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    • 专利摘要:
    polypeptide, its use, in vitro method of inducing differentiation of mesenchymal stem cells into chondrocytes, and pharmaceutical composition for intra-articular application. the present invention relates to processes and compositions for treating and preventing arthritis and joint damage.
    公开号:BR112012000914B1
    申请号:R112012000914-1
    申请日:2010-07-13
    公开日:2021-04-06
    发明作者:Kristen Johnson；Lori Jennings；Peter G. Schultz
    申请人:Novartis Ag；The Scripps Research Institute；
    IPC主号:
    专利说明:

    [0001] This patent application claims the privilege under 35 U. S. C. § 1,119 (e) of the provisional U.S.S. No. 61 / 225,693, deposited on July 14, 2009, the content of which is incorporated as a reference in its entirety. BACKGROUND OF THE INVENTION
    [0002] Osteoarthritis (OA) represents the most common musculoskeletal disorder.
    [0003] Approximately 40 million Americans are currently affected and this number is expected to increase to 60 million within the next twenty years as a result of an aging population and an increase in life expectancy, making it the fourth leading cause of disability. OA is characterized by a slow degenerative rupture of the joint, which includes both the articular cartilage (which contains the cells and the matrix that produce lubrication and cushioning for the joint) and the subchondrial bone that serves as the basis for the articular cartilage. Current therapies for OA include pain relief with oral NSAIDs or selective cyclooxygenase 2 (COX-2) inhibitors, intra-articular (AI) injection with agents such as corticosteroids and hyaluronan and surgical approaches.
    [0004] Mesenchymal stem cells (MSCs) are present in the articular cartilage of adults and by isolation they can be programmed in vitro to undergo differentiation in chondrocytes and other me-senquimal cell lines. This is partly regulated by growth factors (TGFβs, BMPs), serum conditions and cell-cell contact. BRIEF SUMMARY OF THE INVENTION
    [0005] The present invention provides methods of improving or preventing arthritis or joint damage in a mammal. In some embodiments, the process comprises administering to a mammalian joint, a composition comprising an effective amount of a polypeptide comprising an amino acid sequence that has at least 95% identity with SEQ ID NO: 1 or 25 , thereby improving or preventing arthritis or joint damage in the mammal.
    [0006] In some modalities, the individual has arthritis or joint damage. In some modalities, the individual does not have, but is at risk for, arthritis or damage to the joints.
    [0007] In some embodiments, the polypeptide comprises SEQ ID NO: 1 or 25.
    [0008] In some embodiments, the amino acid sequence has at least 95% identity with SEQs ID NOs 2, 3, 4, 26, 27 or 28. In some embodiments, the amino acid sequence comprises SEQs ID NOs: 2, 3, 4, 26, 27 or 28.
    [0009] In some embodiments, the amino acid sequence is at least 80% identity with any of SEQs ID NOs: 5 - 24. In some embodiments, the amino acid sequence comprises any of SEQs ID NOs: 5 - 24.
    [0010] In some modalities, arthritis is selected from the group consisting of osteoarthritis, traumatic arthritis and autoimmune arthritis.
    [0011] In some embodiments, the mammal is a human being.
    [0012] In some embodiments, the composition also comprises hyaluronic acid.
    [0013] The present invention also provides methods of inducing differentiation of mesenchymal stem cells into chondrocytes that form the cartilage matrix. In some embodiments, the process comprises contacting the mesenchymal stem cells with a sufficient amount of a polypeptide that comprises an amino acid sequence that has at least 95% identity with SEQ ID NO: 1 or 25 to induce differentiation of the stem cells. trunk in chondrocytes. In some embodiments, the process is carried out in vitro. In some embodiments, the process is carried out in vivo, stem cells are present in a mammal. In some embodiments, the mammal is a human being.
    [0014] In some embodiments, the polypeptide comprises SEQ ID NO: 1 or 25.
    [0015] In some embodiments, the amino acid sequence has at least 95% identity with SEQ ID NOs: 2, 3, 4, 26, 27 or 28. In some embodiments, the amino acid sequence comprises SEQ ID NOs: 2, 3 , 4, 26, 27 or 28.
    [0016] In some embodiments, the amino acid sequence is at least 80% identical to any of SEQ ID NOs: 5 - 24. In some embodiments, the amino acid sequence comprises any of SEQ ID NOs: 5 - 24.
    [0017] The present invention also provides pharmaceutical compositions for intra-articular application and for systemic application. In some embodiments, the composition comprising a pharmaceutically effective amount of a polypeptide comprising an amino acid sequence that has at least 95% identity with SEQ ID NO: 1 or 25.
    [0018] In some embodiments, the composition also comprises hyaluronic acid.
    [0019] In some embodiments, the polypeptide comprises SEQ ID NO: 1 or 25.
    [0020] In some embodiments, the amino acid sequence has at least 95% identity with SEQ ID NOs 2, 3, 4, 26, 27 or 28. In some embodiments, the amino acid sequence comprises SEQ ID NOs: 2, 3, 4, 26, 27 or 28.
    [0021] In some embodiments, the amino acid sequence is at least 80% identical to any of SEQ ID NOs: 5 - 24. In some embodiments, the amino acid sequence comprises identical to any of SEQ ID NOs: 5 - 24.
    [0022] Additional aspects, advantages and modalities of the invention can be presented or evident by considering the detailed description below and the claims. In addition, it must be understood that both the previous summary of the invention and the detailed description below are examples and are intended to provide further explanation without limiting the scope of the invention as claimed. DEFINITIONS
    [0023] The terms "peptidomimetic" and "mimetic" refer to a synthetic chemical compound that has substantially the same structural and functional characteristics as a non-naturally occurring or naturally occurring polypeptide (for example, ANGPTL3). Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the peptide matrix. These types of non-peptide compounds are called "peptide mimetics" or "peptidomimetics" (Fauchere, J. Adv. Drug Res. 15:29 (1986); Ve-ber and Freidinger TINS p. 392 (1985) and Evans and others , J. Med. Chem. 30: 1229 (1987), which are hereby incorporated by reference). Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce an equivalent or improved therapeutic or prophylactic effect.
    [0024] In general, peptidomimetics are structurally similar to a polypeptide paradigm (that is, a polypeptide that has a biological or pharmacological activity), as found in a polypeptide of interest, but has one or more peptide bonds optionally substituted by a bond selected from the group consisting of, for example, -CH2 NH-, - CH2S-, - CH2- CH2-, -CH = CH- (cis and trans), -CO CH2-, -CH (OH) CH2- and - CH2SO-. The mimetic can be entirely composed of synthetic unnatural amino acid analogs or, it is a partially natural chimeric peptide amino acid molecule and partially unnatural amino acid analogs. Mimetics can also incorporate any number of conservative substitutions of natural amino acid as long as they do not substantially alter the structure and / or activity of the mimetic. For example, a mimetic composition is within the scope of the invention if it is capable of carrying out at least one activity of a polypeptide of interest.
    [0025] The terms "polypeptide", "peptide" and "protein" are used interchangeably in this case to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of an naturally occurring amino acid, as well as to non-naturally occurring amino acid polymers.
    [0026] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that work in a similar way to naturally occurring amino acids. The naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are subsequently modified, for example, hydroxyproline, γ-carboxyglutamate and O-phosphoserine. The amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, that is, an α carbon that is attached to a hydrogen, a carboxyl group, an amino group and an R group, for example, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (eg, norleukin) or modified peptide backbones, but maintain the same basic chemical structure as a naturally occurring amino acid. The naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine) and pyrrolysin and selenocysteine.
    [0027] "Conservatively modified variants" apply to both amino acid and nucleic acid sequences. With respect to nucleic acid sequences in particular, conservatively modified variants refer to those nucleic acids that encode identical or essentially identical amino acid sequences or when the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneration of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each position where an alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such variations of nucleic acid are "silent variations", which are a kind of conservative variations. Each nucleic acid sequence in this case that encodes a polypeptide also describes each possible silent variation of the nucleic acid. One skilled in the art will recognize that each codon is a nucleic acid (except AUG, which is commonly the only codon for methionine and TGG, which is commonly the only codon for tryptophan) can be modified to provide a functionally identical molecule. Consequently, each silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence.
    [0028] As for amino acid sequences, one skilled in the art will recognize that individual substitutions, deletions or additions, to a nucleic acid, peptide, polypeptide or protein sequence that alter, add or delete a single amino acid or a small percentage of amino acids in the The coded sequence is a "conservatively modified variant" in which the change results in the replacement of an amino acid with a chemically similar amino acid. Conservation substitution tables that provide functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologues and alleles of the invention.
    [0029] 1) Alanina (A), Glicina (G); 2) Ácido aspártico (D), Ácido glutâmico (E); 3) Asparagina (N), Glutamina (Q); 4) Arginina (R), Usina (K); 5) Isoleucina (I), Leucina (L), Metionina (M), Valina (V); 6) Fenilalanina (F), Tirosina (Y), Triptofano (W); 7) Serina (S), Treonina (T) e 8) Cisteína (C), Metionina (M) (ver, por exemplo, Creighton, Proteins (1984)). The following eight groups each contain amino acids that are conservative substitutions for each other: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Plant (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T) and 8) Cysteine (C), Methionine (M) (see, for example, Creighton, Proteins (1984)).
    [0030] The "percentage of sequence identity" is determined by comparing two sequences optimally aligned in a comparison window, where the portion of the polynucleotide sequence in the comparison window can comprise additions or deletions (i.e., spaces) compared to the sequence of reference (for example, a polypeptide of the invention), which do not comprise additions or deletions to align the two sequences. The percentage is calculated by determining the number of positions in which the identical nucleic acid base or amino acid residue occurs in both sequences to provide the number of combined positions, dividing the number of combined positions by the total number of positions in the window. comparison and multiplying the result by 100 to provide the sequence identity percentage.
    [0031] The terms "identical" or percent of "identity", in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same sequences. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or the nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90% or 95% identity in a specified region or, when not specified, in the entire sequence), when compared to aligned for maximum match in the comparison window or in the region designated as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. The invention provides polypeptides that are substantially identical to the polypeptides, respectively, exemplified herein (for example, any of SEQ ID NOs: 1 - 28), as well as uses of which include, but are not limited to, use for treatment or treatment. prevention of arthritis or joint damage. Optionally, the identity exists in a region that is at least approximately 50 nucleotides in length or more preferably in a region that is 100 to 500 or 1000 or more nucleotides in length or the entire length of the reference sequence.
    [0032] For sequence comparison, typically a sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, sub-sequence coordinates are designed, if necessary, and sequence algorithm program parameters are designed. The pre-set program parameters can be used or alternative parameters can be designed. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences in relation to the reference sequence, based on the program parameters.
    [0033] The "comparison window", as used in this case, includes reference to a segment of any of the number of contiguous positions selected from the group consisting of from 20 to 600, usually from approximately 50 to approximately 200, more usually from approximately 100 to approximately 150 where a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Sequence alignment methods for comparison are well known in the art. The optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2: 482c, by the Homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, for the search for the similarity method of Pearson and Lip-man (1988) Proc. Natl. Acad. Know. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wl) or by manual alignment and visual inspection (see, for- example, Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).
    [0034] Two examples of algorithms that are suitable for determining percent sequence identity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul and others (1977) Nuc. Acids Res.
    [0035] 25: 3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215: 403-410, respectively. The computer program for performing BLAST analyzes is publicly available from the National Center for Biotechnology Information. This algorithm first involves the identification of some high score sequence pairs (HSPs) by identifying short words of length W in the search sequence, which matches or satisfies some positive T limit value when aligned with a word of the same length in database string. T is called the neighborhood word score limit (Altschul and others, above). These early neighborhood word hits act as seeds to initiate searches to find longer HSPs that contain them. The word matches are extended in both directions along each sequence to the point where the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always> 0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extent of the word coincidences in each direction is interrupted when: the cumulative alignment score decreases by the amount X of its maximum value reached; the cumulative score reaches zero or below, due to the accumulation of one or more negative score residue alignments or when the end of one of the sequences is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as a pre-established data a word length (W) of 11, an expectation (E) or 10, M = 5, N = - 4 and a comparison of both filaments. For the amino acid sequences, the BLASTP program uses as a pre-established data a word length of 3 and an expectation of (E) of 10 and the score matrix of BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M = 5, N = - 4 and a comparison of both filaments.
    [0036] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787). A measure of similarity provided by the BLAST algorithm is the least probability of sum (P (N)), which provides an indication of the probability by which a combination of two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the least probability of sum in a comparison of the test nucleic acid to the reference nucleic acid is less than approximately 0.2, more preferably less than approximately 0.01 and more preferably still less than approximately 0.001.
    [0037] The term "hyaluronic acid" is used in this case to include derivatives of hyaluronic acid that include esters of hyaluronic acid, salts of hyaluronic acid and also includes the term hyaluronan. The designation also includes both low and high molecular weight forms of hyalurananes and cross-linked hyaluronans or hyalanes. Examples of such hyaluronans are Synvisc ™ (Genzyme Corp. Cambridge, Mass.), ORTHO VlSC ™ (Anika Therapeutics, Woburn, Mass.) And HYALGAN ™ (Sanofi-Synthelabo Inc., Malvern, Pa.). BRIEF DESCRIPTION OF THE DRAWINGS
    [0038] Figure 1. Quantitative assessment of type II collagen production induced by MSC during in vitro chondrogenesis. hMSCs (10,000 / 384 wells) were applied for 24 hours in hMSC growth media (Millipore). The cells were treated with the above proteins for an additional 72 hours. The media was replaced with serum-free DMEM and cultured for an additional 14 days in the absence of any additional stimuli. After completion, the cells were fixed with formalin, washed, stained with an antibody to type II collagen and counterstained with DAPI. The amount of collagen type II dye application was quantitatively evaluated by high-level image formation (Opera, Perkin Elmer). Data are collected from 3 experiments in duplicate for each dose (n = 6). Figure 2. Characterization of chondrogenesis induced by ANGTPL3. (A) hMSCs were cultured in pellet culture (1 x 106 cells / pellet) for 21 days in serum-free DMEM, IX ITS and ANGTPL3 (when indicated). The media was replaced every 3 days. The expression of agrecana mRNA was quantitatively evaluated using specific human Taqman probes according to the manufacturer's instructions (data are gathered from 3 experiments in duplicate (n = 6). (B) Bovine cartilage was isolated, perforated in symmetrical circles and slices were treated for 48 hours with 20 ng / ml TNFa and 10 ng / ml OSM (inflammatory mediators) to induce cartilage matrix degradation in the presence or absence of ANGPTL3 to identify the percentage inhibition of glycosaminoglycan release, an indicator of matrix damage (data gathered from 4 donors, n = 12). (C) Normal human primary con-drocytes and synoviocytes were applied and cultured for 24 hours at 37 ° C. an increase in cell growth Specific chondrocyte in response to ANGTPL3 during the 24-hour period (data gathered from 2 experiments, 2 replicates / dose (n = 4)) - Figure 3. mANGTPTL3. (A) Primary protein structure and confirmed glycosylation sites. (B) The atomic structure (1.8 angstrom) of mANGTPL3 is the FLD terminal (225-455). Figure 4: Alignment of ANGPTL3 sequences. (A) Sequence alignment of native human, mouse, bovine and canine ANGPTL3 proteins; (B) Sequence alignment of native human, mouse, bovine, canine and equine ANGPTL3 proteins. Figure 5. In vivo efficiency of mANGPTL3 in a surgical model of osteoarthritis.
    [0039] The surgical transection of the anterior cruciform ligament (ACL), the medial tibial meniscus ligament (MMTL) and the medial collateral ligament (MCL) of the right knee from C57BL / 6 mice (n = 12 / group) was performed to induce instability of the knee joint and thus lead to an OA phenotype. One week after surgery, mice were dosed intra-articularly as indicated once / per for 3 -4 weeks. (THE). Peripheral blood was collected on the 28th day after surgery. Circulating type II collagen fragments (CTX-II) were quantitatively evaluated by an ELISA (Nordic biosciences). Dose of mANGPTL3 = 200 ng / knee, 3 injections weekly (B) Quantitative assessments of the tibial plaque were performed on a scale of 0 - 4, 0 being normal OA and 5 being severe OA (rupture of the entire thickness of the cartilage). Two sections of each mouse were blindly classified by 2 observers. Dose of mANGPTL3 = 200 ng / knee, 3 injections weekly (C) OA pain was measured by disability testing or by determining the percentage of time the mouse was standing on the surgical leg vs. the other leg via the monitoring. The readings represent the pain response on the 36th day after surgery (3 doses of ANGP-TL3 weekly at the indicated concentration). DETAILED DESCRIPTION I. Introduction
    [0040] The present invention is based, in part, on the discovery that Angiopoietin-like (ANGPTL3) stimulates chondrocyte differentiation in mesenchymal stem cells. Consequently, the present invention provides methods of inducing MESENQUIMAL stem cell differentiation into chondrocytes. In addition, the present invention is responsible for administering ANGPTL3 proteins to prevent or ameliorate arthritis or joint damage by administering an ANGPTL3 protein to a joint, to the vertebrae, to the vertebral disc or systemically. II. Similar 3 to Angiopoietin
    [0041] Angiopoietin-like 3 is a member of the family of secreted angiopoietin-like factors. It is predominantly expressed in the liver and has the characteristic structure of angiopoietins, consisting of a signal peptide, N-terminal coiled-spring domain (CCD) and the domain similar to C-terminal fibrinogen (FBN). It has been shown that the FBN-like domain in angiopoietin-like 3 binds to αν / β3 integrins and this binding induces endothelial cell adhesion and migration.
    [0042] A variety of ANGPTL3 proteins can be used according to the present invention. As explained in this case, native ANGPTL3 is generally cleaved in vivo to amino-terminal and carboxyl terminal fragments. The present invention considers the use of several ANGP-TL3 proteins that have chondrogenic activity. In some embodiments, the invention is responsible for the use of full-length native ANGPTL3 protein amino acid sequences (or variants thereof). In some embodiments, the invention is responsible for ANGPTL3 proteins that comprise a part (not the entire full-length native sequence) of the ANGPTL3 sequence or a variant thereof, which maintains chondrogenic activity, that is, not the amino-terminal end of the protein. native. In some embodiments, the ANGPTL3 proteins of the invention do not have the CCD domain and / or do not have significant CCD activity. Thus, in some embodiments, the ANGPTL3 proteins of the invention comprise at least one fragment (for example, at least 50, 100, 150, 200, 250 contiguous amino acids) of the mouse native ANGPTL3 sequence (for example, SEQ ID NO: 12), human (for example, SEQ ID NO: 8), bovine (for example, SEQ ID NO: 16), canine (for example, SEQ ID NO: 20) or equine (for example, SEQ ID NO: 24) or substantially identical sequences, but do not comprise at least 200 contiguous amino-terminal amino acids of a native ANGPTL3 protein.
    [0043] In some embodiments, the ANGPTL3 proteins of the invention comprise a fibrinogen-like domain. In some embodiments, the ANGPTL3 proteins of the invention comprise contiguous amino acids corresponding to amino acids 207 - 455, 207 - 400, 207 - 350, 225 - 455, 225 - 400, 225 - 350, 241 - 455, 241-400, 241-350 from the native mouse (for example, SEQ ID NO: 12), human (for example, SEQ ID NO: 8), bovine (for example, SEQ ID NO: 16), canine (for example, SEQ ID NO: 20) or equine (for example, SEQ ID NO: 24) ANGPTL3 protein sequence or are substantially identical to such sequences, however they do not include the amino acid sequence of the native flanking-angora protein. In some embodiments, the ANGPTL3 proteins of the invention (which include, but are not limited to any of SEQ ID NOs: 1-28), but where at least a part of the C-terminal is missing, for example, 10, 20 are missing , 30, 40, 50 amino acids of the C-terminus.
    [0044] Although the ANGPTL3 proteins of the invention as described above may not include the native protein ANGPTL3 sequences that flank the regions described above, the ANGPTL3 proteins of the invention may include non-native flanking ANGPTL3 protein sequences. For example, the active chondrogenic part of an ANGPTL3 protein can be fused to one or more heterologous amino acids to form a fusion protein. Fusion partner sequences may include, but are not limited to, amino acid labeling, non-L amino acids (e.g., D-) or other amino acid mimics to extend in vivo half-life and / or protease resistance, which address strings or other strings.
    [0045] The ANGPTL3 proteins of the invention encompass variants and truncations of native ANGPTL3 proteins as well as variants and truncations of active fragments described herein. The active variants can be identified in any number of ways known to those skilled in the art. In some embodiments, the amino acid alignments of active proteins can be established to identify those positions that are not variants or that include conserved amino acid variations. SEQ ID NOs: 1, 2, 3 or 4 represent consensus sequences that comprise the amino acids invariant between certain areas (position 241 - 455, 225 - 455 and 207 - 455 and native of full length, respectively) of the human ANGPTL3 proteins, of mouse, bovine and canine. SEQ ID NOs: 25, 26, 27 or 28 represent consensus sequences that comprise the amino acids invariant between certain areas (position 241 -455, 225 - 455 and 207 - 455 and native in full length, respectively) of the native human ANGPTL3 proteins , mouse, bovine, canine and equine. Thus, in some embodiments, the chondrogenic ANGPTL3 proteins of the invention comprise SEQ ID NOs: 1, 2, 3, 4, 25, 26, 27 or 28. In some embodiments, the chondrogenic ANGPTL3 proteins of the invention comprise an amino acid sequence substantially identical to any of SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 ( as measured by the length of SEQ ID NOs).
    [0046] The ANGPTL3 proteins of the invention have chondrogenic activity. As defined in this case, chondrogenesis or chondrogenic activity refers to the development of chondrocytes from MSCs. Indicators of chondrogenic activity include, but are not limited to, cartilage matrix production. The production of cartilage matrix can be measured by several markers, for example, such as the production of Sox9, type II collagen or glycosaminoglycan (GAG). In some modalities, the production of GAG is measured as a marker for the production of cartilage matrix. In some modalities, a 3-fold increase in the production of GAG with the expression of the specific protein for the cartilage indicates the positive production of cartilage matrix.
    [0047] In some embodiments, the ANGPTL3 polypeptides of the invention will comprise at least one non-naturally encoded amino acid. The processes for obtaining and introducing a non-naturally occurring amino acid into a protein are known. See, for example, US Patent Nos. 7,083,970 and 7,524,647. The general principles for the production of orthogonal translation systems that are suitable for obtaining proteins that comprise one or more desired unnatural amino acids are known in the art, as are the general methods for the production of orthogonal translation systems. For example, see the International Publication Numbers WO 2002/086075, entitled "METHODS AND COMPOSITION FOR THE PRODUCTION OF ORTHOGONAL tRNA-AMINOACYL-tRNA SYNTHETASE PAIRS;" WO 2002/085923, entitled "IN VIVO INCORPORATION OF UNNATURAL AMINOACIDS;" WO 2004/094593, entitled "EXPANDING THE EUKARYOTIC GENETIC CODE;" WO 2005/019415, filed on July 7, 2004; WO 2005/007870, filed on July 7, 2004; WO 2005/007624, deposited on July 7, 2004; WO 2006/110182, filed on October 7, 2005, entitled "ORTHOGONAL TRANSLATION COMPONENTS FOR THE VIVO INCORPORATION OF UNNATURAL AMINOACIDS" and WO 2007/103490, filed on March 7, 2007, entitled "SYSTEMS FOR THE EXPRESSION OF ORTHOGONAL TRANSLATION COMPONENTS IN EUBACTERIAL HOST CELLS. " Each of these patent applications is hereby incorporated by reference in its entirety. For discussion of orthogonal translation systems that incorporate unnatural amino acids and their production and use, see also, Wang and Schultz, (2005) "Expanding the Genetic Code." Angewandte Chemie Int Ed 44: 34-66; Xie and Schultz, (2005) "An Expanding Genetic Code." Methods 36: 227-238; Xie and Schultz, (2005) "Adding Amino Acids to the Genetic Repertoire." Curr Opinion in Chemical Biology 9: 548-554 and Wang et al., (2006) "Expanding the Genetic Code." Annu Rev Biophys Biomol Struct 35: 225 - 249; Deiters et al., (2005) "In vivo incorporation of an alkyne into proteins in Escherichia coli." Bioorganic & Medicinal Chemistry Letters 15: 1521-1524; Chin et al., (2002) "Addition of p-Azido-L-phenylalanine to the Genetic Code of Escherichia coli." J Am Chem Soc 124: 9026-9027 and International Publication No. WO 2006/034332, filed on September 20, 2005, the content of which is incorporated as a reference in its entirety. Additional details are found in the Pats. U.S. No. 7,045,337; No. 7,083,970; No. 7,238,510; No. 7,129,333; No. 7,262,040; No. 7,183,082; No. 7,199,222 and No. 7,217,809.
    [0048] A "non-naturally encoded amino acid" refers to an amino acid that is not one of the common amino acids or pyrolysin or sele-nocistein. Other terms that can be used synonymously with the term "non-naturally encoded amino acid" are "unnatural amino acid", "artificial amino acid", "non-naturally occurring amino acid", and variously hyphenated and non-hyphenated versions of it. The term "non-naturally encoded amino acid" also includes, but is not limited to, amino acids that occur by modification (for example, post-translational modifications) of a naturally encoded amino acid (which includes, but is not limited to, 20 amino acids or pyrrolysin and selenocysteine), but they are not themselves naturally incorporated into a growing polypeptide chain by the translation complex.
    [0049] Examples of such naturally occurring amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine and O-phosphotyrosine.
    [0050] A non-naturally encoded amino acid is typically any structure that has any substituting side chain other than one used in the twenty natural amino acids. Since the non-naturally encoded amino acids of the invention typically differ from natural amino acids only in the structure of the side chain, non-naturally encoded amino acids form amide bonds with other amino acids, including, but not limited to, natural or not naturally encoded, in the same way. in which they are formed into naturally occurring polypeptides. However, non-naturally encoded amino acids have side chain groups that distinguish them from natural amino acids. For example, R optionally comprises an alkyl-, aryl-, acyl-, ce-to-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynyl, ether, thiol, seleno-, sulfonyl group -, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino or similar or any combination thereof. Other non-naturally occurring amino acids of interest that may be suitable for use in the present invention include, but are not limited to, amino acids comprising a photoactivable cross-linker, centrifugation-labeled amino acids, fluorescent amino acids, metal-linked amino acids, metal-containing amino acids , radioactive amino acids, amino acids with new functional groups, amino acids that covalently or non-covalently interact with other molecules, photocaptured and / or photo-somerizable amino acids, amino acids that comprise biotin or a bio-thine analogue, glycosylated amino acids such as a serine substituted with sugar, other amino acids modified with carbohydrates, amino acids containing keto, amino acids comprising polyethylene glycol or polyether, amino acids substituted with heavy atom, chemically cleavable and / or photocleavable amino acids, amino acids with elongated side chains compared to natural amino acids, included but not limited to, long chain polyethers or hydrocarbons, including, but not limited to, more than 5 or more than approximately 10 carbons, carbon-containing sugar-containing amino acids, active redox amino acids, amino acids containing thioacid amino acids and amino acids that comprise one or more toxic groups.
    [0051] Examples of non-naturally encoded amino acids which may be suitable for use in the present invention and which are useful for reactions with water-soluble polymers include, but are not limited to those with carbonyl, aminooxy, hydrazine, hydrazide, semicarbazide, azide and acino groups reactive. In some embodiments, non-naturally encoded amino acids comprise a saccharide group. Examples of such amino acids include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosamini-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L -asparagine and O-mannosaminyl-L-serine. Examples of such amino acids also include examples in which the naturally occurring -N or -O bond between the amino acid and saccharide is replaced by a covalent bond that is not commonly found in nature - which includes, but is not limited to, an alkene , an oxime, a thioether, an amide and the like. Examples of such amino acids also include saccharides that are not commonly found in naturally occurring proteins such as 2-deoxy-glucose, 2-deoxy-galactose and the like.
    [0052] Another type of modification that can optionally be introduced in the ANGPTL3 proteins of the invention (for example, within the polypeptide chain or at the -N or -C terminal), for example, to extend the half-life in vivo, is PEGylation or incorporation of long chain polyethylene glycol (PEG) polymers. The introduction of PEG or long-chain PEG polymers increases the effective molecular weight of the present polypeptides, for example, to avoid rapid filtration into the urine. In some embodiments, a Plant residue in the sequence of ANGPTL3 is conjugated to PEG directly or through a ligand. Such a linker can, for example, be a Glu residue or an acyl residue that contains a thiol functional group for attachment to the appropriately modified PEG chain. An alternative method for introducing a PEG chain is to first introduce a Cys residue at the -C terminus or to solvent-exposed residues such as substitutes for the Arg or Lys residues. This Cys residue is then a site specifically linked to a PEG chain that contains, for example, a maleimide function. Methods for incorporating PEG or long-chain PEG polymers are well known in the art (described, for example, in Veronese, FM et al., Drug Disc. Today 10: 1451-8 (2005); Greenwald, RB et al., Adv Drug Deliv. Rev. 55: 217-50 (2003); Roberts, MJ et al., Adv. Drug Deliv. Rev., 54: 459 - 76 (2002)), the content of which is incorporated herein by reference. Other polymer conjugation methods known in the art can also be used in the present invention. In some embodiments, poly (2-methacryloyloxyethyl phosphorylcholine) (PMPC) is introduced as a polymer conjugate with the ANGPTL3 proteins of the invention (see, for example, WO 2008/098930; Lewis et al., Bioconjug Chem., 19: 2144 - 55 (2008)). In some embodiments, a polymer conjugate that contains phosphorylcholine with the ANGP-TL3 proteins can be used in the present invention. One skilled in the art would readily recognize that other biocompatible polymer conjugates can be used.
    [0053] An alternative approach most recently reported for incorporating PEG or PEG polymers by incorporating unnatural amino acids (as described above) can be performed with the present polypeptides. This approach uses a pair of tRNA / tRNA synthase and is encoded in the expression plasmid by the amber suppressor codon (Dei-ters, A et al. (2004). Bio-org. Med. Chem. Lett. 14, 5743-5). For example, p-azidophenylalanine can be incorporated into the present polypeptides and then reacted with a PEG polymer that has an acetylene group in the presence of a reducing agent and copper ions to facilitate an organic reaction known as "Huisgen cicloaddition [3 +2]. "
    [0054] In certain embodiments, the present invention considers specific mutations of the ANGPTL3 proteins in order to alter the glycosylation of the polypeptide. Such mutations can be selected to introduce or eliminate one or more glycosylation sites, including, but not limited to, O-linked or N-linked glycosylation sites. In certain embodiments, the ANGPTL3 proteins of the present invention have glycosylation sites and unchanged patterns regarding naturally occurring ANGPTL3 proteins. In certain embodiments, a variant of ANGPTL3 proteins includes a variant of glycosylation in which the number and / or type of glycosylation sites has been changed from naturally occurring ANGPTL3 proteins. In certain embodiments, a variant of a polypeptide comprises a greater or lesser number of N-linked glycosylation sites relative to a native polypeptide. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, where the amino acid residue designated as X can be any amino acid residue except proline. Substituting amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions that delete this sequence will remove an existing N-linked carbohydrate chain. In certain embodiments, a rearrangement of N-linked carbohydrate chains is provided, in which one or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created .
    [0055] Examples of variants of ANGPTL3 proteins include cysteine variants in which one or more cysteine residues are deleted from or replaced by another amino acid (e.g., serine) in relation to the amino acid sequence of naturally occurring ANGPTL3 proteins. In certain embodiments, cysteine variants can be useful when ANGPTL3 proteins have to be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. In certain embodiments, cysteine variants have less cysteine residues than the native polypeptide. In certain embodiments, cysteine variants have an equivalent number of cysteine residues to minimize the interactions that result from unpaired cysteines.
    [0056] In some embodiments, functional variants or modified forms of ANGPTL3 proteins include fusion proteins of an ANGPTL3 protein of the invention and one or more fusion domains. Well-known examples of fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain (Fc) constant region, binding to maltose (MBP) or human serum albumin. A merger domain can be selected to provide a desired property. For example, some fusion domains are particularly useful for the isolation of fusion proteins by affinity chromatography. For the purpose of affinity purification, matrices relevant to affinity chromatography are used, such as resins conjugated to glutathione, amylase and nickel or cobalt. Many such matrices are available in kit form, such as Pharmacia's GST purification system and the QLAexpress ™ system (Qiagen) useful with fusion partners (ΗΙS6). As another example, a fusion domain can be selected in order to facilitate the detection of ANGPTL3 proteins. Examples of such detection domains include the various fluorescent proteins (e.g., GFP) as well as "epitope tags", which are generally short peptide sequences for which a specific antibody is available. Well-known epitope tags for which monoclonal antibodies are readily available include FLAG, influenza virus (HA) hemagglutinin and c-myc tags. In some cases, the fusion domains have a protease dividing site, such as for Factor Xa or Thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby release the recombinant proteins from them. The released proteins can then be isolated from the fusion domain through subsequent chromatographic separation. In certain embodiments, an ANGPTL3 protein is fused to a domain that stabilizes the ANGPTL3 protein in vivo (a "stabilizing" domain). By "stabilization" is meant anything that increases the half-life in the serum, regardless of whether this is due to less destruction, less elimination by the kidneys or other pharmacokinetic effect. Fusions with the Fc portion of an immunoglobulin are known to impart desirable pharmacokinetic properties to a wide range of proteins. Similarly, fusions with human serum albumin may confer desirable properties. Other types of fusion domains that can be selected include multimerization domains (for example, dimerization, tetramerization) and functional domains (which provide additional biological function, as desired). III. Indications of Protein Diseases Similar to Angiopoietin
    [0057] It is envisaged that the polypeptides, compositions and methods of the present invention can be used to treat or prevent any type of arthritis or joint damage. It is further considered that the polypeptides, compositions and methods of the present invention can be used to treat or prevent various disorders in cartilage. In some embodiments, the proteins of the invention are administered to prevent arthritis or joint damage, for example, when there is a genetic or family history of arthritis or joint damage or before or during joint surgery. Examples of health conditions or disorders that will be treated or prevented with the polypeptides, compositions and methods of the invention, include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, osteoarthritis, degenerative disc disease, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sjor-gen syndrome, systemic vasculitis, sarcoidosis, autoimmune haemolytic anemia (pancito-penia immune, paroxysmal nocturnal hemoglobinuria, trombopathic thrombocytopenia, autoimmune thrombocythemia, thrombocytopathia , immune-mediated thrombocytopenia), thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediated kidney disease (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the central nervous systems peripheral such as multiple sclerosis, p idiopathic demyelinating olineuropathy or Guillain-Barre syndrome and chronic inflammatory demyelinating polyneuropathy, hepatobiliary diseases such as infectious hepatitis (hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis and sclerosing cholanginitis, inflammatory bowel disease (ulcerative colitis: Crohn's disease), gluten-sensitive enteropathy and Whipple's disease, autoimmune or immune-mediated skin diseases including bullous skin diseases, erythema multiforme and contact dermatitis, psoriasis, diseases allergic reactions such as asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity and urticaria, immunological diseases of the lungs such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, transplant-associated diseases including graft rejection and graft-versus-host disease. In some embodiments of the invention, the polypeptides, compositions and methods of the present invention can be used to treat osteoarthritis.
    [0058] In some embodiments, the polypeptides, compositions and methods of the present invention provide a method for stimulating chondrocyte proliferation and cartilage production in cartilage tissues that have been damaged due to traumatic damage or chondropathy. Of particular importance for treatment are tissues that exhibit articulated surfaces such as the spine, shoulder, elbow, wrist, finger joints, hip, knee, ankle and foot joints.
    [0059] Examples of diseases that may benefit from treatment include osteoarthritis, rheumatoid arthritis, other autoimmune diseases, or osteochondritis dissecans. In addition, defective conformation is often seen in forms of dwarfism in humans suggesting that polypeptides, compositions and methods would be useful in these patients.
    [0060] It is envisaged that the polypeptides, compositions and methods of the present invention can be used to treat a mammal. As used in this case, a "mammal" means any mammal classified as a mammal, including humans, domestic and farm animals and zoo, sports or pet animals, such as cattle (for example, cows), horses, dogs, sheep, pigs, rabbits, goats, cats etc. In some embodiments of the invention, the mammal is a human being.
    [0061] In some embodiments, the polypeptides of the invention may be heterologous to the mammal to be treated. For example, a bovine ANGPTL3 protein or fragments thereof, a protein or a peptide derived from a bovine ANGPTL3 protein (for example, a modified bovine ANGPTL3 protein, a conservative variant of bovine ANGPTL3 protein, an imitator of peptide derived from a bovine ANGPTL3 protein) are used in the treatment of a human patient. In some embodiments, a heterologous ANGP-TL3 protein can be used to expand chondrocyte populations in culture for transplantation. In some embodiments, the expanded cultures will then be mixed with polypeptides and compositions homologous to the mammal to be treated and placed in the joint space or directly in the cartilage defect. Alternatively, the polypeptides of the invention are derived from the same species, that is, a human ANGPTL3 protein or fragments thereof, a protein or a peptide derived from a human ANGPTL3 protein (for example, a modified human ANGPTL3 protein, a conservative protein variant Human ANGPTL3, a peptidomimetic derived from a human ANGPTL3 protein) is used in the treatment of a human patient. By using a protein derived from the same species of mammal when being treated, inadvertent immune responses can be avoided.
    [0062] The polypeptides and compositions of the present invention can be applied by direct injection into the synovial fluid of the joint, systemic administration (oral or intravenous) or directly to the cartilage defect, either alone or complexed with a suitable vehicle during prolonged protein release. The polypeptides, compositions and methods of the present invention can also be used to expand chondrocyte populations in the culture for autogenous or allogeneic chondrocyte transplantation.
    [0063] The transplant can optionally be administered with concurrent treatment consisting of administration of the polypeptides and compositions of the present invention. In these procedures, for example, chondrocytes can be harvested arthroscopically from an undamaged area that contains minimal damage to the damaged joint and can be cultured in the presence of the polypeptides and compositions of the present invention to increase the number of cells before transplantation. The expanded cultures will then be mixed with the polypeptides and the compositions of the present invention and placed in the joint space or directly in the defect. The polypeptides and compositions of the present invention can be used in combination with periosteal or perichondrial grafts that contain cells that can form cartilage and / or help to keep transplanted chondrocytes or their precursor cells in place. The polypeptides and compositions of the present invention can be used for repairing cartilage damage in association with joint washing, bone marrow stimulation, abrasion arthroplasty, subchondral perforation or subchondral bone microfracture.
    [0064] In addition, after the growth of cartilage due to administration of the polypeptides and compositions of the present invention, additional surgical treatment may be necessary to adequately contour the newly formed cartilage surface. IV. Pharmaceutical compositions
    [0065] The dose of a compound of the present invention to treat the aforementioned diseases or disorders varies depending on the manner of administration, the age and weight of the subject's body and the condition of the subject to be treated and will ultimately be decided by the physician or a-tendente veterinarian. Such an amount of the compound as determined by the attending physician or veterinarian is referred to in this case as an "effective amount."
    [0066] Formulations suitable for administration include excipients, including, but not limited to, aqueous and non-aqueous solutions, sterile isotonic solutions, which may contain antioxidants, buffers, bacteriostats and solutes that make the formulation isotonic and aqueous suspensions and non-aqueous sterols which may include suspending agents, solubilizers, thickening agents, stabilizers and preservatives.
    [0067] The dose administered to a patient in the context of the present invention would need to be sufficient to effect a beneficial response in the subject over time. The dose will be determined by the effectiveness of the particular protein used and the subject's health status, as well as the body weight or the surface area of the area to be treated. The particle size of the dose will also be determined by the existence, nature and extent of any adverse side effects that accompany the administration of a particular protein or vector to a particular subject. Administration can be carried out in single or divided doses. V. Methods of Administration
    [0068] Any method can be used to apply the proteins to an affected joint. In the practice of this invention, the compositions can be administered, for example, intra-articularly (i.e., in a joint), orally, intravenously. The formulations of the compounds can be presented in sealed single-dose or multi-dose containers, such as ampoules and small vials.
    [0069] Solutions and suspensions can be prepared starting from stable powders, granules and tablets of the kind described above. The proteins of the present invention can also be used effectively in combination with one or more additional active agents (for example, hyaluronic acid or a salt thereof) depending on the therapy or the desired effect. EXAMPLES
    [0070] The following examples are offered to illustrate, but not to limit the claimed invention. Example 1: Sieve for high circulation of material for chondrogenesis inducers.
    [0071] To identify and define a new non-invasive strategy for OA joint repair, a sieve was developed for high circulation of non-induced protein material capable of selectively directing the differentiation of human mesenchymal stem cells (MSCs) to chondrocytes . The dosage system models the MSCs residing in cartilage and identifies mediators that stimulate the potential natural repair and improve the regeneration of the integrated cartilage. It is chosen to test a collection of protein secreted without comparison by cell-based sieves, with large circulation of material from human and mouse MSCs. This approach provides a strategy that allows the rapid identification of uncharacterized ligands and native protein that affect chondrogenesis.
    [0072] To study the secreted proteins, two approaches were considered: the production of conditioned media (CM) by a cell line (HEK293T) from a producing mammal and the generation of purified proteins from free-style HEK-F cells. Together, these complementary approaches were responsible for new target identification in a variety of applications.
    [0073] Protein selection in the MSC assay identified the main candidate, Angiopoeitin-like 3, abbreviated ANGPTL3. ANGPTL3 was identified in two parallel but initial orthogonal screens of both CM and purified proteins. In the proof of concept CM selection, C3H10tl / 2 cells were incubated for 7 days after the transfer of CM. Chondrogenesis has been found to occur in wells containing ANGP-TL3 -75 dosed by Alcian blue stain for the detection of cartilage matrix production. ANGPTL3 -75 was independently identified by selecting 531 purified proteins for chondrocytic differentiation from human MSCs.
    [0074] After the initial selection trials, ANGPTL3 was subsequently characterized in six secondary trials. These assays include: (1) monolayer culture of mouse C3H10tl / 2 mesenchymal cells: type II collagen induction and Sox9 protein expression (chondrogenesis markers), but without osteocalcin induction (osteogenesis markers); (2) inhibition of TNFa / oncostatin M (OSM) - release of induced nitric oxide (NO) in bovine chondrocytes; (3) inhibition of TNFα / OSM-induced glycosaminoglycan (GAG) release in bovine cartilage organ culture; (4) induction of cartilage matrix gene expression (aggrecan) in a human MSC pellet culture system; (5) lack of toxicity in primary human chondrocytes, human synovial fibroblasts and human MSCs; (6) stimulation of proliferation of human chondrocytes.
    [0075] The mouse ANGPTL3 protein works in human and bovine cell types and, therefore, was chosen for further characterization because it is a potently induced chondrogenic differentiation (0.5-5nM) although its human homologue maintains a similar potency for chondrogenesis compared to other inducers of chondrogenesis. chondrogenesis previously known in the type II collagen assay based on quantitative image formation (Figure 1). Both mouse and human MSCs differed in monolayer and pellet cultures (respectively) for 18 to 21 days when treated with 0.5-5nM ANGPTL3 and express three specific cartilage proteins: type II collagen, Sox9 and aggrecan. This was assessed both by immunohistochemical staining and by quantitative evaluation of mRNA by Taqman (Figure 2A). In addition, the data suggest after culturing mouse MSCs For 18 days, ANGPTL3 inhibits the spontaneous tendency to a fibrotic repair response through reduced expression of alkaline phosphatase. To assess the potential ability to prevent tissue damage, bovine cartilage organ cultures were stimulated with TNFa and oncostatin M (OSM). The release of stimulated glycosaminoglcano, an indicator of matrix degeneration, was significantly inhibited by treatment with ANGPTL3 (Figure 2B). In addition, treatment of primary human chondrocytes but not human sino-viocytes led to a 2-fold increase in cell growth within 24 hours (Figure 2C) suggesting a specificity of their action on cartilage. The protein also had no obvious toxic effects in vitro (<100μΜ) on viability in primary human chondrocytes, synovial fibroblasts and MSCs (data are not shown).
    [0076] In a diet comparison between two candidates currently in clinical trials, treatment with 100 ng / ml of FGF 18 or with 100 ng / ml d BMP7 could induce chondrogenic nodules, but less overall matrix production compared to ANGPTL3. FGF 18 lacked the ability to increase the formation of a stain with Alcian Blue, Sox9 or type II collagen, indicating a lack of specificity of a true cartilage matrix. 100 ng / ml of BMP7 increased the staining with Alcian blue, Sox9 and type II collagen, but the significance was not so great at similar concentrations. Example 2: Expression of recombinant full-length ANGPTL3 and analysis of mutant protein and function
    [0077] Mouse ANGPTL3 is provided as a 51 kDa protein. It belongs to a family of 7 identified Angiopoietin-like (ANGPTL) proteins that have structural similarity to angiopoiteins, however there is a lack of the ability to bind to the Tie2 receptor and has distinct functions. These contain a N-terminal coil-wound (CCD) domain and a C-terminal fibrinogen-like (FLD) domain. ANGPTL proteins are rigidly regulated by their microenvironment and interactions with the extracellular matrix (ECM), yet the precise sites of interaction nor the partners have been elucidated in detail.
    [0078] ANGPTL3 is secreted by the liver and circulates systemically. It is controlled by liver X-receptors (LXR), with evidence that LXR-induced hyperglyceridemia is due to the release of ANGPTL3. The interactions between CCD and ECM through a putative heparin binding motif can lead to inhibition of cleavage in the recognition sequence (R221-R224) in the proprotein convertase, similar to that reported for ANGPTL4.
    [0079] The cleavage results in a significant increase in the ability of CCD to inhibit the activity of lipoprotein (LPL) and thereby leads to an increase in triglyceridemia (TG). This represents the main biological function of ANGPTL3 identified prior to the present invention. The C-terminal FLD is sufficient to induce adhesion of the endothelial cell and serves as a mediator for angiogenesis after direct implantation in the rat cornea in vivo. It has also been shown that recombinant ANGPTL3 could bind to purified α / β3 integrin and lead to an increase in FAK, MAPK and ΑΚΤ signaling in endothelial cells. These data suggest the interaction of FLD with integrins to mediate angiogenesis.
    [0080] No expression of ANGPTL3 was reported or observed in our studies using "western blotting" in human chondrocytes, hMSCs or human synovial fibroblasts and no expression of it was found in mouse knee joints. In addition, there are no reported activities for fragment in joint cells that refer to this new chondrogenic function identified in our selection.
    [0081] Our data indicate that full-length ANGPTL3 is a new mediator of chondrogenesis and cartilage protection. We examined which domains of ANGPTL3 are critical for this new chondrogenic function. A series of ANGPTL3 and full-length protein truncations were expressed and purified in an engineered HEK-S cell line that provides limited and homogeneous glycosylation for detailed biophysical characterization. Mass spectrometry approaches confirmed the occupation of four predicted N-linked glycosylations (NI15, N232, N296 and N357, Figure 3A). Starting from HEK fragments processed en-dogenally, the proteolytic cleavage site between the CCD and FLD domain was determined to be R224. The full-length ANGPTL3 elutes by size exclusion chromatography with a mass> 400 kDa, indicating a trimer with heterogeneous glycosylation. The FLD (241-455), presented as a monomer by size exclusion and static light diffusion, was crystallized. The atomic structure of the FLD was determined at a resolution of 1.8 angstroms. The FLD structure revealed a beta heartwood configuration with helical stretches that guide the three closed circuits towards terminal C, typical of other FLD counterparts (Figure 3B). The temperature factors in the C-terminal part of the domain are higher than the rest of the structure, suggesting a high degree of flexibility in this region. The overlap with the ANGPTL2 / Tie2 structure (code: 2GY7) suggests that the C-terminal part of the FLD may be involved in protein-protein interactions.
    [0082] The five truncation products generated were evaluated in multiple chondrogenic assays. The results suggest no retention of activity in the CCD alone, however the activity remains in the FLD. In addition, SMADI phosphorylation is increased after stimulation for 3 days at full length or with G or H mutants only, suggesting activation of chondrogenic signaling cascade.
    [0083] Although systemic exposure of an intra-articularly injected protein is limited due to lymphatic drainage of synovial fluid, minimizing systemic exposure can sometimes be desirable. If there is systemic exposure, the cleavage of ANGPTL3 could lead to the release of the CCD domain. The lower inhibition of LPL activity by CCD could lead to changes in patients' TG levels. Our results indicate potential clinical advantages of ANGPTL3. For example, by researching where the chondrogenic activity in ANGPTL3 is located, undesired systemic effects on lipid metabolism or angiogenic properties would be minimized or excluded. Chondrogenic activity is mainly located at the C-terminus, so the specific use of this domain would alleviate the concerns of regulating TG of the full-length or N-terminal molecule. As full-length ANGPTL3 is normally present in the serum, there is no expected immunogenicity. An additional advantage of using the C-terminus is that it is cleaved and is a monomer (~ 29kDa) compared to trimerized full-length protein, thereby possibly reducing the half-life of any small percentage that could be present in circulation and limitation any potential effect of angiogenesis. Example 3: In vivo analysis of ANGPTL3
    [0084] We performed several in vivo assessments of full-length ANGPTL3 to control potential adverse events and intra-articular retention. After intra-articular (AI) injection of the left knee joints of 8-week-old C57BL / 10 mice with 3.6 μg of full-length ANGPTL3, the expression was immunohistochemically evaluated in the presence and absence of exposure for 24 hours. For 24 hours, very little or no detectable levels of ANGPTL3 would need to remain in the synovial fluid because the typical trans-synovial flow renewal material for synovial lymphatic vessels for proteins and deionized water is approximately 2 hours. The results did not reveal endogenous expression of ANGPTL3 in untreated joints, but significant detection of the protein in the pericellular matrix in the articular cartilage and in the menisci even at the 24-hour time point. In addition, broad cytotoxicity was not detected in vivo for damage to chondrocytes or cartilage. After a series of 3 IA injections in rat knee joints (once / week for three weeks), there was no clinical toxicity (no swelling in the joint or walking alternatives) or evidence of an acute inflammatory reaction in the rat joint . Histologically, there was no increase in synovitis or uncontrolled proliferation in the joints of the five injected rats. As in the mouse, ANGPTL3 could be detected in the cartilage matrix and around the chondrocytes. These results indicate that ANGPTL3 does not cause undesired effects to the cartilage itself and that ANGPTL3 enters and is retained in the cartilage and meniscus in vivo.
    [0085] OA is not a simple disease entity and can be considered the consequence of several etiological factors. In humans it is often caused by abnormal biomechanical stress or by genetic or acquired abnormalities in cartilage or joint bone. Therefore, choosing the "best" small animal model for OA is difficult and multiple models needed to be explored to determine the protective properties of any therapeutic agent. Efficiency studies completed a chronic OA model (induced by collagenase VII based on the research described by van der Kraan and colleagues) and an acute surgical model involving the transection of the three major ligaments (ACL, MCL and MMTL) in the joint based on work Glasson and others. Both models induced pathological changes commonly associated with OA: loss of staining with proteoglycan, erosion of cartilage and bones, formation of osteophytes and metaplastic changes in the synovium and ligaments may be evident 4-8 weeks after onset of OA. Figure 5 represents the regeneration capacity of the OA ANGPTL3 surgical model. To begin examining the potential biomarkers for OA, peripheral blood was collected during the surgical model to measure the type II collagen fragments released due to cartilage damage (Figure 5A). Histological analysis and subsequent grading of the middle tibial plaque revealed regeneration in the cartilage matrix after treatment with 200 ng ANGTPL3 / knee once a week for 3 weeks (Figure 5B).
    [0086] In an 8-week mouse OA surgical set, three doses of ANGPTL3 were examined for relief of OA-induced pain through disability measures. This method measures the weight distribution between surgical and non-surgical legs. On day 36 after surgery and 3 weekly treatments with PROI, a dosage as low as 100 ng / knee demonstrated significant improvement compared to vehicle-treated surgical knees (Figure 5C). These combined data provide concrete evidence that ANGTPL3 has in vivo efficiency in two models of OA (both pathological correction and pain reduction) and supports the advancement of its development as a new therapy for OA.
    [0087] It is understood that the examples and modalities described herein are for illustrative purposes only and that various modifications or changes in the light of them will be suggested to persons skilled in the art and need to be included in the spirit and scope of this patent application and within the scope of the claims attached. All publications, patents and patent applications cited herein are hereby incorporated as a reference in their entirety for all purposes.
    权利要求:
    Claims (13)
    [0001]
    Use of a polypeptide, characterized by the fact that it is used to prepare a drug to improve or prevent arthritis or joint damage in a mammal, in which the polypeptide has chondrogenic activity and is selected from: (a) the polypeptide consisting of an amino acid sequence of SEQ ID NO: 1 or 25; (b) the polypeptide consisting of an amino acid sequence of SEQ ID NO: 2, 3, 4, 26, 27, or 28; or (c) the polypeptide consisting of an amino acid sequence of SEQs ID No. s: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24.
    [0002]
    Use according to claim 1, characterized by the fact that the polypeptide (i) is PEGylated; and / or (ii) is fused to a human serum albumin (HSA), an immunoglobulin heavy chain (Fc) constant region, a polyhistidine, a glutathione S transferase (GST), a thioredoxin, a protein A, a G protein or a maltose-binding protein (MBP).
    [0003]
    Use according to claim 1 or 2, characterized by the fact that (a) the mammal has arthritis or joint damage or (b) the mammal does not, but is at risk of arthritis or joint damage.
    [0004]
    Use according to any one of claims 1 to 3, characterized by the fact that (a) arthritis is selected from the group consisting of osteoarthritis, traumatic arthritis and autoimmune arthritis; and / or (b) the mammal is a human, a dog or a cat; or (c) the drug still comprises hyaluronic acid.
    [0005]
    In vitro method of inducing differentiation of mesenchymal stem cells into chondrocytes, characterized by the fact that it comprises the contact of mesenchymal stem cells with a sufficient amount of a polypeptide consisting of an amino acid sequence of SEQ ID NO: 1 or 25 and with chondrogenic activity to induce the differentiation of mesenchymal stem cells into chondrocytes.
    [0006]
    Method according to claim 5, characterized in that the polypeptide (i) is PEGylated; and / or (ii) is fused to a human serum albumin (HSA), an immunoglobulin heavy chain (Fc) constant region, a polyhistidine, a glutathione S transferase (GST), a thioredoxin, a protein A, a G protein or a maltose-binding protein (MBP).
    [0007]
    Method according to claim 5 or 6, characterized in that the amino acid sequence of the polypeptide: (a) consists of SEQs ID No. s: 2, 3, 4, 26, 27 or 28; or (b) consists of SEQs ID No. s: 5, 9, 13, 17 or 21.
    [0008]
    Use of a polypeptide, characterized by the fact that it is to prepare a pharmaceutical composition to induce repair or production of cartilage in a mammal, in which the polypeptide has chondrogenic activity and consists of an amino acid sequence of SEQ ID NO: 1 or 25.
    [0009]
    Use according to claim 8, characterized in that the polypeptide (i) is PEGylated; and / or (ii) is fused to a human serum albumin (HSA), an immunoglobulin heavy chain (Fc) constant region, a polyhistidine, a glutathione S transferase (GST), a thioredoxin, a protein A, a G protein or a maltose-binding protein (MBP).
    [0010]
    Use according to claim 8 or 9, characterized by the fact that the mammal is a human being, a dog or a cat.
    [0011]
    Use according to any one of claims 8 to 10, characterized in that the amino acid sequence of the polypeptide: (a) consists of SEQs ID No. s: 2, 3, 4, 26, 27 or 28; or (b) consists of SEQs ID No. s: 5, 9, 13, 17 or 21.
    [0012]
    Pharmaceutical composition for intra-articular application, characterized by the fact that it comprises a pharmaceutically effective amount of a polypeptide having chondrogenic activity selected from: (a) a polypeptide consisting of an amino acid sequence of SEQ ID NO: 1 or 25; (b) a polypeptide consisting of an amino acid sequence of SEQs ID No. s: 2, 3, 4, 26, 27 or 28; or (c) a polypeptide consisting of an amino acid sequence of SEQs ID No. s: 5, 9, 13, 17 or 21, and further comprising hyaluronic acid.
    [0013]
    Composition according to claim 12, characterized in that the polypeptide is (i) PEGylated; and / or (ii) is fused to a human serum albumin (HSA), an immunoglobulin heavy chain (Fc) constant region, a polyhistidine, a glutathione S transferase (GST), a thioredoxin, a protein A, a G protein or a maltose-binding protein (MBP).
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    法律状态:
    2017-12-05| B25A| Requested transfer of rights approved|Owner name: THE SCRIPPS RESEARCH INSTITUTE (US) , NOVARTIS INT |
    2017-12-19| B25A| Requested transfer of rights approved|Owner name: THE SCRIPPS RESEARCH INSTITUTE (US) , NOVARTIS AG |
    2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|
    2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-05-28| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |
    2019-06-25| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|
    2020-02-04| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|
    2020-06-30| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-01-26| B09W| Correction of the decision to grant [chapter 9.1.4 patent gazette]|Free format text: REF. A RPI NO 2582 DE 30/06/2020. |
    2021-04-06| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 10 (DEZ) ANOS CONTADOS A PARTIR DE 06/04/2021, OBSERVADAS AS CONDICOES LEGAIS. |
    2021-05-25| B16C| Correction of notification of the grant [chapter 16.3 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 13/07/2010 OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF |
    优先权:
    申请号 | 申请日 | 专利标题
    US22529309P| true| 2009-07-14|2009-07-14|
    US61/225,293|2009-07-14|
    PCT/US2010/041850|WO2011008773A2|2009-07-14|2010-07-13|Mesenchymal stem cell differentiation|
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