ACVR1-FC FUSION PROTEIN, PREPARATION METHOD THEREFOR, AND APPLICATION THEREOF
20190062402 ยท 2019-02-28
Inventors
Cpc classification
C07K19/00
CHEMISTRY; METALLURGY
C12Y207/1103
CHEMISTRY; METALLURGY
A61P19/08
HUMAN NECESSITIES
C07K2319/30
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The present invention provides an ACVR1-Fc fusion protein, a nucleic acid sequence encoding said fusion protein, a vector or a host cell comprising said encoding sequence, a method for producing said fusion protein, and use of any of the above in prevention and/or treatment of diseases or conditions associated with ACVR1 abnormality (e.g. ACVR1 mutation and/or over-activation).
Claims
1. A fusion protein including the following elements: an ACVR1 element having the amino acid sequence ACVR1 or an active fragment thereof, preferably an ACVR1 extracellular segment sequence; and an Fc element.
2. The fusion protein of claim 1, wherein the elements in the fusion protein are independently selected wherein: the ACVR1 element is selected from: a sequence having SEQ ID NO: 4; a sequence as shown in SEQ ID NO: 4 and having one or more amino acid deletions, substitutions, or insertions and having the same biological activity as the sequence shown in SEQ ID NO: 4; and the sequence shown in SEQ ID NO: 4 and having 90% or more homology to the sequence shown in SEQ ID NO: 4 and having the same biological activity as the sequence shown in SEQ ID NO: 4; the Fc element is selected from the group consisting of an Fc fragment comprising human IgG1, IgG2, IgG3, IgG4 the signal peptide element is selected from the group consisting of: a CD33 signal peptide; a surface antigen protein signal peptide; an antibody protein signal peptide; and a secretion signal peptide molecule.
3. The fusion protein of claim 1, wherein the fusion protein is selected from the group consisting of: a sequence having SEQ ID NO: 8; a sequence having one or more amino acid deletions, substitutions, or insertions with the sequence shown in SEQ ID NO: 8, and having the same biological activity as the sequence shown in SEQ ID NO: 8; and a sequence having at least 90% homology to the sequence shown in SEQ ID NO: 8 and having the same sequence as shown in SEQ ID NO: 8 sequences that have the same biological activity.
4. An isolated nucleic acid molecule which is the coding sequence of the fusion protein of claim 1 or is the complement of the coding sequence.
5. The nucleic acid molecule of claim 4, wherein the nucleic acid molecule further comprises: the sequence set forth in SEQ ID NO: 3; and the sequence set forth in SEQ ID NO: 5.
6. A vector wherein the vector contains the nucleic acid molecule according to claim 4.
7. A host cell wherein the host cell comprises the vector of claim 6.
8. The method of producing the fusion protein according to claim 1, the method including the steps of: cultivating a host cell under conditions suitable for the expression of the fusion protein so as to express the fusion protein, the host cell including a vector, the vector containing a nucleic acid molecule that is one of a coding sequence of the fusion protein and a complement of the coding sequence of the fusion protein; and isolating the fusion protein.
9. A medicament or a pharmaceutical composition for at least one of the group consisting of preventing a disease or condition associated with abnormal ACVR1 and treating a disease or condition associated with abnormal ACVR1, the medicament or a pharmaceutical comprising at least one of: the fusion protein of claim 1; a nucleic acid molecule that is one of a coding sequence of the fusion protein and a complement of the coding sequence of the fusion protein; a vector containing the nucleic acid molecule; and a host cell including the vector.
10. (canceled)
11. The fusion protein of claim 1, wherein the FC element is a human IgG Fc fragment.
12. The fusion protein of claim 1, further including a signal peptide element.
13. The fusion protein of claim 1, further including at least one linker peptide sequence located between the ACVR1 element and the Fc element.
14. The fusion protein of claim 2, wherein the Fc element is selected from the group consisting of: an Fc fragment having the sequence of SEQ ID NO: 6; an Fc fragment having one or more amino acid deletions, substitutions, or insertions into the sequence of SEQ ID NO: 6 and having the same biological activity as the sequence of SEQ ID NO: 6; and a sequence having at least 90% homology to the sequence shown in SEQ ID NO: 6 and having the same biological activity as the sequence shown in SEQ ID NO: 6.
15. The fusion protein of claim 14, wherein the a secretion signal peptide molecule is a signal peptide having SEQ ID NO:2.
16. The medicament or pharmaceutical composition of claim 9, further comprising a pharmaceutically acceptable carrier.
17. The medicament or pharmaceutical composition of claim 9, wherein the disease or condition is selected from at least one of the group consisting of: ossification or pleonosteosis associated diseases; and at least one of the group consisting of cancers and over-activation associated with ACVR1.
18. The medicament or pharmaceutical composition of claim 17, wherein the at least one of the group consisting of cancers and over-activation associated with ACVR1 is at least one of: high-grade gliomas, such as diffuse endogenous pontine gliomas; and ovarian cancer.
19. (canceled)
20. A method for at least one of preventing a disease or condition associated with abnormal ACVR1 and for treating a disease or condition associated with abnormal ACVR1 in a subject, wherein the method comprises administering to the subject an effective amount of a medicament or pharmaceutical composition of claim 9.
21. The method of claim 20, wherein the disease or condition is selected from at least one of the group consisting of: ossification or pleonosteosis associated diseases; and at least one of the group consisting of cancers and over-activation associated with ACVR1, the at least one of the group consisting of cancers and over-activation associated with ACVR1 being at least one of: high-grade gliomas, such as diffuse endogenous pontine gliomas; and ovarian cancer.
22. The fusion protein of claim 12, further including: at least one linker peptide sequence disposed between any two of the elements selected from the ACVR1 element; the Fc element; and the signal peptide element.
23. The nucleic acid molecule of claim 5, further comprising the sequence set forth in SEQ ID NO: 1.
24. The nucleic acid molecule of claim 23, wherein the nucleic acid molecule is selected from the group consisting of: a sequence having SEQ ID NO: 7; a nucleotide sequence having one or more nucleotide deletions, substitutions or insertions into the sequence set forth in SEQ ID NO: 7; and a sequence having at least 90% homology to the sequence shown in SEQ ID NO: 7 and having the same biological activity as the sequence shown in SEQ ID NO: 7.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0089] In the following, the inventions are specified with reference to the drawings. It should be understood that the drawings are provided only for description, with no intention to limit the scope of the invention.
[0090]
[0091]
[0092]
[0093] a: construction of the adenovirus plasmid expressing ACVR1 R206H;
[0094] b: an optical-microscopic picture of normally cultured HUVEC cells;
[0095] c: a fluorescent-microscopic picture of HUVECs infected with recombinant adenovirus expressing ACVR1 R206H;
[0096]
[0097]
[0098]
[0099]
[0100]
[0101] a: HUVECs were infected with ACVR1 R206H-adenovirus; 5 days later, the cells were transferred into the osteogenesis inducing medium and incubated for additional 7 days, and then stained with ALP;
[0102] b: HUVECs infected with ACVR1 R206H-adenovirus were incubated in the osteogenesis inducing medium for 21 days, and then stained with Alizarin Red;
[0103] c: HUVECs were infected with ACVR1 R206H-adenovirus; 5 days later, the cells were transferred into the osteogenesis inducing medium and incubated for additional 14 days, and then stained with Alcian Blue. The cells were examined by taking pictures under a bright field microscope.
[0104]
[0105] a: differentiation medium containing 3 g/ml control (recombinant human IgG 1 Fc);
[0106] b: differentiation medium containing 1.5 g/ml ACVR1-Fc fusion protein;
[0107] c: differentiation medium containing 3 g/ml ACVR1-Fc fusion protein.
[0108]
[0109] a: differentiation medium containing 3 g/ml Fc protein as control (recombinant human IgG1Fc);
[0110] b: differentiation medium containing 1.5 g/ml ACVR1-Fc fusion protein;
[0111] c: differentiation medium containing 3 g/ml ACVR1-Fc fusion protein.
[0112]
[0113] a: differentiation medium containing 3 g/ml Fc protein as control (recombinant human IgG1Fc);
[0114] b: differentiation medium containing 1.5 g/ml ACVR1-Fc fusion protein;
[0115] c: differentiation medium containing 3 g/ml ACVR1-Fc fusion protein.
[0116]
[0117]
[0118] a: Western blotting showing ACVR1-Fc's effects on the expression of five osteogenesis markers;
[0119] b: Inhibition of each of the markers was calculated, taking the expression of GAPDH as the baseline.
[0120]
[0121] a: Western blotting of ACVR1-Fc's effects on inhibiting the phosphorylation of Smad-1/5/8 and p38MAP;
[0122] b: quantitative analysis of the inhibition of phosphorylation of Smad-1/5/8 protein by ACVR1-Fc via Western blotting;
[0123] b and c: quantitative analysis of the inhibition of phosphorylation of p38MAP protein by ACVR1-Fc via Western blotting;
[0124] ***: P<0.001.
DETAILED DESCRIPTION OF THE INVENTION
[0125] The inventors, through extensive and intensive studies, constructed an expression vector of ACVR1-Fc fusion protein, obtained an ACVR1-Fc fusion protein and characterized the superior biological activities of the fusion protein, which promises to be a new approach for preventing and treating diseases or conditions associated with ACVR1 abnormality (e.g. ACVR1 mutation and/or over-activation). For instance, the fusion protein of the invention can effectively inhibit the activation of the ACVR1 and the ACVR2 pathways, and thereby inhibit osteogenic differentiation and chondrogenic differentiation, which makes it useful in prevention and/or treatment of pleonosteosis-associated diseases and/or conditions (e.g. fibrodysplasia ossificans progressiva FOP) caused by over-activated ACVR1 and/or ACVR2 signaling pathway(s).
[0126] In the present disclosure, each reference to a range equals to specific disclosure of each of the values in between and also every subsets in between. All the features and elements, though being specified in context an embodiment or example for purpose of explanation and exemplification, can be recombined and reorganized without departing the spirit and scope of invention. In the present disclosure, the features and elements, beyond the specified specific examples, extend to include equivalents in form and in identity, which can be used in an exchangeable way. Unless otherwise indicated, the disclosed features are exemplary examples for the equivalents or similar features.
[0127] As used herein, the terms comprises/comprise/comprising, has/have/having and includes/include/including, including their grammar cognates, are used in an exchangeable way and each include the meaning of or equal to comprise, essentially consist of . . . , substantially consist of . . . and consist of . . .
[0128] As used herein, the term isolated, with reference to nucleic acid molecule and protein, means that the referenced material is separated from and thus substantially free of the substances (e.g. cell components) that are co-present in its naturally occurring environment. The isolated material is preferably homogeneous, in a dry or aqueous state. Purity and homogeneity can be determined via assays like polyacrylamide gel electrophoresis and high performance liquid chromatography. The terms protein, peptide and polypeptide are used in an exchangeable way and refer to a chain of two or more amino acids linked by one or more peptide bond(s) or amido bond(s), which may be optionally modified by, for instance, glycosylation and/or phosphorylation.
[0129] Fusion Protein and Elements Thereof
[0130] As used herein, unless otherwise specified, the term fusion protein refers to an isolated protein, which may be recombinantly produced by a host cell or be extracted in a purified form.
[0131] A fusion protein of the invention may comprise an element (a) and an element (b), optionally, an element (c), and ever further, optionally an element (d), as defined in the followings:
[0132] (a) an ACVR1 element, comprising the amino acid sequence of ACVR1 or a functionally active fragment thereof;
[0133] (b) an Fc element, comprising an Fc fragment of human IgG;
[0134] (c) optionally, a signal peptide element; and
[0135] (d) optionally, linker peptide(s) between any two of the above.
[0136] As used herein, the term element refers to an amino acid sequence incorporated as an integral part of the fusion protein.
[0137] In the present disclosure, the ACVR1 element (a) has an amino acid sequence substantially identical to the full-length sequence of the native ACVR1 or a variant thereof, or to the extracellular domain, and has substantially the same biological activity of the native ACVR1. The element (a) preferably has the sequence of the extracellular domain of ACVR1, and more preferably has the sequence of SEQ ID NO: 4.
[0138] In some embodiments, the ACVR1 element is selected from the group consisting of:
[0139] (i) one having the sequence of SEQ ID NO: 4;
[0140] (ii) one having a sequence containing one or more amino acid deletions, substitutions and/or additions relative to the sequence of SEQ ID NO: 4 and having the same biological activity as the sequence of SEQ ID NO: 4; and
[0141] (iii) one having a sequence more than 90% homologous to the sequence of SEQ ID NO:4 and having the same biological activity as the sequence of SEQ ID NO: 4.
[0142] As used herein, the term Fc region or Fc fragment refers to a fragment consisting of hinge region +CH2 region +CH3 region. In the present disclosure, the Fc element (b) has an amino acid sequence substantially the same as that of a native IgG Fc fragment or a variant thereof, and has substantially the same biological activity as the native Fc fragment. Besides the CH2 and the CH3 regions of IgG, the Fc element may further comprise the hinge region. The element (b) may be the Fc region from IgG1-4, preferably the Fc region from IgG 1, more preferably one having the sequence of SEQ ID NO: 6.
[0143] In some embodiments, the Fc element is selected from the group consisting of:
[0144] (i) one having the sequence of SEQ ID NO: 6;
[0145] (ii) one having a sequence containing one or more amino acid deletions, substitutions and/or additions relative to SEQ ID NO: 6 and have the same biological activity as the sequence of SEQ ID NO: 6; and
[0146] (iii) one having a sequence more than 90% homologous to the sequence of SEQ ID NO:6 and having the same biological activity as the sequence of SEQ ID NO: 6.
[0147] In the present disclosure, signal peptide element (c) refers to an amino acid sequence that directs secretion, location and/or transportation of the fusion protein, which is usually 5-30 amino acids in length.
[0148] In some embodiments, the signal peptide element is the signal peptide of protein CD33 (preferably having the sequence of SEQ ID NO: 2), or any other signal peptide capable of guiding a protein's secretion into extracellular environment.
[0149] In the present disclosure, the linker peptide sequence (d) refers to a short peptide acting as a linkage between any two of the elements in the fusion protein, which is usually 1 to 50 (e.g. 5 to 50, 5 to 40, or 10 to 40) amino acids long. A person of ordinary skills in the art knows how to design a suitable linker peptide using conventional means and tools (e.g. PNAS 1998; 95: 5929-5934; Protein Eng, 2000; 13(5): 309-312; Protein Eng, 2003; 15(11): 871 - 879). Usually, the linker peptide does not or does not substantively interfere the correct folding and spatial confirmation of the fusion protein of the invention.
[0150] In the present disclosure, the fusion protein may comprise the elements arranged in any one of the following orders in the direction of 5 to 3:
[0151] (a)-(b); (b)-(a); (c)-(a)-(b); (c)-(b)-(a); (a)-(d)-(b); (b)-(d)-(a); (c)-(d)-(a)-(b);
[0152] (c)-(a)-(d)-(b); (c)-(d)-(b)-(a); (c)-(b)-(d)-(a); (c)-(d1)-(a)-(d2)-(b); and
[0153] (c)-(d1)-(b)-(d2)-(a),
[0154] Wherein, (a) represents the ACVR1 element; (b) represents the Fc element; (c) represents the signal peptide element; (d) represents a linker peptide sequence; (d), (d1) and (d2) independently represent linker peptide sequences that are identidal or different.
[0155] In the present disclosure, preferably, the fusion protein
[0156] (i) having the sequence of SEQ ID NO: 8;
[0157] (ii) having a sequence containing one or more amino acid deletions, substitutions and/or additions relative to SEQ ID NO: 8 and having the same biological activity as the sequence of SEQ ID NO: 8; or
[0158] (iii) having a sequence more than 90% homologous to the sequence of SEQ ID NO:8 and having the same biological activity as the sequence of SEQ ID NO: 8.
[0159] In some embodiments, the fusion protein posseses one or more of the following activities: binding to the same cytokine(s) as the native ACVR1 does, binding to the complex of cytokine(s) and ACVR2, inhibiting phosphorylation of protein Smad-1/5/8, inhibiting phosphorylation and activation of p38 MAP kinase, inhibiting osteogenic differentiation, inhibiting chondrogenic differentiation, reducing calcium ion level in intercellular matrix.
[0160] In the present disclosure, for each of said elements, also contemplated and included are variants of a protein, polypeptide or peptide designated as any one of the elements, which have the same or equivalent biological activity as the prototype protein, polypeptide or peptide. Variation includes but is not limited to one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid deletions, additions and/or substitutions compared to native amino acid sequence. The deletion(s) or addition(s) (insertion(s)) may occur in the C-terminal region and/or the N-terminal region, and is usually no more than 20 amino acids in number, preferably no more than 10, and more preferably no more than 5. As understood, substitution(s) can be made using amino acids of similary properties to retain activities of the prototype protein. Conservative amino acid substitutions are well-known, for example, as within each of the following five groups: aliphatic amino acids: glycine(G), alanine (A), valine (V), leucine (L), isoleucine (I); aromatic amino acids: phenylalanine (F), tyrosine (Y), tryptophan (W); sulfur-containing amino acids: methionine (M), cysteine (C); basic amino acids: arginine(R), Lysine (K), histidine(H); and acidic amino acids: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q). In addition, also contemplated are the fragments or derivatives of inhibitory factors and human albumin, which preferably retain the desired biological activities.
[0161] Said variants also include analogs of the proteins or polypeptides. Compared to the native protein, the analogs may distinguish in amino acid sequence and/or modification onto the primary sequence. These polypeptides include naturally occurring variants and artificially induced genetic variants. Induced variants can be obtained in various ways, such as radiation or exposure to a mutagenic agent to induce random mutagenesis, site-directed mutagenesis, and other mutagenesis techniques known in molecular biology. Analogs also include those containing a residue that is not a naturally occurring L-amino acid (e.g. a D-amino acid), and those containing a non-natually occuring or artificial amino acid (e.g. or -amino cids). It should be understood that the above specification is exemplary, and has no effect of limiting the scope of polypeptides of the invention. Modification, not in sense of changing primary amino acid sequence, may include in vivo or in vitro chemical derivation of a polypeptide, such as acetylation and carboxylation. Modification also includes glycosylation, including those occuring in synthesis, processing or subsequent processing of a polypeptide, so as to obtain a glycosylated polypeptide. This can be conducted by exposing a polypeptide to an enzyme involved in glycosylation (e.g. a mammalian glycosylase or a deglycosylase). Modified forms include sequences that comprise one or more phosphorylated amino acid residue(s) (e.g. phosphotyrosine, phosphoserine, phosphothreonine).
[0162] Each of the elements of the invention, when being designated with a specified peptide or polypeptide, also includes the polypeptides substantially identical (homologous) to the specified one, such as a polypeptide that has an identity (homology) of at least 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or above to the specified one.
[0163] Based on the amino acid sequences provided herein, a person of ordinary skills in the art would know how to obtain a fusion protein of the invention using conventional means and tools, such as recombinant techniques, artificial synthesis, etc. [e.g. Murray K M, Dahl SLAnn; Pharmacother 1997 November; 31(11):1335-8]. For instance, a fusion protein of the invention may be directly obtained via solid-phase synthesis, or via assembly of synthetic fragments into the full-length molecule.
[0164] Fusion Protein Coding Sequences, Vectors and Host Cells
[0165] The present disclosure provides an isolated nucleic acid molecule having a nucleic acid sequence encoding the fusion protein, or the complementary sequence thereof. The nucleic acid molecule encoding the fusion protein of invention can be fully synthesized or be assembled from fragments each encoding one or more elements of the protein.
[0166] The nucleic acid sequence of the invention can be obtained using any of the conventional means, including PCR amplification, recombinant techniques and artificial synthesis. In the case of PCR amplification, the primers can be designed according to the relevant nucleotide sequence(s) in this disclosure, especially the ORFs, and the templates can be obtained from commercially accessible or self-developed cDNA banks, which then give the target sequence via amplification. In case of a long target sequence, usually overlapping amplification, like two or more PCR processes, are conducted to give batches of different fragments which are then assembled into the full-length target. The sequence thus obtained can then be reproduced in bulk using, for example, recombinant techniques.
[0167] In the present disclosure, the nucleic acid molecule encoding the fusion protein may comprise the sequence of SEQ ID NO: 3, which encodes the ACVR1 element; the sequence of SEQ ID NO: 5, which encodes the Fc element; and optionally, the sequence of SEQ ID NO: 1, which encodes the signal peptide element.
[0168] In a preferred embodiment of the invention, the nucleic acid molecule has a sequence selected from the followings:
[0169] (i) one having the sequence of SEQ ID NO: 7;
[0170] (ii) one having a sequence containing one or more amino acid deletions, substitutions and/or additions relative to SEQ ID NO: 7 and having the same biological activity as the sequence of SEQ ID NO: 7;
[0171] (iii) one having a sequence more than 90% homologous to the sequence of SEQ ID NO:7 and having the same biological activity as the sequence of SEQ ID NO: 7; and
[0172] (iv) one hybridizing to any one of the above sequences under a stringent condition and being correspondingly functionally active.
[0173] The invention also includes nucleic acid sequences that are identical (homologous) or substantially identical (homologous) to a specified one, such as those having an identity (homology) of at least 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or even higher. When two nucleic acid sequences are described as substantially identical/homologous to each other, they hybridize under a high stringent condition. Accordingly, the nucleic acid sequences of invention include those hybridizing to a specified one, especially for example the sequence of SEQ ID NO: 7, under a moderate stringent condition, and more preferably under a high stringent condition.
[0174] As used herein, the term the term stringent condition refers to:
[0175] (1) hybridization and elution at a low ionic strength and a high temperature, such as 0.2SSC, 0.1% SDS, 60 C.; or
[0176] (2) hybridization in the presence of a denaturing agent (e.g. 50%(v/v) formamide), 0.1% calf serum/0.1% Ficoll, 42 C. etc.; or
[0177] (3) hybridization only occurring between sequences that are at least 50%, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more, and more preferably at least 95% homologous to each other.
[0178] The DNA sequence of the fusion protein of invention may be incorporated in a suitable expression vector, which is then transferred into a suitable host cell. The transformed host cells are cultivated in a culture, from which the fusion protein of invention can be separated and purified.
[0179] As used herein, the term vector includes plasmids, cosmids, expression vectors, clonal vectors, viruse vectors, etc. Examples include but are not limited to vectors capable of expression in eukaryotes (e.g. CHO cells, COS cells, etc.), vectors capable of expression in yeasts (e.g. Saccharomyces cerevisiae and Pichia pastoris), vectors capable of expression in insect cells (e.g. silk worms), and vectors capable of expression in prokaryotes. Commercially available vectors are useful here. For example, an expression vector according to the disclosure may be constructed using a commercially available vector by incorporating the nucleotide sequence encoding the fusion protein as operably linked to a regulatory sequence.
[0180] As used herein, the term operably linked refers to the association of DNA sequences in a single chain so that the function of one is affected by the other. In one instance, a DNA sequence of signal peptide (i.e, a leader sequence) is operably linked to a polypeptide-coding sequence, when the DNA sequence of signal peptide is expressed as part of the precursor to mediate secretion of the polypeptide. In another instance, a promoter is operably linked to a coding sequence, when it regulates transcription of the coding sequence. In a further instance, a ribosome bind site is operably linked to a coding sequence, if it is located so that the coding sequence is effectively expressed. Usually, operably linked means being adjacent and, in the case of a leader sequence, it means being adjacent within ORF.
[0181] As used herein, the term host cell includes prokaryotes and eukaryotes. Prokaryotic examples include Escherichia coli, Bacillus Subtilis, etc. Eukaryotic examples include yeasts, insect cells and mammalian cells. Preferably, the host cell is a eukaryote, like CHO DG44.
[0182] As used herein, the term transform refers to introduction of a nucleic acid molecule of interests into a host cell using whatever a suitable means as known in the art. Examples of transformation techniques, each maybe differentially prefer certain type(s) of host, include inter alia electroporation, transfection using chemicals like calcium chloride and DEAE-glucan, particle bombardment, lipofection, and infection (see, Sambrook et al., Molecular Cloning, 2n.sup.d Version, 1989). In an embodiment, electroporation is preferred.
[0183] The host cells are cultivated under a suitable condition such that the fusion protein is expressed. The condition of cultivation, such as medium, temperature and time, can be determined via routine experimentation. The expression of said fusion protein can be detected by any suitable means and tools as known in the field, such as SDS-PAGE, Western blotting, etc. Finally, conventional protein separation and purification techniques may be used to give the purified fusion protein, such as centrifugation, precipitation, filtration, chromatography, etc. Specifically, useful chromatography techniques include affinity assay, gel filtration, ion exchange, hydrophobic chromatography, and reverse chromatography, etc. Isolation and purification of the fusion protein may also include combination between or among any of the above techniques.
[0184] Applications
[0185] The fusion protein, the coding sequence, the vector and the host cell comprising the coding sequence can be used as a medicament for prevention and/or treatment of a disease or a condition associated with ACVR1 abnormality (e.g. mutation and/or over-activation), such as a pleonosteosis-associated disease or condition, and a cancer associated with ACVR1 mutation and/or over-activation, etc.
[0186] Preferably, the pleonosteosis is caused by over-activated ACVR1 and/or ACVR2 signaling pathway(s). In some embodiments, the pleonosteosis-associated disease or condition is selected from the group consisting of fibrodysplasia ossificans progressiva, cartilage hyperostosis, hyperostosis, etc. Preferably, the cancer associated with ACVR1 mutation and/or over-activation is selected from the group consisting of high-grade glioma, such as diffuse endogenous cerebral bridge glioma, ovarian cancer, etc.
[0187] Accordingly, the disclosure also provides a pharmaceutical composition that comprises (a) an effective amount of the fusion protein of the invention, the coding sequence, the vector and/or the host cell comprising said coding sequence of the invention, and (b) a pharmaceutically acceptable vehicle.
[0188] As used herein, the term an effective amount or an effective dosage refers to an amount, which confers an effect or action on the treated subject (human or animal) and which can be tolerated by the subject. The term pharmacologically acceptable means that when a molecule or a composition is appropriately administrated to a subject (human or animal), it does not cause undesired effects, such as toxicity, irritation and allergia, and has a reasonable efficacy/risk ratio.
[0189] A pharmaceutically acceptable vehicle should be compatible with the fusion protein of invention. That is, the vehicle, when being mixed with the protein in a pharmaceutical composition, does not cause a substance reduction in pharmacological efficacy. Examples of pharmaceutically acceptable vehicles or components thereof include those as taught in Remington: The Science and Practice of Pharmacy 21.sup.st Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, Pa., 2005.
[0190] The pharmaceutical composition may be formulated in various dosage forms. A beneficial dosage can be determined by a physician according to known factors, including race, age, body weight, specific condition or state of the disease to be treated and path of administration. Paths of administration include inter alia oral, intranasal, respiratory airway pathways.
[0191] In the instance that the therapeutic agent is a polynucleotide, it can be delivered in the form of a naked polynucleotide, a combination or conjugate with a drug delivery agent, and/or a recombinant plasmid or viral vector comprising same or a component comprised in the plasmid or viral vector. Examples of agents useful for drug delivery include inter alia Mirus M Transit TKO lipothrophic reagents, lipofectin reagents, cellfectins, cationic polymers (e.g. polylysine) and liposomes.
[0192] For an enhanced effect, the fusion protein of invention may be used in combination with an additional medicament or therapy. For instance, prevention and/or treatment of FOP using a fusion protein of the invention may also include simultaneous or sequential administration or application of an additional medicament or therapy of FOP. The additional medicament or therapy may include but not be limited to prevention of a secondary damage, modulation of local functionality, anti-inflammation, administration of glucocorticoids, non-steroidal anti-inflammatory drugs NSAID, bisphosphonates, and/or rosiglitazone and radiotherapy. For instance, prevention and/or treatment of cancers associated with ACVR1 mutation and/or over-activation using a fusion protein of the invention may also include simultaneous or sequential administration or application of an additional medicament or therapy of FOP, wherein the additional medicament or therapy may include but not be limited to radiotherapy, chemotherapy, surgery, etc.
[0193] Advantages
[0194] The present disclosure provides solutions to problems such as difficulty in obtaining effective expression of a recombinant protein, lack of high-expression stains, incorrect configuration and/or conformation leading to insolubility or aggregation or loss in biological activity. The present invention, in one aspect, provides a recombinant protein molecule comprising extracellular domain of a receptor, which allows for effective expression in mammalian cells.
[0195] The fusion proteins of invention have the advantages including stable expression, high yield, easiness in purification and highly biologically active, and can be advantageously used to prevent and/or treat diseases and/or conditions associated with ACVR1 abnormality (e.g. mutation and/or over-activation). For instance, the fusion proteins of invention can effectively inhibit osteogenic differentiation and chondrogenic differentiation, and are thus useful in prevention and treatment of pleonosteosis-associated diseases or condition. In addition, the fusion proteins of the invention are also capable of inhibiting genesis, development and metastasis of tumors (e.g. ovarian cancer) associated with ACVR1 mutation and/or over-activation.
EXAMPLES
[0196] The invention would be further illustrated by referring to the following examples. It should be understood that the examples are provided only for the purpose of illustration, rather than limiting scope of the invention. Various forms of modification and variation would be obvious to the skilled in the art, which are all included in scope of the invention.
[0197] In the following Examples, unless otherwise specified, conventional conditions and methods are used, for example, as taught in Molecular CloningA Laboratory Manual (3.sup.rd version, Cold Spring Harbor Laboratory Press, New York, 1989) or as taught in the manufacturer's instruction. DNA sequencing can be performed using conventional means or by a commercial institute.
[0198] Unless otherwise specified, all the percentages and parts are calculated on a weight basis. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Particularly, the materials and methods as specified are exemplary, and various equivalents can be used.
Example 1
Construction of Expression Plasmid of the Fusion Protein
[0199] The ACVR1-Fc expression gene consists of three fragments (
[0200] Fragment 1: the signal peptide sequence of protein CD33 at the 5 terminus (the coding sequence is as set forth by SEQ ID NO: 1 and the corresponding amino acid sequence is as set forth by SEQ ID NO: 2);
[0201] Fragment 2: expression gene of the ACVR1 extracellular domain (amino acids 21-123) in the middle (the coding sequence is as set forth by SEQ ID NO: 3 and the corresponding amino acid sequence is as set forth by SEQ ID NO: 4); and
[0202] Fragment 3: the sequence encoding the amino acid sequence of human IgG 1 at the 3 terminus (the coding sequence is as set forth by SEQ ID NO: 5 and the corresponding amino acid sequence is as set forth by SEQ ID NO: 6), which encodes the fragment spanning amino acid residues 216 to 447 of human IgG 1, including the hinge region, the CH2 region and the CH3 region (i.e., hinge+CH2+CH3).
[0203] The three genes were individually produced by polymerase chain reaction (PCR), and then assembled via overlap extension PCR. The polymerase chain reaction was conducted using high-fidelity polymerase Plantium pfx (Invitrogen). The PCR condition was designed by following the manufacturer's instruction and adapting to the selected type of reaction. The PCR fragments were purified using the gel DNA fragment kit (Qiagen).
[0204] Synthesis of Fragment 1
[0205] The template for PCR amplification of Fragment 1 comprised the nucleotide sequence (SEQ ID NO: 1) coding for the 16-amino acid signal peptide of CD33 protein.
[0206] The 5-primer CMV-P comes from the plasmid vector, which has the sequence of
TABLE-US-00001 (SEQIDNO:9) 5-CGCAAATGGGCGGTAGGCGTG-3;
and
[0207] The 3-primer SP-3 has the sequence of 5-AGCCAGGGCCCCTGCC-3 (SEQ ID NO: 10).
[0208] Synthesis of the cDNA of Fragment 2
[0209] The template plasmid for PCR amplification of Fragment 2 comprised the full-length gene of ACVR1s extracellular domain (SEQ ID NO: 3).
[0210] The 5-primer ACVR1-5 has the sequence of
TABLE-US-00002 (SEQIDNO:11) 5-GGGCAGGGGCCCTGGCTATGGAGGACGAGAAGCCC-3.
[0211] A seventeen-ribonucleotide sequence complementary to the 3 terminus of Fragment 1 was added at the 5 terminus to facilitate the assembly with Fragment 1.
[0212] The 3-primer ACVR1-3 has the sequence of
TABLE-US-00003 (SEQIDNO:12) 5-TATCACAGCTCTTGGGCTCCTCCAGGTGGAAGTTCTGGG-3.
[0213] Similarly, a nineteen-ribonucleotide sequence complementary to the 5 terminus of Fragment 3 was added at the 3 terminus to facilitate the assembly with Fragment 3.
[0214] Synthesis of the cDNA of Fragment 3
[0215] The template plasmid for PCR amplification of Fragment 3 comprised the gene coding for the amino acid sequence (aa 216 to 447) of human IgG 1 Fc (SEQ ID NO: 5).
[0216] The 5-primer Fc-5 has the sequence of 5-GAGCCCAAGAGCTGTGATA-3 (SEQ ID NO: 13), which is complementary to the 3 terminus sequence of the cDNA of Fragment 2.
[0217] The 3-primer BGH-R has the sequence of
TABLE-US-00004 (SEQIDNO:14) 5-AACTAGAAGGCACAGTCGAGGC-3.
[0218] Assembly of the Fragments
[0219] Overlap extension PCR was conducted to link the cDNAs of Fragments 1 and 2. The two fragments were purified and used as templates, and the 5-prime CMV-P for Fragment 1 and the 3-primer ACVR1-3 for Fragment 2 were used for the PCR. The assembled PCR fragment was then linked to Fragment 3, using CMV-P and BGH-R as the primers.
[0220] Construction of the Recombinant Plasmid
[0221] The obtained PCR fragment was treated with restriction endonucleases NotI and XbaI. The ACVR1-Fc expression gene fragment was cloned into pcDNA3.1(Invitrogen), an expression vector effective in mammalian cells, using T4 DNA ligase. The anti-neomycin gene in pcDNA3.1 was replaced by the DHFR (dihydrofolate reductase) gene, and the modified vector is useful to screen for stably transformed mammalian cells. The recombinant plasmid was tranfected into DH5a competent cells. Clones positive for correct recombinant plasmids was identified by colony PCR. The recombinant plasmids were purified and digested, and correct sequence of the recominant gene was verified by sequencing.
Example 2
Construction of Expression Strain for the Fusion Protein
[0222] Host cell CHO DG44 (Item No. 12609-012, Invitrogen, USA) was cultivated through passages by following the manufacturer's instruction for CHO DG44. Non-transfected cells were cultivated in suspension in a CD DG44 culture medium (Invitrogen) supplemented with 8 mM L-glutamine and 5 g/ml recombinant human insulin.
[0223] A CHO DG44 cell line capable of stable and high expression of the protein was constructed by stable transfection. Thus cloned CHO DG44 cells were cultivated in suspension in a culture medium free of serum and animal proteins.
[0224] The cell line stably expressing the fusion protein was constructed using the methods and steps as described below. Vector plasmids expressing the fusion protein were prepared using the Plasmid Maxi Preparation Kit from TianGen. The plasmids (100 g) were treated with the restriction endonuclease Puvl to give linearized plasmids. The DG44 cells were cultivated through at least three passages before being transfected by the expression vector plasmid. The DG44 cells (110.sup.7 cells in total) were mixed with the digested plasmids in CD DG44 growth medium (0.8 ml), then transferred into a 0.4-cm pulsing cup (Bio-Rad). The cell/plasmid mixture was pulsed using a gene pulser (Bio-Rad, Gene Pulser Xcell). The transfected cells were cultivated in cell culture flask T-75 containing 20 ml growth medium. The flask T-75 with transfected cells was maintained in an incubator at 37 C., 8% CO.sub.2 for 24 hours.
[0225] Transformants were selected by limiting dilution on a 96-well plate. The selection medium was OptiCHO comprising 8 mM L-glutamine, 5 g/ml recombinant human insulin and 100 nM methotrexate (MTX). The cells were cultivated in an incubator at 37 C., 8% CO.sub.2. Three weeks later, the culture fluid in each well positive for clone formation was analyzed by ELISA (alkaline phosphatase-conjugated goat-anti-human IgG Fc antibody, Jackson ImmuneResearch). The clones with high expression level of the protein were amplified and again, analyzed by ELISA. The amplification and analysis were repeated until stable high expression strains were obtained.
Example 3
Protein A Affinity Chromatography and HPLC-SEC of the Fusion Protein
[0226] The ACVR1-Fc fusion protein was purified from the supernate of the culture of stable expression cells using protein A affinity column according to the standard protocol (POROS, Mabcapture A). The purified protein was analyzed using reductive and non-reductive SDS-PAGE electrophoreses, as well as HPLC-SEC (high pressure liquidmolecule sieve).
Example 4a
ELISA Assay of the Fusion Protein's Binding to BMP-2 Protein In Vitro
[0227] In a 50 mM NaCO.sub.3 solution, 3.5 M recombinant human BMP-2 protein (Item No. 10426-HNAE, Sino Biological Inc. China) was dissolved. Aliquots (50 l) of the BMP-2 protein solution were added onto a 96-well ELISA plate, and kept in refrigerator at 4 C. overnight. The next day, the ELISA plate was washed three times with TBST, before addition of 100 l/well TBST with 3% BSA as blocking solution. The same number of blank wells, to which 100 l of the blocking solution was added, were prepared to detect non-specific binding of ACVR1-Fc. The ELISA plate was placed in thermostat at 37 C. for 1 hr.
[0228] Dilutions of the fusion protein (3 serial dilution) were prepared in binding solution of TBST with 1% BSA. The blocking solution was decanted, and 50 l/well of the 3 serial dilution of the fusion protein was added. The plate was placed in thermostat at 37 C. for 1 hr. After the fusion protein solution was decanted, the ELISA plate was washed three times with TBST, and then 50 l/well of the secondary antibody (alkaline phosphatase-conjugated goat-anti-human IgG Fc antibody, Jackson ImmuneResearch) was added to react in thremostat at 37 C. for 1 hr. The developing antibody was removed, and to the ELISA plate, 200 l/well TBST wash solution was added. The plate was then placed on a horizontal rotator at 100 rpm for 5 minutes, after which, the wash solution was decanted. The process was repeated five times. To the ELISA plate, 50 l/well antibody developing solution (PNPP) was added. The ELISA plate was placed in thermostat at 37 C., and was read at 405 nm.
Example 4b
ELISA Assay of the Fusion Protein's Binding to other BMP/TG Family Members In Vitro
[0229] The recombinant human Activin A (Item no. 120-14E), BMP-5(Item no. 120-39), BMP-6 (Item no. 120-06) and BMP-7(Item no. 120-03) were all obtained from Peprotech (USA). These proteins are separately dissolved in a solution of NaCO.sub.3 (20 mM, pH 9.6) to a concentration of 2 g/ml. To a 96-well ELISA plate (Maxisorp, Nunc), 50 l/well of the protein solution was added. The plate was then placed in refrigerator at 4 C. overnight. The next day, the ELISA plate was washed three times with PBST (PBS with 0.05% Tween-20), and then to the plate, 100 l/well PBST (with 3% BSA) was added as blocking solution. The same number of blank wells, to which 100 l of the blocking solution was added, were prepared to detect non-specific binding of ACVR1-Fc.
[0230] The ELISA plate was placed in thremostat at 37 C. for 2hr. Dilutions of the fusion protein (3 serial dilutions) were prepared in binding solution of PBST with 1% BSA. The blocking solution was decanted, and 50 l/well of the 3 serial dilutions of the fusion protein was added. The plate was placed in thermostat at 37 C. for 2 hr. After the fusion protein solution was detanted, the ELISA plate was washed three times with PBST. Then, to the plate, 50 l/well of the 3000 diluted secondary antibody (alkaline phosphatase-conjugated goat-anti-human IgG Fc antibody, Jackson ImmuneResearch) was added to react in thremostat at 37 C. for 2 hr. After the developing antibody was decanted, to the ELISA plate 200 l/well PBST wash solution was added. The plate was placed on a horizontal rotator at 100 rpm for 5 minutes, after which, the wash solution was decanted. The process was repeated five times. To the ELISA plate, 50 l/well antibody developing solution (PNPP) was added. The plate was placed in thermostat at 37 C., and was read on a microplate reader (iMax, Bio-rad) at 405 nm and 490 nm.
Example 5
Construction of the Recombinant Adenovirus Vector
[0231] The ACVR1 gene was cut off from the vector Sport-ACVR1 (human) (Invitrogen) using restriction endonucleases Small and XhoI. G to A shift was made at position 617 in the obtained ACVR1 gene fragment using site-directed mutagenesis to obtain the ACVR1(M). The obtained ACVR1(M) fragment was cloned into plasmid pIRES2-EGFP (Invitrogen), which was then incorporated into pMD18-T simple vector (Takara) to obtain the recombinant plasmid. The obtained recombinant plasmid was packaged into pAd CMV/V5-DEST (Invitrogen) to construct the recombinant adenovirus of ACVR1(M)-IRES-GFP. The obtained virus vector was verified by DNA sequencing, and protein expression was verified by green fluorescence from GFP (
Example 6
Induction of Osteogenic Differentiation and Chondrogenic Differentiation in HUVECs Infected with the Recombinant Virus
[0232] HUVECs (ATCC, CRL-1739) were maintained in EGM medium (Lonza, CC-3162) supplemented with 10% FBS (Gibco, 10099-141) and 1% penicillin-streptomycin (Gibco 15070-063). The cells were starved for 24 hr in Human Endothelial-Serum Free Medium (Gibco, 11111-044) supplemented with 2%FBS, 1% penicillin and streptomycin, as well as two growth factors, i.e., EGF (final concentration: 10 ng/ml) and bFGF (final concentration: 20 ng/ml), before being infected with the recombinant virus. Then, the ACVR1(M)-IRES-GFP adenovirus was added into the cells (MOI: 200, see
Example 7
Evaluation of the Degree of Osteoblast Differentiation in the HUVEC ModelAlkaline Phosphatase Staining and Alizarin Red S Staining
[0233] In order to evaluate the degree of osteoblast differentiation in the HUVEC model obtained in Example 6, at day 7 and day 21 of the osteogenesis progress, the cells were stained with alkaline phosphatase (ALP) and Alizarin Red (Salizarin Red S). At day 7 of the incubation, the cells were washed three times with PBS, and then fixed with 4% formaldehyde. The cells were washed with PBS, before the substrate working solution was added, and the cells were then cultivated in dark for 30 minutes. At the end, the cells were washed with water, then examined and photographed under bright field microscope.
[0234] At day 21 of the osteogenic differentiation incubation, calcification of the cellular matrix was evaluated by Alizarin Red staining. As in the case of alkaline phosphatase staining, the cells were washed with PBS, fixed with 4% formaldehyde, washed with PBS again, and stained with 1% Alizarin Red S staining solution (pH 4.2) for 10 minutes. The cells were examined and photographed under bright field microscope.
Example 8
Evaluation of the HUVEC Model's Potential of Chondrogenic DifferentiationAlcian Blue Staining
[0235] At day 14 of the chondrogenic differentiation according to Example 6, the cells were harvested and washed three times with PBS, and fixed with 4% formaldehyde. The cells were washed three times again with PBS, and 0.3% Alcian Blue Stain 8GX (Sigma) was added for co-incubation. The cellular model's potential of chondrogenic differentiation was evaluated by detecting sulfated proteoglycan.
Example 9
Atomic Absorption Analysis of Calcium
[0236] Osteogenesis was induced on a 6-well plate by following the procedure of Example 6. At day 21 of the incubation, the cells were harvested, and calcium levels determined by atomic absorption were compared between cells.
[0237] The cells were washed three times with PBS (calcium- and magnesium-free), and then 1 ml lysis solution (0.1% Triton X-100, 10 mM Tris, pH 7.5) was added. The cells were decalcified using 11.6 N HCl at room temperature for 16 hr to release calcium as possible. The lysate was pipetted into a 1.5 ml Eppendoff, then centrifuged at 6000 rpm for 10 minutes. The supernate was collected and evaluated for calcium level by atomic emission spectrometer (Agilent, 7200).
Example 10
Levels of Osteogenic Differentiation Marker Proteins and Chondrogenic Differentiation Marker ProteinsWestern Blotting
[0238] Western blotting was conducted to detect levels of osteogenic differentiation marker proteins and chondrogenic differentiation marker proteins, also the phosphorylation of BMP-Smad1/5/8. The cells were treated with RIPA lysis solution (containing the proteinase inhibitor PMSF), and supernate was collected via centrifugation at 13000 rpm for 5 minutes. Protein content in the supernate was determined by BCA assay. Lysates containing equivalent amounts of proteins were subjected to separation by SDS-PAGE electrophoresis. The electrophoresis was transferred onto the PVDF film, and the proteins were detected by Western blotting. The primary antibodies were specific for the marker proteins or phosphorylation of the proteins. The antibodies specific for the osteogenesis markers and the chondrogenesis markers were obtained from Abcam. The antibodies specific for the proteins in signaling pathway and for phosphorylation were obtained from Cell Signaling Technology. The secondary antibody was peroxidase-conjugated goat-anti-rabbit or goat-anti-mouse IgG (Jackson Immunoresearch Laboratories). Detection of the proteins was visualized by ECL Plus (Millipore).
[0239] Results obtained in Examples 1 to 10 are as discussed below:
[0240] Result 1: Identification of CHO DG44 as a Stable Expression Cell Line for ACVR1-Fc
[0241] By following the procedure of Example 2, a stable high expression strain for ACVR1-Fc was obtained, which provided an expression at the level of 600 mg/L.
[0242] The fusion protein was purified through a protein A affinity column and then analyzed by reductive and non-reductive SDS-PAGE electrophoreses as described in Example 3. As shown in
[0243] HPLC-SEC also indicated a MW around 80 kDa (
[0244] Result 2a: ACVR1-Fc Binding to BMP-2 Protein In Vitro
[0245] It has been shown that ACVR1 binds to bone morphogenetic protein-2(BMP-2). The ELISA in Example 4a indicates (
[0246] Result 2b: ACVR1-Fc Binding to other BMP/TGF Family Members In Vitro
[0247] It has been shown that, several bone morphogenetic proteins, including BMP-2, BMP-5, BMP-6 and BMP-7, bind to ACVR1, resulting in activation of the BMP signaling pathway. The ELISA in Example 4b (
[0248] The results indicate that the fusion protein of invention is capable of binding to different BMP/TGF family members, with differential capacity and affinity.
[0249] Up till now, there has only been reported that the protein complex ACVR-1/ACVR-2 binds to the members, like BMP-2, to effect the signaling. There has not been any report about whether ACVR-1 protein by itself is capable of binding to any BMP/TGFb proteins in vitro or in vivo. We find, for the first time, that ACVR-1 by it self is capable of binding to a BMP/TGFb proteins. Further, the in vitro biological activity data as provided suggests that the fusion protein of the invention is capable of inhibiting osteogenesis in cells carrying mutation(s) in ACVR-1. This also supports that the ACVR1-Fc protein of the invention binds to multiple BMP/TGFb proteins and has physiological effects.
[0250] Result 3: Construction of Osteogenic Differentiation Model in HUVEC
[0251] It has been shown that ACVR1 R206H is expressed in epithelia and induces the cells' transformation into stem-like cells, which are then capable of differentiating into osteoblasts and chondrocytes. In the present study, we reconstructed the HUVEC system to investigate the capability of the ACVR-1 fusion protein as a potential therapeutic to inhibit osteogenesis and chondrogenesis and to inhibit phosphorylation of the cells involved in cartilage formation and the BMP signaling pathway.
[0252] As shown in
[0253] Staining of differentiated cells expressing ACVR1 R206H using alkaline phosphatase, Alizarin Red S and Alcian Blue are as shown in
[0254] Result 4: Study of ACVR1-Fc's Inhibition on Osteogenic Differentiation and Chondrogenic Differentiation
[0255] The alkaline phosphatase staining, the Alizarin Red S staining and the Alcian Blue staining (results shown in
[0256] As shown by
[0257] As seen in
[0258] Calcium ion level in intercellular matrix increases during osteoblast differentiation. At day 21, the concentration of calcium ion in intercellular matrix is determined via atomic absorption, as described in Example 9. As shown in
[0259] Result 5: Evaluation of Osteogenic Differentiation Marker Proteins
[0260] By following the procedure in Example 10, Western blotting was conducted to investigate effects the ACVR1-Fc protein has on expression of the markers of osteoblasts. The result is shown in
[0261] Result 6: Phosphorylation of the Proteins Involved in the Osteogenic Differentiation Signaling Pathway
[0262] The protein Smad-1/5/8 and the p38 MAPK signaling pathways are involved in osteogenic differentiation and chondrogenic differentiation. Accordingly, we evaluated whether ACVR1-Fc influences phosphorylation of protein Smad-1/5/8 and phosphorylation in the p38 MAPK pathway. Results are shown in
[0263] As shown in
[0264] All documents identified in this disclosure are incorporated in their entirety herein by reference and as each is individually and specifically cited. In view of the description above, those of ordinary skill in the art will appreciate that various substitutions and/or other alterations may be made to the embodiments without departing from the spirit and scope of the invention as defined by the claims.
APPEDIX 1. Brief Description of Sequence Identity
[0265]
TABLE-US-00005 SEQ ID NO: Identity 1 coding sequence of Fragment 1 (signal peptide of CD33) 2 amino acid sequence of Fragment 1 3 coding sequence of fragment 2 (the extracellular domain of ACVR1) 4 amino acid sequence of Fragment 2 5 coding sequence of Fragment 3(human IgG 1) 6 amino acid sequence of Fragment 3 7 coding sequence of ACVR1-Fc fusion protein 8 amino acid sequence of CVR1-Fc fusion protein 9 5-primer for Fragment 1, CMV-P 10 3-primer for Fragment 1, SP-3 11 5-primer for Fragment 2, ACVR1-5 12 3-primer for Fragment 2, ACVR1-3 13 5-primer for Fragment 3, Fc-5 14 3-primer for Fragment 3, BGH-R 15 full-length amino acid sequence of ACVR1
APPEDIX 2. References
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