Placenta-expressed proteins for use in the treatment of tendon injuries - Patent Application 20070122999

Abstract

The use of Fc-tagged pregnancy-specific glycoprotein 1 (PSG1-Fc) in a method for treating tendon injury in humans is described, wherein PSG1 is administered by intratendinous or peritendinous injection. Also described is the use of CC49 in a method for treating tendonitis, tendinosis, tendinopathy, or tendon injury in equine mammals, wherein CC49 is administered by intratendinous or peritendinous injection.

Description

[Technical Field]

[0001] The present invention relates to the use of placenta-expressed proteins to treat tendon injuries. [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. Although PSG proteins vary considerably in structure among primates, horses, and rodents, they 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.). Humans and mice contain 11 and 17 distinct PSG genes encoding PSG proteins, 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 equine 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 that total PSG protein concentrations exceeded 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 determined by ELISA, as well as the activation of latent TGF-β1 [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)].

[0004] J Biol Chem.2015 Feb 13;290(7):4422-31.doi:10.1074 / jbc.M114.597518.Epub 2014 Dec 29.].

[0005] WO 2017049082 describes one specific PSG protein, PSG1, and its involvement in the pathway responsible for the induction of immune tolerance. PSG1 is involved in the activation of transforming growth factor-β1 (TGFβ1), a cytokine critical for the differentiation of tolerance-inducing CD4+CD25+FoxP3+ regulatory T cells (Tregs), a cell population that is essential for the suppression of inflammatory T cells and has been shown to be important in the prevention of graft-versus-host disease (GvHD). Summary of the Invention

[0006] The present applicant has discovered that pregnancy-specific glycoproteins (e.g., PSG1) enhance the migration of mesenchymal stem cells (MSCs). Migration of MSCs to the injury site is beneficial for the healing of tendon injuries. In one embodiment, PSG1 is administered by injection into or adjacent to the tendon. In one embodiment, the method is a method of halting, slowing, or reversing tendon degeneration. In one embodiment, the treatment is a causal treatment. In another embodiment, the treatment is a symptomatic treatment, e.g., a method of treating symptoms of tendon injury, such as tendinitis, tendinopathy, tendon pain, or stiffness. One aspect of the present invention involves administering a combination of pregnancy-specific glycoproteins (e.g., PSG1) and MSCs to tendon injuries. PSG and MSCs can be administered simultaneously or separately. MSCs can be conditioned prior to administration in a cell culture medium containing PSG.

[0007] In one embodiment, the PSG1 is Fc-tagged PSG1 (PSG1-Fc).In one embodiment, a nucleic acid encoding PSG1 is administered to a mammal.

[0008] In one aspect, the present invention provides Fc-tagged pregnancy-specific glycoprotein 1 (PSG1-Fc) for use as a pharmaceutical.

[0009] In any embodiment, PSG1 is administered by intratendinous or peritendinous injection into the affected area.

[0010] In any embodiment, PSG1 is administered to the mammal by transfecting the mammal with a PSG1 (PSG1-Fc) expression vector.

[0011] In some embodiments, the present invention provides a topical formulation of pregnancy-specific glycoprotein 1 (PSG1) comprising a therapeutically effective amount of PSG1 in combination with a pharmaceutically acceptable excipient. The topical formulation of PSG1 can be a cream, ointment, gel, oil suspension, or lotion.

[0012] In any embodiment, PSG1 is administered to a mammal by administering cells transfected with a PSG1 (or PSG1-Fc) expression vector to the mammal. In any embodiment, PSG1 is administered to a 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, donor cells are obtained from the recipient. In one embodiment, 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.

[0013] In any embodiment, the MSCs are administered to the mammal, generally at the site of the tendon injury, generally by injection.

[0014] In any embodiment, PSG1 (or PSG1-Fc) is co-administered with MSCs, typically in the same injection.

[0015] In any embodiment, PSG1 (or PSG1-Fc) is administered separately to the MSCs.

[0016] In any embodiment, the MSCs are cultured in cell culture with PSG1 (or PSG1-Fc) prior to administration.

[0017] The methods of the invention provided above recite PSG1 and modified versions of PSG1, e.g., PSG1-Fc, in therapy, although the invention also relates to the use of PSG proteins other than PSG1 (and modified versions thereof) in the above-described therapeutic methods.

[0018] The present invention also provides pharmaceutical compositions comprising a pregnancy-specific glycoprotein (eg, PSG1 or PSG-Fc), mesenchymal stem cells (MSCs) and a suitable pharmaceutical excipient.

[0019] In any embodiment, the composition is injectable.

[0020] In any embodiment, the MSCs are conditioned in a cell culture medium containing PSG.

[0021] In any embodiment, PSG1 (or PSG) is modified with 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 purification of the modified PSG1. Examples of modifications of PSG1 polypeptides are provided below, and examples of modifications of PSG1 polypeptides include antibody fragments, e.g., the addition of an Fc portion, the addition of a PEG functional group, and the substitution of native amino acids with L-isomers. In one embodiment, the functional group is an Fc portion. In one embodiment, the Fc portion is modified to have an increased plasma half-life compared to the native Fc portion.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.

[0022] In another aspect, the invention provides CC49 (typically recombinant CC49) for use in a method of treating or preventing tendon injury. In a preferred embodiment, the mammal is an equine (i.e., a horse). In any embodiment, the condition is a degenerative condition of the tendon (e.g., tendinitis, tendinopathy) or a tendon-related condition of unknown etiology (e.g., tendinopathy). In any embodiment, the condition causes lameness due to tissue damage in response to a stimulus (e.g., injury). In any embodiment, CC49 is administered by direct injection into the injured tissue or by injection into the tendon. 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 tendon condition (typically tendinitis) in an equine mammal, wherein CC49 is administered to the equine mammal by intra-articular injection into the affected tendon.

[0023] In any embodiment, the MSCs are administered to the mammal, generally at the site of the tendon injury, generally by injection.

[0024] In any embodiment, CC49 (or CC49-Fc) or a homologue or variant thereof is co-administered with MSCs, typically in the same injection.

[0025] In any embodiment, CC49 (or CC49-Fc) or a homologue or variant thereof is administered separately from the MSCs.

[0026] In any embodiment, the MSCs are cultured in cell culture with CC49 (or CC49-Fc) or a homolog or variant thereof prior to administration.

[0027] 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 derived from equine 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 manufacturing.

[0028] In any embodiment, CC49 is administered parenterally. In any embodiment, CC49 is administered by intra-articular injection.

[0029] In another aspect, the present invention provides recombinant CC49 for use as a medicament.

[0030] In another aspect, the present invention provides a pharmaceutical composition comprising CC49 (or CC49-Fc) or a homologue or variant thereof, mesenchymal stem cells (MSCs) and a suitable pharmaceutical excipient.

[0031] In any embodiment, the composition is injectable.

[0032] In any embodiment, the MSCs are conditioned in a cell culture medium comprising CC49 or a homologue or variant thereof.

[0033] In another aspect, the present invention provides Fc-tagged CC49 (eg, Fc-tagged recombinant CC49).

[0034] In another aspect, the present invention provides an Fc-tagged CC49 for use as a pharmaceutical.

[0035] Other aspects and preferred embodiments of the present invention are defined and described in the other claims set forth below. [Brief explanation of the drawings]

[0036] [Figure 1] PSG1 and CC49 proteins enhance wound closure in a cell line scrape wound assay. Human MSCs (n=3) treated with PSG1-Fc, PSG1-V5His, CC49-Fc, CC49-V5His, or 50 μl PBS after 16 hours. [Figure 2] PSG1 and CC49 proteins enhance wound closure in a cell line scrape wound assay. Equine MSC cells treated with PSG1-Fc, PSG1-V5His, CC49-Fc, CC49-V5His, or 50 μl PBS after 16 hours (n=3). DETAILED DESCRIPTION OF THE INVENTION

[0037] 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 set forth in full.

[0038] 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) meaning that may be enjoyed in the art: Unless the context otherwise requires, the use of the singular herein should be read to include the plural and vice versa. The terms "a" or "an" when 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 specific symptoms. The term is used broadly to encompass any disorder, illness, disorder, condition, illness, state, or syndrome in which physiological function is impaired, regardless of the nature of the etiology (or whether an etiological basis for the disease has actually been established). Thus, the term encompasses 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 context, the term is used interchangeably with the term "therapy."

[0042] Furthermore, 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 or therapeutically effective amount of a drug defines an amount that can be administered to a subject without excessive toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit / risk ratio, yet 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 individual's age and general condition, the mode of administration, and other factors. Therefore, it is not possible to specify an exact effective amount, but one of ordinary skill in the art would 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 outcomes include eradication or alleviation of symptoms, reduction in pain or discomfort, prolonged survival, improved mobility, and other markers of clinical improvement. A therapeutic outcome need not be a complete cure. Improvement can be observed in biological / molecular markers or by 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 connection with the above-defined treatment and effective amount, the term subject (if the context allows, should be read to include " individual ", " animal ", " patient " or " mammal ") defines any subject to which treatment is applied, particularly mammalian subject.Mammalian subject includes but is not limited to human beings, 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 human beings. As used herein, the term "equine" refers to a mammal of the family Equidae, which includes horses, donkeys, asses, Tibetan asses, and zebras.

[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.This protein is found in a subset of mammals, including, for example, primates, rodents, horses, and bats, but is 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] As used herein, the term "tendon injury" refers to an injury to a damaged or diseased tendon that results in one or more of pain, reduced function, and impaired exercise tolerance. The injury may be traumatic or caused by a disease, such as a degenerative condition, or both. Such tendon injuries are characterized by abnormalities in tendon microstructure, composition, and cellularity (Neal L. Millar, Karin G. Silbernagel, et al., Tendinopathy, Nature Reviews, 2021, pages 1-21; Cho et al., Mesenchymal Stem Cells Use in the Treatment of Tendon Disorders: A Systematic Review and Met-Analysis of Prospective Clinical Studies, Annals of Rehabilitation Medicine, 2021, pages 274-283). Examples of tendon injuries include tendonitis (also called "tendinitis"), tendinopathy, tennis elbow, tennis tendinopathy, enthesopathy, tenosynovitis, and rotator cuff injuries.

[0048] As used herein, the term "MSCs" or "mesenchymal stem cells" refers to multipotent stem cells that can be harvested from bone marrow or adipose tissue and have the ability to differentiate into various cell types, including, but not limited to, osteoblasts, chondrocytes, myocytes, and adipocytes. Their ability to suppress the normal process of allogeneic rejection allows for the use of tissue-mismatched cells as a therapeutic approach to regenerative medicine. MSCs have been widely used in tissue engineering and regenerative medicine, including in the field of musculoskeletal repair. Recent studies have demonstrated the effectiveness of MSC therapy in patients with tendon injuries. (English et al. Allogeneic Mesenchymal Stem Cells: Agents of Immune Modulation, Journal of Cell Biochemistry, 2011, 112, pages 1963-1968; Cho et al. Mesenchymal Stem Cells Use in the Treatment of Tendon Disorders: A Systematic Review and Met-Analysis of Prospective Clinical Studies, Annals of Rehabilitation Medicine, 2021, pages 274-283; Costa-Almeida et al. Mesenchymal Stem Cells Empowering Tendon Regenerative Therapies, International Journal of Molecular Sciences, 2019, 20, 3002). The MSCs used in the present application can be obtained from any suitable tissue, such as bone marrow or adipose tissue, or from a cell bank. The MSCs used in the methods and compositions of the present invention can be autologous (e.g., obtained from the subject to be treated) or allogeneic (obtained from a different subject).Allogeneic MSC therapy has been described by English et al. (J Cell Biochem. 2011 Aug;112(8):1963-8. doi:10.1002 / jcb.23119) and Karantalis (Circ Res. 2015 Jan 2;116(1):12-15). MSCs can be conditioned before administration, for example, cultured in cell culture medium containing PSG1 or CC49, or Fc-tagged variants, or variants or homologs thereof. MSCs are administered at a concentration of 1 x 10 per dose. 5 cells ~1×10 10 Cell doses are typically 1 x 10 per dose. 6 cells ~1×10 8 The dose may be administered in cellular amounts. SEQ ID NO: 1 PSG1

number

[0049] The term "PSG1" also includes Fc-tagged PSG1 proteins (PSG1-Fc), an example of which is provided below in SEQ ID NO: 2, in which the Fc tag has been modified by site-directed mutagenesis to introduce MTS mutations M252Y / S254T / T256E and HN mutations H433K / N434F: SEQ ID NO: 2 PSG1-Fc ORF

number

number

number

[0050] 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)) and mature peptide. The term also includes the mature peptide without the signal sequence. SEQ ID NO:4: CC49

number

number

number

[0051] 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 and equine wild-type PSG1 and CC49 proteins. Thus, for example, the terms should be interpreted 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 five or fewer amino acids, more preferably four or fewer, even more preferably three or fewer, and most preferably only one or two. Insertions, additions, and substitutions with natural and modified amino acids are contemplated. Mutants may have conservative amino acid changes, where the introduced amino acid is structurally, chemically, or functionally similar to the amino acid being replaced. Typically, proteins altered by the substitution or deletion of catalytically important residues are excluded from the term "mutant." Details of such catalytically important residues are well known to those skilled in the art of protein modeling. Generally, variants will have at least 70% amino acid sequence identity with the wild-type protein (excluding the signal peptide, as noted above), preferably at least 80%, more preferably at least 90%, and ideally at least 95%, 96%, 97%, 98%, or 99% sequence identity. In this context, sequence identity includes both sequence identity and sequence similarity; i.e., a polypeptide sequence sharing 70% amino acid identity with the wild-type protein will have any 70% of aligned residues that are identical to or conservative substitutions for the corresponding residues in the wild-type protein. With respect to PSG1, specific variants within the scope of the present invention are the mutant PSG1 proteins identified in paragraphs 25-38 of International Patent Application Publication No. WO2017049082. This 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. Fc tags can be modified to exhibit increased plasma half-life and / or stability. Such Fc tags are known from 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.

[0052] 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 readily prepared according to well-established standard liquid-phase or, preferably, solid-phase protein synthesis methods known in the art (see, e.g., J.M. Stewart and J.D. 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 equivalents of proteins characterized by increased in vivo or in vitro stability and / or efficacy and / or half-life in relation to the practice 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 an aldehyde followed by reduction with NaBH4; amidation with methyl acetimidate; 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; mixed disulfide formation with other thiol compounds; maleimide; reaction with maleic anhydride or other substituted maleimides; formation of mercury derivatives using 4-chloromercuribenzoic acid, 4-chloromercuriphenylsulfonic acid, phenylmercuric chloride, 2-chloromercuric-4-nitrophenol, and other mercuric acids; and carbamylation with cyanate at alkaline pH. Tryptophan residues can be modified, for example, by oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfonyl halides. Tyrosine residues can be altered by nitration with tetranitromethane to form 3-nitrotyrosine derivatives. Modification of the imidazole ring of histidine residues can be achieved by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate. Examples of incorporation of unnatural amino acids and derivatives during protein synthesis include, but are not limited to, norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and / or D-isomers of amino acids. Modifications to 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 on related variant / analog proteins.

[0053] As used herein, the term "sequence identity" should be understood to include both sequence identity and sequence similarity; i.e., a variant (or homolog) sharing 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 conservative substitutions for the corresponding residues in the reference sequence over the entire length of the sequence. Sequence identity is the amount of exact match 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 percent similarity or identity with a reference sequence, provided that a percentage of the aligned residues of the variant (or homolog) are identical to or conservative substitutions for the corresponding residues in the reference sequence, and the variant (or homolog) shares the same function as the reference sequence. This 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.

[0054] The term "PSG1" also includes PSG1 proteins (modified proteins) that have been modified by other than insertion, deletion, or substitution of amino acid residues by functional modification. Similarly, "CC49" also includes CC49 proteins (modified proteins) that have been modified by other than insertion, deletion, or substitution of amino acid residues by functional modification.

[0055] As used herein, the term "tendon injury" should be understood to mean damage to the tendon as a result of tendonitis, tendinosis, or tendinopathy, or damage due to trauma, such as a fall or sports injury, wear and tear, or other disease process. The methods of the present invention relate to treating conditions by slowing or inhibiting tendon damage and / or by causing tendon growth and / or repair.

[0056] 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 consisting of creams, multiple emulsions, anhydrous compositions, aqueous dispersions, oils, milks, balms, balsams, foams, lotions, gels, cream gels, water-alcohol solutions, water-glycol solutions, cosmetics, personal care products, hydrogels, liniments, serums, soaps, powders, pastes, semisolid formulations, liniments, serums, shampoos, conditioners, ointments, any rinse-off formulation, talc, mousses, powders, sprays, aerosols, solutions, suspensions, emulsions, syrups, elixirs, polysaccharide films, patches, gel patches, bandages, adhesive systems, water-in-oil emulsions, oil-in-water emulsions, and silicone emulsions. The topical compositions of the present invention are administered in a cosmetically or pharmaceutically effective amount, i.e., a non-toxic but sufficient amount to provide the desired effect. It will be appreciated that one of ordinary skill in the art can determine the appropriate dosage of the topical compositions of the present invention to administer without undue experimentation. Alternatively, a physician will determine the actual dosage most appropriate for a patient, depending on the particular condition, disease, or disorder being treated or cared for, as well as the person's age, weight, and / or health. This will depend on a variety of factors, including the activity of the particular compound used, the compound's metabolic stability and duration of action, 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 merited, and these are within the scope of the present invention. For example, the compositions 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 (intra-articular, intratendinous, etc.), 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 a 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 a lipid or oil. The emulsion can be, but is not limited to, an oil-in-water, water-in-oil, water-in-oil-in-water, or oil-in-water-in-silicone emulsion. The emulsion can contain a moisturizing agent. The emulsion can contain an anti-foaming agent such as silicone. The emulsion can have any suitable viscosity. The emulsion can further contain an emulsifier and / or an anti-foaming agent. Methods for preparing emulsions are known to those skilled in the art.

[0057] The active agent (PSG1 or CC49) is used in the topical or pharmaceutical compositions of the present invention at a pharmaceutically or therapeutically effective concentration to achieve the desired effect, preferably from 0.00000001% (by weight) to 100% (by weight), typically from 0.00000001% (by weight) to 40% (by weight), preferably from 0.000001% (by weight) to 15% (by weight), more preferably from 0.0001% (by weight) to 10% (by weight), and even more preferably from 0.0001% (by weight) to 5% (by weight) of the total weight of the composition. Ideally, from about 0.00001% w / w to about 0.5% w / w of the composition, more preferably from 0.00005 w / w to about 0.05 w / w, and most preferably from 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.

[0058] The composition of the present invention can be administered individually or in combination with other pharmacologically active agents (such as MSCs).It is understood that such combined therapy includes but is not limited to various treatment regimens, including the administration of multiple agents in a single dosage form or separate individual dosage forms together.When agents are present in different dosage forms, administration can be simultaneous or nearly simultaneous, or can follow any predetermined regimen that includes the administration of different agents.Suitable active agents can be as described herein.

[0059] In some embodiments of the present 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, surfactant mixed micelles, surfactant-phospholipid mixed micelles, millispheres, spheres, lipospheres, particles, nanospheres, nanoparticles, milliparticles, solid nanoparticles, and microemulsions, including water-in-oil microemulsions with reverse micelle internal structures, and nanoemulsion microspheres, microparticles.

[0060] Various methods for preparing liposomes are available, e.g., Szoka et al. al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Patent No. 4,186,183, U.S. Patent No. 4,217,344, U.S. Patent No. 4,235,871, U.S. Patent No. 4,261,975, U.S. Patent No. 4,485,054, U.S. Patent No. 4,501,728, U.S. Patent No. 4,774,085, U.S. Patent No. 4,837,028, U.S. Patent No. 4,946,787, PCT Publication No. WO 91 / 17424, Deamer & Bangham, Biochem. Biophys. Acta 443:629-634(1976);Fraley,et al.,PNAS 76:3348-3352(1979);Hope et al.,Biochim.Biophys.Acta 812:55-65(1985);Mayer et al.,Biochim.Biophys.Acta 858:161-168(1986);Williams et al.,PNAS 85:242-246(1988);Liposomes(Ostro(ed.),1983,Chapter 1);Hope et al.,Chem.Phys.Lip.40:89(1986);Gregoriadis,Liposome Technology (1984) and Lasic, Liposomes: from Physics to Applications (1993)). Suitable methods include, for example, sonication, extrusion, high pressure / homogenization, microfluidization, detergent dialysis, calcium-induced fusion of small liposomal vesicles, and ether fusion methods, all of which are well known in the art.

[0061] These delivery systems can be adapted to achieve greater penetration of the compounds and / or peptides of the present invention, which 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 over a certain period of time, preferably at a constant release rate over a certain 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.

[0062] The compounds of the present invention can be administered orally. The compounds (and optionally other ingredients) 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 compounds can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. The compounds can be coated or co-administered with some material to prevent their inactivation. The coating can be configured to protect the active agent during passage through the stomach and release the active agent in the ileum.

[0063] The methods of the present invention may involve administering a nucleic acid construct configured to express an active agent (PSG1 or CC49) in vivo, optionally in combination with MSCs. As used herein, the term "PSG1 expression vector" or "CC49 expression vector" refers to any suitable vector, including chromosomal vectors, non-chromosomal vectors, and synthetic nucleic acid vectors (nucleic acid sequences comprising a suitable set of expression control elements) suitable for intracellular expression of PSG1 or CC49 (or modified versions thereof, such as Fc-tagged proteins). 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 methods for their use are well known in the art (see, e.g., U.S. Pat. Nos. 5,589,466 and 5,973,972). In one embodiment, the DNA comprises an expression control sequence.

[0064] 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 another embodiment, 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).

[0065] Expression control sequences are engineered to control and promote transcription of the gene of interest and subsequent expression of the protein in various cell systems. Plasmids combine an expressible gene of interest with expression control sequences (i.e., expression cassettes) containing desired elements, such as a promoter, enhancer, selection marker, operator, etc. In the expression vectors of the present invention, the nucleic acid molecule encoding the PSG1 amino acid sequence or CC49 amino acid sequence can contain or be associated with any suitable promoter, enhancer, selection marker, operator, repressor protein, polyA termination sequence, and other expression-promoting elements.

[0066] 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 in a manner that allows transcription of the protein-coding nucleotide sequence, when the appropriate signals are present. Expression of the 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., the human CMV IE promoter / enhancer, or the 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.

[0067] The nucleic acid molecules of the invention may also be operably linked to an effective 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).

[0068] Selection markers are elements well known in the art. Under selection conditions, only cells that express the appropriate selection marker can survive. Generally, in cell culture, the selection marker gene expresses a protein, usually an enzyme, that confers resistance to various antibiotics. 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).

[0069] 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), cyanofluorescent protein (CFP), enhanced cyanofluorescent protein (eCFP), or yellow fluorescent protein (YFP).

[0070] For 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 may be a recombinant "regulatory fusion protein" (RFP). RFPs consist essentially of a transcription blocking domain and a ligand binding domain that regulates their activity. An example of such an expression system is described in U.S. Patent No. 20090162901 A1, which is incorporated herein by reference in its entirety.

[0071] As used herein, "operator" refers to a DNA sequence introduced within or near a gene such that the gene can be regulated by RFP binding to the operator, thereby preventing or allowing 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 Escherichia coli (E. coli), which binds to the LexA peptide, and the lactose and tryptophan operators, which bind to the repressor proteins encoded by the Lad and trpR genes of E. coli. These also include 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 be within a specific 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.

[0072] In an exemplary cellular expression system, cells are engineered to express the 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 the gene encoding the protein of interest, driven 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, and therefore translation (expression) of the protein of interest occurs after transcription. (See, e.g., U.S. Patent No. 7,435,553, incorporated herein by reference in its entirety.)

[0073] The vector of the present invention can also use the Cre-lox recombination tool 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 recombination between two loxP sites; and 2) a loxP site (e.g., a specific 34 base pair sequence consisting of an 8-bp core sequence where recombination occurs and two adjacent 13-bp inverted repeats) or a mutant loxP site. (See, for example, 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 U.S. Patent No. 20100291626 A1, all of which are incorporated herein by reference.) Another recombination strategy may utilize the yeast-derived FLP recombinase in conjunction with the consensus sequence FRT (see also, for example, Dymecki, SM, 1996, PNAS 93(12):6191-6196).

[0074] As used herein, the term "host cell" includes any cell suitable for expressing a recombinant nucleic acid sequence, including 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 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, tumor cells, and cell lines derived from the foregoing cells. In some embodiments, the cell comprises one or more viral genes, e.g., a retinal cell (e.g., a PER.C6® cell) that expresses 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.

[0075] As used herein, the term "transformed cell" refers to a host cell that contains a nucleic acid stably integrated into the cellular genome, the nucleic acid comprising a nucleotide sequence encoding expression of a PSG1 protein or a CC49 protein. In another embodiment, the invention provides a cell that contains a non-integrated (i.e., episomal) nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, that comprises a sequence encoding expression of a PSG1 protein or a CC49 protein. In another embodiment, the invention provides a cell line produced by stably transfecting a host cell with a plasmid comprising an expression vector of the invention.

[0076] As used herein, the term "engineered" as applied to a cell means genetically manipulated using recombinant DNA techniques, generally involving the synthesis of a suitable expression vector (see above) and then transfecting the expression vector into a host cell (generally stable transfection).

[0077] As used herein, the term "heterologous expression" refers to the expression of a nucleic acid in a host cell that does not naturally harbor the nucleic acid. The insertion of a nucleic acid into a heterologous host is accomplished by recombinant DNA techniques.

[0078] As used herein, in the context of treatment, the term "administering" should be interpreted to include any form of delivery capable of delivering an active agent, including intravenous, oral, intramuscular, 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 pump. (See Ignacio et al., Ann. NYAcad. 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). Intraperitoneal - for systemic administration - administered directly into the peritoneum by 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 - can be prepared with implants (e.g., small silicone implants) that release active substances. The implants can be placed in the muscle (Kieran and Greensmith, 2004 Neurosci 125(2):427-39).

[0079] Modified proteins In any embodiment, the PSG1 protein or CC49 protein (including protein fragments and variants) can 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: increased plasma half-life; increased lipophilicity of the protein; decreased renal clearance of the modified protein; increased activity of the modified protein; and increased resistance of the modified protein to proteolysis (i.e., by mammalian and particularly human gastrointestinal proteases). Various methods for modifying the proteins of the invention to exhibit these properties are disclosed herein, including conjugating the protein to a binding partner (e.g., an albumin-binding small molecule, a large polymer, a long-life plasma protein, or an antibody or antibody fragment), cyclization, N- or C-terminal or side chain additions, protecting groups, replacing one or more L-amino acids with D-isomers, amino acid modifications, increased plasma protein binding, and increased albumin binding. Modified proteins include, but are not limited to, the 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.

[0080] In any embodiment, the modification may be any modification that provides the protein and / or composition of the present invention with 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.

[0081] In some embodiments, 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.

[0082] Those skilled in the art will recognize suitable techniques for reacting amino acids with these protecting groups. These groups can be added by preparative methods known in the art, such as those outlined in paragraphs

[0104] to

[0107] of U.S. Patent No. 2014120141. The groups can remain on the protein or can be removed. Protecting groups can be added during synthesis.

[0083] In any embodiment of the present invention, the protein may be substituted with one or more groups selected from straight-chain or branched, long-chain or short-chain, saturated or unsaturated, hydroxyl, amino, aminoacyl, sulfate, or sulfide groups, or may be 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 with hydroxyl or sulfur-containing groups, such as, but not limited to, SO3H, SH, or SS.

[0084] In any embodiment of the invention, the protein is R1-X-R2.

[0085] The R1 and / or R2 groups were attached to the amino terminus (N-terminus) and carboxyl terminus (C-terminus) of the protein sequence, respectively.

[0086] In one embodiment, the protein is R1-X. Alternatively, the protein is X-R2.

[0087] 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.

[0088] In any embodiment, R1 is selected from the group formed by H, an acyclic substituted or unsubstituted aliphatic group, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc), and R5-CO-, wherein R5 is selected from the group formed by H, an acyclic substituted or unsubstituted aliphatic group, 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.

[0089] According to another preferred embodiment, R2 is -NR3R4, -OR3 or -SR3, wherein 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, substituted or unsubstituted 3-10 membered heterocyclyl ring, and substituted or unsubstituted heteroarylalkyl of 2 to 24 carbon atoms and 1 to 3 atoms other than carbon, wherein the alkyl chain is of 1 to 6 carbon atoms. Optionally, R3 and R4 can be joined 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.

[0090] 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.

[0091] In a preferred embodiment, the acyl (or acetyl) group is attached to the N-terminus of at least one amino acid of the protein.

[0092] In any embodiment of the present invention, the protein is modified to include side chain protecting groups. The side chain protecting groups can be one or more of the following: benzyl or benzyl-based groups, t-butyl-based groups, benzyloxycarbonyl (Z) groups, and allyloxycarbonyl (alloc) protecting groups. The side chain protecting groups can be derived from achiral amino acids, such as achiral glycine. The use of achiral amino acids can help stabilize the resulting protein as well as facilitate the synthetic pathways of the present invention. Preferably, the protein further includes a modified C-terminus, preferably an amidated C-terminus. The achiral residue can be alpha-aminoisobutyric acid (methylalanine). It is understood that the specific side chain protecting groups used will depend on the protein sequence and the type of N-terminal protecting group used.

[0093] 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 by typically 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, PEG homopolymers or copolymers, monomethyl-substituted polymers of PEG (mPEG) and polyoxyethyleneglycerol (POG), polyamino acids such as polylysine, polyglutamic acid, and polyaspartic acid, particularly those in the L-configuration, pharmacologically inactive proteins such as albumin, gelatin, fatty acids, polysaccharides, lipid amino acids, and dextran. The polymer moiety may be linear or branched and may have a molecular weight of 500 to 40,000 Daltons (DA), 5,000 to 10,000 Da, or 10,000 to 5,000 Da. The compound may be any suitable cell-permeable compound, such as tat protein, penetratin, or pep-1. The compound may be an antibody molecule. The compound may be a lipophilic moiety or a polymer moiety.

[0094] Lipophilic substituents and polymeric substituents are known in the art. Lipophilic substituents include acyl groups, sulfonyl groups, N atoms, O atoms, or S atoms that form 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, branched, saturated, or unsaturated. The hydrocarbon chain may be further substituted. The hydrocarbon chain may be a cycloalkane or a heterocycloalkane.

[0095] 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 via 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.

[0096] 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.

[0097] The polymer or compound may be conjugated via an ester, sulfonyl ester, thioester, amide, carbamate, urea, sulfonamide.

[0098] Those skilled in the art will be aware of suitable means for preparing the described conjugates.

[0099] Proteins can be chemically modified by covalent conjugation with polymers, for example, to increase their circulating half-life.The exemplary polymers and methods of binding such polymers to proteins are exemplified 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.

[0100] The proteins of the present invention can be subjected to one or more modifications to manipulate storage stability, pharmacokinetics, and / or any aspect of the protein's biological activity, such as potency, selectivity, and drug interactions. Chemical modifications that can be performed on proteins 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.

[0101] Modified proteins can also include sequences in which one or more residues have been modified (i.e., by phosphorylation, sulfation, acylation, amidation, PEGylation, etc.), and mutants containing one or more modified residues relative to the parent sequence. Amino acid sequences can also be modified with labels capable of directly or indirectly providing 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, e.g., U.S. Pat. Nos. 3,654,090, 3,850,752, and 4,016,043). Enzymes can be conjugated by reaction with cross-linking 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.).

[0102] In one embodiment, the protein, variant, and / or composition is modified to increase drug performance. 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 the 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), fusion to the Fc domain and human serum albumin, fusion with hydrophilic amino acid polymers such as XTEN or PAS, and / or fusion with half-life extending proteins.

[0103] Proteins or proteins may contain weak sites within their sequences that are susceptible to proteolytic cleavage when in an environment with enhanced proteolytic degradation, such as in the blood or gastrointestinal tract. In one embodiment, proteins, variants, and / or compositions contain modifications of one or more weak sites such that the protein, variant, and / or composition is not subject to proteolytic breakdown / cleavage or the amount of proteolytic breakdown / cleavage of the protein, variant, and / or composition is reduced compared to one or more unmodified proteins. Thus, proteins can be modified to increase the resistance of the modified protein to proteolysis by mammalian gastrointestinal proteases. Suitable modifications are described in Diao et al. (Clinical pharmacokinetics 52.10(2013):855-868).

[0104] Protein modifications to extend the in vivo half-life of proteins are described in the literature, for example, 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.

[0105] Due to the obvious advantages of long-acting proteins and protein drugs, strategies to extend the plasma half-life of such compounds are highly sought after. Short plasma half-lives are generally due to rapid renal clearance and enzymatic degradation during systemic circulation. Protein / protein modifications can result in extended plasma half-lives. By reducing the total amino acid content of somatostatin and substituting D-amino acids for L-analogs, the plasma half-life of the derivative octreotide was 1.5 hours, compared with only a few minutes for somatostatin. A PEG (2.40K) conjugate of INF-alpha-2b exhibited a 330-fold longer plasma half-life than the native protein. The purpose of this review was to provide an overview of possible strategies for extending plasma half-life, such as N- and C-terminal modifications or PEGylation, as well as methods for evaluating the effectiveness of drug modifications. Furthermore, basic data on the most important proteolytic enzymes in human blood, liver, and kidney, as well as their cleavage specificities and inhibitors, are provided to predict the enzymatic cleavage of proteins and protein drugs during systemic circulation.

[0106] Strategic Approaches to Optimizing Protein ADME Properties.Li Di AAPS J.2015 Jan;17(1):134-143.

[0107] Strategies for stabilizing proteins against proteolysis Many methods are available for enhancing protein stability through structural modification. Some methods not only improve stability but also enhance other ADME properties, for example, cyclization can increase stability and permeability, and 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.

[0108] N- and C-terminal protection Several proteolytic enzymes in blood / plasma, liver, and kidneys, including exopeptidases, aminopeptidases, and carboxypeptidases, degrade protein sequences from the N- and C-termini. N- and / or C-terminal modifications can often improve protein stability. In many cases, N-acetylation and C-amidation have been reported to increase resistance to proteolysis.

[0109] Substitution of L-amino acids with D-amino acids Substitution of natural L-amino acids with unnatural D-amino acids reduces substrate recognition and binding affinity of proteolytic enzymes and increases their stability. One example is vasopressin, which contains L-Arg and has a half-life of 10 to 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 with D-serine not only improved potency in mouse plasma by 1.8-fold but also increased stability by 4-fold.

[0110] 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 by an ethylamide, has a much longer half-life in humans.

[0111] ·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-rich proteins, such as linaclotide, lepirudin, and ziconotide, are commercially available or in preclinical or clinical development.

[0112] Conjugation to macromolecules Conjugation to macromolecules (e.g., polyethylene glycol (PEG), albumin) is an effective strategy to improve protein stability and reduce renal clearance.

[0113] Renal clearance Many proteins exhibit promising in vitro pharmacological activity but fail to demonstrate in vivo efficacy due to their very short in vivo half-lives (minutes). The rapid clearance and short half-lives of proteins prevent their successful development into drugs. The primary causes of rapid clearance of proteins from the systemic circulation are enzymatic proteolysis and / or renal clearance. Glomeruli have pore sizes of approximately 8 nm, and hydrophilic proteins with molecular weights <2–25 kDa are prone to rapid filtration through the glomerulus of the kidney. Because proteins are not readily reabsorbed through the renal tubules, they often have high renal clearance and short half-lives. 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 regarding whether renal clearance is likely to be the primary elimination route.

[0114] In patients with renal impairment, dose adjustments may be necessary to avoid protein drug accumulation and increased drug exposure, as inappropriate dosing in these patients can cause toxicity or render treatment ineffective. Several strategies have been developed to reduce protein renal clearance and extend half-life. These are outlined below.

[0115] Increased plasma protein binding Renal clearance of proteins is reduced when they bind to membrane or serum proteins. One example is the cyclic protein drug octreotide, a drug used to treat endocrine tumors, which has a half-life of approximately 100 minutes in humans (unbound fraction 0.65) due to binding to lipoproteins.

[0116] 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 via the highly conjugated small molecule.

[0117] Conjugation to large polymers Conjugation of proteins to large synthetic or natural polymers or carbohydrates can increase their molecular weight and hydrodynamic volume, thus decreasing their renal clearance. Common polymers used for protein conjugation are PEG, polysialic acid (PSA), and hydroxyethyl starch (HES).

[0118] Fusion to long-lived plasma proteins Plasma proteins such as albumin and immunoglobulin (IgG) fragments have long half-lives of 19–21 days in humans. Due to their high molecular weight (67–150 kDa), these proteins have low renal clearance, and their binding to neonatal Fc receptors (FcRn) reduces their excretion via pinocytosis by vascular epithelia. Covalent binding of proteins to albumin or IgG fragments can reduce renal clearance and extend their half-lives. Fusion Proteins for Half-Life Extension of Biologics as a Strategy to Make Biobetters William R. Strohl BioDrugs.2015;29(4):215-239.

[0119] 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.

[0120] 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.

[0121] Zhang, L, Bulaj, G. Converting Proteins into Drug Leads by Lipidation. Curr Med Chem. 2012;19(11):1602-18.

[0122] 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.

[0123] 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

[0124] 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 use as therapeutics. Antibodies against the unmodified drug, once formed, could rapidly neutralize and eliminate protein drugs. Unexpectedly, PEGylation improved the pharmacokinetics of proteins, even in the absence of anti-drug antibodies. Simply by making the drug molecule larger, PEGylation resulted in drugs being filtered more slowly by the kidney. The empirical observation that increasing size or hydrodynamic radius resulted in decreased renal clearance and increased half-life became the primary rationale for PEGylation of proteins and protein drugs. PEGylation can have various effects on molecules, including making 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. Alterations in the size, structure, and linkage mode of the PEG polymer can affect the biological activity of the attached drug.

[0125] First-generation PEGylation methods have been fraught 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. For example, PEG readily binds to the amino groups of lysines on the surface of proteins. 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 product. 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 leukemia. Although these drugs were complex mixtures of various PEGylated species, they possess improved therapeutic properties over native enzymes, including increased serum half-life and reduced protein immunogenicity. Due to the inherent polydispersity of PEG, quality and batch-to-batch reproducibility have been difficult. Despite this limitation, two PEGylated interferons (PEG-interferon alpha-2b and PEG-interferon alpha-2a), which are heterogeneous populations of multiple mono-PEGylated positional isomers, have been approved by the FDA for the treatment of hepatitis C. These drugs were launched in 2001 and 2002, respectively.

[0126] Various improvements and modifications have been made to the basic PEGylation technology. Second-generation PEGylation processes have introduced branched structures and alternative chemistries for PEG conjugation. In particular, PEGs with cysteine-reactive groups, such as maleimide or iodoacetamide, allow for targeted PEGylation to a single residue within a protein, reducing heterogeneity of the final product but not eliminating it due to the polydispersity of PEG itself.

[0127] Although the original rationale for PEGylation was to reduce immunogenicity, several examples of immunogenic PEGylated proteins exist. One example is PEGylated urate oxidase, an enzyme that reduces plasma uric acid levels in patients with gout. In clinical trials, a relatively high percentage of gout patients failed to respond to treatment and developed antibodies specific to PEG but not to uricase protein 2. PEGylated liposomes, 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 production of specific anti-PEG IgM antibodies, thus accelerating the clearance of PEG-modified proteins from the body.

[0128] The main potential drawback of using PEG as a modifier is its non-biodegradability. The U.S. Food and Drug Administration (FDA) has approved its use 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 After repeated administration of some PEGylated proteins to animals, vacuolation of renal tubular cells has been observed. Recently, toxicity studies using proteins conjugated with large (≥40 kDa) PEG have also shown vacuolation of choroid plexus epithelial cells. Choroid plexus epithelial cells produce cerebrospinal fluid and form the blood-CSF barrier. While the long-term negative consequences of cell vacuolation are unknown, it represents an undesirable outcome for several potential therapeutic approaches. One possible alternative is the substitution of biodegradable polymers for PEG. Polymers such as hydroxyethyl starch (HES) are possible alternatives. HES is nontoxic, biodegradable, and used as a blood volume expander. The HES conjugation process functions similarly to PEGylation, which reduces renal clearance by increasing the hydrodynamic radius of proteins, 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 the use of defined polyproteins as polymer components. This approach is described later in this article.

[0129] Lipidization The second major chemical modification method for increasing 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 for extending insulin half-life, shares the same fundamental mechanism of half-life extension as PEGylation: increasing the hydrodynamic radius and decreasing renal filtration. However, the lipid moiety itself is relatively small, and its effect is indirectly mediated through noncovalent 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 proteins from attack by peptidases through steric hindrance, similar to that seen with PEGylation. One consequence of lipidation is that it reduces the water solubility of proteins, but this can be regulated by manipulating the linker between the protein and the fatty acid, for example, by using glutamate or mini-PEGs within the linker. Manipulation of the linker and alteration of the lipid moiety can affect self-aggregation, which can contribute to increased half-life by slowing biodistribution independent of albumin.

[0130] Following pioneering studies with insulin, lipidation of various proteins has been investigated, particularly in the diabetes field, 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: the GLP-1 analog liraglutide and insulin detemir, both long-acting antidiabetic drugs.

[0131] A potentially pharmacologically important difference between PEGylation and lipidation is that the therapeutically active protein is covalently attached to the much larger PEG, while the smaller fatty acyl-protein conjugate is non-covalently attached to the larger bound and unbound forms of albumin present in equilibrium. This can lead to differences in biodistribution that can result in different pharmacology, as access to receptors localized in different tissues can induce different effects. In some cases, more limited biodistribution may be desirable, while in other cases, greater tissue penetration may be important. An interesting variation of the PEG approach that addresses this issue, utilizing releasable PEG conjugates with predictable cleavage rates, has been developed by Santi et al.

[0132] 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 modified protein, whether biologically or synthetically produced. The advantage of using synthetic proteins is that they can incorporate unnatural amino acids designed to address certain issues, including instability due to known proteolytic cleavage drawbacks. They also offer greater flexibility in selecting conjugation sites, which is important when activity or efficacy is highly dependent on modified residues such as free termini or C-terminal amides.

[0133] Traditional gene fusions: Fc and HSA Traditional genetic fusion to long-lived serum proteins offers an alternative method for 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 (HSA). Fc fusion involves fusing the extracellular domain of a protein, protein, or receptor 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 utilize 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 undergoing late-stage clinical trials. Human serum albumin, the same protein used by fatty acylated proteins, 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, as opposed to the monomeric structure of HSA, which results in the fusion protein being presented as a dimer or monomer depending on the choice of fusion partner. The dimeric nature of protein-Fc fusions can have avidity effects if the target receptors are sufficiently closely spaced or are themselves dimeric. This may or may not be desirable depending on the target. In one embodiment, the Fc domain is engineered to contain mutations. Methods for engineering mutations into the Fc domain are described in Rath et al. (2013; doi=10.3109 / 07388551.2013.834293).

[0134] Engineered polyprotein fusions: XTEN and PAS An interesting variation on the recombinant fusion concept has been the development of engineered low-complexity sequences as fusion partners—basically, 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, as opposed 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 higher temperatures or HPLC purification.

[0135] The most advanced polyprotein in this class, called XTEN (Amunix), is 864 amino acids long and composed of six amino acids (A, E, G, P, S, and T). This is made possible by the polymer's biodegradability, but it is much larger than the typically used 40 kDa PEG, conferring a concomitantly longer half-life extension. The fusion of XTEN to protein drugs results in a 60- to 130-fold increase in half-life compared to the native molecule. Two fully recombinantly produced XTENylated products, VRS-859 (Exenatide-XTEN) and VRS-317 (Human Growth Hormone-XTEN), are in clinical trials. In a Phase Ia trial, VRS-859 was found to be well tolerated and effective in patients with type 2 diabetes. VRS-317 has been reported to have superior pharmacokinetic and pharmacodynamic properties compared to previously tested rhGH products and has the potential for once-monthly administration.

[0136] A second polymer based on similar conceptual considerations is PAS (XL-Protein GmbH), a random coil polymer 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 unclear, but this will become clear as these polyproteins are incorporated into more therapeutics and the behavior of the fusions is characterized.

[0137] Protein-protein fusions, whether the partner is Fc, HSA, XTEN, or PAS, are all genetically encoded and therefore suffer from similar constraints. One limitation is that only naturally occurring amino acids can be incorporated, unlike methods using chemical conjugation, which 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 and 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. Protein termini are often involved in receptor interactions, and genetic fusion to one or both termini can significantly impair activity. The site of PEG or lipid conjugation can be anywhere on the protein, allowing it to be optimized to maximize the biological activity of the resulting therapeutic.

[0138] A hybrid method for integrating half-life-extending proteins with 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 conjugation sites and in incorporating unnatural amino acids or modifications into the protein backbone. One of the first attempts to integrate the benefits of gene fusion with chemical conjugation for half-life extension was made by researchers at the Scripps Research Institute in La Jolla, using technology that later formed the basis of the biotechnology company CovX. These researchers developed a platform using catalytic aldolase antibodies to form reversible covalent enamine bonds between the active site lysine of the antibody and a beta-diketone incorporated into a protein or small molecule. The resulting conjugate is called CovXBody™. This approach combines the functional properties of protein drugs or small molecules with the long serum half-life of antibodies through chemical conjugation rather than through gene fusion. Following the initial demonstration of this technology, 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.

[0139] Recently, XTEN polyproteins have also been used in chemical conjugation modes,12 providing a more direct analogy to PEG. The first example of an XTENylated protein made using this method is GLP2-2G-XTEN, in which the protein is chemically conjugated to an XTEN protein polymer using maleimide-thiol chemistry. The chemically conjugated GLP2-2GXTEN molecule demonstrated comparable in vitro activity, in vitro plasma stability, and pharmacokinetics in rats to recombinant fusion GLP2-2G-XTEN.

[0140] The number and spacing of reactive groups, such as lysine or cysteine ​​side chains, within the fully designed sequence of an XTEN or PAS polyprotein can be precisely controlled by site-specific variation due to the limited set of amino acids from which they are constructed. 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.

[0141] In summary, hybrid protein half-life extension methods are emerging that combine the advantages and overcome the individual limitations of chemical conjugation and gene fusion methods. These methods 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 exclusively of natural L-amino acids or as linear unidirectional polyproteins fused at either the N- or C-terminus, thereby opening the door to a wider range of longer-acting protein-based drugs.

[0142] 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.). A detailed explanation of dosage principles is now further provided to better illustrate certain embodiments.

[0143] 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 treating a tissue degenerative condition in an equine mammal.

[0144] 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 kilogram of body weight per day, given in divided doses about 1 to about 6 times daily, or in sustained-release form, can be effective to achieve desired results. In one embodiment, the dosage is 10 to 100, 30 to 70, 40 to 60, or ideally about 50 μg / ml.

[0145] As a non-limiting example, treatment of a disease in a human or animal can include administration of a single or divided dose about every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof, 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 1 week, 2 weeks, 3 weeks, 4 weeks, 5 days, or 6 days. The present invention may be provided by 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 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.

[0146] Example The present invention will now be described with reference to specific examples, which are merely illustrative and for illustrative purposes only and are not intended to limit in any way the scope of the claimed exclusive rights or the invention described, and which constitute the best modes presently contemplated for carrying out the invention.

[0147] Example 1 Mesenchymal stem cells (MSCs) from various tissue sources are being developed for use as therapies for tendon diseases and injuries (e.g., Costa-Almeida et al., 2019; Cho et al., 2021). PSG1 and CC49 proteins modify MSC phenotype as evidenced by enhanced MSC migration in an in vitro scrape wound assay (Figure 1), justifying their use either as independent treatments or in combination therapy with MSCs.

[0148] Scraping wound assay Human and equine 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. For the abrasion wound assay, human and equine MSCs (2 × 10 5 Cells (1000 cells / ml) were seeded in 1 ml into 6-well plates using one IBIDI culture insert per well. After 24 hours, the inserts and medium were removed, and each well was treated with 1 ml of cell culture medium supplemented with 50 μg of PSG1-V5His or PSG1-FC or 50 μl of PBS. Scraping wounds were imaged 16 hours after treatment using EVOS FL Auto and Wimasis WimScratch analysis, or the extent of wound closure was determined using ImageJ analysis.

[0149] Example 2 Construction of pTT3 expression vector Both vectors contained 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.

[0150] Fc tag cloning The human Fc tag was PCR amplified from a sample of Epstein-Barr virus (EBV)-transformed lymphocyte cDNA. The equine Fc tag was amplified from the pcDNA-IGHG1 vector, developed by Bettina Wagner. Both the human and equine Fc tags were subcloned into the pTT3 vector using engineered HindIII sites in the primer tails, resulting in in-frame insertion at the 3' end of the PSG1 / CC49 ORF. After insertion of the Fc tag, site-directed mutagenesis was used to remove the internal HindIII site to allow in-frame transcription of the PSG1 or CC49 ORF with the Fc tag. Site-directed mutagenesis was used to introduce a 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 (Rath et al., 2015; for Fc modifications, see https: / / doi.org / 10.3109 / 07388551.2013.834293). Site-directed mutagenesis was used (Phusion Site-Directed Mutagenesis Kit - Thermo Fisher Scientific) to introduce a human Fc tag.

[0151] Example 3 PSG1 for the treatment of tendon injuries A mouse model of tendonitis was generated according to the method of Cho et al. (Cho Y, Kim HS, Kang D, Kim H, Lee N, Yun J, Kim YJ, Lee KM, Kim JH, Kim HR, Hwang YI, Jo CH, Kim JH. CTRP3 exacerbates tendinopathy by dysregulating tendon stem cell differentiation and altering extracellular matrix composition. Sci Adv. 2021 Nov 19;7(47):eabg6069. doi:10.1126 / sciadv.abg6069. Epub 2021 Nov 19. PMID:34797714; PMCID:PMC8604415).

[0152] Protocol for inducing tendon injury in mice (CIOA model). 1. Up to nine 8-week-old C57BL / 6J male mice per treatment group are anesthetized with isoflurane. 2. Shave the surgical area and wipe with alcohol. 3. The skin and synovial sheath of the right ankle are incised using a scalpel blade. 4. The Achilles tendon is then separated from the soleus muscle. 5. Place a 1.5 mm diameter ear punch lateral to the Achilles tendon, fitting approximately half the diameter. 6. The Achilles tendon is then cut with a punch, leaving a 0.75 mm semicircular defect on the lateral side.

[0153] Experimental protocol timeline (days): D-7: Receipt of mice and 1 week of acclimatization D1: Treatment groups receive a peritendinous injection of either PBS, 100 μg of PSG1-Fc, or 100 μg of CC49-Fc around the severed Achilles tendon. On days 8 and 15, the treated animals received repeated peritendinous injections of either PBS, 100 μg of PSG1-Fc, or 100 μg of CC49-Fc around the transected Achilles tendon. On day 20, the animals were euthanized and autopsied. The Achilles tendons, along with the attached whole muscle and bone, were harvested and preserved to prevent tissue shrinkage.

[0154] At necropsy, Achilles tendons were collected, harvested, and preserved with attached muscle and bone to prevent tissue shrinkage during tissue processing. For histological analysis, tissues were embedded in paraffin, stained, and analyzed using a modified Bonar scoring system. This system assesses the severity of tendon damage based on the categories of cellularity, cell morphology, collagen organization, and ground substance. This method was adapted from Fearon et al. (Fearon A, Dahlstrom JE, Twin J, Cook J, Scott A. The Bonar score revisited: region of evaluation significantly influences the standardized assessment of tendon degeneration. J Sci Med Sport. 2014;17(4):346-350. doi:10.1016 / j.jsams.2013.07.008).

Claims

1. A pharmaceutical composition comprising pregnancy-specific glycoprotein 1 (PSG1) or Fc-tagged PSG1 (PSG1-Fc), or an expression vector of said PSG1 or PSG1-Fc, for the treatment of tendon injury in humans, said pharmaceutical composition being administered by intratendinous or peritendinous injection.

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

3. A pharmaceutical composition described in claim 1 for administration to humans, comprising an expression vector for PSG1 or PSG1-Fc.

4. A pharmaceutical composition described in any one of claims 1 to 3, used in combination with administering mesenchymal stem cells to an injured tendon by intratendinous or peritendinous injection.

5. The pharmaceutical composition described in claim 4, which is administered simultaneously with the mesenchymal stem cells to the injured tendon by intratendinous or peritendinous injection.

6. The pharmaceutical composition according to claim 4, wherein the mesenchymal stem cells are preconditioned in a cell culture medium containing the PSG1.

7. A pharmaceutical composition comprising CC49 or Fc-tagged CC49 (CC49-Fc), or an expression vector for said CC49 or CC49-Fc, for the treatment of tendon injury, which is administered to an equine mammal by intratendinous or peritendinous injection.

8. 8. The pharmaceutical composition of claim 7, comprising the Fc-tagged CC49 (CC49-Fc), wherein the Fc tag is optionally an equine Fc tag.

9. The pharmaceutical composition described in claim 7, which contains an expression vector for CC49 or CC49-Fc and is administered to the equine mammal.

10. A pharmaceutical composition described in any one of claims 7 to 9, used in combination with administering mesenchymal stem cells to an injured tendon by intratendinous or peritendinous injection.

11. The pharmaceutical composition of claim 10, wherein the composition is co-administered with the mesenchymal stem cells to the injured tendon by intratendinous or peritendinous injection.

12. The pharmaceutical composition of claim 10, wherein the mesenchymal stem cells are preconditioned in a cell culture medium containing the CC49.

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