PSG1 for use in the treatment of osteoarthritis

Abstract

The use of Fc-tagged pregnancy-specific glycoprotein 1 (PSG1-Fc) in the treatment of osteoarthritis or damaged cartilage in humans is described, where PSG1 is administered by intra-articular injection. Also described is the use of CC49 in a method of treatment of osteoarthritis or damaged cartilage in equine mammals, where CC49 is administered by intra-articular injection. (Figure 3A-C) The use of PSG1 in a method of treatment of wounds, scarring, burns and diabetic ulcers in mammals is also described.

Description

[Technical field]

[0001] The present invention relates to the use of placenta-expressed proteins to treat disease. [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).

[0005] Jones et al. (Biology of Blood and Marrow Transplantation, 1 September 2018) described a protective role for recombinant PSG1 in a mouse model of acute graft-versus-host disease.

[0006] Blois et al. (Mucosal Immunology, Vol. 7, No. 2, 14 August 2013) describe the use of PSG1 in preventing DSS-induced colitis in mice. Summary of the Invention

[0007] In one aspect, the invention provides a pregnancy specific glycoprotein (e.g., PSG1) for use in a method of treating or preventing a condition characterized by loss of or damage to cartilage. In one embodiment, the condition is a degenerative joint condition. In one embodiment, the degenerative joint condition is osteoarthritis. In another embodiment, the condition is damaged cartilage caused by trauma (e.g., a fall or a sports injury). In one embodiment, the PSG1 is administered by intra-articular injection. In one embodiment, the method is a method of slowing, stopping, or reversing the loss of cartilage in an affected joint. In one embodiment, the method is for treating osteoarthritis of the knee, hip, or hand. In one embodiment, the treatment is a causal treatment. In another embodiment, the treatment is a symptomatic treatment, e.g., a method of treating a symptom of osteoarthritis, e.g., joint pain or stiffness. In one embodiment, the method is a method of repairing damaged cartilage in a subject. Figures 3A-C demonstrate the effective treatment of osteoarthritis using PSG1-Fc and CC49-Fc.

[0008] According to another aspect of the present invention, there is provided a pregnancy specific glycoprotein, such as pregnancy specific glycoprotein 1 (PSG1), for use in a method of treating a wound in a mammal. PSG1 has been shown to promote wound contracture, or re-epithelialization of wounds, and promote wound closure in mouse and porcine animal models compared to untreated wounds.

[0009] Typically, the method of the invention is for promoting wound closure compared to an untreated wound.

[0010] Typically, the wound is a skin wound. However, the present invention also applies to the treatment of non-skin wounds, for example wounds to organs other than the skin. Such wounds can be caused, for example, by surgery, trauma, drug use or disease. In one embodiment, the wound is a diabetic ulcer, for example a diabetic food ulcer.

[0011] Typically, PSG1 is administered locally to the wound.Typically, the method comprises applying a topical formulation of PSG1 to the wound, particularly to the periphery of the wound.

[0012] In any embodiment, PSG1 may be administered subcutaneously to the wound, typically by subcutaneous injection.

[0013] PSG1 is typically administered in an amount effective to promote epithelialization by keratinocytes at the site of a skin wound.

[0014] In any embodiment, the wound comprises a scar, hi any embodiment, PSG1 is administered to a scar.

[0015] In any embodiment, the wound is a wound resulting from excision of a hypertrophic scar.

[0016] In any embodiment, the wound comprises or consists of a keloid scar.

[0017] According to another aspect of the present invention there is provided a pregnancy specific glycoprotein, such as pregnancy specific glycoprotein 1 (PSG1), for use in a method of treating keloid scars in a mammal.

[0018] According to another aspect of the invention there is provided a pregnancy specific glycoprotein, such as pregnancy specific glycoprotein 1 (PSG1), for use in a method of treating or preventing hypertrophic scarring in a mammal. In any embodiment the method comprises excising a hypertrophic scar and then administering the pregnancy specific glycoprotein to a wound created by excision of the scar.

[0019] In a further aspect, the present invention provides an Fc-tagged pregnancy-specific glycoprotein 1 (PSG1-Fc) for use in a method for treating a wound, particularly a skin wound, in a mammal. In one embodiment, PSG1-Fc is administered topically to the wound or by intradermal (IV) administration to the wound.

[0020] In a further aspect, the present invention provides a topical formulation of pregnancy specific glycoprotein 1 (PSG1) comprising a therapeutically effective amount of PSG1 in combination with a pharma- ceutically acceptable excipient. The topical formulation of PSG1 can be a cream, ointment, gel, oil suspension or lotion.

[0021] In a further aspect, the present invention provides Fc-tagged pregnancy specific glycoprotein 1 (PSG1-Fc) for use as a medicament.

[0022] In one embodiment, PSG1 is administered by subcutaneous, intradermal, intravenous or intraperitoneal delivery, or by injection into the affected area. In one embodiment, PSG1 is administered by intra-articular injection.

[0023] In any embodiment, PSG1 is administered to the mammal by transfecting the mammal with a PSG1 (or PSG1-Fc) expression vector.

[0024] In any embodiment, PSG1 is administered to the mammal by administering cells transfected with a PSG1 (or PSG1-Fc) expression vector to the mammal. In any embodiment, PSG1 is administered to the mammal by administering cells pretreated with PSG1 (e.g., cells incubated with or cultured in the presence of PSG1) to the mammal. In any embodiment, PSG1 and cells (e.g., mesenchymal stem cells) are co-administered to the mammal. In any embodiment, the cells are stem cells. In any embodiment, the cells are mesenchymal stem cells. In any embodiment, the donor cells are obtained from the recipient. In one embodiment, the donor cells are obtained from a mammal of the same species (e.g., human to human or horse to horse) (allogeneic cell therapy). In any embodiment, the cells are transfected ex vivo.

[0025] 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.

[0026] In any 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).(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.

[0027] In another aspect, the present invention provides CC49 (typically recombinant CC49) for use in a method of treating a wound in a mammal, typically a non-human mammal. In a preferred embodiment, the mammal is an equine (i.e., a horse). Data provided herein show that CC49 exhibits a range of gene regulatory activities consistent with tissue repair actions similar to PSG1. For example, CC49, like PSG1, regulates cytokine expression in human cell lines (HaCaT, Jurkat) representing keratinocyte and lymphocyte lineages, respectively. CC49 enhances migration of HaCaT and mesenchymal stem cells (MSC) in in vitro abrasion wound assays (Figures 1A-C).

[0028] In another aspect, the invention provides CC49 (typically recombinant CC49) for use in a method of treating a condition in a mammal characterized by loss of or damage to cartilage. In a preferred embodiment, the mammal is an equine (i.e., a horse). In any embodiment, the condition is a degenerative condition of the joint (e.g., a joint disease such as osteoarthritis). In any embodiment, the condition is a traumatic injury to the cartilage, e.g., caused by a fall or other type of trauma. In any embodiment, the condition causes lameness due to tissue damage in response to a stimulus (e.g., injury). In any embodiment, CC44 is administered by direct injection into the damaged tissue or for intra-articular injection. In one particular aspect, the invention provides CC49, particularly a modified CC49 such as CC49-Fc, for use in a method of treating or preventing a joint condition (typically osteoarthritis) in an equine mammal, wherein CC49 is administered to the equine mammal by intra-articular injection into the affected joint.

[0029] In any embodiment, CC49 is modified with a functional group. In any embodiment, the functional group is configured to increase the plasma half-life of the modified CC49. In any embodiment, the functional group is an Fc portion from horse IgG1 (an example is described in Wagner B1, Robeson J, McCracken M, Wattrang E, Antczak DF. Horse cytokine / IgG fusion proteins--mammalian expression of biologically active cytokines and a system to verify antibody specificity to equine cytokines. Vet Immunol Immunopathol. 2005 May 1;105(1-2):1-14. DOI:10.1016 / j.vetimm.2004.11.010). Fc tags are also useful for purifying proteins during pharmaceutical production.

[0030] In some embodiments, CC449 is administered parenterally. In some embodiments, CC449 is administered by intra-articular injection.

[0031] In another aspect, the present invention provides recombinant CC49 for use as a medicament.

[0032] In another aspect, the present invention provides Fc-tagged CC49 (eg, Fc-tagged recombinant CC49).

[0033] In another aspect, the present invention provides an Fc-tagged CC49 for use as a medicament.

[0034] 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]

[0035] [Figure 1]PSG1 and CC49 proteins enhance wound closure in cell line scrape wound assays. A) Equine MSCs (n=3) treated with PSG1-Fc, PSG1-V5His, CC49-Fc, CC49-V5His or 50 μl PBS after 16 hours. B) Human MSCs (n=3) treated with PSG1-Fc, PSG1-V5His, CC49-Fc, CC49-V5His or 50 μl PBS after 16 hours. C) HaCaT cells (n=3) treated with PSG1-Fc, PSG1-V5His, CC49-Fc, CC49-V5His or 50 μl PBS after 16 hours. (*P<0.05, **P<0.01, ***P<0.001). D) Human HaCaT cells treated with PSG1-V5His or 50 μl PBS were analyzed for gene expression changes using the RT2 Profiler™ PCR Array Human Wound Healing (330231, Cat. No. PAHS-121ZE-4; Qiagen, UK). [Diagram 2] A) and B) PSG1 enhances epithelialization of porcine skin wounds compared to controls. C) and D) PSG1 enhances closure of mouse skin wounds compared to controls. Similar results were obtained in normal and diabetic mice. [Diagram 3] PSG1-Fc and CC49-Fc reduce osteoarthritis in a murine collagenase-induced osteoarthritis (CIOA) model. A) PSG1-Fc and CC49-Fc increase trabecular bone thickness in the treatment group compared to the PBS-treated (control) group. B) PSG1-Fc and CC49-Fc increase mineral density in the treatment group compared to the PBS-treated group. C) PSG1-Fc and CC49-Fc reduce osteophyte density in the treatment group compared to the PBS-treated group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] 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.

[0037] 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:

[0038] 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.

[0039] 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.

[0040] 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.

[0041] As used herein, the term "treatment" or "treating" refers to an intervention (e.g., administration of a drug to a subject) that cures, ameliorates, or alleviates the symptoms of a disease, or eliminates its cause (or reduces the effects of its cause). In this case, the term is used synonymously with the term "therapy."

[0042] 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.

[0043] 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).

[0044] 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.

[0045] 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.

[0046] 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.

[0047] SEQ ID NO:1: JPEG2024514194000002.jpg66138

[0048] 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:

[0049] SEQ ID NO:2 JPEG2024514194000003.jpg240160

[0050] 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:

[0051] SEQ ID NO:3 Modified human Fc ORF JPEG2024514194000004.jpg86159

[0052] As used herein, the term "CC49" refers to the horse PSG-like CEACAM49 full-length protein, represented by SEQ ID NO: 4 below, including the signal sequence (amino acid residues, usually residues 1 to 32 or 38), the mature peptide. The term also includes the mature peptide without the signal sequence.

[0053] SEQ ID NO:4: JPEG2024514194000005.jpg33137

[0054] The term "CC49" also includes Fc-tagged CC49 proteins (CC49-Fc), an example of which is provided below in SEQ ID NO:5:

[0055] SEQ ID NO:5 JPEG2024514194000006.jpg153160

[0056] SEQ ID NO:6 provides the open reading frame for the horse Fc tag which forms part of SEQ ID NO:5

[0057] SEQ ID NO:6 Horse Fc ORF JPEG2024514194000007.jpg147160

[0058] The terms "PSG1" and "CC49" also include mutants, which are proteins having substantially identical amino acid sequences to wild-type PSG1 or CC49 proteins, typically human wild-type PSG1 and horse wild-type CC49 proteins. Thus, for example, the terms should be construed to include proteins or polypeptides 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 introduced amino acid is structurally, chemically or functionally similar to the amino acid that is replaced. Typically, proteins altered by 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, a variant will 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 or CC49 proteins modified with a tag, such as an Fc tag, including a human Fc tag or an equine Fc tag. The Fc tag may be modified to exhibit increased plasma half-life and / or stability. Such Fc tags are known in the literature and described herein.Examples of modifications to the human Fc tag include the triple substitution YTE (M252Y / S254T / T256E) in the CH2 domain and (H433K / N434F) in the CH3 domain to increase stability and half-life. In one embodiment, the invention provides a CC49 protein modified with an Fc tag, typically an Fc tag derived from an equine antibody, typically an equine IgG antibody.

[0059] PSG1 or CC49 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 produced 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 to impose conformational constraints on proteins or their analogs. Examples of side chain modifications include, for example, reductive alkylation by reaction with aldehydes followed by reduction with NaBH4; 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 modification of amino groups by pyridoxylation of lysine with 5'-pyridoxal phosphate followed by reduction with NaBH4.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-chloromercuric-4-nitrophenol and other mercuric acids; carbamylation with cyanate at alkaline pH. Tryptophan residues may 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 may 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, the use of 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 D-isomers of amino acids. Modifications of protein structure include the generation of retro-inverso proteins containing reverse sequences encoded by D-amino acids. Changes may reduce susceptibility to proteolysis, reduce susceptibility to oxidation, alter (typically desirably increase) the binding affinity of the variant sequence, and / or confer or modify other physicochemical or functional properties to the variant / analog protein of interest.

[0060] 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.

[0061] The term "PSG1" also includes PSG1 proteins (modified proteins) that have been modified other than by insertion, deletion, or substitution of amino acid residues through functional group modification. Similarly, "CC49" also includes CC49 proteins (modified proteins) that have been modified other than by insertion, deletion, or substitution of amino acid residues through functional group modification.

[0062] As used herein, the term "wound" should be understood to mean a wound or a scar formed as a result of a wound. The scar may be a hypertrophic scar or a keloid scar.

[0063] As used herein, the term "skin wound" refers to a wound in the skin and optionally the underlying tissue of a mammal.

[0064] As used herein, the terms "epithelialization" and "re-epithelialization" applied to wounds refer to the process in which open wounds or scars, such as keloid scars of the skin, are covered by epithelial (keratinocyte) cells that may migrate from the wound periphery.The method of the present invention relates to the treatment of wounds, scars left by wounds, keloid scars, and scars, particularly hypertrophic scars or wounds caused by excision of scars.The method of the present invention also includes the treatment of burns or scars caused by burns.

[0065] As used herein, the term "PSG1 topical formulation" refers to a formulation of PSG1 suitable for topical administration to mammalian skin. The topical composition may be provided in a formulation selected from the group including cream, multiple emulsion, anhydrous composition, aqueous dispersion, oil, milk, balsam, foam, lotion, gel, cream gel, water-alcohol solution, water-glycolic solution, cosmetic, personal care product, hydrogel, liniment, serum, soap, powder, paste, semi-solid formulation, liniment, serum, shampoo, conditioner, ointment, optional rinse-off formulation, talc, mousse, powder, spray, aerosol, solution, suspension, emulsion, syrup, elixir, polysaccharide film, patch, gel patch, bandage, adhesive system, water-in-oil emulsion, oil-in-water emulsion, and silicone emulsion. The topical composition of the present invention is administered in a cosmetically or pharma-ceutically effective amount. In other words, in an amount that is non-toxic but sufficient to provide the desired effect. It is understood that one of ordinary skill in the art can determine the appropriate dose of the topical composition of the present invention to administer without undue experimentation. Alternatively, a physician would determine the actual dose most suitable 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 depends 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, excretion rate, 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 are 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., 0.1 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 exemplary embodiments, one or more doses of 10-300 mg / day, or more preferably 10-150 mg / day, are administered to the patient. For injection into tissues (such as intra-articularly), doses of 0.01-1.0 mg, preferably 0.05-0.5 mg, and ideally about 0.1 mg are contemplated.This amount and frequency are 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.

[0066] The active agent (PSG1 or CC49) is used in the topical or pharmaceutical compositions 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), and 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, about 0.0001% w / w to about 0.004% w / w of the composition of PSG1 or CC49 is preferably used.

[0067] 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.

[0068] 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.

[0069] 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 See 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.

[0070] 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.

[0071] 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.

[0072] As used herein, the term "conditions characterized by loss of or damage to cartilage" refers to osteoarthritis or cartilage damaged by trauma, such as falls or sports injuries, wear and tear, or other disease processes.The method of the present invention relates to treating conditions by slowing or inhibiting the loss of cartilage and / or by causing cartilage growth and / or repair.Treatment of conditions characterized by damage to or degeneration of articular cartilage is envisioned.

[0073] The method of the present invention may involve administering a nucleic acid construct configured to express an active agent (PSG1 or CC49) in vivo. As used herein, the term "PSG1 expression vector" or "CC49 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 expression of PSG1 or CC49 (or modified versions thereof, such as Fc-tagged proteins) 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 or CC49 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.

[0074] In any 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 any 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.)

[0075] 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., an 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 or CC49 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.

[0076] 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.

[0077] 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).

[0078] 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).

[0079] 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).

[0080] 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.

[0081] 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.

[0082] 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).

[0083] 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).

[0084] 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.

[0085] 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 or CC49 protein. In another embodiment, the present 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 or CC49 protein. In another embodiment, the present invention provides a cell line produced by stably transfecting a host cell with a plasmid that contains an expression vector of the present invention.

[0086] 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).

[0087] 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.

[0088] As used herein, in the context of treatment, the term "administer" should be interpreted to include any form of delivery that can deliver an active agent to a subject, including intravenous delivery, oral delivery, intramuscular delivery, 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). · Implants - Implants that can be prepared with implants that release active substances (e.g. small silicone implants) can be placed into the muscle (Kieran and Greensmith, 2004 Neurosci 125(2):427-39).

[0089] Modified Proteins In any embodiment, the PSG1 protein or CC49 protein (including protein fragments and variants) may be a modified protein. The term "modified protein" is used interchangeably with the term "protein derivative". In any 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 group, 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.

[0090] In any 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 any 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 any embodiment, the modification may be any modification that increases the selectivity of the composition or protein.

[0091] In any 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.

[0092] 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.

[0093] In any embodiment of the invention, the protein may be substituted by one or more groups selected from straight or branched chain, 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.

[0094] In any embodiment of the invention, the protein is R1-X-R2.

[0095] The R1 and / or R2 groups were attached to the amino terminus (N-terminus) and carboxyl terminus (C-terminus) of the protein sequence, respectively.

[0096] In one embodiment, the protein is R1-X. Alternatively, the protein is X-R2.

[0097] Preferably, R1 is H, C1-4 alkyl, acetyl, benzoyl or trifluoroacetyl; X is a protein active agent of the invention (e.g., PSG1 or Fc-tagged PSG1, CC49 or Fc-tagged CC49); R2 is OH or NH2.

[0098] In any 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-, where 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.

[0099] 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 -OH and -NH2.

[0100] According to another embodiment of the present invention, R1 is selected from the group formed by H, acetyl, lauroyl, myristoyl or palmitoyl, and R2 is -NR3R4 or -OR3, where R3 and R4 are independently selected from H, methyl, ethyl, hexyl, dodecyl and hexadecyl, and preferably R2 is -OH or -NH2.More preferably, R1 is acetyl or palmitoyl, and R2 is -NH2.

[0101] In a preferred embodiment, the acyl (or acetyl) group is attached to the N-terminus of at least one amino acid of the protein.

[0102] In any embodiment of the 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 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] The polymer or compound may be conjugated via an ester, sulfonyl ester, thioester, amide, carbamate, urea, sulfonamide.

[0108] Those skilled in the art will be aware of suitable means for preparing the described conjugates.

[0109] 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.

[0110] 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.

[0111] 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.).

[0112] 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.

[0113] Proteins 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 comprises 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 unmodified protein. Thus, proteins can 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).

[0114] 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.

[0115] 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.

[0116] Strategic Approaches to Optimizing Protein ADME Properties.Li Di AAPS J.2015 Jan;17(1):134-143.

[0117] 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.

[0118] ·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.

[0119] 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.

[0120] 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.

[0121] ·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.

[0122] · Conjugation to macromolecules Conjugation to macromolecules (eg, polyethylene glycol (PEG), albumin) is an effective strategy to improve protein stability and reduce renal clearance.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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).

[0128] 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.

[0129] Fusion Proteins for Half-Life Extension of Biologics as a Strategy to Make Biobetters William R. Strohl BioDrugs.2015;29(4):215-239.

[0130] 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.

[0131] 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.

[0132] Zhang, L, Bulaj, G. Converting Proteins into Drug Leads by Lipidation. Curr Med Chem. 2012;19(11):1602-18.

[0133] 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.

[0134] 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

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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).

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] Example 1 Construction of pTT3 expression vector All vectors contain the relevant PSG1 or CC49 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). The CC49 sequence was obtained by PCR and directionally subcloned into pTT3 using PCR primers containing EcoRI and HindIII restriction sites. Site-directed mutagenesis was used to remove the previously engineered V5-His tag.

[0159] Fc tag cloning The human Fc tag was PCR amplified from a sample of Epstein-Barr virus (EBV) transformed lymphocyte cDNA. The horse Fc tag was amplified from the pcDNA-IGHG1 vector, kindly provided by Bettina Wagner. Both the human and horse Fc tags were subcloned into the pTT3 vector using HindIII sites engineered into the primer tails to insert in-frame at the 3' end of the PSG1 / CC49 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 or CC49 ORF with the Fc tag.

[0160] 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).

[0161] Example 2 Human immortalized keratinocyte cell line (HaCaT; Boukamp et al., 1988) was purchased from the German Cancer Research Center (DKFZ). Cells were grown in DMEM (D6429, Sigma-Aldrich, UK) supplemented with 10% FBS; 100 μg / ml streptomycin; 100 U / ml penicillin; 2 mM L-glutamine and cultured at 37 °C and 5% CO2. Human mesenchymal stem cells (MSCs) were grown in MEM (M2279, Sigma-Aldrich, UK) supplemented with 10% FBS; 100 μg / ml streptomycin; 100 U / ml penicillin; 1 ng / ml FGF2 (SRP4037, Sigma-Aldrich, UK) and cultured at 37 °C and 5% CO2. Equine MSCs were grown in DMEM (D6429, Sigma-Aldrich, UK) supplemented with 10% FBS; 100 μg / ml streptomycin; 100 U / ml penicillin; 2 mM L-glutamine and cultured at 37 °C and 5% CO2. HaCaT cells (1 × 10 5 cells / ml) and human or equine MSC cells (2 × 10 5 Cells / ml) were seeded in 1 ml in 6-well plates. After 24 h, the inserts and medium were removed and each well was treated with 1 ml of cell culture medium supplemented with 50 μg of PSG1-Fc, PSG1-V5His, CC49-Fc, CC49-V5His, or 50 μl of PBS. To determine the extent of wound closure, the abrasion wounds were imaged 16 h after treatment using EVOS FL Auto and Wimasis WimScratch analysis or ImageJ analysis. Using the human HaCaT keratinocyte cell line as well as human and equine mesenchymal stem cells (MSCs), we determined that PSG1 or CC49 enhance the migration of cell types associated with wound healing. The results are shown in Figure 1A-C.

[0162] Example 3 Effect of PSG1 on wound closure in pigs Six 2×2 cm (4 cm) holes were placed on each flank of five pigs. 2 ) Full-thickness excision wounds were created and treated with 1 ml of PBS, or 250 μg of PSG1 in PBS by intradermal injection at eight sites around the wound edge on day 0, with repeat treatment on day 3. Dressings were changed on days 3 and 7, and wounds were photographed on days 0, 3, 7, and 10, and sampled on day 10. Representative photographs are shown in Figure 2A. The extent of wound epithelialization on day 10 is shown in Figure 2B.

[0163] Example 4 Effect of PSG1 on wound closure in mice Adult male mice of the C57Bl / 6J strain were created with circular full-thickness excision wounds 0.4 cm in diameter and injected intradermally at four equidistant sites around the wound edge with either 100 ul PBS or 50 ug PSG1 in 100 ul PBS. Preliminary dose-response experiments showed that the lowest effective dose of PSG1 to achieve maximal wound closure was 50 ug, and this dose was used in subsequent experiments. Wounds were measured and photographed immediately after PSG1 administration (day 1) and 5 days after wounding. Results are shown in Figure 2C and Figure 2D (% of wounds at day 5 that were not healed).

[0164] Example 5 Effect of PSG1 on gene expression in HaCaT human cell line HaCaT human keratinocyte cell line was 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 PSG1. 5 Cells were seeded at a density of 1000 cells / ml into 6-well plates for 24 h, and cDNA was prepared and analyzed at 24 h post-treatment. Results of two replicate experiments are shown in Figure 1D.

[0165] Example 6 Mouse models of osteoarthritis A mouse model of osteoarthritis was created according to the method of Farrell et al. (Farrell E, Fahy N, Ryan AE, Flatharta CO, O'Flynn L, Ritter T, Murphy JM. vIL-10-overexpressing human MSCs modulate naive and activated T lymphocytes following induction of collagenase-induced osteoarthritis. Stem Cell Res Ther. 2016 May 18;7(1):74. doi:10.1186 / s13287-016-0331-2. PMID:27194025).

[0166] Protocol for intra-articular injection of collagenase into the knee joint (CIOA model) 1. Anesthetize adult male and female mice of the C57Bl / 6 strain, up to 9 per treatment group, with isoflurane. 2. Shave the hind legs and apply a depilatory cream to ensure all hair is removed. 3. Disinfect the injection area with an iodine solution. Using a 10 μl Hamilton syringe with a 4.27G needle, 7 μl of collagenase, phosphate buffered saline (PBS), or PSG protein in PBS (100 ug / ml) is drawn into the syringe. 5. With the knee slightly flexed, locate the intra-articular space by palpation. 6. Insert the needle laterally to avoid damaging the patellar ligament. 7. To avoid puncturing the posterior part of the bursa, point the needle upwards and into the kneecap, contacting the femoral condyle. 8. After placement in the synovial cavity, slowly push the piston rod to administer the fluid. 9. Retract the needle and gently move the hindlimb to encourage fluid distribution. 10. After the procedure, the animals are kept on a heating pad and monitored until they are fully mobile and have recovered from anesthesia.

[0167] Experimental protocol timeline (days): D-7: Receipt of mice and 1 week of acclimation D0: First collagenase injection into the knee D1: Second collagenase injection into the knee D7: Injection of PBS or PSG1-Fc or CC49-Fc into the knee (maximum of 18 mice per treatment) D42: Euthanasia and necropsy

[0168] At necropsy, both knee joints (untreated and treated) are harvested for histological analysis and blood samples are taken for flow cytometry and cytokine ELISA assays to identify systemic changes to immune cell populations. Experimental outcome measurements are primarily focused on histological examination of treated joints using a semi-quantitative scoring system to assess cartilage metrics, synovial thickening and ectopic bone formation. A subset of analyses involves flow cytometry to investigate immune cell profiles in the blood and lymph nodes and ELISAs for a cytokine panel. The results are shown in Figure 3A-C and demonstrate the effective treatment of osteoarthritis using PSG1-Fc and CC49-Fc.

[0169] 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]

[0170] Sequence Listing 3 <223> Modified human Fc ORF Sequence Listing 6 <223> Horse Fc ORF

Claims

1. A pharmaceutical composition comprising pregnancy specific glycoprotein 1 (PSG1) for treating osteoarthritis or damaged cartilage in humans, administered by intra-articular injection.

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

3. A pharmaceutical composition for treating osteoarthritis or damaged cartilage in humans, administered by intra-articular injection, comprising an expression vector for pregnancy specific glycoprotein 1 (PSG1) or Fc-tagged PSG1 (PSG1-Fc).

4. A pharmaceutical composition for treating osteoarthritis or damaged cartilage in an equine mammal comprising CC49, administered by intra-articular injection.

5. 5. The pharmaceutical composition of claim 4, wherein said CC49 is an Fc-tagged CC49 (CC49-Fc), and said Fc tag is optionally an equine Fc tag.

6. A pharmaceutical composition for treating osteoarthritis or damaged cartilage in an equine mammal, comprising an expression vector for CC49 or Fc-tagged CC49 (CC49-Fc).

7. A pharmaceutical composition comprising pregnancy specific glycoprotein 1 (PSG1) for treating diabetic ulcers in humans.

8. The pharmaceutical composition described in claim 7, wherein the PSG1 is Fc-tagged PSG1 (PSG1-Fc).

9. The pharmaceutical composition of claim 7 or 8, administered locally to the diabetic ulcer.

10. A pharmaceutical composition comprising pregnancy specific glycoprotein 1 (PSG1) for treating a mammalian wound.

11. The pharmaceutical composition of claim 10, which is applied topically to the wound.

12. 12. The pharmaceutical composition of claim 10 or 11, wherein the wound is caused by excision of a hypertrophic scar.

13. 12. The pharmaceutical composition of claim 10 or 11, wherein the wound comprises a keloid scar.

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