Treatment of cerebrovascular events and neurological disorders

Abstract

The present invention provides a pregnancy-specific glycoprotein (e.g., PSG1) for use in a method for treating neurological disorders or stimulating the survival or recovery of tissue damaged by cerebrovascular events or brain trauma in a subject. PSG1 can be Fc-tagged PSG1.

Description

[Technical field]

[0001] The present invention relates to the use of placenta-expressed proteins to treat cerebrovascular events, such as acute ischemic stroke, and neurological disorders. [Background technology]

[0002] Pregnancy-specific glycoproteins (PSGs) are thought to be involved in regulating immune, angiogenic, and platelet responses at the maternal-fetal interface and in the maternal circulation during pregnancy. PSG proteins are part of the carcinoembryonic antigen cell adhesion molecule (CEACAM) family, which are themselves members of the immunoglobulin superfamily. PSG proteins vary considerably in structure among primates, horses and rodents, but retain conserved functions (Aleksic D, et al. Convergent evolution of pregnancy-specific glycoproteins in human and horse. Reproduction. 2016 Sep;152(3):171-84. doi:10.1530 / REP-16-0236. Epub 2016 Jun 8. Moore T, Dveksler GS. Pregnancy-specific glycoproteins: complex gene families regulating maternal-fetal interactions. Int J Dev Biol. 2014;58(2-4):273-80. doi:10.1387 / ijdb.130329gd. Review. PMID:25023693.). There are 11 and 17 distinct PSG genes encoding PSG proteins in humans and mice, respectively. Human PSGs consist of one N-terminal immunoglobulin variable (IgV)-like domain (N domain) followed by generally two to three Ig constant (IgC)-like domains of two different types (designated A and B), whereas rodent PSGs contain two to nine consecutive N domains followed by one IgC-like domain. Seven horse CEACAM-derived PSG-like proteins have a single N domain and an A2 domain (Aleksic et al., 2016).

[0003] PSG1 is an abundantly expressed member of 11 distinct human PSG genes, and one study estimated total PSG protein concentrations to exceed 100 μg / ml during the third trimester of pregnancy. During pregnancy, transforming growth factor beta (TGF-β) regulates trophoblast invasion, angiogenesis, and extracellular matrix production. Treatment of various cells with PSG1 or other PSGs increased the secretion of total TGF-β1 in the supernatant as well as the activation of latent TGF-β1, as determined by ELISA [Ballesteros A, Mentink-Kane MM, Warren J, Kaplan GG, Dveksler GS. Induction and activation of latent transforming growth factor-β1 are carried out by two distinct domains of pregnancy-specific glycoprotein 1(PSG1). J Biol Chem.2015 Feb 13;290(7):4422-31.doi:10.1074 / jbc.M114.597518.Epub 2014 Dec 29.].

[0004] WO2017049082 describes one specific PSG protein, PSG1, and its involvement in the pathways involved in the induction of immune tolerance. PSG1 is involved in the activation of transforming growth factor-β1 (TGFβ1), a cytokine important for the differentiation of tolerance-inducing CD4+CD25+FoxP3+ regulatory T cells (Tregs), a cell population that has been shown to be essential for the suppression of inflammatory T cells and important for the prevention of graft-versus-host disease (GvDH). Summary of the Invention

[0005] The applicant has found that PSG1 can modify the phenotype of microglial cells in mammals from M1 pro-inflammatory phenotype to M2 anti-inflammatory phenotype (Figure 1). Microglia are resident immune cells of the CNS and are involved in the pathogenesis of various neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, prion disease, and multiple sclerosis and amyotrophic lateral sclerosis. Thus, the present invention relates to the use of PSG1 in the treatment of neurological disorders. The applicant has also found that PSG1 provides improved behavioral outcomes in mouse stroke models after treatment (foot injury test) (Figure 2), and is therefore applicable to the treatment of stroke and other cerebrovascular events and traumatic brain injury in mammals.

[0006] In a first aspect, the present invention provides a pregnancy specific glycoprotein (eg, PSG1) for use in a method of stimulating survival or recovery of tissue damaged by a cerebrovascular event or traumatic brain injury.

[0007] In another aspect, the present invention relates to the use of a pregnancy specific glycoprotein (eg, PSG1) as a neuroprotective agent in a subject.

[0008] In some embodiments, the PSG1 is Fc-tagged PSG1 (PSG1-Fc).

[0009] In any embodiment, the cerebrovascular event is an acute ischemic stroke.

[0010] In any embodiment, the cerebrovascular event is a hemorrhagic stroke.

[0011] In any embodiment, the cerebrovascular event is a transient ischemic attack (TIA).

[0012] In any embodiment, PSG1 is administered to the subject intravenously.

[0013] In any embodiment, PSG1 is administered locally to the site of brain injury using a drug delivery catheter.

[0014] In any embodiment, PSG1 is administered using a drug delivery system described in Pharm Res 2013 Oct;30(10):2429-44.

[0015] In any embodiment, PSG1 is administered to a subject at a dosage of 0.01-100, typically 0.01-0.1, 0.1-1.0, 1.0-10, or 10-100 mg / kg body weight.

[0016] In any embodiment, PSG1 is administered to the subject within 72, 48, or 24 hours, preferably within 18 hours, more preferably within 12 hours, more preferably within 6 hours, more preferably within 4 hours, and ideally within 2 hours, of a cerebrovascular event.

[0017] In any embodiment, the method includes administering another drug, such as a thrombolytic drug, a blood thinner, or an antiplatelet agent. Examples of blood thinners include apixaban (Eliquis), dabigatran (Pradaxa), edoxaban (Savaysa), fondaparinux (Arixtra), heparin (Fragmin, Inohep, and Lovenox), rivaroxaban (Xarelto), warfarin (Coumadin, Jantoven), and antiplatelet agents. Examples of thrombolytic drugs include tissue plasminogen activator (tPA), tenecteplase, alteplase, urokinase, reteplase, and streptokinase.

[0018] In any embodiment, the method includes performing an endovascular thrombectomy on the subject.

[0019] In any embodiment, the method results in the amelioration of one or more symptoms of the cerebrovascular event or brain trauma. Exemplary symptoms include: Difficulty walking, dizziness, Loss of balance and coordination, Difficulty speaking or understanding others who are speaking, numbness or paralysis in the face, legs, or arms (most likely on only one side of the body); Blurred or dark vision, as well as Sudden headache, especially if accompanied by nausea, vomiting or dizziness.

[0020] In a further aspect, the present invention provides pregnancy specific glycoprotein 1 (PSG1) for use in a method of treating a neurological disease / disorder, such as a disease or condition characterized by neurodegeneration, neuronal cell death or demyelination in a mammal. Examples include diseases characterized by altered regulation of neural stem cells or accessory cells such as microglia and oligodendrocytes, resulting in neuronal cell death or demyelination. PSG1 can promote neuronal repair through direct effects on neural cell lineages, including stem cells, by modulating the phenotype (including cytokine production) of accessory cells such as microglia and oligodendrocytes, and by modulating the central and peripheral immune systems. Jurkat and microglial cells treated with PSG1 have been found to exhibit altered cytokine profiles consistent with delayed disease progression. In one embodiment, PSG1 is Fc-tagged pregnancy specific glycoprotein 1 (PSG1-Fc).

[0021] In one embodiment, the disease or condition characterized by neurodegeneration causes neuronal cell death. In one embodiment, the neurodegenerative disease is ALS (also known as motor neuron disease or Lou Gehrig's disease) or its variants, including primary lateral sclerosis and spinal muscular atrophy. Other neurodegenerative diseases include Alzheimer's disease and other memory disorders, ataxia, Huntington's disease, Parkinson's disease, multiple system atrophy, and progressive supranuclear palsy.

[0022] In one embodiment, the disease or condition characterized by neurodegeneration is a demyelinating disease, hi one embodiment, the neurodegenerative disease is MS (also known as multiple sclerosis) or a variant thereof.

[0023] In any embodiment, the neurological disorder is a seizure disorder, for example, epilepsy.

[0024] In one aspect, the invention provides PSG1, particularly Fc-tagged PSG1, for use in the causative treatment or prevention of diseases or conditions associated with demyelination of the mammalian central nervous system (CNS) or peripheral nervous system (PNS) by maintaining the integrity of myelination of axonal cells.

[0025] As used herein, the term "maintaining the integrity of myelination of axonal cells" should be understood to mean effecting remyelination of axonal cells, inhibiting or preventing demyelination of axonal cells, or both. The term "causal" treatment should be understood to mean treatment of demyelination as the underlying cause of disease and to exclude treatments intended to directly address the clinical symptoms of disease (symptomatic treatments).

[0026] In another aspect, the present invention broadly relates to a method for treating or preventing a disease or condition associated with demyelination in the mammalian central nervous system (CNS) or peripheral nervous system (PNS) by maintaining the integrity of myelination of axonal cells, the method comprising administering a clinically effective amount of PSG1, particularly Fc-tagged PSG1, to an individual in need thereof.

[0027] In another aspect, the present invention relates to a method for treating recurrence, particularly acute recurrence, of a disease or condition associated with demyelination of the central nervous system (CNS) or peripheral nervous system (PNS) of a mammal by maintaining the integrity of myelination of axonal cells, comprising administering a clinically effective amount of PSG1, particularly Fc-tagged PSG1, to the mammal upon recurrence of the disease or condition.Preferably, administration of a clinically effective amount of the active agent is continued during the period of recurrence of the disease or condition, and is preferably discontinued upon remission of the disease or condition.

[0028] In another aspect, the present invention relates to a method for prolonging the remission period of a disease or condition associated with demyelination of the central nervous system (CNS) or peripheral nervous system (PNS) of a mammal by maintaining the integrity of myelination of axonal cells, comprising administering a clinically effective amount of PSG1, particularly Fc-tagged PSG1, to the mammal during the remission period of the disease or condition.Preferably, the administration of a clinically effective amount of the active agent is continued during the relapse period of the disease or condition.

[0029] In another aspect, the present invention relates to a non-immune-mediated therapy (preferably a non-immune-mediated causal therapy) for diseases or conditions associated with demyelination of the central nervous system (CNS) or peripheral nervous system (PNS) of a mammal, particularly multiple sclerosis (MS), particularly primary progressive MS, the method comprising administering to the mammal a clinically effective amount of PSG1, particularly Fc-tagged PSG1, in an amount sufficient to cause clinically significant remyelination of axonal sheaths of the mammal, or inhibition of demyelination. The term "non-immune-mediated therapy" should be understood to mean a therapy that is not aimed at attenuating the immune response of the mammal.

[0030] In a further aspect, the present invention provides a pregnancy specific glycoprotein (e.g., PSG1) for use in a method of treating traumatic brain injury in a mammal, such as a method of stimulating survival or recovery of tissue damaged by traumatic brain injury. In any embodiment, the pregnancy specific glycoprotein is administered to tissue damaged by traumatic brain injury.

[0031] In a further aspect, the present invention provides a pregnancy-specific glycoprotein (e.g., PSG1) for use in a method for treating spinal cord injury in a mammal, for example, a method for stimulating survival or recovery of spinal cord tissue damaged by traumatic spinal cord injury or traumatic spinal cord disease.In any embodiment, the method is a method for treating spinal cord pathology, such as glial scarring of the spinal cord, caused by traumatic spinal cord injury or traumatic spinal cord disease.In any embodiment, the method is a method for regenerating spinal cord tissue of the damaged spinal cord.

[0032] In a further aspect, the present invention provides a composition comprising Fc-tagged pregnancy-specific glycoprotein 1 (PSG1-Fc) and a cell graft comprising neural stem cells and mesenchymal stem cells.

[0033] In one embodiment, PSG1 is administered by intramuscular, intrathecal, subcutaneous, intravenous or intraperitoneal delivery, or by injection into the affected area.

[0034] In one embodiment, PSG1 is administered to a mammal by transfecting the mammal with a PSG1 (or PSG1-Fc) expression vector.

[0035] In one embodiment, PSG1 is administered to the mammal by administering to the mammal cells transfected with a PSG1 (or PSG1-Fc) expression vector. In one embodiment, the cells are stem cells. In one embodiment, the cells are brain cells (e.g., neurons, oligodendrocytes, astrocytes, or microglial cells) or mesenchymal stem cells. In one embodiment, donor cells are obtained from the recipient. In one embodiment, donor cells are obtained from a mammal (e.g., human) different from the recipient (allogeneic cell therapy). In one embodiment, the cells are transfected ex vivo.

[0036] The methods of the invention provided above recite PSG1 and modified versions of PSG1, such as PSG1-Fc, in the treatment, although the invention also relates to the use of PSG proteins other than PSG1 (and modified versions thereof) in the above treatment methods.

[0037] In one embodiment, PSG1 (or PSG) is modified by a functional moiety. The functional moiety may be configured to increase the plasma half-life of the modified PSG1. The functional moiety may be configured to increase the cell-penetrating functionality of the modified PSG1. The functional moiety may be configured to increase the activity of the modified PSG1. The functional moiety may be configured to facilitate the purification of the modified PSG1. Examples of modifications of PSG1 polypeptides are provided below, and examples of modifications of PSG1 polypeptides include antibody fragments, such as the addition of an Fc moiety, the addition of a PEG functional group, and the replacement of natural amino acids with L-isomers. In one embodiment, the functional group is an Fc moiety. In one embodiment, the Fc moiety is modified to have an increased plasma half-life compared to the natural Fc moiety.Altered Fc tags are described in the following papers: Algirdas Grevys, Malin Bern, Stian Foss, Diane Bryant, Terje Bratlie, Anders Moen, Kristin Stoen Gunnarsen, Audun Aase, Terje Einar Michaelsen, Inger Sandlie and Jan Andersen. Fc Engineering of Human IgG1 for Altered Binding to the Neonatal Fc Receptor Affects Fc Effec-tor Functions.Journal of Immunology June 1,2015,194(11)5497-5508.Dall'Acqua WF,Kiener PA,Wu H.Properties of human IgG1s engineered for enhanced binding to the neonatal Fc receptor(FcRn).The Journal of Biological Chemistry.281:23514-23524(2006).Abhishek Saxena and Donghui Wu. Advances in Therapeutic Fc Engineering-Modulation of IgG-Associated Effector Functions and Serum Half-life. Frontiers in Immunology. 2016;7:580. Exemplary modifications to the human Fc tag include triple substitutions YTE (M252Y / S254T / T256E) in the CH2 domain and (H433K / N434F) in the CH3 domain to increase stability and half-life (SEQ ID NO:3). These modifications can be made using site-directed mutagenesis. In one embodiment, the present invention provides a PSG1 protein conjugated with an Fc tag encoded by SEQ ID NO:3.

[0038] Further aspects and preferred embodiments of the present invention are defined and described in the other claims set forth below. [Brief description of the drawings]

[0039] [Figure 1] Phenotypic modulation of C20 microglial cell line ( Garcia-Mesa et al., 2017 ) by recombinant PSG1-V5His protein at 50ug / ml for 5 hours. [Diagram 2] Improved behavioral outcome (paw injury test) in a mouse stroke model following treatment with 100ug of recombinant PSG1-Fc. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] All publications, patents, patent applications, and other references mentioned herein are incorporated by reference in their entirety for all purposes as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference and the contents of which were cited in full.

[0041] Definitions and general preferences As used herein, unless specifically indicated otherwise, the following terms are intended to have the following meanings, in addition to any broader (or narrower) meanings that may be enjoyed in the art:

[0042] Unless otherwise required by context, the use of the singular herein should be read to include the plural and vice versa. The term "a" or "an" used in reference to an entity should be read to refer to one or more of that entity. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0043] As used herein, the term "comprise" or variations thereof, such as "comprises" or "comprising," should be read to indicate the inclusion of any enumerated integer (e.g., feature, element, characteristic, property, method / process step, or limitation) or group of integers (e.g., feature, element, characteristic, property, method / process step, or limitation) but not the exclusion of any other integer or group of integers. Thus, as used herein, the term "comprising" is inclusive or open-ended and does not exclude additional unenumerated integers or method / process steps.

[0044] As used herein, the term "disease" is used to define any abnormal condition that impairs physiological function and is associated with a specific symptom.The term is used broadly to include any disorder, disease, disorder, condition, illness, state or syndrome in which physiological function is impaired, regardless of the nature of etiology (or whether or not the etiological basis of the disease is actually established).The term therefore includes conditions resulting from infection, trauma, injury, surgery, radiological ablation, age, poisoning or nutritional deficiency.

[0045] As used herein, the term "treatment" or "treating" refers to an intervention (e.g., administration of a drug to a subject) that cures, improves or alleviates the symptoms of a disease, or eliminates its cause (or reduces the effects of its cause) (e.g., reducing the accumulation of pathological levels of lysosomal enzymes). In this case, the term is used synonymously with the term "therapy."

[0046] Additionally, the terms "treatment" or "treating" refer to an intervention (e.g., administration of a drug to a subject) that prevents or delays the onset or progression of a disease, or reduces its incidence (or eradicates) within the treated population. In this context, the term treatment is used synonymously with the term prophylaxis.

[0047] As used herein, an effective amount or therapeutically effective amount of a drug defines an amount that may be administered to a subject without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit / risk ratio, but sufficient to provide the desired effect, e.g., treatment or prevention, manifested by a sustained or temporary improvement in the subject's condition. The amount will vary from subject to subject, depending on the age and general condition of the individual, the mode of administration, and other factors. Thus, it is not possible to specify an exact effective amount, but one of ordinary skill in the art will be able to determine an appropriate "effective" amount in any individual case, using routine experimentation and general knowledge of the background art. In this context, therapeutic results include eradication or relief of symptoms, reduction in pain or discomfort, prolonged survival, improved mobility, and other markers of clinical improvement. A therapeutic result need not be a complete cure. Improvement may be observed in biological / molecular markers, clinical or observational improvement. In a preferred embodiment, the method of the present invention is applicable to humans, large racing animals (horses, camels, dogs) and domestic companion animals (cats and dogs).

[0048] In the context of the above-defined treatment and effective amount, the term subject (which should be read as including "individual", "animal", "patient" or "mammal" if the context allows) defines any subject, particularly a mammalian subject, to which treatment is applied.Mammalian subjects include, but are not limited to, humans, farm animals, livestock, zoo animals, sports animals, pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, camels, bison, cattle, dairy cows; primates, such as apes, monkeys, orangutans and chimpanzees; canines, such as dogs and wolves; felines, such as cats, lions, tigers; equines, such as horses, donkeys and zebras; food animals, such as dairy cows, pigs and sheep; ungulates, such as deer and giraffes; and rodents, such as mice, rats, hamsters and guinea pigs.In a preferred embodiment, the subject is a human. As used herein, the term "equine" refers to a mammal of the family Equidae, which includes the horse, donkey, ass, Tibetan ass, and zebra.

[0049] As used herein, the term "cerebrovascular event" or "CVA" refers to a subject's cerebral ischemia or hemorrhage, which causes a neurological symptom or group of neurological symptoms. It is also commonly referred to as "stroke". The main clinical features are sudden or subacute onset and focal neurological deficits (except for subarachnoid hemorrhage). Depending on when the patient is seen and the underlying cause, the deficits may be stable, progressive, or completely resolved. Apart from these common features, cerebrovascular diseases are a highly diverse group of disorders that are further classified according to etiology, location, and duration of symptoms. Cerebrovascular diseases are subdivided into ischemic events and cerebral hemorrhage, each of which has many etiologies. Ischemic events are further classified according to whether symptoms occur in the carotid or vertebrobasilar distribution, and by the duration of symptoms. Transient ischemic attacks (TIAs) rarely last more than a few minutes and never last more than 24 hours. In ischemic strokes, neurological deficits are present for more than 24 hours and may be progressive, stable, or resolving.

[0050] As used herein, the term PSG protein refers to the CEACAM-related protein that lacks cell membrane anchor and is mainly expressed in placental tissue.Such protein is found in a subset of mammals, including, for example, primates, rodents, horses, and bats, but not found in, for example, ungulates and canines (Robert Kammerer, Wolfgang Zimmermann.Coevolution of activating and inhibitory receptors within mammalian carcinoembryonic antigen families.BMC Biol.2010;8:12.Published online 2010 Feb 4.doi:10.1186 / 1741-7007-8-12). Examples of PSG gene and protein sequences are available (McLellan AS, Fischer B, Dveksler G, Hori T, Wynne F, Ball M, Okumura K, Moore T, Zimmermann W. Structure and evolution of the mouse pregnancy-specific glycoprotein (Psg) gene locus. BMC Genomics. 2005 Jan 12;6:4. PMID:15647114; Kammerer & Zimmermann, 2010; Aleksic et al., 2016). In general, PSGs (i.e., PSG1) are recombinant proteins.

[0051] As used herein, the term "pregnancy-specific glycoprotein 1" or "PSG1" refers to the full-length protein represented by SEQ ID NO:1 below, including the signal sequence (amino acid residues 1-34), the mature peptide (residues 35-419), and the mature peptide without the signal sequence. The term also includes the mature peptide without the signal sequence.

[0052] SEQ ID NO:1: JPEG2024514195000002.jpg66138

[0053] The term "PSG1" also includes Fc-tagged PSG1 proteins (PSG1-Fc), an example of which is provided in SEQ ID NO:2 below, in which the Fc tag has been modified by site-directed mutagenesis to introduce the MTS mutations M252Y / S254T / T256E and the HN mutations H433K / N434F:

[0054] SEQ ID NO:2 JPEG2024514195000003.jpg240160

[0055] SEQ ID NO:3 provides the open reading frame for a modified Fc tag incorporating the MTS mutations M252Y / S254T / T256E and the HN mutations H433K / N434F:

[0056] SEQ ID NO:3 Modified human Fc ORF JPEG2024514195000004.jpg86159

[0057] The term "PSG1" also includes mutants, which are proteins with substantially identical amino acid sequences to wild-type PSG1 protein, typically human wild-type PSG1 protein. Thus, for example, the term should be interpreted to include proteins or polypeptides that are altered with respect to one or more amino acid residues. Preferably, such alterations involve the insertion, addition, deletion and / or substitution of no more than 5 amino acids, more preferably no more than 4, even more preferably no more than 3, and most preferably only 1 or 2 amino acids. Insertions, additions and substitutions with natural and modified amino acids are envisaged. Mutants may have conservative amino acid changes, where the amino acid introduced is structurally, chemically or functionally similar to the amino acid that is replaced. Typically, proteins that are altered by the substitution or deletion of catalytically important residues are excluded from the term "mutant". Details of such catalytically important residues are well known to those skilled in the art of protein modelling. In general, the variants have at least 70% amino acid sequence homology with the wild-type protein (excluding the signal peptide as described above), preferably at least 80% sequence homology, more preferably at least 90% sequence homology, ideally at least 95%, 96%, 97%, 98% or 99% sequence homology. In this context, sequence homology includes both sequence identity and sequence similarity, i.e., a polypeptide sequence that shares 70% amino acid homology with the wild-type protein is one in which any 70% of the aligned residues are identical to or conservative substitutions for the corresponding residues in the wild-type protein. With respect to PSG1, specific variants included within the scope of the present invention are the variant PSG1 proteins identified in International Patent Application Publication No. WO2017049082, paragraphs 25-38. The term also includes PSG1 proteins modified with a tag, such as an Fc tag, including a human Fc tag. The Fc tag may be modified to exhibit increased plasma half-life and / or stability. Such Fc tags are known from the literature and are described herein.Examples of modifications to the human Fc tag include triple substitutions MST (M252Y / S254T / T256E) in the CH2 domain and (H433K / N434F) in the CH3 domain to increase stability and half-life.

[0058] PSG1 for use in the present invention can be produced in whole or in part by chemical synthesis or by expression from nucleic acid (i.e., recombinant). For example, the proteins of the present invention and for use in the present invention can be easily prepared according to well-established standard liquid phase protein synthesis methods known in the art or preferably solid phase protein synthesis methods (see, for example, JM Stewart and JD Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984), in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984)). If necessary, any of the proteins used in the present invention can be chemically modified to increase their stability. Chemically modified proteins or protein analogs include any functional chemical equivalent of a protein that is characterized by increased stability and / or efficacy and / or half-life in vivo or in vitro with respect to the implementation of the present invention. The term protein analog also refers to any amino acid derivative of the proteins described herein. Protein analogs can be generated by procedures including, but not limited to, side chain modifications, incorporation of unnatural amino acids and / or their derivatives during protein synthesis, and the use of crosslinkers and other methods that impose conformational constraints on proteins or their analogs. Examples of side chain modifications include, for example, reaction with an aldehyde followed by NaBH 4These include reductive alkylation by reduction with methylacetimidate; amidation with methylacetimidate; acetylation with acetic anhydride; carbamylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6,trinitrobenzenesulfonic acid (TNBS); alkylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with 5'-pyridoxal phosphate followed by reduction with NaBH. The guanidino group of arginine residues can be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal, and glyoxal. Carboxyl groups can be modified by carbodiimide activation via o-acylisourea formation followed by subsequent derivatization, for example, to the corresponding amide. Sulfhydryl groups can be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimides; formation of mercuric derivatives using 4-chloromercuribenzoic acid, 4-chloromercuriphenylsulfonic acid, phenylmercuric chloride, 2-chloromerc-4-nitrophenol and other mercuric acids; carbamylation with cyanate at alkaline pH. Tryptophan residues can be modified, for example, by oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfonyl halides. Tyrosine residues may be altered by nitration with tetranitromethane to form 3-nitrotyrosine derivatives. Modification of the imidazole ring of histidine residues can be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.Examples of incorporation of unnatural amino acids and derivatives during protein synthesis include, but are not limited to, norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and / or the use of D-isomers of amino acids. Modification of protein structure includes the generation of retro-inverso proteins that contain reverse sequences encoded by D-amino acids. Changes can be those that reduce susceptibility to proteolysis, reduce susceptibility to oxidation, alter the binding affinity of the variant sequence (typically, desirably, increase affinity), and / or confer or modify other physicochemical or functional properties to the variant / analog protein of interest.

[0059] In this specification, the term "sequence identity" should be understood to include both sequence identity and sequence similarity, i.e., a variant (or homolog) that shares 70% sequence identity with a reference sequence is one in which any 70% of the aligned residues of the variant (or homolog) are identical to or are conservative substitutions of the corresponding residues in the reference sequence over the entire length of the sequence. Sequence identity is the amount of exact matched characters between two different sequences. This does not count gaps, and the measurement relates to the shorter of the two sequences. With respect to "sequence homology", the term should be understood to mean a variant (or homolog) that shares a defined percentage similarity or percentage identity with a reference sequence, where the percentage of aligned residues of the variant (or homolog) are identical to or are conservative substitutions of the corresponding residues in the reference sequence, and the variant (or homolog) shares the same function as the reference sequence. The alignment and percent homology or percent sequence identity can be determined using software programs known in the art, for example, one alignment program is BLAST using default parameters. Details of these programs can be found at the following internet address: http: / / www.ncbi.nlm.nih.gov / blast / Blast.cgi.

[0060] The term "PSG1" also includes PSG1 proteins that have been modified other than by insertion, deletion or substitution of amino acid residues by functional group modification (modified proteins).

[0061] A physician will determine the actual dosage that is most appropriate for a patient depending on the particular condition, disease or disorder being treated or cared for, as well as the age, weight and / or health of the person. It will depend on a variety of factors, including the activity of the particular compound used, the metabolic stability and length of action of that compound, the age, weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual being treated. Of course, there may be individual instances where higher or lower dosage ranges are warranted, and this is within the scope of the present invention. For example, the composition may be administered at a dose of 0.01 to 50 mg / kg body weight, e.g., 0.01 to 1.0 mg / kg, typically 0.1 to 30 mg / kg, more preferably 0.1 to 20 mg / kg body weight, more preferably 0.1 to 10 mg / kg body weight, and preferably 0.1 to 5 mg / kg body weight. In an exemplary embodiment, one or more doses of 10 to 300 mg / day, or more preferably 10 to 150 mg / day, are administered to the patient. This amount and frequency is most suitable for this purpose. The frequency of application or administration can vary widely depending on the needs of each subject, with the recommended range of application or administration being once a month to 10 times a day, preferably once a week to 4 times a day, more preferably 3 times a week to 3 times a day, and even more preferably once or twice a day. In one embodiment of the present invention, the emulsion contains lipids or oils. The emulsion can be, but is not limited to, oil-in-water, water-in-oil, water-in-oil-in-water, and oil-in-water-in-silicone emulsions. The emulsion can contain a moisturizing agent. The emulsion can contain an antifoaming agent, such as silicone. The emulsion can have any suitable viscosity. The emulsion can further contain an emulsifier and / or an antifoaming agent. Methods for preparing emulsions are known to those skilled in the art.

[0062] The active agent (PSG1) is used in the pharmaceutical composition of the present invention in a pharma- ceutical or therapeutically effective concentration to achieve the desired effect, preferably in the form of 0.00000001% (by weight) to 100% (by weight), typically 0.00000001% (by weight) to 40% (by weight), preferably 0.000001% (by weight) to 15% (by weight), more preferably 0.0001% (by weight) to 10% (by weight), even more preferably 0.0001% (by weight) to 5% (by weight) with respect to the total weight of the composition. Ideally, about 0.00001% w / w to about 0.5% w / w of the composition, more preferably 0.00005 w / w to about 0.05 w / w, and most preferably about 0.0001 w / w to about 0.01 w / w of PSG1 is preferably used. Ideally, from about 0.0001% w / w to about 0.004% w / w of the composition of PSG1 is preferably used.

[0063] The composition of the present invention can be administered individually or in combination with other pharmacologically active agents.It is understood that such combination therapy includes, but is not limited to, various therapeutic regimens, including administration of multiple agents together in a single dosage form or separate individual dosage forms.When agents are present in different dosage forms, administration can be simultaneous or near simultaneous, or can follow any predetermined regimen that includes administration of different agents.Suitable active agents can be as described herein.

[0064] In some embodiments of the invention, the compositions may be delivered via any one of liposomes, mixed liposomes, oleosomes, niosomes, ethosomes, millicapsules, capsules, macrocapsules, nanocapsules, nanostructured lipid carriers, sponges, cyclodextrins, vesicles, micelles, mixed surfactant micelles, mixed surfactant-phospholipid micelles, millispheres, spheres, lipospheres, particles, nanospheres, nanoparticles, milliparticles, solid nanoparticles, and microemulsions including water-in-oil microemulsions having a reverse micelle internal structure, and nanoemulsions, microspheres, microparticles.

[0065] A variety of methods are available for preparing liposomes, see, for example, Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, and 4,837,028. No. 4,235,871, U.S. Pat. No. 4,261,975, U.S. Pat. No. 4,485,054, U.S. Pat. No. 4,501,728, U.S. Pat. No. 4,774,085, U.S. Pat. No. 4,837,028, U.S. Pat. No. 4,946,787, PCT Publication No. WO 91 / 17424, Deamer & Bangham, Biochim. Biophys. Acta 443:629-634(1976);Fraley,et al.,PNAS 76:3348-3352(1979);Hope et al.,Biochim.Biophys.Acta 812:55-65(1985);Mayer et al.,Biochim.Biophys.Acta 858:161-168(1986);Williams et al.,PNAS 85:242-246(1988);Liposomes(Ostro(ed.),1983,Chapter 1);Hope et al.,Chem.Phys.Lip.40:89(1986);Gregoriadis,Liposome Technology (1984) and Lasic, Liposomes: from Physics to Applications (1993)). Suitable methods include, for example, sonication, extrusion, high pressure / homogenization, microfluidization, detergent dialysis, calcium-induced fusion of small liposomal vesicles, and ether fusion methods, all of which are well known in the art.

[0066] These delivery systems can be adapted to achieve greater penetration of the compounds and / or peptides of the present invention. This can improve pharmacokinetic and pharmacodynamic properties. The delivery system can be a sustained release system in which the compounds or peptides of the present invention are gradually released during a period of time, preferably at a constant release rate over a period of time. The delivery system is prepared by methods known in the art. The amount of peptide contained in the sustained release system depends on where the composition is delivered and the duration of release, as well as the type of condition, disease and / or disorder being treated or cared for.

[0067] The compound of the present invention can be administered by oral administration. The compound (and other ingredients, if desired) can also be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or directly incorporated into the subject's diet. For oral therapeutic administration, the compound can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The compound can be coated or co-administered with any material to prevent its inactivation. The coating can be configured to protect the active agent during passage through the stomach and release the active agent in the ileum.

[0068] The method of the present invention may involve administering a nucleic acid construct configured to express an active agent (PSG1) in vivo. As used herein, the term "PSG1 expression vector" may be any suitable vector, including chromosomal vectors, non-chromosomal vectors and synthetic nucleic acid vectors (nucleic acid sequences that include a suitable set of expression control elements) suitable for expressing PSG1 (or its modified version, such as Fc-tagged protein) in cells. Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid molecule encoding the PSG1 amino acid sequence is contained in a naked DNA or RNA vector, including, for example, a linear expression element (e.g., as described in Sykes and Johnston, Nat Biotech 12, 355-59 (1997)), a compacted nucleic acid vector (e.g., as described in U.S. Pat. No. 6,077,835 and / or WO 00 / 70087), or a plasmid vector such as pBR322, pUC 19 / 18, or pUC 118 / 119. Such nucleic acid vectors and their use are well known in the art (see, for example, U.S. Pat. Nos. 5,589,466 and 5,973,972). In one embodiment, the DNA comprises an expression control sequence.

[0069] In one embodiment, the vector is suitable for expressing proteins in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, 1989, J Biol Chem 264, 5503-5509), pET vectors (Novagen, Madison, Wis.), and the like. In one embodiment, the expression vector may also or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters such as yeast alpha factor, alcohol oxidase, and PGH (reviewed in F. Ausubel et al., ed., 1987, Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York; and Grant et al., 1987, Methods in Enzymol 153, 516-544). In other embodiments, the expression vector is suitable for expression in baculovirus-infected insect cells. (Kost, T; and Condreay, JP, 1999, Current Opinion in Biotechnology 10(5):428-33.)

[0070] Expression control sequences are engineered to control and promote the transcription of the gene of interest and the subsequent expression of the protein in various cell systems. The plasmid combines the expressible gene of interest with an expression control sequence (i.e., expression cassette) that includes desired elements such as, for example, a promoter, an enhancer, a selection marker, an operator, etc. In the expression vector of the present invention, the nucleic acid molecule encoding the PSG1 amino acid sequence can include or be associated with any suitable promoter, enhancer, selection marker, operator, repressor protein, polyA termination sequence, and other expression promoting elements.

[0071] As used herein, a "promoter" refers to a DNA sequence sufficient to direct transcription of a DNA sequence to which it is operably linked, i.e., linked to allow transcription of a protein-coding nucleotide sequence, when appropriate signals are present. Expression of a protein-coding nucleotide sequence can be placed under the control of any promoter or enhancer element known in the art. Examples of such elements include strong expression promoters (e.g., human CMV IE promoter / enhancer, or CMV major IE (CMV-MIE) promoter, as well as RSV, SV40 late promoter, SL3-3, MMTV, ubiquitin (Ubi), ubiquitin C (UbC) and HIV LTR promoters). In some embodiments, the vector comprises a promoter selected from the group consisting of SV40, CMV, CMV-IE, CMV-MIE, RSV, SL3-3, MMTV, Ubi, UbC and HIV LTR.

[0072] The nucleic acid molecules of the invention may also be operably linked to an efficient poly(A) termination sequence, an origin of replication for the plasmid product in E. coli, an antibiotic resistance gene as a selectable marker, and / or a convenient cloning site (e.g., a polylinker). The nucleic acid may also include a regulatable inducible promoter (developmentally regulated, inducible, repressible) as opposed to a constitutive promoter such as CMV IE (one of skill in the art will recognize that such terms are actual descriptors of the degree of gene expression under particular conditions).

[0073] Selection markers are elements well known in the art. Under selection conditions, only cells that express appropriate selection markers can survive. In general, selection marker genes express proteins, usually enzymes, that confer resistance to various antibiotics in cell culture. Under other selection conditions, cells that express fluorescent protein markers can be visualized and therefore selected. Embodiments include beta-lactamase (bla) (beta-lactam antibiotic resistance gene or ampicillin resistance gene, i.e., ampR), bls (blasticidin resistance acetyltransferase gene), bsd (blasticidin-S deaminase resistance gene), bsr (blasticidin-S resistance gene), Sh ble (Zeocin® resistance gene), hygromycin phosphotransferase (hpt) (hygromycin resistance gene), tetM (tetracycline resistance gene, i.e., tetR), neomycin phosphotransferase II (npt) (neomycin resistance gene, i.e., neoR), kanR (kanamycin resistance gene), and pac (puromycin resistance gene).

[0074] In certain embodiments, the vector comprises one or more selectable marker genes selected from the group consisting of bla, bls, bsd, bsr, Sh ble, hpt, tetR, tetM, npt, kanR and pac. In other embodiments, the vector comprises one or more selectable marker genes encoding green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyano fluorescent protein (CFP), enhanced cyano fluorescent protein (eCFP) or yellow fluorescent protein (YFP).

[0075] For the purposes of the present invention, gene expression in eukaryotic cells can be tightly regulated using a strong promoter controlled by an operator, which in turn is regulated by a regulatory protein, which can be a recombinant "regulatory fusion protein" (RFP). RFPs essentially consist of a transcription blocking domain and a ligand binding domain that regulates its activity. Examples of such expression systems are described in US20090162901, the entirety of which is incorporated herein by reference.

[0076] As used herein, "operator" refers to a DNA sequence introduced within or near a gene such that the gene can be regulated by the binding of RFP to the operator, thereby preventing or allowing the transcription of the gene of interest, i.e., the nucleotides encoding the polypeptide of the present invention. In prokaryotic cells and bacteriophages, several operators have been well characterized (Neidhardt, ed., Escherichia coli and Salmonella; Cellular and Molecular Biology 2d. Vol 2 ASM Press, Washington DC 1996). These include, but are not limited to, the operator region of the LexA gene of E. coli, which binds to the LexA peptide, and the lactose operator and tryptophan operator, which bind to the repressor proteins encoded by the Lad and trpR genes of E. coli. These also include the bacteriophage operators from the lambda PR gene and phage P22 ant / mnt genes, which bind to the repressor proteins encoded by lambda cI and P22 arc. In some embodiments, when the transcription blocking domain of RFP is a restriction enzyme such as NotI, the operator is a recognition sequence for that enzyme. Those skilled in the art will recognize that the operator must be located adjacent to or 3' from the promoter so that it can control transcription by the promoter. For example, U.S. Patent No. 5,972,650, incorporated herein by reference, specifies that the tetO sequence is within a certain distance from the TATA box. In certain embodiments, the operator is preferably located immediately downstream of the promoter. In other embodiments, the operator is located within 10 base pairs of the promoter.

[0077] In an exemplary cell expression system, cells are engineered to express tetracycline repressor protein (TetR), and a protein of interest is placed under the transcriptional control of a promoter whose activity is regulated by TetR. Two tandem TetR operators (tetO) are placed immediately downstream of the CMV-MIE promoter / enhancer in a vector. Transcription of a gene encoding a protein of interest, directed by the CMV-MIE promoter in such a vector, can be blocked by TetR in the absence of tetracycline or some other suitable inducer (e.g., doxycycline). In the presence of an inducer, the TetR protein cannot bind to tetO, so translation (expression) of the protein of interest occurs after transcription. (See, e.g., U.S. Patent No. 7,435,553, which is incorporated herein by reference in its entirety).

[0078] The vector of the present invention can also use Cre-lox recombination tools to facilitate the integration of a gene of interest into the host genome. The Cre-lox strategy requires at least two components: 1) Cre recombinase, an enzyme that catalyzes the recombination between two loxP sites; and 2) loxP sites (e.g., a specific 34 base pair per sequence, consisting of an 8 bp core sequence where recombination occurs and two adjacent 13 bp inverted repeats) or mutant lox sites. (See, e.g., Araki et al., 1995, PNAS 92:160-4; Nagy, A. et al., 2000, Genesis 26:99-109; Araki et al., 2002, Nuc Acids Res 30(19):e103; and US20100291626, both of which are incorporated herein by reference). Another recombination strategy may utilize the yeast-derived FLP recombinase with the consensus sequence FRT (see also, for example, Dymecki, SM, 1996, PNAS 93(12):6191-6196).

[0079] As used herein, the term "host cell" includes any cell suitable for expressing a recombinant nucleic acid sequence. Cells include prokaryotic and eukaryotic cells (single or multicellular), bacterial cells (e.g., strains of E. coli, Bacillus species, Streptomyces species, etc.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. partoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, mammalian cells, human cells, or cell fusions, such as hybridomas or quadromas. In certain embodiments, the cell is a human, horse, dog, cat, supine, monkey, ape, hamster, rat or mouse cell. In other embodiments, the cell is a eukaryotic cell and is selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK 293, 293 EBNA, MSR 293, MDCK, HaK, BHK21), HeLa, HepG2, WI38, MRC5, Colo25, HB 8065, HL-60, Jurkat, Daudi, A431 (epidermis), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, tumor cells, and cell lines derived from the aforementioned cells. In some embodiments, the cell comprises one or more viral genes, e.g., a retinal cell (e.g., a PER.C6® cell) expressing a viral gene. In some embodiments, the cell is a CHO cell. In other embodiments, the cell is a CHO K1 cell. In one embodiment, the host cell is a bacterium.

[0080] As used herein, the term "transformed cell" refers to a host cell that contains a nucleic acid stably integrated into the cell genome, comprising a nucleotide sequence that codes for the expression of PSG1 protein. In another embodiment, the invention provides a cell that contains a non-integrated (i.e., episomal) nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, comprising a sequence that codes for the expression of PSG1 protein. In another embodiment, the invention provides a cell line that is produced by stably transfecting a host cell with a plasmid that contains an expression vector of the invention.

[0081] As used herein, the term "engineered" as applied to a cell means genetically engineered using recombinant DNA techniques, and generally involves synthesizing a suitable expression vector (see above) and then transfecting the expression vector into a host cell (generally stable transfection).

[0082] As used herein, the term "heterologous expression" refers to the expression of a nucleic acid in a host cell that does not naturally possess the nucleic acid. The insertion of a nucleic acid into a heterologous host is accomplished by recombinant DNA techniques.

[0083] As used herein, the term "neural stem cells" refers to self-renewing multipotent cells that generate neurons and glia of the nervous system. They are described below: Clarke, D et al. (2000). “Generalized Potential of Adult Neural Stem Cells”.Science.288(5471):1660-63.Bibcode:2000Sci...288.1660C.doi:10.1126 / science.288.5471.1660.PMID 10834848; Gilbert et al.Helsinki,the University of(2014).Developmental biology(Tenth edition.ed.).Sunderland,Mass.:Sinauer.ISBN 978-0878939787;and Temple et al. (1989). “Division and differentiation of isolated CNS blast cells in microculture”.Nature.340(6233):471-73.Bibcode:1989 Natur.340..471T.doi:10.1038 / 340471a0.PMID 2755510.

[0084] As used herein, in the context of treating neurodegeneration, the term "administering" should be interpreted to include any form of delivery that can deliver an active agent to the CNS or PNS, including intravenous, oral, intramuscular, intrathecal and inhalation delivery. Methods for achieving these delivery means are well known to those skilled in the art of drug delivery and include the following: Delivered intrathecally by mini-osmotic pumps. (See Ignacio et al., Ann. NY Acad. Sci. 2005, 1053:121-136.) Direct delivery into muscle via syringe or mini-osmotic pump (Azzouz et al., Nat Med. 2005;11(4):429-33). Intraperitoneally - for systemic administration - administered directly into the peritoneum via syringe or mini-osmotic pump (Kieran et al., Nat Med 2004;10(4):402). Subcutaneous - for systemic administration - administered directly under the skin by syringe (Reinholz et al., Exp Neurol. 1999;159(1):204-16). Intraventricular - directly into the ventricles of the brain by injection or using a small catheter attached to an osmotic pump (Sathasivam et al.,2005 Neuropath App Neurobiol;31(5):467). Implants - Implants can be prepared that release active substances (e.g. small silicone implants) and can be placed into muscles or directly into the spinal cord (Kieran and Greensmith, 2004 Neurosci 125(2):427-39).

[0085] Modified Proteins In one embodiment, the PSG1 protein (including protein fragments and variants) can be a modified protein. The term "modified protein" is used interchangeably with the term "protein derivatives". In one embodiment, the term "modified protein" refers to a protein that has been modified to exhibit one or more of the following properties compared to the unmodified protein: increase plasma half-life; increase protein lipophilicity; decrease renal clearance of the modified protein; increase activity of the modified protein and increase resistance of the modified protein to proteolysis (i.e., by mammalian and especially human gastrointestinal proteases). Various methods of modifying the proteins of the present invention to exhibit these properties are disclosed herein, including conjugating the protein to a binding partner (e.g., albumin-binding small molecule, large polymer, long life plasma protein, or antibody or antibody fragment), cyclization, addition of N-terminus or C-terminus or side chain, protecting groups, replacement of one or more L-amino acids with D-isomers, amino acid modification, increased plasma protein binding, increased albumin binding. Modified proteins include, but are not limited to, proteins that are substituted with one or more groups as defined herein, or that are conjugated with binding partners, or that are cyclized.Generally, proteins are modified to increase their half-life in vivo in animals.Various modification methods are provided below.

[0086] In one embodiment, the modification may be any modification that provides the protein and / or composition of the present invention with an increased ability to penetrate cells. In one embodiment, the modification may be any modification that increases the half-life of the composition or protein of the present invention. In one embodiment, the modification may be any modification that increases the activity of the composition or protein. In one embodiment, the modification may be any modification that increases the selectivity of the composition or protein.

[0087] In one embodiment, the group is a protecting group. The protecting group may be an N-terminal protecting group, a C-terminal protecting group, or a side chain protecting group. The protein may have one or more of these protecting groups.

[0088] Those skilled in the art will be aware of suitable techniques for reacting amino acids with these protecting groups. These groups can be added by preparation methods known in the art, such as those outlined in paragraphs

[0104] to

[0107] of US2014120141. The groups can remain on the protein or can be removed. Protecting groups can be added during synthesis.

[0089] In one embodiment of the present invention, the protein may be substituted by one or more groups selected from linear or branched, long or short chain, saturated or unsaturated, hydroxyl, amino, aminoacyl, sulfate or sulfide groups, or unsubstituted having 1 to 29 carbon atoms. N-acyl derivatives include acyl groups derived from acetic acid, capric acid, lauric acid, myristic acid, octanoic acid, palmitic acid, stearic acid, behenic acid, linoleic acid, linolenic acid, lipoic acid, oleic acid, isostearic acid, elaidic acid, 2-ethylhexanoic acid, coconut oil fatty acid, tallow fatty acid, hardened tallow fatty acid, palm kernel fatty acid, lanolin fatty acid or similar acids. These may be substituted or unsubstituted. If substituted, they are preferably substituted by hydroxyl groups, or sulfur-containing groups, such as, but not limited to, SO3H, SH or SS.

[0090] In one embodiment of the invention, the protein is R1-X-R2.

[0091] The R1 and / or R2 groups are attached to the amino terminus (N-terminus) and carboxyl terminus (C-terminus) of the protein sequence, respectively.

[0092] In one embodiment, the protein is R1-X. Alternatively, the protein is X-R2.

[0093] Preferably, R1 is H, C1-4 alkyl, acetyl, benzoyl or trifluoroacetyl; X is a protein of the invention, R2 is OH or NH2.

[0094] In one embodiment, R1 is selected from the group formed by H, acyclic substituted or unsubstituted aliphatic groups, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc), and R5-CO-, wherein R5 is selected from the group formed by H, acyclic substituted or unsubstituted aliphatic groups, substituted or unsubstituted alicyclyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heterocyclyl, and substituted or unsubstituted heteroarylalkyl; R2 is selected from the group formed by -NR3R4, -OR3 and -SR3, where R3 and R4 are independently selected from the group formed by H, acyclic substituted or unsubstituted aliphatic groups, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted aralkyl, with the proviso that R1 and R2 are not α-amino acids.

[0095] According to another preferred embodiment, R2 is -NR3R4, -OR3 or -SR3, where R3 and R4 are independently selected from the group formed by H, substituted or unsubstituted C1-C24 alkyl, substituted or unsubstituted C2-C24 alkenyl, tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc), substituted or unsubstituted C2-C24 alkynyl, substituted or unsubstituted C3-C24 cycloalkyl, substituted or unsubstituted C5-C24 cycloalkenyl, substituted or unsubstituted C8-C24 cycloalkynyl, substituted or unsubstituted C6-C30 aryl, substituted or unsubstituted C7-C24 aralkyl, 3-10 membered substituted or unsubstituted heterocyclyl ring, and substituted or unsubstituted heteroarylalkyl of 2-24 carbon atoms and 1-3 atoms other than carbon, the alkyl chain being of 1-6 carbon atoms. Optionally, R3 and R4 can be linked by a saturated or unsaturated carbon-carbon bond to form a ring with the nitrogen atom. More preferably, R2 is -NR3R4 or -OR3, where R3 and R4 are independently selected from the group formed by H, substituted or unsubstituted C1-C24 alkyl, substituted or unsubstituted C2-C24 alkenyl, substituted or unsubstituted C2-C24 alkynyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C6-C15 aryl, and 3-10 membered substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl having a 3-10 membered ring and an alkyl chain of 1-6 carbon atoms. More preferably, R3 and R4 are selected from the group formed by H, methyl, ethyl, hexyl, dodecyl or hexadecyl. Even more preferably, R3 is H and R4 is selected from the group formed by H, methyl, ethyl, hexyl, dodecyl or hexadecyl. According to an even more preferred embodiment, R2 is selected from the group formed by -OH and -NH 2 is selected from.

[0096] According to another embodiment of the invention, R1 is selected from the group formed by H, acetyl, lauroyl, myristoyl or palmitoyl, R2 is -NR3R4 or -OR3, where R3 and R4 are independently selected from H, methyl, ethyl, hexyl, dodecyl and hexadecyl, preferably R2 is -OH or -NH 2 More preferably, R1 is acetyl or palmitoyl and R2 is -NH 2 It is.

[0097] In a preferred embodiment, the acyl (or acetyl) group is attached to the N-terminus of at least one amino acid of the protein.

[0098] In one embodiment of the present invention, the protein is modified to include a side chain protecting group. The side chain protecting group may be one or more of the following: benzyl or benzyl-based groups, t-butyl-based groups, benzyloxy-carbonyl (Z) groups, and allyloxycarbonyl (alloc) protecting groups. The side chain protecting group may be derived from an achiral amino acid, such as achiral glycine. The use of an achiral amino acid helps to stabilize the resulting protein as well as facilitate the synthesis route of the present invention. Preferably, the protein further includes a modified C-terminus, preferably an amidated C-terminus. The achiral residue may be alpha-aminoisobutyric acid (methylalanine). It is understood that the particular side chain protecting group used will depend on the sequence of the protein and the type of N-terminal protecting group used.

[0099] In one embodiment of the present invention, the protein is conjugated, linked or fused to one or more polyethylene glycol polymers or other compounds, such as molecular weight increasing compounds. The molecular weight increasing compound is any compound that increases the molecular weight of the resulting conjugate, typically by 10% to 90%, or 20% to 50%, and may have a molecular weight of 200 to 20,000, preferably 500 to 10,000. The molecular weight increasing compound may be PEG, any water-soluble (amphiphilic or hydrophilic) polymer moiety, homo- or copolymers of PEG, monomethyl-substituted polymers of PEG (mPEG) and polyoxyethyleneglycerol (POG), polyamino acids, such as polylysine, polyglutamic acid, polyaspartic acid, especially those in the L-configuration, pharmacologically inactive proteins, such as albumin, gelatin, fatty acids, polysaccharides, lipid amino acids, as well as dextran. The polymer moiety may be linear or branched and may have a molecular weight of 500-40000 Daltons (DA), 5000-10000 Da, 10000-5000 Da. The compound may be any suitable cell-permeable compound, for example, tat protein, penetratin, pep-1. The compound may be an antibody molecule. The compound may be a lipophilic moiety or a polymer moiety.

[0100] Lipophilic and polymeric substituents are known in the art. Lipophilic substituents include acyl groups, sulfonyl groups, N-, O- or S-atoms forming part of esters, sulfonyl esters, thioesters, amides or sulfonamides. The lipophilic moiety may comprise a hydrocarbon chain having 4 to 30 C-atoms, preferably 8 to 12 C-atoms. The lipophilic moiety may be linear or branched, saturated or unsaturated. The hydrocarbon chain may be further substituted. The hydrocarbon chain may be a cycloalkane or a heterocycloalkane.

[0101] Proteins can be modified at the N-terminus, C-terminus, or both. The polymer or compound is preferably linked to an amino group, a carboxyl group, or a thiol group, and can be linked by the N-terminus or C-terminus of the side chain of any amino acid residue. The polymer or compound can be conjugated to the side chain of any suitable residue.

[0102] The polymer or compound may be conjugated via a spacer, which may be a natural or unnatural amino acid, succinic acid, lysyl, glutamyl, asparagyl, glycyl, beta-alanyl, gamma-aminobutanoyl.

[0103] The polymer or compound may be conjugated via an ester, sulfonyl ester, thioester, amide, carbamate, urea, sulfonamide.

[0104] Those skilled in the art will be aware of suitable means for preparing the described conjugates.

[0105] Proteins can be chemically modified by covalent conjugation to polymers, for example, to increase their circulatory half-life.Exemplary polymers and methods for attaching such polymers to proteins are illustrated in, for example, U.S. Patent No. 4,766,106, U.S. Patent No. 4,179,337, U.S. Patent No. 4,495,285 and U.S. Patent No. 4,609,546.Additional exemplary polymers include polyoxyethylated polyol moieties and polyethylene glycol (PEG) moieties.

[0106] The proteins of the present invention may be subjected to one or more modifications to manipulate the storage stability, pharmacokinetics, and / or any aspect of the protein's biological activity, such as potency, selectivity, and drug interactions. Chemical modifications to which the protein may be subjected include, but are not limited to, conjugation to the protein of one or more of polyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran, poly-(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polypropylene glycol, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, colominic acid or other carbohydrate-based polymers, polymers of amino acids, and biotin derivatives. PEG conjugation of proteins at Cys residues is disclosed, for example, in Goodson, RJ & Katre, NV (1990) Bio / Technology 8, 343 and Kogan, TP (1992) Synthetic Comm. 22, 2417.

[0107] Modified proteins may also include sequences in which one or more residues have been modified (i.e., by phosphorylation, sulfation, acylation, amidation, PEGylation, etc.), and variants that contain one or more modified residues relative to the parent sequence. Amino acid sequences may also be modified with a label that can directly or indirectly provide a detectable signal, including, but not limited to, radioisotope labels, fluorescent labels, and enzyme labels. Fluorescent labels include, for example, Cy3, Cy5, Alexa, BODIPY, fluorescein (e.g., FluorX, DTAF, and FITC), rhodamine (e.g., TRITC), auramine, Texas Red, AMCA Blue, and Lucifer Yellow. Preferred isotopic labels include 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, and 286Re. Preferred enzyme labels include peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plus peroxidase, and alkaline phosphatase (see, for example, U.S. Pat. Nos. 3,654,090, 3,850,752, and 4,016,043). Enzymes can be conjugated by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde, and the like. Enzyme labels can be detected visually or measured by calorimetric, spectrophotometric, fluorospectrophotometric, amperometric, or gasometric techniques. Other labeling systems, such as avidin / biotin, Tyramide Signal Amplification (TSA™), etc., are known in the art and are commercially available (see, e.g., ABC kit, Vector Laboratories, Inc., Burlingame, Calif.; NEN® Life Science Products, Inc., Boston, Mass.).

[0108] In one embodiment, the protein, variant and / or composition is modified to increase drug performance capacity. In one embodiment, the protein, variant and / or composition is modified to increase stability, permeability, maintain efficacy, avoid toxicity, and / or increase half-life. Modifications can be as described above. For example, modifications can be to protect N-terminus and C-terminus, modified amino acids, cyclization, amino acid substitution, and / or conjugation to macromolecules or large polymers or long-lived plasma proteins. Strategies to extend half-life can be as described by Strohl, et al (BioDrugs, 2015), Schlapschy, et al (Protein Eng Des Sel. 2013), Podust, VN, et al (Protein Eng Des Sel. 2013), Zhang, L et al (Curr Med Chem. 2012), Gaberc-Porekar, V, et al (Curr Opin Drug Discov Devel. 2008). Examples include the use of PEGylation, lipidation (covalent attachment of fatty acids to protein side chains), fusions to the Fc domain and human serum albumin, fusions with hydrophilic amino acid polymers such as XTEN or PAS, and / or fusions with half-life extending proteins.

[0109] The protein or proteins may contain weak sites in their sequences that are susceptible to proteolytic cleavage when in an environment with enhanced proteolytic activity, such as in blood or in the gastrointestinal tract. In one embodiment, the protein, variant and / or composition contains one or more weak site modifications such that the protein, variant and / or composition does not undergo proteolytic destruction / cleavage or the amount of proteolytic destruction / cleavage of the protein, variant and / or composition is reduced compared to the unmodified protein or proteins. Thus, the protein may be modified to increase the resistance of the modified protein to proteolytic degradation by mammalian gastrointestinal proteases. Suitable modifications are described in Diao et al. (Clinical pharmacokinetics 52.10(2013):855-868).

[0110] Modifications of proteins to increase their in vivo half-life have been described in the literature, for example in: Strategies to improve plasma half-life time of protein and protein drugs.Werle M, Bernkop-Schnurch A. Amino Acids.2006 Jun;30(4):351-67.

[0111] Due to the obvious advantages of long-acting proteins and protein drugs, strategies to extend the plasma half-life time of such compounds are highly demanding. Short plasma half-life times are generally due to rapid renal clearance and enzymatic degradation occurring during systemic circulation. Protein / protein modifications can result in an extension of the plasma half-life time. By reducing the total amino acid content of somatostatin and replacing L:-analog amino acids by D:-amino acids, the plasma half-life time of the derivative octreotide was 1.5 hours compared to only a few minutes for somatostatin. The PEG(2.40K) conjugate of INF-alpha-2b showed a 330-fold longer plasma half-life time compared to the native protein. The aim of this review was to provide an overview of possible strategies to extend plasma half-life time, such as N- and C-terminal modifications, or PEGylation, as well as methods to evaluate the efficacy of drug modifications. Furthermore, basic data are provided on the most important proteolytic enzymes of human blood, liver and kidney, as well as their cleavage specificities and inhibitors, in order to predict the enzymatic cleavage of proteins and protein drugs in the systemic circulation.

[0112] Strategic Approaches to Optimizing Protein ADME Properties.Li Di AAPS J.2015 Jan;17(1):134-143.

[0113] Strategies for stabilizing proteins against proteolysis Many approaches are available for enhancing protein stability through structural modification. Some approaches not only improve stability but also enhance other ADME properties, for example, cyclization can increase stability and permeability, conjugation with macromolecules can improve stability and reduce renal clearance. It is important to maintain efficacy and avoid toxicity while improving protein stability and ADME properties.

[0114] N- and C-terminal protection Several proteolytic enzymes in blood / plasma, liver or kidney include exopeptidases, aminopeptidases and carboxypeptidases, which degrade protein sequences from the N-terminus and C-terminus. Modifications of the N-terminus or / and C-terminus can often improve protein stability. In many instances, N-acetylation and C-amidation have been reported to increase resistance to proteolysis.

[0115] Substitution of L-amino acids with D-amino acids Substitution of natural L-amino acids by unnatural D-amino acids reduces substrate recognition and binding affinity of proteolytic enzymes and increases stability. One example is vasopressin, which contains L-Arg and has a half-life of 10-35 minutes in humans. The D-Arg analog, desmopressin, has a half-life of 3.7 hours in healthy human volunteers. In studies of bicyclic protein inhibitors of the cancer-associated protease urokinase-type plasminogen activator (uPA), substitution of a specific glycine by D-serine not only improved potency in mouse plasma by 1.8-fold, but also increased stability by 4-fold.

[0116] Amino acid modification Modification of natural amino acids can improve protein stability by introducing steric hindrance or interfering with enzyme recognition. For example, gonadotropin releasing hormone has a very short half-life (minutes), whereas buserelin, in which one Gly is replaced by t-butyl-D-Ser and the other Gly is replaced by an ethylamide, has a much longer half-life in humans.

[0117] ·Cyclization Cyclization introduces conformational constraints, reducing protein flexibility and increasing stability and permeability. Depending on the functional groups, proteins can be cyclized head-to-tail, head / tail-to-side chain, or side chain-to-side chain. Cyclization is commonly achieved by lactamization, lactonization, and sulfide-based bridging. Disulfide bridges introduce folding and conformational constraints that can improve potency, selectivity, and stability. Several disulfide bond-rich proteins, such as linaclotide, lepirudin, and ziconotide, are commercially available or in preclinical or clinical development.

[0118] · Conjugation to macromolecules Conjugation to macromolecules (eg, polyethylene glycol (PEG), albumin) is an effective strategy to improve protein stability and reduce renal clearance.

[0119] Renal clearance Many proteins show promising in vitro pharmacological activity but fail to show in vivo efficacy due to very short in vivo half-life (minutes). The rapid clearance and short half-life of proteins prevent them from being developed into good drugs. The main causes of rapid clearance of proteins from the systemic circulation are enzymatic proteolysis or / and renal clearance. Glomeruli have a pore size of approximately 8 nm, and hydrophilic proteins with MW < 2-25 kDa are prone to rapid filtration through the glomerulus of the kidney. Proteins are not easily reabsorbed through the renal tubules, and therefore often have high renal clearance and short half-life. Other minor pathways of protein clearance include endocytosis, as well as degradation by the proteasome and liver. Comparison of systemic and renal clearance in animal models provides useful information as to whether renal clearance is likely to be the major elimination route.

[0120] In patients with renal impairment, dose adjustments may be required to avoid protein drug accumulation and increased drug exposure, as inappropriate dosing in patients with renal impairment may cause toxicity or render treatment ineffective. Several strategies have been developed to reduce protein renal clearance and extend half-life. These are reviewed below.

[0121] Increased plasma protein binding Renal clearance of proteins is decreased when they bind to membrane or serum proteins. One example is the cyclic protein drug octreotide, a treatment for endocrine tumors, which has a half-life of about 100 minutes in humans (unbound fraction 0.65) due to binding to lipoproteins.

[0122] Covalent binding to albumin-binding small molecules Covalent attachment of albumin-binding small molecules to proteins can reduce glomerular filtration, improve proteolytic stability, and extend half-life by indirectly interacting with albumin through the highly conjugated small molecule.

[0123] Conjugation to large polymers Conjugation of proteins to large synthetic or natural polymers or carbohydrates can increase their molecular weight and hydrodynamic volume and therefore decrease their renal clearance. Common polymers used for protein conjugation are PEG, polysialic acid (PSA) and hydroxyethyl starch (HES).

[0124] Fusion to long-lived plasma proteins Plasma proteins such as albumin and immunoglobulin (IgG) fragments have a long half-life of 19-21 days in humans. Due to their high MW (67-150 kDa), renal clearance is low for these proteins, and their binding to the neonatal Fc receptor (FcRn) reduces excretion via pinocytosis by the vascular epithelium. Covalent binding of proteins to albumin or IgG fragments can reduce renal clearance and extend half-life.

[0125] Fusion Proteins for Half-Life Extension of Biologics as a Strategy to Make Biobetters William R. Strohl BioDrugs.2015;29(4):215-239.

[0126] Schlapschy,M,Binder,U,Borger,C et al.PASYlation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins.Protein Eng Des Sel.2013;26(8):489-501.

[0127] Podust, VN, Sim, BC, Kothari, D et al. Extension of in vivo half-life of biologically active proteins via chemical conjugation to XTEN protein polymer. Protein Eng Des Sel.2013;26(11):743-53.

[0128] Zhang, L, Bulaj, G. Converting Proteins into Drug Leads by Lipidation. Curr Med Chem. 2012;19(11):1602-18.

[0129] Gaberc-Porekar, V, Zore, I, Podobnik, B et al. Obstacles and pitfalls in the PEGylation of therapeutic proteins. Curr Opin Drug Discov Devel. 2008;11(2):242-50.

[0130] Dr Ronald V.Swanson-Long live protein evolution of protein half-life extension technologies and emerging hybrid approaches.From Drug Discovery World on line. Spring 2014

[0131] PEGylation PEGylation, the attachment of long chains of the hydrophilic polymer polyethylene glycol to molecules of interest, was originally conceived as a modification to prevent recognition of foreign proteins by the immune system, thereby enabling their utility as therapeutics. Antibodies to the unmodified drug, once formed, could rapidly neutralize and clear protein drugs. Unexpectedly, PEGylation improved the pharmacokinetics of proteins, even in the absence of anti-drug antibodies. By simply making the drug molecule larger, PEGylation resulted in drugs that were filtered more slowly by the kidney. The empirical observation that an increase in size or hydrodynamic radius resulted in reduced renal clearance and increased half-life became the primary rationale for PEGylation of proteins and protein drugs. PEGylation can have a variety of effects on molecules, including making the protein or proteins more water soluble and protecting them from degradation by proteolytic enzymes. PEGylation can also affect the binding of therapeutic proteins to their cognate cellular receptors, usually reducing affinity. Changes in the size, structure, and mode of attachment of the PEG polymer can affect the biological activity of the attached drug.

[0132] First generation PEGylation methods were riddled with challenges. However, the chemistry of PEGylation is quite simple. The process involves the covalent attachment of polyethylene glycol chains to reactive side chains of proteins or proteins. For example, PEG readily attaches to the -amino groups of lysines on the surface of proteins or proteins2. The reaction is pH dependent. At high pH (above 8.0), lysine side chain amino groups are covalently attached to PEG via N-hydroxysuccinimide. This method typically results in a family of products containing various numbers of PEG chains attached to various sites on the protein, rather than a single distinct product3. The first approved PEGylated pharmaceuticals were Pegademase bovine (PEGylated bovine adenosine deamidase) as enzyme replacement therapy for severe combined immunodeficiency, and Pegaspargase (PEGylated asparaginase) for the treatment of acute lymphoblastic leukemia1. Although these drugs were complex mixtures of various PEGylated species, they have improved properties for therapy over native enzymes, including increased serum half-life, and reduced immunogenicity of the protein. Due to the inherent polydispersity of PEG, quality and batch-to-batch reproducibility have been difficult. Despite this limitation, two PEGylated interferons, a heterogeneous population of multiple mono-PEGylated positional isomers (peginterferon alpha-2b and peginterferon alpha-2a), have been approved by the FDA for the treatment of Hepatitis C. These drugs were launched in 2001 and 2002, respectively.

[0133] Various improvements and modifications have been made to the basic PEGylation technique. Second generation PEGylation processes introduced the use of branched structures and alternative chemistries for PEG attachment. In particular, PEGs with cysteine-reactive groups, such as maleimide or iodoacetamide, allow for the targeting of PEGylation to a single residue within a protein or protein, reducing the heterogeneity of the final product but not eliminating it due to the polydispersity of PEG itself.

[0134] Although the original rationale for PEGylation was to reduce immunogenicity, several examples of immunogenic PEGylated proteins nevertheless exist. One example is PEGylated urate oxidase, an enzyme that reduces plasma uric acid levels in gout patients. In clinical trials, a relatively high percentage of gout patients did not respond to treatment and developed antibodies specific to PEG but not to the uricase protein. 2 PEGylated liposomes, which are also generally considered non-immunogenic, have been found to be immunogenic in some studies. PEGylated liposomes induce a strong anti-PEG immunoglobulin M (IgM) response. Furthermore, multiple injections of PEG-glucuronidase have been shown to induce the generation of specific anti-PEG IgM antibodies, thus accelerating the clearance of PEG-modified proteins from the body.

[0135] The main potential drawback of using PEG as a modifier is that it is non-biodegradable. The U.S. Food and Drug Administration (FDA) has approved the use of PEG as a vehicle for pharmaceuticals, including injectable, topical, rectal and nasal formulations. PEG exhibits little toxicity and is eliminated intact from the body by the kidney (for PEG < 30 kDa) or in the feces (for PEG > 20 kDa). 1 Renal tubular cell vacuolation has been observed following repeated administration of some PEGylated proteins to animals. Recently, choroid plexus epithelial cell vacuolation has also been seen in toxicity studies using proteins conjugated with large (≥ 40 kDa) PEG. Choroid plexus epithelial cells produce cerebrospinal fluid and form the blood-CSF barrier. The long-term negative consequences of cell vacuolation are unknown, but it represents an undesirable outcome for some potential therapeutics. One possible alternative would be the substitution of a biodegradable polymer for PEG. Polymers such as hydroxyethyl starch (HES) are possible alternatives. HES is non-toxic and biodegradable and is used as a blood expander. The process of HESylation works similarly to PEGylation, decreasing renal clearance by increasing the hydrodynamic radius of the protein, but due to its biodegradability, it may be less prone to accumulation. However, HES and other proposed biodegradable polymeric PEG alternatives are polydisperse, like PEG, making characterization of the final product and metabolites difficult. One emerging solution that alleviates both concerns is to use defined polyproteins as the polymeric components. This approach is described later in this editorial.

[0136] Lipidization The second major chemical modification method to increase protein half-life is lipidation, which involves the covalent attachment of fatty acids to protein side chains. Lipidation, originally conceived and developed as a method to extend insulin half-life, shares the same basic mechanism of half-life extension as PEGylation, namely, increasing the hydrodynamic radius to decrease renal filtration. However, the lipid moiety itself is relatively small, and its effect is indirectly mediated through the non-covalent attachment of the lipid moiety to circulating albumin. Albumin, a large (67 kDa) and highly abundant protein in human serum (35-50 g / L), naturally functions to transport lipid-containing molecules throughout the body. Binding to plasma proteins can also protect the protein from attack by peptidases through steric hindrance, again similar to that seen with PEGylation. One consequence of lipidation is that it reduces the water solubility of the protein, but this can be modulated by manipulation of the linker between the protein and the fatty acid, for example, by the use of glutamates or mini-PEGs in the linker. Manipulation of the linker and alteration of the lipid moiety can affect self-aggregation, which may contribute to increased half-life by slowing biodistribution independent of albumin.

[0137] Following pioneering work with insulin,6 lipidation of various proteins has been investigated, particularly in the diabetes area, including human glucagon-like protein-1 (GLP-1) analogs, glucose-dependent insulinotropic polyproteins, and GLP-1R / glucagon receptor coagonists. Two lipidated protein drugs are currently approved by the FDA for human use, both of which are long-acting antidiabetic drugs: the GLP-1 analog liraglutide and insulin detemir.

[0138] A potentially pharmacologically important difference between PEGylation and lipidation is that the therapeutically active protein is covalently attached to the much larger PEG, whereas the smaller fatty acyl-protein conjugate is non-covalently attached to the larger bound and unbound forms of albumin present in equilibrium. This may result in differences in biodistribution that may result in different pharmacology, since access to receptors localized in different tissues may induce different effects. In some cases, a more restricted biodistribution may be desirable, whereas in other cases, greater tissue penetration may be important. An interesting variant of the PEG approach that addresses this issue has been developed by Santi et al.7, in which releasable PEG conjugates with predictable cleavage rates are utilized.

[0139] Both PEGylation and lipidation provide protection against proteases and peptidases by shielding through steric hindrance, and directly or indirectly extend circulating half-life through increasing hydrodynamic radius. Both methods utilize chemical conjugation and are flexible in that they are independent of the means used to generate the protein they are modifying, whether biologically or synthetically generated. The advantage of using synthetic proteins is that they can incorporate unnatural amino acids designed to address some specific issues, including instability due to known proteolytic cleavage drawbacks. They can also be more flexible in terms of the choice of conjugation site, which is important when activity or potency is highly dependent on the free end, or modified residues such as C-terminal amides.

[0140] Traditional gene fusions: Fc and HSA Traditional genetic fusion to long-lived serum proteins offers an alternative method of half-life extension that differs from chemical conjugation to PEG or lipids. Two major proteins have traditionally been used as fusion partners: antibody Fc domains (especially human IgG1 Fc tag and equine IgG1 Fc tag), and human serum albumin (HAS). Fc fusion involves the fusion of a protein, protein or receptor extracellular domain to the Fc portion of an antibody. Both Fc fusion and albumin fusion not only extend half-life by increasing the size of the protein drug, but also take advantage of the body's natural recycling mechanism, namely, the neonatal Fc receptor, FcRn. The pH-dependent binding of these proteins to FcRn prevents degradation of the fusion protein within the endosome. Fusions based on these proteins can have half-lives ranging from 3 to 16 days, much longer than typical PEGylated or lipidated proteins. Fusion to antibody Fc can improve the solubility and stability of the protein or protein drug. An example of a protein Fc fusion is dulaglutide, a GLP-1 receptor agonist currently in late-stage clinical trials. Human serum albumin is the same protein utilized by fatty acylated proteins and is another common fusion partner. Albiglutide is a GLP-1 receptor agonist based on this platform. The main difference between Fc and albumin is the dimeric nature of Fc versus the monomeric structure of HAS, resulting in the presentation of the fusion protein as a dimer or monomer depending on the choice of fusion partner. The dimeric nature of protein Fc fusions can result in avidity effects if the target receptors are spaced sufficiently close to each other or are themselves dimeric. This may or may not be desirable depending on the target. In one embodiment, the Fc domain is engineered to include mutations. Methods for engineering mutations into the Fc domain are described in Rath et al (2013;doi=10.3109 / 07388551.2013.834293).

[0141] Engineered polyprotein fusions: XTEN and PAS An interesting variation of the recombinant fusion concept has been the development of engineered low-complexity sequences as fusion partners, essentially unstructured hydrophilic amino acid polymers that are functional analogs of PEG. The inherent biodegradability of the polyprotein platform makes it attractive as a potentially more benign alternative to PEG. Another advantage is the precise molecular structure of the recombinant molecule, in contrast to the polydispersity of PEG. Unlike HSA and Fc protein fusions, where the three-dimensional folding of the fusion partner needs to be maintained, recombinant fusions to unstructured partners can often be subjected to harsh conditions, such as high temperatures, or HPLC purification.

[0142] The most advanced of this class of polyproteins, called XTEN (Amunix), is 864 amino acids long and composed of 6 amino acids (A, E, G, P, S and T). This is much larger than the typically used 40 KDa PEG, which is made possible by the biodegradability of the polymer, and concomitantly confers a longer half-life extension. The fusion of XTEN to protein drugs results in a 60-130-fold half-life extension over the native molecule. Two fully recombinantly produced XTENylated products, namely VRS-859 (Exenatide-XTEN) and VRS-317 (Human Growth Hormone-XTEN), have entered clinical trials. In a Phase Ia study, VRS-859 was found to be well tolerated and effective in type 2 diabetes patients. For VRS-317, superior pharmacokinetic and pharmacodynamic properties were reported compared to previously tested rhGH products, and it has the potential for once-monthly administration.

[0143] A second polymer based on similar conceptual considerations is PAS (XL-Protein GmbH).9 The random coil polymer is composed of a more restricted set of only three small uncharged amino acids, proline, alanine and serine. Whether differences in the biophysical properties of PAS and the highly negatively charged XTEN may contribute to differences in biodistribution and / or in vivo activity remains to be seen, but will become clear as these polyproteins are incorporated into more therapeutics and the behavior of the fusions is characterized.

[0144] All protein-protein fusions, whether the partner is Fc, HSA, XTEN or PAS, are genetically encoded and therefore suffer from similar constraints. One limitation is that only naturally occurring amino acids are incorporated, unlike methods using chemical conjugation that allow the use of synthetic proteins incorporating unnatural amino acids. Methods that overcome this by expanding the genetic code have been developed by companies such as Ambrx or Sutro, but are not yet widely used. A second limitation is that either the N- or C-terminus of the protein must be fused to the partner. In many cases, the protein termini are involved in receptor interactions, and genetic fusion to one or both termini can greatly impair activity. The site of PEG or lipid conjugation can be anywhere on the protein and therefore can be optimized to maximize the biological activity of the resulting therapeutic.

[0145] A hybrid approach to integrate half-life extension proteins and synthetic proteins Gene fusion has traditionally offered the potential for even longer half-life extension, but it lacks the advantages offered by methods utilizing chemical conjugation, PEGylation, and lipidation, in terms of flexibility in binding sites and incorporation of unnatural amino acids or modifications into the protein backbone. One of the first attempts to integrate the advantages of gene fusion with chemical conjugation for half-life extension was made by researchers at the Scripps Research Institute in La Jolla, using a technology that later formed the basis of the biotechnology company CovX. These researchers developed a platform using catalytic aldolase antibodies in which the active site lysine of the antibody forms a reversible covalent enamine bond with a beta-diketone incorporated into a protein or small molecule. The resulting conjugate is called CovXBody™. This approach combines the functional properties of a protein drug or small molecule with the long serum half-life of an antibody, not by gene fusion, but rather by chemical conjugation. Following the initial demonstration of this technology, the researchers expanded the use of the CovX-Body™ prototype, based on a peptidomimetic pharmacophore that targets integrins. At least three molecules based on this structure are in clinical development: CVX-096, a Glp-1R agonist; CVX-060, an angiopoietin-2 binding protein; and CVX-045, a thrombospondin mimetic.

[0146] Recently, XTEN polyproteins have also been used in a chemical conjugation fashion12 that makes them even more directly similar to PEG. The first example of an XTENylated protein made using this method is GLP2-2G-XTEN, where the protein is chemically conjugated to the XTEN protein polymer using maleimide-thiol chemistry. The chemically conjugated GLP2-2GXTEN molecule showed comparable in vitro activity, in vitro plasma stability and pharmacokinetics in rats comparable to recombinant fusion GLP2-2G-XTEN.

[0147] The number and spacing of reactive groups, such as lysine or cysteine ​​side chains, within the fully designed sequence of the XTEN or PAS polyprotein can be precisely controlled by site-specific variation due to the limited set of amino acids from which they are composed. This provides additional flexibility over methods that can utilize Fc or albumin, whose sequences naturally contain many reactive groups, and contrasts with CovX technology, which relies on reactive residues within highly specialized active sites. Furthermore, the lack of tertiary structure in XTEN or PAS should provide greater flexibility in the conditions and chemistries used for coupling and purification of the conjugates.

[0148] In summary, hybrid protein half-life extension methods have emerged that combine the advantages and overcome the individual limitations of chemical conjugation and gene fusion methods. These methods open the door to a wide range of longer-acting protein-based drugs, since they allow the creation of molecules based on recombinant polyprotein-based partners that confer longer half-lives but free the therapeutic protein moiety from the limitations of being composed only of natural L-amino acids or as linear unidirectional polyproteins fused at either the N- or C-terminus.

[0149] Exemplary Dosages and Administration Strategies As noted above, the compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of a protein of the invention (or in the case of a combination composition comprising a protein of the invention and a second component, a first and second amount; in the case of a combination composition comprising two proteins of the invention and a second agent of the invention or a protein and two second agents, a first, second and third amount, etc.). In order to better illustrate certain embodiments, a detailed explanation of the dosing principles is now further provided.

[0150] In practicing the present invention, the amount or dosage range of protein used is typically one that effectively induces, promotes or enhances epithelialization of a wound (in the context of wound treatment), or the amount or dosage range of protein used is typically one that effectively modifies the gene expression profile of cells of the nervous system so as to slow the progression of a neurodegenerative condition, or the amount or dosage range of protein used is typically one that modifies the gene expression profile of cells in the context of the treatment of a tissue degenerative condition in an equine mammal.

[0151] In yet another aspect, a daily dosage of about 0.01 to 100 milligrams of active ingredient (e.g., a protein of the invention) per kilogram of body weight is provided to the patient. Typically, about 1 to about 5, or about 1 to about 10 milligrams per day given in divided doses about 1 to about 6 times daily, or in sustained release form, may be effective to obtain the desired results. In one embodiment, the dosage is 10 to 100, 30 to 70, 40 to 60, and ideally about 50 μg / ml.

[0152] As a non-limiting example, treatment of a human or animal disease can include administration of the compound for at least one of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, or 40 days, or alternatively, for at least one of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 days, or 6 days, using single or divided doses about every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof. In some embodiments, the administration of a daily dosage of a protein of the invention in an amount of about 0.1 to 100 mg / kg, e.g., 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg.

[0153] Example The present invention will now be described with reference to specific examples, which are merely illustrative and for illustrative purposes only, and are in no way intended to limit the scope of the claimed exclusivity or the invention described. These examples constitute the best modes currently contemplated for carrying out the invention.

[0154] Example 1 Construction of pTT3 expression vector All vectors contain the relevant PSG1 open reading frame (ORF) subcloned into the pTT3 expression vector in frame with a carboxy-terminal V5-His tag obtained from the pBlueBac4.5-V5-His vector. Vectors expressing full-length PSG1 were previously described ( Shanley et al., 2013 ; Houston et al., 2016 ).

[0155] Fc tag cloning The human Fc tag was PCR amplified from a sample of Epstein-Barr virus (EBV) transformed lymphocyte cDNA. The human Fc tag was subcloned into the pTT3 vector using a HindIII site engineered into the primer tail to insert the tag in frame at the 3' end of the PSG1 ORF. After insertion of the Fc tag, an internal HindIII site was removed using site-directed mutagenesis to allow in-frame transcription of the PSG1 ORF with the Fc tag.

[0156] Site-directed mutagenesis was used to modify the human Fc tag in the CH2 domain (triple substitution YTE (M252Y / S254T / T256E) and in the CH3 domain (H433K / N434F) to increase stability and half-life (see Rath et al., 2015, for Fc modifications: https: / / doi.org / 10.3109 / 07388551.2013.834293). Site-directed mutagenesis was used to modify the human Fc tag (Phusion Site-Directed Mutagenesis Kit - Thermo Fisher Scientific).

[0157] Example 2 Effect of PSG1 on gene expression in human cell lines C20, a human microglial cell line, cells were treated in vitro with PSG1 and gene expression changes were analyzed using Qiagen Wound Healing Profiler qRT-PCR arrays. Cells were incubated at 1 × 10 with 50ug / ml recombinant PSG1-V5His. 5 Cells were seeded at a density of 1000 cells / ml into 6-well plates for 5 h, and cDNA was prepared and analyzed at 5 or 24 h post-treatment. The results are shown in Figure 1.

[0158] Example 3 Effect of PSG1 on behavioral outcomes (paw injury test) in a mouse stroke model Permanent middle cerebral artery clotting The procedure was performed under sterile conditions. Mice were anesthetized using isoflurane (3–4% for induction and 1–3% for maintenance). During the procedure, pedal withdrawal reflex and spontaneous movement were monitored at minimum 5-min intervals. After induction, anesthesia was maintained using 1–2% isoflurane using a face mask. Rectal temperature was maintained at 37 °C using a homeothermic blanket system. Before incision, the skin between the right ear and eye was infiltrated with 0.5% bupivacaine (0.1 ml), and the temporalis muscle was retracted to expose the temporal and parietal bones. A small craniotomy was made over the arterial bifurcation, and the meninges were cut, exposing the middle cerebral artery (MCA) with its parietal and frontal branches. The distal portion of the MCA was ligated using a miniature vascular cautery. The incision was sutured using monofilament sutures and closed using intermittent closure. Anesthesia was discontinued, warm lactated Ringer's solution was administered i.p. (0.5 ml), and the animals were allowed to recover in a small ThermoCare® Water Jacketed Warmer until reperfusion. During recovery from anesthesia, respiratory rate, movement, and ability to maintain sternal recumbency were monitored at 15 min intervals until the animals were ambulatory.

[0159] Subcutaneous injection of vehicle (PBS) or PSG1-Fc Using sterile 25-27 gauge needles and syringes, and using aseptic procedures, mice were injected at room temperature with 50-100 µl volumes of sterile solutions: phosphate-buffered saline (PBS), or 100 micrograms of PSG1-Fc protein purified from transiently transfected HEK293 cells in PBS.

[0160] Blood collection by tail snip Conscious mice were gently restrained manually. The tail was cleaned using a 70% alcohol prep pad. A tail amputation was performed by removing less than 1 mm of the tip of the tail using a sterile scalpel. The tail was gently massaged and 0.1 ml of blood was collected into a heparinized capillary tube. The animals were allowed free movement in their cage during collection. After collection was completed, the wound was gently compressed to stop bleeding and the animals were returned to their home cage. This procedure has been demonstrated to be relatively stress-free. (Guidelines taken from LAST course handbook (page 164; Handbook of Laboratory Animal Management & Welfare; Wolfensohn & Lloyd)).

[0161] Grid Walking Mice were placed on the wire grid and allowed to explore freely for 5 min. Mice were videotaped and later scored by an experimenter blinded to treatment group for the percentage of paw slips by the left and right forepaws during the first 100 steps. Paw slips are scored if the paw completely misses the rung, thus causing the limb to fall between the rungs, or if the paw is correctly placed on the rung but slips off during weight bearing. Animals do not require pre-training, but are placed on the grid once prior to surgery to allow for familiarization and to obtain a baseline score.

[0162] Experimental timeline Day 7: Receipt of mice and acclimatization for 7 days D0: Permanent MCA coagulation D0+1 h: Subcutaneous administration of PSG1-Fc (21 mice) or vehicle (21 mice) D2: Blood collection D3: Grid Walking D5: Grid walking followed by euthanasia

[0163] Equivalent The above description details presently preferred embodiments of the invention. It is anticipated that those skilled in the art who consider these descriptions will recall numerous modifications and variations in their practice. These modifications and variations are intended to be encompassed within the scope of the appended claims. [Sequence List Free Text]

[0164] Sequence Listing 3 <223> Modified human Fc ORF

Claims

1. A pharmaceutical composition comprising pregnancy specific glycoprotein 1 (PSG1) for stimulating survival or recovery of tissue damaged by a cerebrovascular event in humans.

2. The pharmaceutical composition of claim 1, wherein the PSG1 is Fc-tagged PSG1 (PSG1-Fc).

3. The pharmaceutical composition according to claim 1 or 2, wherein the cerebrovascular event is acute ischemic stroke.

4. The pharmaceutical composition according to claim 1 or 2, wherein the cerebrovascular event is hemorrhagic stroke.

5. The pharmaceutical composition according to claim 1 or 2, wherein the cerebrovascular event is a transient ischemic attack (TIA).

6. 3. The pharmaceutical composition of claim 1 or 2, which is administered to a subject by intravenous or intrathecal administration.

7. The pharmaceutical composition according to claim 1 or 2, which is administered to the subject at a dosage of 0.1 to 100 mg / kg body weight.

8. The pharmaceutical composition of claim 1 or 2, wherein the composition is administered to the subject within 6 hours of the cerebrovascular event.

9. A pharmaceutical composition comprising pregnancy specific glycoprotein 1 (PSG1) for treating human neurological disorders characterized by altered microglial regulation leading to neuronal cell death or demyelination.

10. The pharmaceutical composition described in claim 9, wherein the PSG1 is Fc-tagged PSG1 (PSG1-Fc).

11. 11. The pharmaceutical composition according to claim 9 or 10, wherein the neurological disorder characterized by altered microglial regulation leading to neuronal cell death or demyelination is a neurodegenerative disease.

12. A pharmaceutical composition comprising pregnancy specific glycoprotein 1 (PSG1) for treating traumatic brain injury in a mammal by stimulating survival or recovery of tissue damaged by traumatic brain injury.

13. The pharmaceutical composition described in claim 12, wherein the PSG1 is Fc-tagged PSG1 (PSG1-Fc).

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