain atlas by applying a macroscopic anatomical method (automated anatomic labeling), and will be reoriented and reformatted to obtain a 2.64-cm thick reference section. A template of eight irregular regions of interest (ROIs) will be manually drawn on this section to assess the anatomical extent of striatal and occipital structures having both specific and nonspecific uptake of [123 I] FP-CIT, respectively. The ROI template will be also positioned on the reference SPECT section and adjusted on both striatal and occipital cortex. Striatal ROIs will be also segmented into their anterior (caudate nucleus) and posterior (putamen) portions. Specific striatal dopamine uptake transporter (DAT) binding of [123 I] FP-CIT will be calculated in the whole striatum, putamen and caudate nucleus using the formula: [(mean counts in specific ROI)—(mean counts in occipital ROI)]/(mean counts in occipital ROI). Putamen/caudate ratios for each subject will also be calculated.
All patients will also undergo F-Fluoro-2-deoxyglucose positron emission tomography scanning (FDGPET) at rest, after intravenous injection of 170 MBq. Each acquisition will include a computed tomography (CT) transmission scan of the head (50 mAs lasting 16 s) followed by a three dimensional (3D) static emission of 15 min using a Biograph Truepoint 64 PET/CT scanner (Siemens). PET sections will be reconstructed using an iterative algorithm (OS-EM), corrected for scatter and for attenuation, using density coefficients derived from the low dose CT scan of the head obtained with the same scanner. Images will be reconstructed in the form of transaxial images of 128 Ř 128 pixels of 2 mm, using an iterative algorithm, ordered-subset expectation maximization (OSEM). The resolution of the PET system will be 4-5 mm FWHM.
Biomechanical evaluation: Biomechanical evaluation will be assessed at baseline and at six and 12 months after CAR-Treg cell administration. Two specific sets of parameters, one for standing and one for gait initiation, will be automatically extracted by means of ad hoc algorithms (Carpinella et al., IEEE Trans Neural Syst Rehabil Eng. 2007, incorporated herein by reference). For standing, the center of pressure (CoP) mean velocity and spatial displacement will be measured (Canesi et al., J. Transl. Med. 2016, incorporated herein by reference). To examine gait initiation, anticipatory postural adjustment will be analyzed (Canesi et al., J. Transl. Med. 2016, incorporated herein by reference) (i.e. imbalance and unloading phases) and measure the following parameters: (1) the duration of both phases, (2) the antero-posterior (AP) and mediolateral (ML) shift and velocity of the CoP, (3) the CoP mean length and velocity. The (4) length and (5) velocity of the first step will also be measured. Spatial parameters will be normalized on the basis of body height (% BH).
Treg isolation and expansion: PolyTregs will be selected and expanded from five individuals with PSP based on three cell surface markers—CD4, CD25, and CD127—to purify the FOXP3+ Tregs present in the peripheral blood as described previously (Putnam et al., Diabetes 2009; Bluestone et al., Sci. Transl. Med., 2015, incorporated herein by reference).
400 ml of fresh peripheral blood will be collected into blood pack units containing citrate phosphate dextrose and processed within 24 hours for isolation of PBMCs via Ficoll density gradient. Tregs will be isolated on a high-speed cell sorter with the following GMP-grade lyophilized antibodies: CD4-PerCP (peridinin 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 no. 04-418Q) containing 10% human heat-inactivated pooled AB serum (Valley Biomedical). Tregs will be analyzed for purity after sorting. The expected purity of CD4+CD127lo/—CD25+ cells is more than 96% (Bluestone et al., Sci. Transl. Med., 2015, incorporated herein by reference).
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. Transl. Med., 2015, incorporated herein by reference). A unit of blood is expected to yield between 4.2×106 and 11.8×106 purified CD4+CD127lo/—CD25+ Tregs, (Bluestone et al., Sci. Transl. Med., 2015, incorporated herein by reference). The expanded Treg preparations are expected to be around 90% FOXP3+. Treg preparations will be checked for viability, CD4+ percentage, and CD8+ cell contamination (Bluestone et al., Sci. Transl. Med., 2015, incorporated herein by reference).
Phenotypic and TCR analysis of expanded polyTregs: Key cell surface markers, CD4 and CD127, used to isolate the Tregs, will be checked after expansion.
Previous data have shown that the naïve CD45RA+ Tregs preferentially expand in these cultures and CD45RA+RO− cells down-regulate CD45RA and up-regulate CD45RO over the expansion period (Bluestone et al., Sci. Transl. Med., 2015, incorporated herein by reference). CCR7, a Treg trafficking receptor, CD38, a multifunctional ectoenzyme associated with enhanced Treg function and CD45 RO will be determined before and after expansion. The TCRβ repertoire of the expanded Tregs will be also analyzed and compared to the freshly isolated populations to determine the polyclonality of the expanded Tregs. The expanded cells are expected to exhibit polyclonality indistinguishable from the preexpansion cultures and that the Tregs remain a highly diverse population after expansion (Bluestone et al., Sci. Transl. Med., 2015, incorporated herein by reference).
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):
Production and functional analysis of CAR-Treg cells: CAR RNA will be optimized for anti-MOG CAR expression on Tregs after electroporation of human Tregs and on mouse Tregs after adoptive transfer to PSP mouse models as per published protocols (Zhao, Y et al., 2010 Cancer Res and Beatty, G L et al., 2014 Cancer Immunol Res; Singh, N et al., 2014 Oncoimmunol; the contents of each of which are incorporated herein by reference). Delivery of anti-MOG CAR using second-generation lentivirus vectors and standard protocols (Levine, B. L. et al., 2017 Mol Ther Methods & Clin Dev, incorporated herein by reference) will also be optimized. Human Tregs will be either transduced under GMP condition using electroporation of RNA (about 1 μg/3×106 Treg) or by lentivirus transduction (1×106 pfu/3×106 Treg). Clinical grade RNA for anti-MOG CAR will be produced. About 0.9 mg RNA per patient for 2.6×109 Treg cells; 4.5 mg for 5 patients will be required. Clinical grade lentivirus will be produced. About 8.7×109 pfu per patient for 2.6×109 Treg cells; 4.3×1010 pfu for 5 patients will be required. Functional analysis of MOG-specific CAR-Tregs will be performed as described above in 3.3.
One single administration of MOG-specific CAR-Tregs will be performed for each patient (2.6×109 CAR-Tregs per patient). Cells will be administered to at least 5 PSP patients. Patients will receive premedication with acetaminophen and diphenhydramine. CAR-Tregs will be infused via a peripheral intravenous line over 10 to 30 min. Vital signs will be taken before and after infusion, then every 15 min for at least 1 hour, then every hour for the first 4 hours, and every 4 hours for 20 hours. Chemistries and complete blood count with differential blood count will be repeated the next day before discharge from the clinical research unit. Patients will be seen for follow-up assessments on day 4 after infusion, then weekly for 4 weeks, then every 13 weeks for 1 year, and then every 26 weeks for 2 years. Telephone monitoring for adverse events will continue every 6 months for 5 years after infusion followed by a final clinic visit.
Patient Assessment after Car-Treg Cell Infusion
The effect of CNS-specific CAR-Tregs on clinical, neuropsychological, radiological and biomechanical parameters in PSP patients will be assessed as described above. All the tests will be performed at each follow-up point: 1, 3, 6 and 12 months after cell administration.
Drugs for Multiple Sclerosis (MS) that exploit components of immunosuppressive T regulatory lymphocytes (Tregs) to shut down the damaging immune responses that cause disease will be developed and tested. The worldwide MS market is about $21.5 billion but the approved drugs for the most common form of MS give only modest disease modification with significant side effects. For more severe forms of MS the treatment options are limited to only 1 recently approved drug.
There are 11 FDA approved drugs for Relapsing Remitting form of MS (RRMS—85% of diagnosed MS). There are several orally available and antibody-based drugs currently approved or under clinical evaluation for RRMS. In March 2017 the FDA approved the use of Ocrelizumab (anti-CD20 antibody, Roche) for Primary Progressive MS (PPMS— about 10% of diagnosed MS). Ocrelizumab gave a 25% reduction in symptoms and is at present the only immunomodulatory for PPMS in the USA. Secondary Progressive MS (SPMS) invariably develops in patients with RRMS, for which there are also limited disease modification options.
Anti-MOG hybridomas will be produced by immunization of mice with recombinant human MOG through CROs. VH and VL genes will be cloned and anti-scFv molecules. The orientation of VH and VL as well as the linkers (between the scFv or inside each scFv) may greatly affect the stability, expression level and binding ability 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 scale-up production. An expression construct will be generated encoding 4 anti-MOG scFv with connecting linkers, a central linker, then connecting to Treg-associated enzymes or mimetics.
GTIPs will be tested in mouse, acute and chronic EAE models of MS. The levels of Th1, Th17, CTL (blood and CNS) specific for myelin basic protein (MBP), myeloid inflammatory cells (macrophages and neutrophils) and anti-MBP antibodies will be measured. Immunological responses will be correlated with disease progression. Dosing will be varied to gain insights into potential use in late stage MS. Products will be dosed in normal mice to gain insights into any potential off target effects.
Clinical studies will be conducted to test the efficacy the GTIPs as disease modifying agents in MS. The products will first be tested be in RRMS patients who are unresponsive to first use drugs. Safety and tolerability will be measured using dosing regimes similar to those for antibody therapies (e.g. first 3 i.v. doses every 2 weeks then every 4 weeks for 20 weeks). In phase 2 studies, primary measures will be decreased disease relapse frequency and brain lesions. Secondary measures will be decreased inflammatory cytokines, Th1/Th17 cells and other leukocytes levels in the blood. Side effects may include increases susceptibility to infection. These studies will allow us to benchmark the effectiveness of the compounds against other second use drugs that give up to 49% reduction in relapse frequency. If the products show acceptable levels of efficacy then they will be entered into the longer-term clinical phase 2 studies in PPMS patients. Primary measures will be delayed decline in motor function and reduced brain lesions and secondary measures will be reduced levels of inflammatory cytokines, Th1/Th17 cells and other leukocytes in the blood.
All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g. Genbank sequences or GeneID entries), patent application, or patent, was specifically and individually indicated incorporated by reference in its entirety, for all purposes. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. § 1.57(b)(1), to relate to each and every individual publication, database entry (e.g. Genbank sequences or GeneID entries), patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. § 1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.
While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it is understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.
This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/031,261, filed on May 28, 2020, which is incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/034968 | 5/28/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63031261 | May 2020 | US |
