Recombinant CCN domain proteins and fusion proteins

Abstract

The present invention relates to a recombinant protein having a thrombospondin type 1 repeat homology domain corresponding to a CCN family protein member or an amino acid sequence related thereto and its use. In addition, the present invention relates to a fusion protein comprising a thrombospondin type 1 repeat homology domain corresponding to a CCN family protein member or an amino acid sequence related thereto in combination with a fusion partner and an optional linker region. In addition, a novel protease-resistant Fc fragment is disclosed herein.

Description

Technical Field

[0001] The present invention relates to a recombinant protein having a thrombospondin type 1 repeat homology domain (domain III) corresponding to a CCN family protein member or an amino acid sequence related thereto and its use, in particular including such a protein that is truncated and / or comprises certain amino acid modifications. In addition, the present invention relates to such a fusion protein, which comprises a thrombospondin type 1 repeat homology domain corresponding to a CCN family protein member or an amino acid sequence related thereto, optionally combined with a fusion partner via a linker peptide. In particular, the fusion partner is a monomeric protein, and the fusion protein is monomeric.

[0002] Furthermore, disclosed herein are novel protease-resistant Fc fragments. Background Art

[0003] CCN proteins are one of a family of secreted glycoproteins.CCN was originally coined as an acronym derived from the first three identified members of the gene family; C yr61, C TGF and N OV. However, the acronym was recently adapted to an abbreviation for Cellular Communication Network factor and approved by the HUGO Gene Nomenclature Committee (Perbal B, Tweedie S and Bruford E, J Cell Commun Signal. 2019 Sep; 13 (3): 435). The protein is generally classified as a matricellular protein associated with the extracellular matrix (ECM). CCN proteins are not part of the scaffolding function of organizing cells into tissues, but are considered to be signaling proteins and can be used as independent autocrine or paracrine signaling factors, or as modifiers of other extracellular signaling proteins. Together with a group of three Wnt-inducible signaling pathway proteins (WISP1 / CCN4, WISP2 / CCN5, and WISP3 / CCN6), they contain a family of six homologous cysteine-rich proteins in mammals, which have been renamed CCN1 to 6.

[0004] The original members of the CCN family share a modular structure with an N-terminal peptide signal for secretion followed by four conserved domains. The first domain shows sequence homology with insulin-like growth factor binding protein (IGFBP) and is therefore called the IGF binding protein homology domain, but has only negligible affinity for IGF. The second domain is called the von Willebrand factor C-type repeat (VWC) homology domain, which is commonly found in extracellular matrix (ECM) proteins. The third domain is called the platelet response protein type I repeat homology domain (TSP-1), which can be involved in the connection of CCN proteins to integrins. The fourth domain is the cysteine-rich C-terminal repeat or cystine knot homology domain, which is the domain reported to bind heparin. The fifth member of the CCN protein family, WISP2 (Wnt1-inducible signaling pathway protein 2) (also known as CCN5) is atypical in lacking a carboxy-terminal cystine knot domain (domain 4). The TSP-1 homology domains of the CCN protein family share 34% amino acid sequence identity and 25% sequence similarity (as analyzed by ClustalOmega, see below for reference). The four-domain CCN proteins contain 38 conserved cysteines throughout the primary sequence, with the exception of CCN6, where 4 cysteines of the VWC homology domain are not conserved in other family members. In addition, for CCN5, which lacks a carboxy-terminal cystine knot homology domain, all cysteines of the IGFBP, VWC, and TSP-1 homology domains are conserved compared to other CCN family members.

[0005] A non-conserved, protease-sensitive central region, often called the hinge region, bisects the protein. The expression of CCN proteins is regulated at the transcriptional, post-transcriptional, and translational levels in response to changes in environmental stimuli.

[0006] Information on the domain organization of the CCN protein family can be found, for example, in Liu et al, 2017, Journal of Diabetes, 9, pp. 462-474.

[0007] At the cellular level, CCN proteins can have multiple regulatory effects at the interface of the extracellular matrix and the cell surface. CCN proteins can regulate cell adhesion, migration, proliferation, differentiation, apoptosis, survival, aging and gene expression. By regulating one or more aspects of these cell functions in a cell type-specific manner, CCN coordinates complex biological processes, including cardiovascular and bone development during embryogenesis, and inflammation, wound healing and tissue damage and repair in adults. In general, the 4 domains CCN 1 to 4 and CCN6 (particularly CCN1, CCN2 and CCN4) can exert profibrotic activity, but CCN5, which only contains 3 domains I to III, has antifibrotic activity.

[0008] CCN proteins are also involved in a wide variety of pathological conditions, such as organ failure due to progressive fibrosis, such as liver fibrosis and idiopathic pulmonary fibrosis, and in cancer invasion and metastasis. Reference is made in this regard to Jun and Lau, 2011, Nat. Rev. Drug. Discovery, 10 (12), pp. 945-963. Similarly, in general, it has been shown that 4-domain CCN proteins, especially CCN2, are involved in the mechanisms of various fibrotic diseases, however, in preclinical disease models of such diseases, it has been shown that increased levels of CCN5 can be beneficial.

[0009] exist et al., J. Biol. Chem, 293:46, pp.17953-17970, reported that connective tissue growth factor (CTGF) (also known as CCN2) is synthesized and secreted as an inactive preprotein that requires proteolytic cleavage to release the biologically active CCN2, and that homodimers of the C-terminal fragment containing domains III to IV represent the biologically fully active form of CCN2, and finally, all major reported activities of CCN2 can be summarized by the homodimer of the C-terminal domain III to IV fragment. The activity assay reported by et al. revealed that both the unprocessed full-length CCN2 and the N-terminal fragment containing domains I to II were biologically inactive. In addition, it was found that proteolytic processing of full-length CCN2 by matrix metalloproteinase (MMP) activity released its potential activity. The findings reported by et al. imply that prepro-CCN2 is autoinhibited by N-terminal domains I and II. It has also been found that C-terminal domains III and IV fragments of CCN1 and CCN3 are sufficient to activate rapid cell signaling and elicit cellular physiological responses. However, the extent to which endopeptidase cleavage of the hinge region of CCN1 and / or CCN3 or any other CCN protein family member is required to release biological activity is unknown.

[0010] CCN2 is known to be highly expressed during development, in a variety of pathological conditions involving enhanced fibrogenesis and tissue fibrosis, and in several cancers (Jun and Lau, 2011, supra). The fact that CCN proteins are involved in a wide variety of pathological conditions, are extracellular proteins that are mechanistically involved in the development of fibrosis, and show limited expression in healthy organisms, makes them attractive therapeutic targets.

[0011] Jeong et al., 2016, J. American College of Cardiology, 67: 13, pp. 1557-1568 reported a study that examined the effects of adeno-associated virus-mediated CCN5 gene transfer to the mouse heart after experimentally induced pressure overload. The study concluded that CCN5 can reverse established cardiac fibrosis by inhibiting the production of myofibroblasts and enhancing their apoptosis in the myocardium, indicating that CCN5 may provide a platform for the development of anti-cardiac fibrosis treatments.

[0012] In US2008 / 0207489, methods for treating smooth muscle proliferation-based disorders are disclosed, which comprise the expression of CCN5 or the administration of CCN5 protein to smooth muscle cells.

[0013] In EP 2 556 839, compositions comprising genetic vectors containing nucleotide sequences encoding full-length CCN5 or CCN2ΔCT are provided and their use in the treatment of heart failure is suggested. CCN2ΔCT in EP 2 556 839 is defined as the amino acid sequence of CCN2 truncated after K251 (uniprot numbering).

[0014] Although it has been reported in some experimental systems that overexpression of CCN5 leads to a phenotype opposite to that of overexpression of CCN2 (Jeong et al., supra, Yoon et al., J Mol Cell Cardiol, 49(2), 294-303 Aug 2010), to the best of the authors' knowledge, direct antagonism of CCN5 against four-domain CCN proteins has not been reported. In particular, the structural basis for CCN5 / WISP2-mediated antagonism of other CCN family members was unknown prior to the work presented in the present invention.

[0015] The present inventors have shown at an earlier stage that full-length CCN2 (FL-CCN2) is a preproprotein, an inactive precursor, and that fragments comprising domains III and IV were shown to convey all biologically relevant activities of CCN2. To what extent CCN proteins in general are secreted as inactive preproproteins that require proteolytic activation remains unknown. However, the susceptibility of full-length CCN proteins to a variety of proteases, as shown by the present inventors ( et al., J. Biol. Chem. (2018) 293 (46) 17953-17970) and others (Butler, G Set al. Matrix Biol 59, 23-38 (2017) and Guillon-Munos, A. et al. J Biol Chem 286, 25505-25518 (2011)), suggesting that the unmodified full-length CCN protein would be highly unsuitable as a drug due to stability issues both during recombinant protein manufacturing and after in vivo administration. This unsuitability of using unmodified full-length CCN proteins as therapeutic proteins also applies to fusion proteins of full-length CCN proteins, such as described for full-length CCN1 (Schutze, N. et al. (2005) Protein Expr Purif 42, 219-225) and full-length CCN6 (Schutze, N et al. (2007) BMC Cell Biol 8, 45). It is well known in the field of CCN proteins that the susceptibility of these proteins to proteolysis is one of the reasons why it is difficult to produce recombinant CCN proteins. In addition, based on et al. (J Biol Chem 2018; 293(46): 17953-17970), recombinant full-length CCN protein can be a far from ideal biopharmaceutical because its activity can depend on prior proteolytic processing, making it unpredictable in pharmacokinetics and pharmacodynamics. In addition, in the case of Fc fusion proteins, in addition to the proteolytic susceptibility of the components (e.g., peptide linkers, CCN fragments, and Fc fragments), the arrangement of the components has also been shown to be important for the potency and efficacy of the recombinant fusion protein. One example is the paper published by the inventors ( et al. (2018)), where variants of an Fc fusion protein containing domains III to IV of CCN2 were found to have widely varying activities in a manner that was not easily predicted a priori.

[0016] The effects of CCN proteins are reported to be susceptible to the effects of high concentrations of synthetic peptides that antagonize the primary sequence of CCN proteins. One example is the inhibition of AKT phosphorylation stimulated by recombinant CCN2 in Rat2 fibroblasts by peptides derived from domain I (IGFBP homology domain) of CCN2 and, to a lesser extent, domain III (TSP-1 repeat homology domain) of CCN2 (Moe et al., J. Cell Commun. Signal. (2013) 7: 31-47). Another example is the inhibition of CCN2 (domain IV)-stimulated adhesion of hepatic stellate cells by peptides derived from domain IV (cystine knot homology domain) of CCN2 (Gao R and Brigstock DR., J Biol Chem. 2004 Mar 5; 279 (10): 8848-55). In addition, peptides from domain III of CCN1 (Leu et al. J. Biol. Chem, 2003, Vol. 278, No. 36, Issue of September 5, pp. 33801-33808, 2003) and peptides from domain III of CCN1, CCN2, CCN3, CCN5 and CCN6 (Karagiannis EG and Popel The International Journal of Biochemistry & Cell Biology 39 (2007) 2314-2323) have been reported to have some anti-angiogenic effects in in vitro assays with HUVEC cells (Leu et al. J. Biol. Chem, 2003 and Karagiannis EG and Popel, Int J Biochem Cell Biol 39 (2007)) and anti-adhesion effects on 1064SK human foreskin fibroblasts (Leu et al. J. Biol. Chem, 2003 and Karagiannis EG and Popel, Int J Biochem Cell Biol 39 (2007)). et al. J. Biol. Chem, 2003), these peptides contain only one conserved cysteine ​​(Leu et al. J. Biol. Chem, 2003) or two conserved cysteines (Karagiannis EG and Popel, Int J Biochem Cell Biol 39, 2007), which are the core of the invention described in this document. It is known that the cysteines in domain III of CCN proteins will generate disulfide bridges, such as CCN2 endogenously expressed from HUVEC cells (Lu, S et al. (2015) Nat methods 12, 329-331) and from purified recombinant CCN2 ( et al., J.Biol.Chem.2018). The disulfide bridge across C199 to C228 (uniprot numbering) shown in CCN2 confers a complex 3D structure in which the amino acid chain folds toward itself. This means that the complete domain III of the CCN protein cannot be expected to be replicated by a short peptide that is not structurally restricted by the disulfide bridge between cysteines, as in the complete domain III of the CCN protein produced in the eukaryotic system. In addition, the inhibition of CCN2 activity by peptides derived from the primary sequences of domains I, III, and IV illustrates the lack of knowledge in the art about whether peptides derived from specific domains of CCN2 can confer inhibition on the four-domain CCN protein.

[0017] The present inventors have now discovered, based on structure-activity analysis of CCN family proteins and the observation that CCN2 requires proteolytic processing to become biologically active, that the biologically active portion of the CCN5 protein is domain III, the thrombospondin type I repeat homology domain. This new knowledge has led to the provision of biologically active structures based on domain III of CCN5 as well as domain III of other members of the CCN protein family. SUMMARY OF THE INVENTION

[0019] The inventors' insight into the structure-activity relationship of CCN5 and other CCN proteins has led to the provision of novel biologically active recombinant proteins that recapitulate cell signaling and cell physiological functions attributed to CCN5 signaling, and which can also antagonize other four-domain CCN proteins (Cyr61 (also known as CCN1), CTGF (also known as CCN2), NOV (also known as CCN3), WISP1 (also known as CCN4) and WISP3 (also known as CCN6)). In other words, a protein in the form of a fusion protein comprising domain III (TSP-1 homology domain) based on a CCN protein is provided, which recapitulates the biological activity of CCN5 or has the biological activity of CCN5, and which is able to antagonize or inhibit the effects of the four-domain CCN proteins CCN1 to 4 or CCN6. In particular, the proteins herein have anti-fibrotic activity and may also have direct anti-cancer activity.

[0020] As described above, Domain III (TSP-1 homology domain) of other CCN proteins (i.e. 4-domain CCN proteins) was unexpectedly found to be sufficient to recapitulate the activity reported for CCN5 when provided as a separate domain in the absence of other CCN domains. Thus, in other words, Domain III of a 4-domain CCN protein has the same activity as CCN5 when provided as a separate domain (i.e. as an isolated domain) in the absence of other CCN domains, or alternatively, expressed as Domain III / TSP-1 homology domain of CNN5 (which is an activity that is the opposite of the activity of the full-length 4-domain CCN protein). Thus, based on the experiments disclosed in this document, it is clear that an isolated TSP-1 homology domain of any CCN protein can exert the same activity as the activity of the TSP-1 homology domain of CCN5. Except that in the case of CCN5, this may be different from the activity exerted by the full-length CCN protein.

[0021] It has been discovered that, in accordance with the present invention and disclosure herein, monomeric fusion proteins in which domain III of a CCN protein is fused to a monomeric fusion partner have particular benefits and utility.

[0022] According to a first aspect, the present invention provides a monomeric fusion protein comprising:

[0023] (i) a polypeptide corresponding to at least a portion of the thrombospondin type 1 repeat (TSP-1) homology domain of a CCN family protein;

[0024] (ii) a monomeric fusion partner fused to the amino acid sequence of (i) at the N-terminus or C-terminus; and

[0025] (iii) optionally a peptide linker between the polypeptide of (i) and the monomeric fusion partner of (ii),

[0026] wherein the polypeptide of (i) is 40 to 60 amino acids in length and comprises: an amino acid sequence selected from SEQ ID NO: 37 or 2 to 6, or a sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 37 or 2 to 6, wherein all cysteine ​​residues in the sequence selected from SEQ ID NO: 37 or 2 to 6 are conserved,

[0027] And wherein the monomeric fusion partner of (ii) and the peptide linker of (iii) are not or do not comprise the following: an IGF binding protein homology domain of a CCN family protein, a von Willebrand factor C-type repeat homology domain or a cysteine ​​knot domain.

[0028] As will be described in more detail below, SEQ ID NO.37 and 2 to 6 represent 44 amino acid truncated fragments of domain III of CCN5, CCN3, CCN2, CCN1, CCN4 and CCN6, respectively, which contain 6 conserved cysteine ​​residues of the domain. In particular, the flanks of the fragment are the first and last cysteine ​​residues of the domain. Such fragments have been found to be particularly effective and resistant to proteolytic degradation.

[0029] In one embodiment, the polypeptide of (i) comprises or consists of:

[0030] (a) an amino acid sequence selected from SEQ ID NO: 1 or 8 to 12; or

[0031] (b) an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 1 or 8 to 12; or

[0032] (c) a portion of the amino acid sequence of (a) or (b), wherein the portion comprises at least 44 amino acid sequences of SEQ ID NO: 37, 6, 2, 3, 4 or 5, respectively, or comprises a sequence having at least 80% sequence identity with a sequence selected from SEQ ID NO: 37, 6, 2, 3, 4 or 5, respectively.

[0033] SEQ ID NO. 1 and 8 to 12 represent slightly longer N-terminal truncated fragments of domain III of CCN5, CCN6, CCN3, CCN2, CCN1 and CCN4, respectively. These fragments contain the corresponding sequences of SEQ ID NO. 37, 6, 2, 3, 4 and 5, respectively, as well as some additional C-terminal sequences from the corresponding domain III.

[0034] In another embodiment, the polypeptide of (i) comprises an alanine residue at the position corresponding to position 2 of the sequence selected from SEQ ID NO: 37 or 2 to 6, or SEQ ID NO: 1 or 8 to 12. In some embodiments, the amino acid sequence of (i) comprises the following: an amino acid sequence selected from SEQ ID NO: 38 or 42 to 46, or an amino acid sequence having at least 80% sequence identity thereto. In another embodiment, the amino acid sequence of (i) comprises the following: an amino acid sequence selected from SEQ ID NO: 7, or 47 to 51, or an amino acid sequence having at least 80% sequence identity thereto. In this regard, it has been found that substitution of this residue at position 2 is beneficial for promoting protein stability.

[0035] According to another aspect of the present invention, the monomeric fusion protein has the following: an amino acid sequence selected from SEQ ID NO: 84, 85, 88, 89, 97, 98, 102, 103, 106, 107, 110, 111, or an amino acid sequence having 80% sequence identity thereto.

[0036] In some embodiments of these aspects, the monomeric fusion partner is selected from serum albumin, transferrin, and a monomeric Fc fragment, particularly a monomeric Fc fragment of IgG, more particularly a monomeric Fc fragment of human IgG.

[0037] As described above, substitution at position 2 of the domain III fragment herein improves the stability of the fragment, in particular resistance to protease degradation. It is considered and proposed herein that such sequence-modified variants of the domain III fragment represent proteins useful per se, without being linked to a fusion partner.

[0038] Therefore, another aspect of the present invention also provides a protein with a length of 40 to 60 amino acids, comprising or consisting of the amino acid sequence shown in SEQ ID NO: 7, 38, 42 to 46 or 47 to 51, or a sequence having at least 80% sequence identity thereto, wherein the protein comprises an alanine residue at the position corresponding to position 2 of the sequence of SEQ ID NO: 7, 38, 42 to 46, 47 to 51, and wherein all cysteine ​​residues in the sequence are conserved.

[0039] Additional proteins and fusion proteins are provided as further aspects of the invention, as described in detail below.

[0040] According to another aspect of the present invention, there is provided a recombinant protein comprising formula (I)

[0041] Cys-A-Cys-B-Cys-C-Cys-D-Cys-E-Cys-F (Formula (I))

[0042] in:

[0043] A is a peptide of formula II

[0044] A1-A2-A3-A5-A6-A7-A8-A9

[0045] wherein A1 is selected from P, A, V, I and L; A2 is selected from E, D, A, I, L and V; A3 is selected from G, Q, Y, S, N, W, F; A4 is selected from A, I, L, V, S, T; A5 is an amino acid selected from T, Y, N, G, Q and S; A6 is an amino acid selected from A, V, I, L, P, S, E, D, K, R and H; A7 is W; A8 is selected from G, T, S, Q, Y, N, P, A, V, I and L; A9 is an amino acid selected from A, P, L, I, V, Q; and

[0046] B is a peptide of formula III

[0047] B1-B2-B3

[0048] wherein B1 is an amino acid selected from the group consisting of G, Q, N, S, Y and T; B2 is an amino acid selected from the group consisting of T, S, N, F, Q, H, R and K; B3 is an amino acid selected from the group consisting of G, Q, N, S, Y, T; wherein one of B1 to B3 is absent; and

[0049] C is a peptide of formula IV

[0050] C1-C2-C3-C4-C5-C6-C7-C8-C9-C10-C11-C12-C13-C14

[0051] wherein C1 is an amino acid selected from the group consisting of G, Q, N, S, Y and T; C2 is an amino acid selected from the group consisting of K, R, H, M, T, S, A, L, I and V; C3 is an amino acid selected from the group consisting of G, Q, N, S, Y and T; C4 is an amino acid selected from the group consisting of M, F, A, I, L, V and W; C5 is an amino acid selected from the group consisting of G, Q, N, S, T, Y, A, I, L and V; C6 is an amino acid selected from the group consisting of G, Q, N, S and T; C7 is an amino acid selected from the group consisting of H, R and L; C8 is an amino acid selected from the group consisting of A, L, I and V; C9 is an amino acid selected from the group consisting of G, Q, N, S, T and Y; C10 is an amino acid selected from the group consisting of G, Q, N, S, T, Y (preferably N); C11 is an amino acid selected from the group consisting of V, P, A, I, L, G, Q, N, S, T, Y, R, K, D and E; C12 is an amino acid selected from the group consisting of G, Q, N, S, Y and T; C13 is an amino acid selected from the group consisting of H, K, R, A, L, I, V, P, G, Q, N, S, Y and T; C14 is an amino acid selected from the group consisting of F, P, W, G, Q, N, S, Y, T, E and D; and

[0052] D is a peptide of formula V

[0053] D1-D2-D3-D4-D5-D6-D7-D8

[0054] wherein D1 is an amino acid selected from R, K, H, D, E, W, P; D2 is an amino acid selected from P, A, L, I, V, M, W, D and E; D3 is an amino acid selected from D, E, A, L, I, V, R, K and H; D4 is an amino acid selected from G, Q, S, Y, T, R, L, K and H; D5 is an amino acid selected from G, Q, N, S, Y, T, D and E; D6 is an amino acid selected from H, R, K, G, Q, N, S, Y and T; D7 is an amino acid selected from L, H and R; D8 is an amino acid selected from A, L, I and V; and

[0055] E is a peptide of formula VI

[0056] E1-E2-E3-E4

[0057] wherein E1 is an amino acid selected from the group consisting of P, A, L, I, V, M, W, G, Q, N, S, T, Y, D and E; E2 is an amino acid selected from the group consisting of P, A, L, I, V, M, W, G, Q, N, S, T, Y; E3 is an amino acid selected from the group consisting of R, K, H, G, Q, N, S, T and Y; E4 is an amino acid selected from the group consisting of P, A, L, I and V; F is absent or is an amino acid sequence of up to about 13 amino acids,

[0058] The recombinant protein contains 40 to 60 amino acids.

[0059] According to one embodiment of the above aspect, a recombinant protein of formula (I) is provided, wherein:

[0060] A1 is selected from P, I and L; A2 is selected from E, V and A; A3 is selected from W, Q and Y; A4 is selected from S, T and A; A5 is an amino acid selected from T and S; A6 is an amino acid selected from A, E, P, S and K; A7 is W; A8 is selected from G, S and T; A9 is an amino acid selected from P, Q and A; and

[0061] B1 is serine (S); B2 is an amino acid selected from T, K and R; B3 is an amino acid selected from T and S; and

[0062] C1 is the amino acid G; C2 is an amino acid selected from the group consisting of T, L and M; C3 is G; C4 is an amino acid selected from the group consisting of M, F, I and V; C5 is an amino acid selected from the group consisting of S and A; C6 is an amino acid selected from the group consisting of T and N; C7 is R; C8 is an amino acid selected from the group consisting of V and I; C9 is an amino acid selected from the group consisting of S and T; C10 is asparagine N; C11 is an amino acid selected from the group consisting of Q, R, D, V and E; C12 is asparagine N; C13 is an amino acid selected from the group consisting of R, A, P and S; C14 is an amino acid selected from the group consisting of F, Q, S, E and N; and

[0063] D1 is an amino acid selected from R, E and W; D2 is an amino acid selected from L, M and P; D3 is an amino acid selected from E, L, V and R; D4 is an amino acid selected from T, K and Q; D5 is an amino acid selected from Q and E; D6 is an amino acid selected from R, T, S and K; D7 is arginine (R); D8 is an amino acid selected from L and I; and

[0064] E1 is an amino acid selected from L, M, E, N and Y; E2 is an amino acid selected from S, V, L and I; E3 is an amino acid selected from Q and R; E4 is P; F is absent or is a peptide of up to 13 amino acids and comprises an amino acid sequence selected from the group consisting of PPSRGRSPQNSAF, GQPVYSSL, EADLEEN, EQEPEQPTD, DVDIHTLI, and DSNILKTIKIP,

[0065] The recombinant protein contains a total of 44 to 57 amino acids.

[0066] According to yet another embodiment of the above aspect, a recombinant protein of Formula I is provided, wherein F is completely absent, partially absent, or is a peptide of about 13 amino acids comprising the amino acid sequence of PPSRGRSPQNSAF.

[0067] More particularly, a recombinant protein is provided, wherein the protein comprises an amino acid sequence selected from the group consisting of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.37, SEQ ID No.38; and variants or fragments thereof having a sequence identity greater than 50% with the following amino acid sequences: SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.37, SEQ ID No.38.

[0068] According to another aspect of the present invention, there is provided a recombinant protein as defined above, wherein the protein is pegylated.

[0069] According to another aspect, the present invention provides a fusion protein comprising

[0070] (i) thrombospondin type 1 repeat (TSP-1) homology domain of CCN family proteins;

[0071] (ii) a fusion partner fused to the TSP-1 repeat homology domain of (i) at the N-terminus or the C-terminus, and wherein the fusion partner is selected from the group consisting of serum albumin, transferrin and the Fc fragment of human IgG;

[0072] (iii) optionally a peptide linker between the TSP-1 repeat homology domain and the Fc fragment (fused to the TSP-1 repeat homology domain of (i) at the N-terminus or C-terminus).

[0073] According to one embodiment of the above aspect, a fusion protein comprising the recombinant protein according to the present invention as described above is provided as another aspect of the present invention.

[0074] The fusion partner of the fusion protein according to the invention is according to one embodiment selected from the group consisting of the Fc fragment of IgG1, IgG2 or IgG4, serum albumin and transferrin.

[0075] According to another embodiment, a fusion protein is provided, wherein the fusion partner (ii) is an Fc fragment of IgG1, IgG2 or IgG4 containing a stabilizing disulfide bridge. Such mutations can improve the thermal stability of the protein. Stabilizing mutations are known and have been reported in the art.

[0076] According to another embodiment, a fusion protein is provided, wherein the fusion partner (ii) is an Fc fragment of IgG1, IgG2 or IgG4 comprising one or more mutations selected from the group consisting of S228P (involving IgG4), E233P (involving IgG1 and IgG4), F234A (involving IgG4), L234A (involving IgG1), L234V (involving IgG1), F234V (involving IgG4), L235A (involving IgG1 and IgG4), ΔG236 (involving IgG1 and IgG4)) and ΔK447 (involving IgG1, IgG2 and IgG4).

[0077] According to another embodiment, the fusion protein may comprise an Fc fragment selected from the group consisting of: SEQ ID No.15, SEQ ID No.16, SEQ ID No.17, SEQ ID No.18 and SEQ ID No.19.

[0078] According to another embodiment, the fusion protein comprises a linker selected from the group consisting of SEQ ID No.20, SEQ ID No.21, SEQ ID No.22, SEQ ID No.23, SEQ ID No.24, SEQ ID No.25 and SEQ ID No.39.

[0079] According to one embodiment, the linker comprises the amino acid sequence (EAAAK)n, wherein n is at least 4, preferably n is 8.

[0080] According to another embodiment, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID No.26, SEQ ID No.27, SEQ ID No.28, SEQ ID No.29, SEQ ID No.30, SEQ ID No.31, SEQ ID No.40 and SEQ ID No.41.

[0081] According to another embodiment, the fusion partner of (ii) is serum albumin.

[0082] According to another embodiment of the second aspect of the present invention, the fusion partner of (ii) is transferrin.

[0083] Furthermore, according to yet another aspect, the present invention provides a nucleic acid molecule (eg, DNA) encoding the recombinant protein, protein or fusion protein according to the present invention.

[0084] According to one embodiment of this aspect, a DNA sequence is provided comprising the following: a nucleic acid sequence shown in SEQ ID No.34, SEQ ID No.35, SEQ ID No.36 or SEQ ID NO:86, 87, 90, 91, 99, 100, 104, 105, 108, 109, 112 or 113, and a nucleic acid sequence having about 80% sequence identity with SEQ ID NO.34, SEQ ID No.35, SEQ ID No.36 or SEQ ID NO:86, 87, 90, 91, 99, 100, 104, 105, 108, 109, 112 or 113.

[0085] In addition, according to another aspect of the present invention, an expression vector comprising the DNA sequence according to the present invention is provided. A host cell comprising the expression vector according to the present invention is also provided.

[0086] Finally, provided are thrombospondin type 1 repeat (TSP-1) homology domains of CCN family proteins, proteins and fusion proteins as defined above for use as medicaments for treating or preventing disorders by inhibiting or antagonizing cell signaling and cell physiological functions attributed to the four-domain CCN family proteins.

[0087] In one aspect, there is provided a fusion protein or protein as defined herein for use in therapy.

[0088] The fusion protein or protein can be used to treat a condition associated with an activity of a 4-domain CCN protein, particularly an undesirable or abnormal activity of a 4-domain CCN protein. The activity can be associated with fibrosis. The activity can be a pro-fibrotic activity.

[0089] In another aspect, a fusion protein or protein as defined herein is provided for use in treating or preventing fibrosis or any condition exhibiting fibrosis (i.e., a fibrotic condition or disease). In another aspect, a fusion protein or protein as defined herein is provided for use in treating cancer. Also provided is a fusion protein or protein as defined herein for use in treating inflammatory diseases or autoimmune diseases or metabolic diseases.

[0090] According to such aspect of the invention there is also provided the use of a protein or fusion protein as defined herein for the manufacture of a medicament for the treatment or prevention of a disorder or disease as defined or described herein.

[0091] Such aspects also include compositions (eg pharmaceutical compositions) comprising a protein or fusion protein as defined herein for use in treating or preventing a disorder or disease as defined or described herein.

[0092] Such aspects also include methods of treating or preventing a disorder or disease as defined or described herein, said method comprising administering to a subject in need thereof a protein or fusion protein as defined herein, in particular an effective amount of said protein or fusion protein.

[0093] Attached photos

[0094] Figure 1 The cell physiology and cell signaling of CCN5(dIII)-Fcv2 (one embodiment of the present invention defined in sequence SEQ ID No. 28) are shown.

[0095] A) shows that the CCN5(dIII)-Fcv2 fusion protein of SEQ ID No. 28 causes concentration-dependent inhibition of Akt (serine-473) phosphorylation in A549 lung cancer cells.

[0096] B) shows that the CCN5(dIII)-Fcv2 fusion protein of SEQ ID. No. 28 inhibits proliferation in the human lung fibroblast cell line IMR90.

[0097] C) shows that the CCN5(dIII)-Fcv2 fusion protein of SEQ ID No. 28 inhibits the sphere-forming ability (archorage-independent growth) of the estrogen receptor-positive breast cancer cell line MCF-7 and the triple-negative breast cancer cell line MDA-MB-231.

[0098] D) shows that the CCN5(dIII)-Fcv2 fusion protein of SEQ ID No. 28 dose-dependently inhibits TGF-β-induced SMAD reporter activity (SMAD proteins are typical TGF-β-regulated transcription factors).

[0099] All error bars show SD. Statistical significance (p<0.05, indicated by *) was calculated by one-way ANOVA and Dunnetts post hoc test.

[0100] Figure 2 The effect of different variants of the hinge region of the Fc fragment on the protease susceptibility of some embodiments of the present invention in which CCN5 (dIII) is fused to the Fc fragment of IgG is shown, wherein the tested fusion proteins comprise the sequences shown in SEQ ID No. 28, SEQ ID No. 29 and SEQ ID No. 30, respectively, see Example 6 below.

[0101] Figure 3 The aggregation tendency of one embodiment of the present invention depending on the structure of the peptide linker connecting CCN5(dIII) and the Fc fragment of IgG is shown, wherein the tested fusion protein comprises the sequences shown in SEQ ID No.30 and SEQ ID No.31.

[0102] Figure 4 A fusion protein according to the invention is shown comprising a TSP-1 repeat homology domain connected to a peptide linker at the C-terminus and to an Fc fragment via an Fc hinge.

[0103] Figure 5It is shown that relative to the wild-type P195 variant of the CCN5 TSP-1 repeat homology domain (Fc-HLn8-CCN5(dIII), SEQ ID No.40), the susceptibility to endopeptidase cleavage is reduced when an embodiment of the present invention incorporates a mutation of proline 195 of the CCN5 TSP-1 repeat homology domain shown in SEQ ID No 7 (Fc-HLn8-CCN5(dIII)-P195A, SEQ ID No.41).

[0104] Figure 6 The production of a protein corresponding to SEQ ID NO: 58 purified by protein A capture chromatography is shown. It can be seen that dimers are present in the absence of the reducing agent β-mercaptoethanol (- lanes). However, in the presence of β-mercaptoethanol (+ lanes), it can be seen that the primary product is a cleavage fragment containing only the Fc fragment, rather than a complete fusion protein containing all of the portions encoded by SEQ ID NO: 58 (TSP-1 homology domain fragment, peptide linker and Fc fragment).

[0105] Figure 7 The production of a protein corresponding to SEQ ID NO: 27 with a truncated C-terminal tail purified by Protein A capture chromatography is shown. As can be seen, the protein is significantly more resistant to protease degradation than the protein corresponding to SEQ ID NO: 58 with a C-terminal tail.

[0106] Figure 8 Shown is the production of a protein corresponding to SEQ ID NO: 73 similar to the protein corresponding to SEQ ID NO: 27 purified by Protein A capture chromatography. Again, it can be seen that in the presence of β-mercaptoethanol (+ lanes), the protein is more resistant to protease degradation than the protein corresponding to SEQ ID NO: 58.

[0107] Fig. 9 Shown are the results of an assay measuring the level of phosphorylated AKT (Ser-473) in A549 lung cancer cells after administration of different concentrations of a protein corresponding to SEQ ID NO: 41 produced in stably transfected cells. It can be seen that the protein has no inhibitory effect on the phosphorylation of AKT.

[0108] Fig.10Shown are the results of an assay measuring the levels of phosphorylated AKT (Ser-473) in A549 lung cancer cells following administration of different concentrations of a protein corresponding to SEQ ID NO: 80 produced in stably transfected cells. As can be seen, the protein does not show a significant inhibitory effect on the phosphorylation of AKT, and in fact can even lead to an increase in phosphorylated AKT at higher concentrations.

[0109] Fig.11 Shown are the results of an assay measuring the level of phosphorylated AKT (Ser-473) in A549 lung cancer cells after administration of different concentrations of a protein corresponding to SEQ ID NO: 80 produced in transiently transfected cells. It can be seen that the protein has a concentration-dependent inhibitory activity on the phosphorylation of AKT when produced in transiently transfected cells.

[0110] Fig.12 Shown are the results of an assay measuring the level of phosphorylated AKT (Ser-473) in A549 lung cancer cells after administration of different concentrations of proteins corresponding to SEQ ID NOs: 84, 94 and 106. It can be seen that each of these proteins has a concentration-dependent inhibitory activity on the phosphorylation of AKT.

[0111] Fig.13 Shown are the results of an assay measuring the levels of phosphorylated AKT (Ser-473) in A549 lung cancer cells following administration of different concentrations of the protein corresponding to SEQ ID NO: 88. As can be seen, the protein is able to inhibit the phosphorylation of AKT at concentrations above 10 ug / ml.

[0112] Fig.14 Shown are the results of an assay measuring the level of phosphorylated AKT (Ser-473) in A549 lung cancer cells after administration of different concentrations of proteins corresponding to SEQ ID NOs: 102 and 97. As can be seen, both proteins have concentration-dependent inhibitory activity on the phosphorylation of AKT.

[0113] Fig.15 Shown are the results of an assay measuring the level of phosphorylated AKT in A549 lung cancer cells after administration of different concentrations of the protein corresponding to SEQ ID NO: 110. It can be seen that the protein has a concentration-dependent inhibitory activity on the phosphorylation of AKT.

[0114] Fig.16 Shown are the results of multiple experiments involving the protein corresponding to SEQ ID NO: 106.

[0115] A) shows that the protein inhibits the migration of human lung fibroblasts induced by both TGF-β and CCN2.

[0116] B) shows that the protein inhibits the closure of scratch wounds induced by both TGF-β and CCN2.

[0117] C) shows that this protein causes a partial inhibition of TGF-β-induced expression of the gene COL1A1, which is known to be profibrotic.

[0118] D) shows that the protein causes a partial inhibition of TGF-β-induced expression of the gene FN1, which is known to be profibrotic.

[0119] E) shows that the protein causes partial inhibition of TGF-β-induced expression of the gene ACTA2, which is known to be profibrotic.

[0120] F) shows that this protein causes partial inhibition of TGF-β-induced expression of the gene CCN2, which is known to be profibrotic. DETAILED DESCRIPTION OF THE INVENTION

[0122] The present invention is based on the unexpected discovery that the thrombospondin type 1 repeat (TSP-1) homology domain of CCN5 is a fully active structure that confers the cell signaling function of CCN5 / WISP2. Based on this new insight into the activity of the TSP-1 repeat homology domain of CCN5, the inventors provide proteins, recombinant proteins and fusion proteins according to the present invention that can be used to inhibit or antagonize cell signaling and cell physiological functions attributed to the four-domain CCN proteins, namely CCN1, CCN2, CCN3, CCN4 and CCN6. This new insight is believed to be crucial to allowing the formation of stable, homogeneous, drug-like molecules based on CCN5, because it has not been previously revealed which part of the full-length CCN5 is necessary to recapitulate CCN5 activity, and because the full-length CCN molecules are highly susceptible to proteolysis and are difficult to produce in an active, homogeneous form. Furthermore, the implication that specific portions of CCN5 may be sufficient to recapitulate the activity observed, for example, following transient overexpression of the full-length protein is also inconsistent with the prevailing view in the art that CCN proteins function as matricellular proteins. The prevailing view of the mechanism of action of CCN proteins and matricellular proteins in general is that different segments of CCN proteins interact with a variety of other ECM proteins and cell surface receptors, thereby modulating their activity, rather than acting as direct regulators of cell signaling. New knowledge of the activity of the TSP-1 repeat homology domain of CCN5, and knowledge of the structural close relationship to other members of the CCN protein family, also suggests that the TSP-1 repeat homology domains of other CCN family proteins may also be used to inhibit the cell signaling function of the four-domain CCN family proteins. According to one aspect, the recombinant proteins and fusion proteins of the present invention inhibit phosphorylation of AKT (Ser473) in A549 cells.

[0123] Inhibition of such cell signaling is relevant to the treatment of a variety of disorders. For example, CCN2 is associated with several diseases, particularly those in which enhanced fibrogenesis and tissue fibrosis are characteristic pathophysiological features.

[0124] For example, it has been shown that CCN2 overexpression alone is sufficient to induce fibrosis in the lung (see Sonnylal et al., Arthritis Rheum 62, 1523-1532 (2010)). CCN2 was also found to be essential for bleomycin-induced pulmonary fibrosis (Bonniaud, P. et al. Am J Respir Cell Mol Biol 31, 510-516 (2004)), radiation-induced pulmonary fibrosis (Bickelhaupt, S. et al. J Natl Cancer Inst 109 (2017)), and pulmonary fibrosis caused by loss of PTEN (Phosphatase And Tensin Homolog) expression (Parapuram, SK et al. Matrix Biol 43, 35-41 (2015)). In addition, in the absence of other inducers, when CCN2 is removed from lung Clara cells (pulmonary Clara cells) (Wu, S. et al. Am J Respir Cell Mol Biol 42, 552-563 (2010)), alveolar type II epithelial cells (Chen, S. et al. Am J Physiol CCN2 has been found to induce pulmonary fibrosis when expressed and secreted from a fibroblast-specific promoter (Sonnylal et al (2010), supra, Sonnylal, S. et al., J Cell Sci 126, 2164-2175 (2013)) or delivered by adenovirus (Bonniaud, P. et al., Am J Respir Crit Care Med 168, 770-778 (2003)). Thus, multiple reports support the conclusion that CCN2 is not only sufficient to cause fibrosis in the skin or lungs, but is also required for a fully mature fibrotic phenotype in several disease models. Pulmonary fibrosis is a hallmark of the human disease idiopathic pulmonary fibrosis (IPF), but it also occurs in chronic obstructive pulmonary disease (COPD) (Jang, J Het al., COPD 14, 228-237 (2017)) and systemic sclerosis. In fact, pulmonary fibrosis is reported to be the main cause of death in up to 40% of patients with systemic sclerosis (Tyndall AJ et al., Ann Rheum Dis. 2010 Oct; 69(10): 1809-15).CCN2 and other CCN proteins, such as WISP1, have also been implicated in the pathophysiology of both IPF (Konigshoff, M. et al., J Clin Invest 119, 772-787 (2009)) and COPD (Jang et al, supra) in human patients.

[0125] Another example is tumorous disease.For example, in the case of breast cancer, it has been shown that CCN2 promotes bone metastasis (Kang, Y.et al., Cancer Cell 3, 537-549 (2003)) in triple negative breast cancer model (MDA-MB-231). In addition, knocking down CCN2 (Chen, P et al., J Cell Sci 120, 2053-2065 (2007)) in triple negative breast cancer cells (MDA-MB-231) (cell lines expressing high levels of CCN2) reduces the migration ability of these cells, and the overexpression of CCN2 in hormone receptor positive MCF-7 breast cancer cell lines with low endogenous CCN2 expression (Chen et al, the same) improves the migration ability of the latter cells (Chen et al, the same, Chien, W.et al., Iht J Oncol 38, 1741-1747 (2011)). Later reports also found that overexpression of CCN2 in MCF-7 cells increased chemoresistance, while knockdown of CCN2 in MDA-MB-231 cells reduced chemoresistance (Wang, MY et al., Cancer Res 69, 3482-3491 (2009)). Improved chemoresistance conferred by CCN2 was also reported for other breast cancer cells (Lai, D et al., Cancer Res 71, 2728-2738 (2011)). In addition, through overexpression or knockdown studies, it was also shown that CCN2 promoted epithelial-to-mesenchymal transition (EMT) and breast cancer cell anchorage-independent growth (mammosphere formation) (Chen et al, supra, Zhu, X. et al., Oncotarget 6, 25320-25338 (2015)). The findings of both increased chemoresistance and enhanced EMT induced by CCN2 are consistent with the link between EMT and chemoresistance also in other cancer types (Fischer, K. et al., Nature 527, 472-476 (2015), Zheng, X. et al., Nature 527, 525-530 (2015)).

[0126] In a specific aspect, the present invention provides a monomeric fusion protein as defined above, comprising a polypeptide corresponding to at least a portion of a thrombospondin type 1 repeat (TSP-1) homology domain of a CCN family protein, wherein the TSP-1 homology domain sequence may be truncated and / or modified, but wherein the cysteine ​​residues of the domain are conserved. For convenience, the polypeptide may be referred to herein as a "TSP-1 polypeptide", and thus the term is understood not to convey or imply any limitation to only a specific native TSP-1 homology domain sequence. The term "TSP-1 polypeptide" may be used synonymously or interchangeably with "TSP-1 domain protein" or "TSP-1 domain sequence".

[0127] As demonstrated in the examples below, it was unexpectedly found that monomeric fusion partners are advantageous in producing active and stable proteins compared to dimeric fusion partners, such as Fc fragments derived from IgG proteins, which produce dimeric fusion proteins. Monomeric fusions retain the activity of the TSP-1 domain polypeptides they contain. In addition, the proteins are stable, including with respect to proteolytic degradation. As further described below, resistance to proteolytic degradation can be increased by modifying the amino acid sequence of the TSP-1 polypeptide, particularly including the Ala substitutions mentioned above.

[0128] Therefore, relative to the sequence selected from SEQ ID NO: 37 or 2 to 6, or the sequence selected from SEQ ID NO: 1 or 8 to 12, the polypeptide of component (i) of the fusion protein may contain insertions, deletions, substitutions, mutations or any combination thereof, provided that the polypeptide retains at least 80% sequence identity with the sequence and all cysteine ​​residues in the sequence are conserved.

[0129] In another aspect, the present invention provides a protein (e.g., a recombinant protein) consisting of or comprising, but not being a fusion protein: a polypeptide corresponding to at least a portion of a thrombospondin type 1 repeat (TSP-1) homology domain of a CCN family protein, wherein the TSP-1 domain sequence may be truncated and / or modified and comprises an Ala substitution at a position corresponding to position 2 of SEQ ID NO: 37 or 2 to 6 or SEQ ID NO: 1 or 8 to 12, but wherein the cysteine ​​residue of the domain is conserved. In other words, the TSP-1 domain protein may be provided in the absence of or independently of another component, such as a fusion partner. Thus, the TSP-1 domain protein is not fused or linked to another protein domain or component or other functional or structural protein sequence. For convenience, such a protein may be referred to as an "Ala-substituted protein."

[0130] The term "conservative" as used herein means that the residues in a given sequence are not deleted or replaced. In other words, the term "conservative" is used synonymously (and interchangeably) with the term "retained". It simply means that no cysteine ​​residues are removed from the sequence. Therefore, in the above context, it means that none of the cysteine ​​residues in the sequence selected from SEQ ID NO: 37 or 2 to 6 or 1 or 8 to 12 are deleted or replaced. It should be noted that inserting additional residues between conserved residues (e.g., between conserved cysteines), or deleting non-conserved (e.g., non-cysteine) residues, may change their position in the polypeptide sequence relative to the position of the conserved residue in the original reference sequence (e.g., a sequence selected from SEQ ID NO: 37 or 2 to 6). However, such residues are still considered to be "conserved", as defined herein. Therefore, the term "conservative" does not imply any definition or restriction of the position (or more particularly, the position number) of the cysteine ​​residue.

[0131] In some embodiments, the polypeptide of (i) comprises or consists of:

[0132] (a) an amino acid sequence selected from SEQ ID NO: 1 or 8 to 12; or

[0133] (b) an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NOs: 1 or 8 to 12; or

[0134] (c) a portion of the amino acid sequence of (a) or (b), wherein the portion comprises at least 44 amino acid sequences of SEQ ID NO: 37, 6, 2, 3, 4 or 5, respectively, or comprises a sequence having at least 80% sequence identity with a sequence selected from SEQ ID NO: 37, 6, 2, 3, 4 or 5, respectively.

[0135] As described above, the monomeric fusion partner of (ii) and the peptide linker of (iii) are not or do not comprise an IGF binding protein homology domain, a von Willebrand factor C-type repeat homology domain or a cysteine ​​knot domain of a CCN family protein. Alternatively, the only domain of a CCN family protein that may be present in the fusion protein of the present invention is a TSP-1 homology domain.

[0136] Similarly and similarly, in the case of an Ala-substituted protein that is not a fusion protein, the protein does not contain any other CCN domains (except for the TSP-1 domain protein).

[0137] In some embodiments, the polypeptide or Ala-substituted protein of (i) may comprise only a portion of the TSP-1 homology domain as defined above. The inventors have found that the minimum fragment of the desired TSP-1 domain is the 44 amino acid sequence of SEQ ID NO: 37, 6, 2, 3, 4 or 5. Thus, in some embodiments, the polypeptide of (i) is at least 44 amino acids in length. In some embodiments, the polypeptide of (i) is 44 to 57 amino acids in length. However, as described above, one or more amino acid deletions may be present in the 44 amino acid minimum fragment located between the cysteine ​​residues. Thus, in some embodiments, the TSP-1 polypeptide may be less than 44 residues in length, i.e., 40 to 43 residues in length.

[0138] In some embodiments, the polypeptide of (i) consists of an amino acid sequence selected from SEQ ID NO: 37 or 2 to 6, or a sequence having at least 80% sequence identity with a sequence selected from SEQ ID NO: 37 or 2 to 6.

[0139] As described above, the proteins of the present invention, including fusion proteins, exhibit (or in other words, show or have) the activity of CCN5, more particularly the biological activity of CCN5. In one embodiment, the protein may retain or exhibit or have the activity of the TSP-1 homology domain of CCN5. Alternatively, the protein may be defined as exhibiting (or showing or having) the activity of the isolated TSP-1 homology domain of the CCN protein, particularly its biological activity. The foregoing may apply to any activity of the domain, and in particular reflects the activity of the anti-fibrotic effect of the TSP-1 homology domain. Based on any specific biological effect of the domain, any convenient assay or method may be used to determine (or test or detect) such activity.

[0140] It is noteworthy that by analyzing the effect of a given protein on AKT phosphorylation, the activity of the protein can be conveniently assessed. In particular, the ability of a given protein to inhibit the phosphorylation of AKT (Ser-473) in A549 human lung cancer cells can be determined, as described in Example 2. The skilled person will appreciate that other similar assays can be designed to assess the same activity, or to assess other related anti-fibrotic activities.

[0141] As described above, in other aspects of the present invention, a recombinant protein that inhibits or antagonizes cell signaling and cell physiological functions attributed to a four-domain CCN protein is provided, which comprises an amino acid sequence according to Formula I above.

[0142] Cys-A-Cys-B-Cys-C-Cys-D-Cys-E-Cys-F

[0143] wherein A, B, C, D, E and F are as defined above and in the appended claims.

[0144] Formula I is the result of an alignment of the TSP-1 repeat homology domains of structurally related CCN family proteins (CCN 1 to CCN6), all of which contain six conserved cysteines, and take into account amino acids that can be substituted without affecting protein activity (conservative substitutions, as discussed further below). The position of the first conserved cysteine ​​of the TSP1 repeat homology domain of the different CCN proteins is defined as position #1 of the recombinant protein of Formula I.

[0145] The five segments between the conserved cysteines are A, B, C, D and E.

[0146] The first segment A is defined by the formula A1-A2-A3-A5-A6-A7-A8-A9, wherein A1 to A9 are defined above. The amino acid at position #7 (A7) of segment A is tryptophan (W) in all members of the CCN family proteins and is considered conserved.

[0147] The second segment B is defined by the formula B1-B2-B3, wherein B1 to B3 are as defined above. According to one embodiment, B1 and B3 are serine or threonine.

[0148] The third segment C is defined by the formula C1-C2-C3-C4-C5-C6-C7-C8-C9-C10-C11-C12-C13-C14, wherein amino acids C1 to C14 are as defined above. According to one embodiment, amino acids C1 and C3 are glycine (G).

[0149] According to another embodiment, C7 is arginine (R) and both C10 and C12 are asparagine (N).

[0150] The fourth segment D is defined by the formula D1-D2-D3-D4-D5-D6-D7-D8, wherein amino acids D1 to D8 are as defined above. According to one embodiment, D7 is arginine (R).

[0151] The fifth segment E is defined by the formula E1-E2-E3-E4, wherein amino acids E1 to E4 are as defined above. According to one embodiment of the invention, E4 is proline.

[0152] Following the last cysteine ​​is a carboxy-terminal peptide segment (F) of variable length comprising 0 to 13 amino acids.

[0153] Compared to the amino acid sequence of the TSP-1 repeat homology domain of the CCN family protein, F may be deleted or shortened. According to one embodiment, F is absent. According to another embodiment, F consists of a peptide selected from the group consisting of:

[0154] PPSRGRSPQNSAF, GQPVYSSL, EADLEEN, EQEPEQPTD, DVDIHTLI, and DSNILKTIKIP.

[0155] According to one aspect of this embodiment, the recombinant protein may take the form of the amino acid sequence shown in SEQ ID Nos. 8 to 12.

[0156] According to another aspect, the present invention provides a recombinant protein comprising: an amino acid sequence selected from the group consisting of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.37, SEQ ID No.38; and fragments thereof or variants thereof having at least 50% sequence identity with the following amino acid sequences: SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.37, SEQ ID No.38.

[0157] According to one aspect, a recombinant protein is provided, which is composed of an amino acid sequence selected from the group consisting of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.37, SEQ ID No.38; and a fragment thereof or a variant thereof having more than 50% sequence identity with the following amino acid sequences: SEQ ID No.1, SEQ ID No.2, SEQID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7, SEQID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.37, SEQID No.38.

[0158] As used herein, "recombinant proteins" are proteins encoded by recombinant nucleic acids. They are expressed by recombinant nucleic acids in host cells, as further disclosed below.

[0159] "Recombinant nucleic acid" is used herein to describe a nucleic acid molecule that, due to its origin or manipulation, is not associated with all or part of the polynucleotides with which it is naturally associated and / or is linked to polynucleotides other than those with which it is naturally linked, as further disclosed below.

[0160] The skilled person will recognize that modifications to the amino acid sequence of the protein can be introduced without changing the activity of the recombinant protein and fusion protein according to the present invention. Amino acids are generally classified as hydrophobic or hydrophilic and / or as having polar or non-polar side chains. Substitution of one amino acid for another having the same biochemical properties is generally referred to as a conservative substitution.

[0161] Conservative substitutions of amino acids include substitutions between amino acids within the following groups:

[0162] MILV

[0163] ·FYW

[0164] ·KRH

[0165] AG

[0166] ·ST

[0167] ·QN

[0168] ·ED

[0169] Generally speaking, conservative amino acid substitutions are those that do not change the relative charge or size characteristics of the protein in which the amino acid substitution is made, and thus rarely change the structure of the protein, which is why biological activity is not significantly altered.

[0170] The skilled person will recognize that if one or several amino acids are deleted, inserted or added to the amino acid sequence, the biological activity of the protein may be retained as long as the structure and chemical-physical properties are conserved.

[0171] The symbol "Δ" when used before an amino acid herein refers to the deletion of the specified amino acid, for example, ΔK447 is to be understood as a protein in which K447 is absent. Alternatively, the deletion of a specific amino acid is indicated herein by "-", for example, K447- is also to be understood as a protein in which K447 is absent.

[0172] Therefore, it should be understood that the present invention covers recombinant proteins and fusion proteins as disclosed in the appended claims, into which such modifications (amino acid substitutions, deletions, insertions and additions) as described above can be introduced without substantially changing their biological activity (i.e., the ability to inhibit or antagonize cell signaling and cellular physiological functions attributed to the four-domain CCN family proteins CCN1, CCN2, CCN3, CCN4 and CCN6).

[0173] Throughout this specification, amino acid sequences are mentioned. When an amino acid sequence is mentioned herein, the modification of the amino acid sequence or protein in question is sometimes mentioned by reference to the "uniprot number" or Eu number in this specification. Uniprot numbering refers to the numbering used in the Uniprot database (UniProt Consortium, Nucleic Acids Res. 2019 Jan 8; 47 (D1): D506-D515). When referring to the amino acid count of a CCN protein, Uniprot numbering is used. Eu numbering refers to the numbering of Eu antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63: 78-85), and Eu numbering is used when referring to amino acids in a human IgG subclass Fc fragment with or without a mutant or chimera different from the wild type. The Eu numbering system can be accessed, for example, from the International ImMunoGeneTics Information System (IMGT) in the IMGT Scientific Chart. IMGTs are described in Lefranc MP, Biomolecules. 2014 Dec; 4(4): 1102-1139.

[0174] As used herein, when referring to "sequence identity", a sequence that has at least x% identity to a second sequence means that x% represents the number of amino acids in the first sequence that are identical to their matching amino acids in the second sequence relative to the total length of the second amino acid sequence when the two sequences are optimally aligned by global alignment. When x is the maximum value, both sequences are optimally aligned. The alignment and determination of percent identity can be performed manually or automatically.

[0175] Alignment for determining the percentage of amino acid sequence identity can be achieved in a variety of ways within the technical scope of the art, for example, using publicly available computer software such as ClustalOmega (Sievers F, Higgins DG (2018) Protein Sci 27: 135-145), Protein BLAST (from the National Center for Biotechnology Information (NCBI)) or commercially available software such as Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences. NCBI BLAST is another example of software for determining amino acid sequence identity (MacWilliam et al., Nucleic Acids Res. 2013 Jul; 41 (WebServer issue): W597-W600).

[0176] According to one aspect of the present invention, a recombinant protein is provided, which comprises an amino acid sequence selected from the group consisting of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.37 and SEQ ID No.38 and fragments or variants thereof, which have at least 50% sequence identity with the amino acid sequence SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.37 and SEQ ID No.38.

[0177] According to another aspect, a recombinant protein is provided, which comprises an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% sequence identity with an amino acid sequence selected from SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.37 and SEQ ID No.38.

[0178] Bioactive proteins and peptides play an important role in the clinical management of human diseases. However, many proteins and peptides face challenges due to their less than ideal pharmacokinetic properties or because they are removed by renal filtration due to their small size and / or due to proteolytic metabolism. Such factors may limit or challenge the administration of drugs to objects in need of treatment, such as the necessity of continuous infusion administration or frequent subcutaneous administration to maintain the circulating concentration of proteins or peptides at effective therapeutic levels. Due to the obvious challenges and inconveniences to both patients and doctors, the need to continuously or very frequently administer drugs is clinically undesirable.

[0179] One strategy to extend the half-life of biologically active peptides or proteins is to attach polyethylene glycol (PEG) groups to the peptide or protein of interest through a process called PEGylation (see, e.g., Dozie et al. (2015), Int. J. Mol Sci, 16 (10) 25831-25864). The general strategy for protein PEGylation is to react a functional group on the protein with a complementary group on the PEG molecule to form a protein-PEG conjugate. The PEG moiety provides several advantages for improving the stability and circulation half-life of proteins due to its flexibility, hydrophilicity, variable size, and low toxicity.

[0180] Therefore, in one embodiment, the present invention provides a recombinant protein as described above, wherein the protein is pegylated.The fusion protein according to the present invention may also be pegylated.

[0181] Fusion Protein

[0182] Another approach to eliminating the challenges associated with the medical use of peptides and proteins is to obtain an extension of the half-life of biologically active proteins or peptides by preparing fusion proteins (see, e.g., Valeria et al. (2017), "A New Approach to Drug Therapy: Fc-Fusion Technology), Prim Health Care, 7: 255, doi: 10.4172 / 2167-1079.1000255). By covalently fusing a protein or peptide to a carrier protein via genetic recombination, the molecular weight of the protein of interest can be increased to approximately 60 to 70 kDa, which is the threshold for renal filtration.

[0183] The present invention provides a fusion protein comprising:

[0184] (i) thrombospondin type 1 repeat (TSP-1) homology domain of CCN family proteins;

[0185] (ii) a fusion partner fused to the TSP-1 repeat homology domain of (i) at the N-terminus or the C-terminus, wherein the fusion partner is selected from the group consisting of serum albumin, transferrin and immunoglobulin Fc fragment.

[0186] (iii) Optionally, a peptide linker between the TSP-1 repeat homology domain of (i) and the Fc fragment (fused to the TSP-1 repeat homology domain at the N-terminus or C-terminus).

[0187] Throughout this specification, the TSP-1 repeat homology domain may also be indicated and referred to as domain III, which refers to the third domain of the CCN family proteins.

[0188] In a preferred aspect, the fusion partner is a monomeric fusion partner and results in a monomeric fusion protein.Such fusion proteins, and in particular the TSP-1 domains thereof, are defined above and further described below.

[0189] However, the present disclosure also includes additional embodiments involving both the TSP-1 domain protein component and the fusion partner component.

[0190] According to one such embodiment, the TSP-1 repeat homology domain is a recombinant protein of formula I as defined above.

[0191] According to another embodiment, the TSP-1 repeat homology domain is a recombinant protein having an amino acid sequence as defined in any one of the sequences shown in SEQ ID No. 1 to 12, 37 and 38, or a recombinant protein of formula I as defined above.

[0192] According to one embodiment, the TSP-1 repeat homology domain is a recombinant protein comprising an amino acid sequence having at least 50%, 60%, 70%, 80%, 90% or 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.37 and SEQ ID No.38.

[0193] Proteins are inherently susceptible to protease degradation. In order to prevent protease degradation of the recombinant proteins and fusion proteins according to the present invention, modifications to the amino acid sequence can be introduced, for example, by site-directed mutagenesis, to provide protease-resistant recombinant proteins and fusion proteins. For example, point mutations can be introduced in the thrombospondin type 1 repeat (TSP-1) homology domain of the CCN family proteins as defined in SEQ ID No. 1 to 12, 37 or 38 of the fusion protein, or more particularly, in the recombinant protein. According to one embodiment, the introduction of point mutations reduces the susceptibility to proteolytic degradation. A non-limiting example of a point mutation that results in less proteolysis of the recombinant proteins and fusion proteins of the present invention is by introducing a point mutation corresponding to the replacement of proline (P195A) with alanine at position 195 of domain III of CCN5, such as shown in SEQ ID No. 7. Similar mutations can also be introduced in the amino acid sequence derived from domain III of other CCN family members. SEQ ID NO.38 represents a truncated 44 amino acid sequence of the TSP-1 domain of CCN5 comprising an Ala substitution. SEQ ID Nos.42 to 46 represent truncated 44 amino acid sequences of the TSP-1 homologous domains of CCN1, 2, 3, 4 and 6 comprising an Ala substitution, respectively. SEQ ID Nos.47 to 51 represent longer TSP-1 homologous sequences of CCN1, 2, 3, 4 and 6 comprising an Ala substitution, respectively. According to the present invention, any such sequence, or a sequence having at least 80% sequence identity therewith, may be used.

[0194] As mentioned above, in a preferred embodiment, the fusion partner (ii) of the fusion protein according to the invention is monomeric. Any monomeric fusion partner can be used. Thus, the fusion partner can be any protein or part thereof (e.g., a protein domain) that appears and remains in monomeric form when fused to the TSP-1 homology domain protein component. Thus, the fusion protein comprising the monomeric fusion partner and the TSP-1 homology domain protein remains a monomer. That is, it does not dimerize with itself or form higher multimers.

[0195] A variety of proteins are known as possible fusion partners and may include natural proteins or fragments thereof or variants with modified amino acid sequences, as well as synthetic proteins or amino acid homopolymers. Such proteins include, in particular, the Fc fragment of IgG, serum albumin or transferrin.

[0196] A fusion partner is broadly defined herein as a second polypeptide (or second amino acid sequence) that is not naturally present in combination (e.g. adjacent or directly or indirectly linked) with a first CCN TSP-1 homologous polypeptide, but is instead synthetically or artificially linked to a first CCN TSP-1 homologous polypeptide. Thus, a fusion protein comprises a non-natural combination of at least two amino acid sequences or polypeptides linked or fused together.

[0197] The fusion partner can be an amino acid sequence of at least 6, 8, 9, 10, 15, 20, 25, 30, 40 or 50 or more amino acids in length. Typically, the fusion partner is a functional polypeptide, or in other words, it is a polypeptide that imparts a function or property to the fusion protein (e.g., stabilizing the fusion protein (making the first polypeptide more stable) or increasing its serum half-life). Therefore, the fusion partner can be a structural protein or have a structural function, or it can impart an activity or property to the fusion protein, such as binding activity (e.g., the fusion partner can be a member of a binding pair, or it can be an affinity binding partner, etc.). In representative examples, the fusion partner can be albumin (particularly serum albumin), fibrinogen, glutathione S-transferase, transferrin, streptavidin or streptavidin-like protein, or an immunoglobulin or a portion thereof, particularly an Fc portion of an immunoglobulin (e.g., IgG1, IgG2, IgG3 or IgG4) or a portion thereof or a modification thereof. Suitable serum albumins include bovine serum albumin (BSA), mouse serum albumin (MSA), and in particular human serum albumin (HSA). Other possible fusion partners include polypeptides that can be used to improve the pharmacokinetic properties of the fusion protein, such as synthetic polypeptides, such as homoamino acid polymers, proline-alanine-serine polymers or elastin-like peptides, such as Strohl, 2015, BioDrugs 29, described in 215-239. Any fusion partner known in the art for use with therapeutic proteins can be used.

[0198] In one embodiment, the fusion partner (ii) of the fusion protein according to the present invention may be the Fc fragment of IgG (any subclass or chimera of any subclass), serum albumin or transferrin.

[0199] As defined herein, the fusion partner may be coupled to the TSP-1 homology domain protein component of the fusion protein at the N-terminus or C-terminus, for example to the TSP-1 repeat homology domain of CCN5 or any other CCN protein. It may be directly or indirectly linked via a linker, as further described below.

[0200] Fc fragments tend to form dimers, and when used in fusion proteins, the fusion protein construct tends to contain two copies of the fusion protein. However, it is known in the art that monomeric Fc fragments and monomeric fusion proteins containing them can be prepared.

[0201] Thus, in case the fusion partner is an Fc fragment, the Fc fragment is preferably a monomeric Fc fragment of any class, such as a monomeric Fc fragment of human IgG. Chimeric Fc fragments comprising portions of Fc regions from different classes, as well as Fc fragments with modified sequences are contemplated.

[0202] Fc fusion proteins are a growing class of protein therapeutics based on chimeric proteins consisting of an effector domain coupled to an Fc fragment of the IgG isotype. A typical example of a biopharmaceutical product is etanercept (TNF-α receptor coupled to an Fc fragment) for the treatment of, for example, rheumatoid arthritis. Another example of an Fc-fused biopharmaceutical protein is aflibercept. Aflibercept is a VEGF receptor Fc fusion protein used to treat wet macular degeneration and metastatic colorectal cancer. The main principle for preparing Fc fragment fusion proteins is to obtain an extension of half-life due to the increase in molecular weight sufficient to exclude renal excretion and enhance reabsorption in the renal proximal tubules through the neonatal Fc receptor. In addition, the pH-dependent binding of Fc fusion proteins to neonatal Fc receptors (FcRn) on endothelial cells allows Fc-based fusion proteins that were originally destined for endocytosis and subsequent lysosomal degradation to be recycled and released back into the circulation.

[0203] According to one embodiment, a fusion protein is provided, wherein the fusion partner (ii) is an Fc fragment from human IgG (immunoglobulin G, also referred to as immunoglobulin gamma) (including all subclasses of human IgG). According to another embodiment of the present invention, a fusion protein is provided, wherein the fusion partner is an Fc fragment of IgG1, IgG2 or IgG4. Preferably, the Fc fragment of human IgG is an Fc fragment of subclass IgG4 (SEQ ID. No 13) or IgG2 (SEQ ID. No 14).

[0204] IgG1, IgG2 and IgG4 are often superior to IgG3 due to their longer half-life of about 3 weeks. The technician will admit that the selection of the IgG isotype of a specific subclass as an Fc fusion partner will depend on the half-life extension and cytotoxic activity level of the desired final compound. Most of the therapeutic antibodies designated for the treatment of cancer or autoimmune diseases belong to the IgG1 subclass because they have high affinity to Fc receptors and have the powerful ability to exert immune effector functions. On the other hand, when it is expected to lack immune effector functions, IgG2 and IgG4 are preferred IgG subclasses as the skeleton of therapeutic candidates because immune effector functions can cause adverse effects. The tendency of the Fc fragment to activate immune effector functions depends on Ig isotypes and subclasses, and is different because of the difference in immune effector functions. Except selecting the Fc fragment of a suitable IgG subclass, the amino acid sequence of the IgG subclass can be modified, for example, by site-directed mutation, to reduce the ability of the Fc fragment to activate immune effector functions.

[0205] Such Fc fragments can be selected, which form monomers or more precisely, which retain or have monomeric form, or can be modified to introduce mutations that allow or promote monomeric structure. Such mutations are referred to herein as "monomer-generating mutations". Examples of Fc fragments comprising monomer-generating mutations are SEQ ID No. 54 and 55. Those skilled in the art know how to introduce such mutations and select monomeric Fc mutants.

[0206] Avoidance of immune effector activation function of FC fragments

[0207] For example, in the biopharmaceutical fusion protein dulaglutide (Trulicity TM In ) (GLP-1 agonist-Fc fragment fusion protein for once-weekly treatment of type 2 diabetes), the well-characterized mutations F234A and L235A were introduced into the hinge region of the IgG4 Fc fragment to reduce the ability to activate immune effector functions.

[0208] According to one embodiment of the present invention, the Fc fusion partner is the Fc fragment of IgG4, wherein the IgG4 Fc fragment is modified (eg, comprises the above F234A and L235A mutations) to avoid immune effector functions.

[0209] Protease-resistant Fc fragment

[0210] Another factor that can reduce both the yield and the biological half-life in the preparation is the endopeptidase cleavage of the fusion protein. In order to reduce or eliminate the risk of proteolytic degradation, modifications can be introduced into the amino acid sequence of the Fc fragment, particularly by introducing mutations in sites susceptible to proteolytic cleavage. In European patent application EP2654780B1, the Fc domain of the IgG1 constant region is modified by replacing E233-L234-L235-G236 with P233-V234-A235 (missing G236) (EU numbering) to make the resulting protein containing the modified Fc resistant to proteolytic degradation.

[0211] Incorporating the amino acid modifications disclosed in EP2654780B1 into the Fc fragment of IgG4 coupled to domain III of CCN5 was not thought to provide adequate resistance to endopeptidases. However, according to the present invention, an improvement in protease resistance is achieved by further modifying the IgG subtype used as a fusion partner.

[0212] More specifically, it has been found that a fusion protein comprising the entire hinge region of IgG2 and constant heavy chains 2 and 3 of IgG4 exhibits superior resistance to proteolysis.

[0213] In Mueller JP et al., Mol. Immunol. (1997), 34 (6), pp. 441-452, the use of IgG2 / IG4 chimeras in IgG antibodies is disclosed. Another biopharmaceutical monoclonal antibody named eculizumab, which is used to treat paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome, has been shown to be useful, for example, for avoiding the ability of IgG4 to activate FcγR-dependent immune effects. In addition, the IgG4 constant domains 2 and 3 of such chimeric Fc fragments also show the ability to avoid IgG2 activating complement-dependent immune effector functions. In this regard, reference is made to Rother et al., (2007), see Nat. Biotechnol., 25 (11), pp. 1256-1264 and the reports of Mueller JP et al. (supra).

[0214] In addition, Borrok et al. (2017), J. Pharm. Sci. 106; 1008-1017 discloses the introduction of modifications in the Fc fragment to study its effect on the antibody immune effector function (FQQ-YTE mutation). In WO2017158426A1, it is disclosed to modify antibodies by introducing mutations in the Fc fragment to improve the half-life of the antibody. Specifically, modifications of one or more of positions 311, 434, 428, 438 and 435 in the Fc region of an immunoglobulin are disclosed.

[0215] In addition, Kinder et al. J Biol Chem. 2013 Oct 25; 288(43): 30843-54 reported that mutations in the lower hinge of IgG1 (ie, E233P, L234V, L235A, G236-, Eu numbering) resulted in protease resistance of IgG1 antibodies.

[0216] According to one embodiment, the Fc fragment of the fusion protein according to the present invention consists of an Fc fragment of the IgG4 subclass into which the following mutations are incorporated: S228P, F234A, L235A, K447-, Eu numbering, see SEQ ID No.15. Jacobsen et al. J Biol Chem. 2017 Feb 3; 292 (5): 1865-1875 reported that the mutation of Asn297 resulted in the Fc fragment being aglycosylated, which further resulted in the lack of IgG effector function. Jacobsen also found that some variants (N297G) resulted in antibodies having better stability and developability compared to other variants (N297Q or N297A). In addition, other modifications (disulfide bridges) were introduced, which resulted in better stability than parental IgG1.

[0217] According to the present invention, when the fusion partner is an Fc fragment, it may be aglycosylated without or with stable disulfide bridges, such as in SEQ ID No.16.

[0218] To the best of the present inventors' knowledge, an Fc fragment consisting of the entire hinge region of IgG2 and constant heavy chains 2 and 3 of IgG4 has not been previously used to prepare a fusion protein by linking the Fc fragment to an effector protein.

[0219] According to one embodiment, the fusion partner of the fusion protein of the present invention is an aglycosylated IgG1 Fc fragment and is stabilized by a disulfide bridge, and the following mutations are introduced into the lower hinge: E233P, L234V, L235A, G236-(Eu numbering) (SEQ ID NO.17).

[0220] According to one embodiment, the fusion partner of the fusion protein comprising the TSP-1 repeat homology domain of a CCN family protein is the Fc fragment of IgG4, and in addition to the S228P and K477-mutations, the following mutations are introduced into the lower hinge: E233P, L234V, L235A, G236-(Eu numbering) (SEQ ID NO.18).

[0221] In a preferred embodiment, the Fc fragment is a chimera of the hinge region of IgG2 (216ERKCCVECPPCPAPPVA-GP238, Eu numbering) and any other IgG subclass. Most preferably, as shown in SEQ ID.No.19, the Fc fragment is a chimera of the hinge region of IgG2 and the constant heavy domains 2 and 3 of IgG4, wherein the carboxyl terminal K477 (Eu numbering) is deleted. This embodiment of the present invention has been shown to have improved protease resistance characteristics (see Example 6).

[0222] In one embodiment, the fusion partner of the monomeric fusion protein of the present invention is an Fc fragment of IgG1 with disulfide bridge stabilization (R292C, V302C), aglycosylation (N297G) and monomer generation mutations (C220Q, C226Q, C229Q, T366R, L368H, P395K, K409T, M428L), Eu numbering), as provided in SEQ ID NO: 54.

[0223] In another embodiment, the fusion partner of the monomeric fusion protein of the present invention is an Fc fragment that is a chimera of the hinge region of IgG2 and the constant heavy domains 2 and 3 of IgG4, which has a carboxyl-terminal 477-deletion and has monomer-generating mutations (C219Q, C220Q, C226Q, C229Q, L351F, T366R, P395K, F405R, Y407E) and half-life extension mutations (M252Y, S254T, T256E) (Eu numbering), as provided in SEQ ID NO: 55.

[0224] Although the fusion protein according to the present invention is exemplified by using an Fc fragment consisting of the entire hinge region of IgG2, constant heavy chains 2 and 3 of IgG4, and domain III of a member of the CCN protein family, it is believed that favorable protease resistance can also be achieved if such an Fc fragment chimera is coupled to other effector molecules (e.g., VEGFR, FGF-21, or GLP1). The effector molecule is part of the Fc fusion protein, providing the desired pharmacodynamic properties, while the Fc fragment contributes to the pharmacokinetic properties.

[0225] Serum albumin as fusion partner

[0226] An alternative strategy to extend the half-life of peptides and proteins is to use serum albumin (SA) as a fusion partner. IgG and SA share an extended half-life of about 19 days, compared to the half-life of several days or less for most other circulating proteins. SA also has affinity for the neonatal Fc receptor (FcRn) and is rescued from intracellular degradation (see Andersen et al. (2014), J Biol Chem, 289 (19); pp 13492-13502).

[0227] In one embodiment of the present invention, there is provided a fusion protein as described above, wherein the fusion partner is serum albumin, preferably human serum albumin.

[0228] In one embodiment, a monomeric fusion protein is provided as described above, wherein the fusion protein comprises amino acids 25 to 606 of human serum albumin as provided in SEQ ID NO:101.

[0229] In another embodiment of the invention, albumin (eg, human serum albumin) is modified to increase or decrease half-life, for example by altering its FcRn affinity with or without pH dependency, which results in an increase or decrease in half-life.

[0230] Transferrin as fusion partner

[0231] In addition, an alternative strategy to extend the half-life of peptides and proteins is to use transferrin as a fusion partner, taking advantage of the naturally long half-life of transferrin. (Strohl W. BioDrugs. 2015; 29(4): 215-239). Transferrin can be used in its glycosylated or non-glycosylated form.

[0232] In one embodiment of the present invention, a fusion protein is provided as described above, wherein the fusion partner is transferrin, preferably human transferrin.

[0233] In one embodiment, a monomeric fusion protein is provided as described above, wherein the fusion protein comprises amino acids 20 to 698 of human transferrin as provided in SEQ ID NO:53.

[0234] Connectors

[0235] According to another embodiment, the fusion protein according to the present invention may optionally comprise a peptide linker between the fusion partner and the effector molecule, i.e. the linker is fused to the N-terminus or C-terminus of the TSP-1 repeat homology domain (TSP-1 domain protein / polypeptide) of the CCN protein.

[0236] Any peptide linker can be used (as long as it is not a CCN protein sequence), many of which are known and described in the art. The linker can be a flexible linker sequence (which can include repeats of a flexible linker sequence motif). Typical linkers known in the art are rich in small non-polar (e.g., glycine) or polar (e.g., serine or threonine) residues, and are usually composed of glycine and serine residues (GS) or other amino acid residues (e.g., alanine, lysine and / or glutamic acid (A, K and / or E)) or even a stretch of any amino acids. A commonly used linker is the (GGGGS) linker (SEQ ID NO: 121), which can be provided as a repeating unit in the linker (e.g., (GGGGS)n, where the number of copies of n can be adjusted, e.g., 1 to 10, 1 to 6, 1 to 4, etc.). The linker can be 1 to 50, 1 to 45, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 12, 1 to 10, for example 1 to 8, 1 to 6, 1 to 5 or 1 to 4 amino acids long. A variety of different linkers are described and used in the examples below, and any of these can be used in any fusion protein of the present invention.

[0237] In some embodiments, the linker comprises no more than 50 amino acids.

[0238] The properties of peptide linkers can further improve the maintenance of effector function. However, peptide linkers may be susceptible to endopeptidase cleavage and fusion protein elimination. Peptide linkers with glycine with or without serine residues interspersed are generally used, but this design does not always produce fusion proteins with desired activity and endopeptidase resistance. In US20180273603, neurotrophin binding protein-Fc-fusion proteins are disclosed, and it is recommended to use a repeated α-helical linker comprising the sequence A(EAAAK)A (SEQ ID No. 14 therein). In addition, US2018 / 0127478 discloses an amino acid linker consisting of one to three repeats of the sequence EAAAK that is recommended for use in Fc fusion proteins.

[0239] According to the present invention, a linker consisting of the peptide sequence EAAAK (SEQ ID No. 21 herein) can also be incorporated between the TSP-1 homology domain and the fusion partner (Fc fragment). More preferably, the linker consists of repeats of the amino acid sequence EAAAK.

[0240] If a linker is included in the fusion protein of the present invention, the linker is located between the fusion partner and the effector molecule (i.e., domain III of the CCN protein). The linker can be introduced at the C-terminus or N-terminus of domain III of the CCN protein.

[0241] In addition, the helical linker is resistant to endopeptidase cleavage after expression of the recombinant protein in suspension CHO cells. This is important both for manufacturing purposes and in vivo efficacy. In addition, the incorporation of an α-helical linker between the Fc fragment and the effector domain in an Fc fusion protein has been shown to reduce the aggregation tendency of the Fc fusion protein.

[0242] Although these findings were shown in the context of a fusion protein comprising domain III of a CCN family protein as an effector protein, it is believed that advantageous reduced aggregation tendency and protease resistance effects may also be obtained if other effector molecules are combined with the Fc fragment α-helical linker according to the present invention.

[0243] Therefore, the present invention provides an Fc fusion protein comprising an Fc fragment, which has a peptide linker sequence of the formula aa1-aa2-(EAAAK)n-aa3-aa4-aa5 between the Fc fragment and the effector molecule, wherein n≥4, and wherein aa1, aa2, aa3, aa4, aa5 are independently absent or are amino acids. The linker can be located at the N-terminus or C-terminus of the Fc fragment. According to one embodiment, n is 8. According to another embodiment, aa1 is threonine (T), and aa1, aa2, aa3, aa4 and aa5 are Ala (A). According to one embodiment, the linker of the above Fc fusion protein is selected from SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24 and SEQ ID NO.25. In particular, it has been shown that the use of a fusion protein comprising a linker consisting of (EAAAK)-repeats (i.e., for example (EAAAK)n, wherein n is 8) according to the present invention advantageously leads to less aggregation.

[0244] An alternative linker that can be used according to the present invention is a linker having an amino acid sequence as shown in SEQ ID No. 20 (TEGRMD).

[0245] Thus, in one embodiment, the present invention may include incorporating a linker peptide between the fusion partner and domain III on CCN5 (e.g., SEQ ID. No 1-SEQ ID. No. 12, 37 or 38). Non-limiting examples of fusion proteins incorporating a linker of SEQ ID No. 20 are shown in SEQ ID No. 28, SEQ ID No. 29 and SEQ ID No. 30, respectively.

[0246] In the case of an embodiment in which the present invention employs domain III of CCN5 with an N-terminal gene fusion peptide linker (as in SEQ ID. No 20) and an Fc fragment of IgG subtype IgG4 (as in SEQ ID.: No. 15) incorporating the following mutations (S228P, F234A, L235A, K447-, Eu numbering), the complete sequence will be as in SEQ ID. No. 28, also referred to as CCN5 (dIII) -Fcv2.

[0247] In the case of an embodiment in which the present invention adopts domain III of CCN5 with an N-terminal gene fusion peptide linker (as in SEQ ID.No.20) and an Fc fragment of IgG subtype IgG4 (as shown in SEQ ID.No.18) incorporating the following mutations (S228P, E233P, F234V, L235A, G236-, K447-, Eu numbering), the resulting sequence will be as shown in SEQ ID.No.29, also referred to as CCN5(dIII)-Fcv2.1.

[0248] In the case of an embodiment of the present invention using domain III of CCN5 with an N-terminal gene fusion peptide linker (as in SEQ ID. No 20) and a chimeric Fc fragment of IgG subtype IgG2 / 4 subtype (as shown in SEQ ID. No 19), the resulting sequence will be as shown in SEQ ID. No. 30, also referred to as CCN5(dIII)-Fcv2.3.

[0249] In the case of an embodiment in which the present invention employs domain III of CCN5 (as in SEQ ID.No 1) with an N-terminal gene fusion peptide linker (as in SEQ ID.No 25) and a chimeric Fc fragment of IgG subtype IgG2 / 4 subtype (as shown in SEQ ID.No 19), the resulting sequence will be as shown in SEQ ID.No 31, also referred to as CCN5(dIII)-HLn8-Fcv2.3.

[0250] According to a preferred embodiment of the present invention, there is provided a fusion protein according to the present invention, comprising:

[0251] 1) Point mutations in domain III of CCN family proteins (especially CCN5) (see SEQ ID No. 7, resulting in reduced susceptibility to proteolysis of domain III;

[0252] 2) an engineered chimera of the Fc fragment of human IgG4 and human IgG2 (SEQ ID No. 19, which has reduced proteolytic susceptibility relative to the previously described Fc fragment backbone for Fc fusion proteins; and

[0253] 3) A composition comprising an optimized peptide linker (see SEQ ID No. 21 to 25) which reduces the susceptibility to proteolysis, enhances the biological activity of the fusion protein and reduces the aggregation tendency of the fusion protein.

[0254] In some embodiments, the peptide linker between the amino acid sequence of (i) and the monomeric fusion partner has an amino acid sequence selected from SEQ ID NO: 20 to 25 or 39, or an amino acid sequence having 80% sequence identity thereto.

[0255] Alternative linker sequences that may be used according to the present invention are provided in SEQ ID NOs: 57, 63, 65, 67 and 121.

[0256] Recombinant expression

[0257] Recombinant protein and fusion protein according to the present invention can be prepared by cultivating host cells that can express the nucleotide sequence encoding the protein. The technician is very familiar with a variety of available biotechnology techniques, which use commonly available genetic engineering techniques and recombinant DNA expression systems to provide the expression of isolated nucleic acid sequences for preparing recombinant proteins in a variety of host cell systems by heterologous expression, see for example "Recombinant Gene Expression Protocols, in Methods in Molecular Biology, 1997, Ed. Rocky S Tuan, Human Press (ISSN 1064-3745) or Sambrook et al., Molecular Cloning: A laboratory Manual (3rd edition), 2001, CSHL Press, (ISBN 978-087969577-4). For example, the nucleotide sequence encoding the recombinant protein according to the present invention can be inserted into a suitable expression vector comprising all necessary transcription and translation regulatory sequences, which are particularly suitable for instructing the expression of the nucleic acid sequence encoding the desired protein in a suitable host cell. Suitable expression vectors are for example plasmids, cosmids, viruses or artificial yeast chromosomes (YAC's).

[0258] The DNA sequence encoding the recombinant protein of the present invention can be synthesized by methods well known to technicians or commercial suppliers well known to technicians, such as Genscript, Thermo Fisher Scientific, etc.

[0259] According to one embodiment of this aspect, a DNA molecule is provided, comprising a nucleic acid sequence as shown in SEQ ID NO: 86, 87, 90, 91, 99, 100, 104, 105, 108, 109, 112 or 113, or a sequence having at least 80% sequence identity with any of the foregoing sequences. An expression vector comprising such a DNA molecule is also provided. According to another embodiment of this aspect, a host cell comprising such a vector is also provided.

[0260] The DNA sequence to be expressed and used to prepare the recombinant protein can be inserted into a vector generally referred to as an entry vector using a Gateway cloning system (Esposito et al, 2009, "Gateway Cloning for Protein Expression", in Methods in Molecular Biology, 498, pp. 31-54). The gene cloned into the entry vector can be easily introduced into a variety of expression vectors by recombination. For example, the synthetic sequence encoding the recombinant protein or fusion protein according to the present invention can be recombined by BP pathway recombinase cloning to generate an entry vector that can be used to propagate the plasmid in a suitable host cell (e.g., E. coli cells). In a preferred embodiment, E. coli cells that have been mutated to allow efficient propagation of the plasmid are used, such as One Shot Top 10 TM cell.

[0261] According to one embodiment of the present invention, an expression vector comprising a DNA sequence is prepared, wherein the DNA sequence encodes a recombinant protein or fusion protein according to the present invention that is operably connected to a promoter. The technician will recognize that "promoter" as used herein refers to the DNA upstream (5'-prime) region of a DNA coding sequence that controls and causes transcription of a specific gene. The promoter controls the recognition and combination of RNA polymerase and other proteins to cause transcription. "Operably connected" refers to the functional connection between a promoter and a second sequence, wherein the promoter sequence causes and mediates the transcription of a DNA sequence corresponding to the second sequence. In general, operably connected means that the connected nucleotide sequence is continuous.

[0262] Entry vectors and expression vectors (eg, generated from the destination vectors described below) can be isolated using standard plasmid isolation techniques well known to those of skill in the art (eg, using plasmid isolation kits from Qiagen TM or QIAGEN TM QIAprep Plasmid Plus Maxi Kit TM Spin Miniprep Kit).

[0263] If an entry vector containing a DNA sequence encoding a recombinant protein or fusion protein according to the present invention is used, the entry vector can be further recombined with a destination vector using an LR pathway recombinase to generate an expression vector. The expression vector can then be used to express the protein-encoding DNA sequence in an appropriate host cell. Non-limiting examples of suitable destination vectors include pUCOE-DHFR-DEST, such as et al., J. Biol. Chem, 293:46, pp.17953-17970.

[0264] Additionally, the resulting expression vector can be verified by standard restriction enzyme digestion and DNA gel electrophoresis.

[0265] According to one aspect of the present invention, an expression vector is provided, which comprises a nucleic acid sequence encoding a recombinant protein of formula (I). According to another aspect of the present invention, an expression vector is provided, which comprises a nucleic acid sequence encoding a protein, the protein comprising an amino acid sequence selected from the group consisting of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8 to 12, 37, 38, 84, 85, 88, 89, 97, 98, 102, 103, 106, 107, 110 and 111; and amino acid sequences SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.37 and SEQ ID No.38 and SEQ ID Nos. 84, 85, 88, 89, 97, 98, 102, 103, 106, 107, 110 and 111 are fragments or variants thereof having at least 50% sequence identity.

[0266] According to another aspect, an expression vector is provided, which encodes a recombinant protein, wherein the recombinant protein comprises an amino acid sequence having at least 60%, 70%, 80%, 90% or 95% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.37 and SEQ ID No.38 and SEQ ID Nos:84, 85, 88, 89, 97, 98, 102, 103, 106, 107, 110 and 111.

[0267] According to another embodiment of the present invention, an expression vector encoding the fusion protein according to the present invention is provided.

[0268] The skilled person will be aware of the degeneracy of the genetic code, and the preference for specific codons in a variety of organisms. Therefore, depending on the choice of host cell, the nucleic acid sequence encoding the recombinant protein and fusion protein of the present invention can be adapted to the preferred codons of the host cell. Therefore, the amino acids of the protein of the present invention can be encoded by any combination of the codons shown in the following table:

[0269]

[0270]

[0271] Preferably, codons are further optimized to achieve high expression according to the selected host cell.

[0272] For expressing proteins by recombinant DNA technology, in addition to specific embodiments of the present invention, it is preferred that a DNA sequence encoding a signal peptide is attached to the N-terminus of the protein sequence. The signal peptide can be used to instruct the localization of the fusion protein during and / or after synthesis in the host cell. Therefore, it can be a sequence that instructs the secretion of the fusion protein. The use of such signal peptide sequences is well known in the art. The signal peptide can take any form, for example, it can constitute an IgG kappa chain signal peptide, or it can constitute a signal peptide from human serum albumin (SEQ ID. No 32).

[0273] In the case where a signal peptide from human serum albumin (SEQ ID. No 32) is appended to the N-terminus of SEQ ID. No 28, the protein sequence to be expressed may be as shown in SEQ ID. No 33 or SEQ ID No: 85, 89, 98, 103, 107 or 111.

[0274] In addition, for expressing proteins by recombinant DNA technology, according to a specific embodiment of the present invention, a protein having an amino acid sequence as shown in SEQ ID NO. 33, such as SEQ ID NO 34 or a nucleotide sequence as shown in SEQ ID NO: 86, 90, 99, 104, 108 or 112, wherein a translation stop codon is attached to the 3' end of the coding sequence, can be used.

[0275] In the case where the present invention is embodied by the nucleotide sequence in SEQ ID. No. 34, the nucleotide sequence is preferably appended to the immediate 5' end of the coding sequence via a Kozak sequence (e.g., GCCACC), such as in SEQ ID No. 35 or SEQ ID No: 86, 90, 99, 104, 108 or 112. The DNA sequence may further be flanked by DNA elements to enable subcloning, such as pathway recombinase attB sites. However, any cloning or synthesis strategy may be used to generate the DNA sequence and facilitate subcloning into an expression vector. In the case where the DNA sequence incorporates a pathway recombinase site to enable subcloning, the nucleotide sequence may be as shown in SEQ ID. No 36 or SEQ ID No: 87, 91, 100, 105, 109 or 113.

[0276] The obtained expression vector containing the nucleic acid sequence encoding the recombinant protein of the fusion protein of the present invention can be introduced into a suitable host cell to produce the desired protein. A variety of commercially available or proprietary host cells can be used. For example, the expression vector can be transferred to a eukaryotic host cell, such as a CHO cell, such as CHO DG44 DHFR (dihydrofolate reductase - / - ) Adapted to suspension culture of CHO cells. Transfection of host cells with expression vectors can be performed by methods well known to technicians, such as electroporation.

[0277] After culturing the host cells in a suitable medium, the recombinant protein or fusion protein according to the present invention encoded by the expression vector will be produced in the host cells, and the resulting protein can be collected and purified by methods well known to those skilled in the art.

[0278] Expression vectors may include a signal sequence, often referred to as a "signal peptide," for secretion of the expressed protein or fusion protein into the culture medium.

[0279] In order to separate and purify secreted recombinant proteins from cell culture media, one or more pretreatment or clarification steps are usually first used to remove large particles and biomass. Non-limiting examples of applicable pretreatment steps are, for example, reverse osmosis, centrifugation, filtration methods and diafiltration, or combinations thereof. The obtained protein is then usually purified by one or more of a variety of chromatography methods well known to the skilled person, such as by affinity chromatography, ion exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography, size exclusion chromatography or other chromatography techniques, or combinations thereof.

[0280] For example, recombinant proteins or fusion proteins expressed by appropriate host cells can be purified using affinity chromatography methods, such as using MabSelect TM SuRe TMMedia, e.g. 5 ml HiTrap MabSelect mounted on FPLC chromatography system TM SuRe TM Column, such as BioRad NGC Discover equipped with a 5 mm UV flow cell TM 10 Pro system. After loading the sample containing the protein to be purified, the column is usually washed one or more times using one or more suitable wash buffers, and then the protein is eluted using a suitable elution buffer. The obtained protein can be further purified using one or more of the chromatographic methods listed above.

[0281] Should be understood that, can utilize the technology well-known to the technician to introduce multiple modifications in the nucleotide sequence of encoding recombinant protein of the present invention for example to promote expression.By using site-directed mutagenesis, can introduce modification so that the encoding sequence is suitable for the desired host of the expressed sequence, and thereby produce recombinant protein.The technician will know the existence of host-specific codons and the fact that the heterologous nucleic acid sequence is adapted to the host-specific codons and has improved expression efficiency as described above.Other modifications can also be introduced, for example, to promote separation and purification, i.e., by adding the sequence of peptides or proteins that can be used for such purpose.In addition, the nucleotide sequence of the signal peptide that coding is provided for the recombinant protein of expectation from host cell secretion also can be connected with nucleotide sequence of the present invention.

[0282] The present invention also provides host cells suitable for producing recombinant proteins or fusion proteins according to the present invention. A variety of commercially available host cells, both prokaryotic host cells and eukaryotic host cells, particularly suitable for producing recombinant proteins can be used. Non-limiting examples of suitable host cells are, for example, CHO cells, HEK293 cells, Pichia pastoris cells, NSO cells or Escherichia coli cells.

[0283] Finally, the present invention also relates to a thrombospondin type 1 repeat (TSP-1) homology domain of a CCN family protein and a fusion protein comprising the TSP-1 repeat homology domain, which are used as drugs for treating or preventing diseases by inhibiting or antagonizing cell signaling and cell physiological functions attributed to CCN family proteins.

[0284] In one aspect, the invention provides a protein, such as a fusion protein, as defined herein for use in therapy.

[0285] In some aspects, the protein, such as a fusion protein, can be used to treat or prevent fibrosis, or to treat or prevent any condition that exhibits fibrosis (i.e., any fibrotic condition or disorder). Fibrosis can affect any tissue or organ, including, for example, the lungs, eyes, heart, skeletal muscle, peritoneum, kidneys, liver, pancreas, bile ducts, skin, blood vessels, or more systemic systems. In particular, the condition exhibiting fibrosis may be selected from pulmonary fibrosis (including idiopathic pulmonary fibrosis), bronchopulmonary dysplasia, retinal fibrosis, diabetic retinopathy, age-related macular degeneration, retinal detachment, oxygen-induced retinopathy, glaucoma, cardiac fibrosis, post-transplant graft fibrosis, cardiomyopathy-associated fibrosis, muscle fibrosis, Duchenne muscular dystrophy, peritoneal fibrosis, diabetic nephropathy, chronic kidney disease (renal fibrosis), acute kidney injury, tubulointerstitial fibrosis, chronic allograft nephropathy, liver fibrosis, non-alcoholic steatohepatitis, fatty liver disease, chronic pancreatitis, biliary fibrosis, keloids, scarring, systemic sclerosis, atherosclerosis, epidural fibrosis.

[0286] In the case of cardiac fibrosis, the condition to be treated or prevented may include cardiac hypertrophy and heart failure with or without preserved ejection fraction.

[0287] In another aspect, the invention provides a protein, such as a fusion protein, as defined herein for use in treating an inflammatory disease or an autoimmune disease. In some embodiments, the inflammatory disease is selected from rheumatoid arthritis, amyotrophic lateral sclerosis (ALS), inflammatory bowel disease, ulcerative colitis, Crohn's disease.

[0288] On the other hand, the invention provides proteins, such as fusion proteins defined herein, for use in treating cancer. In this regard, it is known that 4-domain CCN proteins can both induce carcinogenic responses in isolated cancer cells and contribute to metastasis, chemo-resistance and immunotherapy resistance by acting directly on cancer cells or tumor stroma. Therefore, the activity of proteins herein in inhibiting the effects or activities of 4-domain CCN proteins provides a basic principle for treating cancer. Cancer can be any malignant or pre-malignant tumor condition. It can belong to any tissue or organ. In one embodiment, cancer can be manifested as a solid tumor. In another embodiment, cancer can belong to or be located in the hematopoietic system. It can be a primary cancer or a secondary cancer or a metastatic cancer. Therefore, cancer can be pancreatic cancer, breast cancer, prostate cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, brain cancer, blood cancer, bone cancer, skin cancer, lung cancer or gastric cancer. In some embodiments, the cancer is selected from pancreatic cancer, pancreatic ductal adenocarcinoma, breast cancer, prostate cancer, cervical cancer, ovarian cancer, liver cancer, hepatocellular carcinoma, urothelial bladder cancer, brain cancer, glioblastoma, acute lymphoblastic leukemia, osteosarcoma, melanoma, mesothelioma, gastric cancer, oral squamous cell carcinoma, esophageal cancer, colorectal cancer, lung cancer.

[0289] On the other hand, the invention provides proteins as defined herein, for example fusion proteins, for use in treating metabolic diseases. Metabolic diseases can be insulin resistance or glucose intolerance or can be associated with them. In some embodiments, metabolic diseases are selected from type 2 diabetes and metabolic syndrome.

[0290] The fusion protein of the present invention can also be used in the treatment methods of the above-mentioned diseases. Similarly, the fusion protein of the present invention can be used in the method of preparing the medicine for treating the above-mentioned diseases. Example

[0291] Example 1

[0292] Expression of the fusion protein according to the invention

[0293] In this example, a fusion protein (CCN5 (dIII) -Fcv2) (i.e., a fusion protein according to SEQ ID No. 28) is provided comprising amino acids 194 to 246 of CCN5 (SEQ ID No. 1) and an Fc fragment of the IgG subclass IgG4 (S228P, F234A, L235A, K447-, Eu numbering) of SEQ ID No. 15, comprising an N-terminal fusion peptide linker (SEQ ID No. 20). The fusion protein is also supplemented with an albumin-derived N-terminal signal sequence of SEQ ID No. 32 and expressed in mammalian cells as disclosed below.

[0294] The DNA sequence shown in SEQ ID NO.36 was synthesized by a commercial supplier and sequence verified. The synthesized sequence was recombined with pDonrZeo by BP pathway recombinase cloning to generate an entry vector. The competent E. coli (One Shot Top10 TM Cells) were then isolated using standard plasmid isolation techniques using TM QIAprep TM The entry vector was isolated using the Spin Miniprep Kit. After plasmid isolation, the entry vector was verified by restriction enzyme digestion followed by DNA gel electrophoresis according to standard techniques well known to the skilled artisan.

[0295] The entry vector containing the sequence SEQ ID NO.35 was further recombined with the destination vector using the LR pathway recombinase. The destination vector used was pUCOE-DHFR-DEST, such as et al., 2018 (supra).

[0296] In transfecting competent E. coli (One Shot Top10 TM After PCR, standard plasmid isolation techniques were used using QIAGEN TM The expression vector was isolated by Plasmid Plus Maxi kit. The resulting expression vector was verified by standard restriction enzyme digestion and DNA gel electrophoresis according to standard techniques well known to technicians. TM The "Maximum Titer" protocol provided by the manufacturer of the CHO Transfection Kit (Gibco Catalog No. A29129) and as et al., 2018 (supra), the resulting expression vector was transferred into ExpiCHO adapted suspension culture CHO cells. Six days after transfection, the cells were precipitated by centrifugation at 4750g for 20 minutes at 4°C, and the supernatant cell culture medium was harvested. 0.1M PMSF in 100% isopropanol was added to a concentration of 1mM, and 0.5M EDTA was added to a concentration of 2mM. Then, 96% ethanol was added to a final concentration of about 3%. 1M TrisHCl pH 7.4 was added to a final concentration of 25mM, followed by chromatographic purification.

[0297] The capture step of the purification was performed by affinity chromatography with Protein A chromatography medium. The medium used in this experiment was rProtein A FF (GE Healthcare). A 5 mL HiTrap TMThe expressed recombinant protein was purified from 60 mL of cell culture medium harvested and supplemented as described above using an rProtein A FF column (GE Healthcare). TM The rProtein A FF column was installed in a FPLC chromatography system (BioRad NGC Discovery TM 10Pro system) and equilibrated with a buffer containing 25mM TrisHCl pH 7.4, 25mM NaCl and 3% ethanol. The harvested cell culture medium containing the recombinant protein was loaded with a sample pump at a rate of 2.5ml / min, followed by washing with 6 column volumes of wash buffer (25mM TrisHCl pH 7.4, 25mM NaCl and 3% ethanol), and then eluted with 0.1M sodium citrate (pH 3.0) in 3% ethanol. The eluate with a UV 280Bm absorbance exceeding 100mAU was collected in 3mL fractions in a low protein binding tube prefilled with 1mL 1M TrisHCl pH 9.0. Use The fraction containing the UV absorption peak was concentrated to 500 μL using a 20 mL, 30 kDA MWCO concentrator. After concentration, the sample was loaded onto an FPLC chromatography system (BioRad NGC Discover TM The FPLC system was equipped with a 50 mM NaCl, 20 mM HEPES pH 7.0 equilibrated 200Increase 10 / 300GL column (GE Healthcare). The sample was injected and the column was perfused with pre-equilibrated buffer (50 mM NaCl, 20 mM HEPES pH 7.0) at a flow rate of 0.25 mL / min. The major UV 280 nm absorption peak was found to contain the purified recombinant protein (CCN5(dIII)-Fcv2, SEQ ID No. 28). TGX Stain-Free TM 10 μL of the collected fractions were subjected to SDS-PAGE on a precast gel and analyzed using ChemiDoc TM The separated recombinant proteins were visualized using the Imaging System (BioRad).

[0298] It is well known to the skilled artisan that recombinant proteins can be produced in a variety of expression systems and purified by a variety of chromatographic methods with similar results.

[0299] Example 2

[0300] A DNA sequence encoding a fusion protein comprising amino acids 194 to 246 of CCN5 (SEQ ID No. 1) with an N-terminal fusion peptide linker (SEQ ID No. 20) and an Fc fragment of the IgG subclass IgG4 of SEQ ID No. 15 (S228P, F234A, L235A, K447-, Eu numbering) (CCN5 (dII'-Fcv2) was expressed to produce a recombinant protein according to SEQ ID NO. 28.

[0301] The resulting proteins were tested for their ability to inhibit pro-survival signaling (serine-473 phosphorylation of AKT) in A549 human lung cancer cells ( Figure 1 A). Fibronectin (Sigma catalog number F1141, Dulbecco's phosphate buffered saline (Lonza catalog number 17-512F, hereinafter referred to as PBS) diluted to 10 μg / mL) coated with tissue culture treated Corning Incorporated 96-well sterile polystyrene plates. A coating solution containing fibronectin was dispensed into the wells at a volume of 100 μL / well, incubated at room temperature for 1 hour, and then the coating solution was decanted, and 100 μL PBS was also dispensed into the fibronectin-coated wells that were also decanted. A549 cells that were subcultured to maintain a maximum confluence of 80% were treated with enzymes (from of L0950-100), diluted to a concentration of 110,000 cells / mL in Dulbecco's modified Eagle's medium (Gibco catalog number: 41965-039) containing high glucose supplemented with 10% heat-inactivated fetal bovine serum (FBS) (500 mL flasks with FBS (from Gibco, catalog number 16000-044) equilibrated to room temperature were incubated with shaking in a water bath at 60°C for 30 minutes) and 50 μg / ml gensumycin (Sanofi), and 100 μL of the cell solution was dispensed into the fibronectin-coated wells. All cell incubations were performed in a cell culture incubator maintained at a temperature of 37°C, a humidified room air atmosphere, and 5% CO2. After overnight incubation, A549 cells were washed twice in PBS, and 90 μL Dulbecco's modified Eagle medium (DMEM, Gibco catalog number: 41965-039) containing high glucose and 50 μg / ml gensumycin (Sanofi) without FBS was distributed to the wells. After incubation for 18 hours in a medium without FBS, the cells were stimulated with a solution of 10 μL of the recombinant protein in question. After 60 minutes of stimulation, the culture medium was decanted and the cells were harvested by adding 50 μL of lysis buffer and a blocking reagent provided according to the Cisbio phosphorylated AKT (Ser473) kit (Cisbio Inc, catalog number: 64AKSPEG). After adding lysis buffer and blocking reagent, the 96-well plate was incubated on a PST-60HL plus (ThermoFisher) plate shaker at 500 rpm for 60 minutes. After shaking, the lysed samples were triturated and 16 μL from each well was transferred to a white-walled HTRF 96-well low-volume plate (Cisbio Inc., catalog number: 66PL96025). To determine the amount of phosphorylated AKT (Ser473), 4 μL of a mixture of labeled antibodies (50 / 50 volume / volume mixture of phosphorylated AKT d2 and phosphorylated AKT cryptate from Cisbio Inc, catalog number: 64AKSPEG) was added to each well (only cryptate antibody was used for negative control wells), the plate was sealed with adhesive plastic film and incubated overnight at 4°C, and then read on a PolarStar Omega plate reader (BMG Labtech, Germany) equipped with a TR-FRET recording head and 337 nm emission and 615 nm and 665 nm excitation filters. The ratio between the 665 nm and 615 nm excitation recordings was blank corrected, and the values ​​of the wells stimulated by the recombinant protein were expressed as a percentage of the wells stimulated by the vehicle.

[0302] Example 3

[0303] A DNA sequence encoding a fusion protein comprising amino acids 194 to 246 of CCN5 (SEQ ID No. 1) with an N-terminal fusion peptide linker (SEQ ID No. 20) and the Fc fragment of the IgG subclass IgG4 of SEQ ID No. 15 (S228P, F234A, L235A, K447-, Eu numbering) (CCN5(dIII)-Fcv2) was expressed to produce a recombinant protein according to SEQ ID NO. 28.

[0304] The resulting proteins were tested for their ability to inhibit profibrotic TGF-β-stimulated transcription (from SMAD2 / 3 binding cis-elements) in IMR90 human lung fibroblasts ( Figure 1 D) Determination of technical et al. (2018) (supra), except that 2500 IMR90 lung fibroblasts / well were used instead of Rat2 cells. Proteins used for stimulation were as Figure 1 As shown in D. IMR90 cells were subcultured as described above for A549 cells before use. IMR90 cells were used before passage 20, i.e. before reaching replicative senescence.

[0305] Example 4

[0306] A DNA sequence encoding a fusion protein comprising amino acids 194 to 246 of CCN5 (SEQ ID No. 1) with an N-terminal fusion peptide linker (SEQ ID No. 20) and the Fc fragment of the IgG subclass IgG4 of SEQ ID No. 15 (S228P, F234A, L235A, K447-, Eu numbering) (CCN5(dIII)-Fcv2) was expressed to produce a recombinant protein according to SEQ ID NO. 28.

[0307] The obtained proteins were tested for their ability to inhibit the proliferation of the human lung fibroblast cell line IMR90 ( Figure 1 B). IMR90 cells were subcultured as described above for A549 cells before use. IMR90 cells were used before passage 20, i.e. before reaching replicative senescence. For experiments, IMR90 cells were harvested as described above for A549 cells, washed in PBS, diluted in DMEM containing 1% FBS and gensumycin, as described in Experiment 2 above, and seeded at a density of 12,000 / well in xCELLigence impedance plates. After 2 hours, cells were stimulated with 10 μL of a solution of the recombinant protein in question or FBS and incubated for an additional 72 hours before being treated with (Promega Inc.) harvested, e.g. et al. (2018) (supra).

[0308] Example 5

[0309] A DNA sequence encoding a fusion protein comprising amino acids 194 to 246 of CCN5 (SEQ ID No. 1) with an N-terminal fusion peptide linker (SEQ ID No. 20) and the Fc fragment of the IgG subclass IgG4 of SEQ ID No. 15 (S228P, F234A, L235A, K447-, Eu numbering) (CCN5(dIII)-Fcv2) was expressed to produce a recombinant protein according to SEQ ID NO. 28.

[0310] The obtained proteins were tested for their ability to inhibit the sphere-forming capacity (anchorage-independent growth) of the estrogen receptor-positive breast cancer cell line MCF-7 and the triple-negative breast cancer cell line MDA-MB-231. Figure 1 C) et al. (supra). MDA-MB-231 cells were treated identically to those described for the MCF-7 cell line in et al. (supra). MCF-7 and MDA-MB-231 cell lines were subcultured as described above for the A549 cell line.

[0311] Example 6

[0312] A DNA sequence encoding a fusion protein was expressed, the fusion protein comprising amino acids 194 to 246 of CCN5 (SEQ ID No. 1) with an N-terminal fusion peptide linker (SEQ ID No. 20) and an Fc fragment of the IgG subclass IgG4 of SEQ ID No. 15 (S228P, F234A, L235A, K447-, Eu numbering) (CCN5 (dIII) -Fcv2), an Fc fragment of the IgG subclass IgG4 of SEQ ID No. 18 (S228P, E233P, F234V, L235A, G236-, K447-, Eu numbering) (CCN5 (dIII) -Fcv2.1) or a chimeric Fc fragment of the IgG2 / 4 subclass (SEQ ID No. 19) (CCN5 (dIII) -Fcv2.3) to produce a chimeric Fc fragment according to SEQ ID NO. 28 (CCN5 (dIII) -Fcv2), SEQ ID The recombinant proteins of SEQ ID No. 29 (CCN5(dIII)-Fcv2.1) and SEQ ID No. 30 (CCN5(dIII)-Fcv2.3).

[0313] Specifically, according to Expifectamine TMThe "Maximum Titer" protocol provided by the manufacturer of the CHO Transfection Kit (Gibco Catalog No. A29129) and as et al., 2018 (supra), expression vectors encoding for expression of SEQ ID NO.28 (CCN5 (dIII) -Fcv2), SEQ ID NO.29 (CCN5 (dIII) -Fcv2.1) and SEQ ID NO.30 (CCN5 (dIII) -Fcv2.3) were transfected into ExpiCHO suspension-adapted CHO cells. Six days after transfection, cells were pelleted by centrifugation at 13000 rpm for 5 minutes in a Heraeus biofuge pico benchtop centrifuge, and the supernatant cell culture medium was harvested. TGX Stain-Free TM Precast gels were used to separate harvested cell culture supernatant samples by SDS-PAGE and analyzed using ChemiDoc TM Recombinant proteins were visualized using the ELISA Imaging System (BioRad). The separated proteins were then transferred to a PVDF membrane using the Trans-Blot-Turbo Semi-Dry Blotting System (Bio-Rad) for Western blot analysis. The blot was probed with an anti-human IgG4 antibody conjugated to horseradish peroxidase (Invitrogen catalog number: A10654) and the antibody was used with SuperSignal TM West Femto Maximum Sensitivity Substrate (ThermoFisherScientific) and ChemiDoc TM An imaging system (BioRad) was used in conjunction for visualization.

[0314] exist Figure 2 , data demonstrating improved protease resistance of the Fc fragment backbone (shown in SEQ ID No. 19) composed of IgG2 / 4 chimera are shown.

[0315] CCN5 / WISP2 (domain III) fused to an IgG4 Fc fragment with the following: an immune effector-silenced IgG4 hinge (as defined by SEQ ID No. 28), CCN5 (domain III) -Fcv2; the same IgG4 backbone incorporating IgG2-based mutations (as defined by SEQ ID No. 29), CCN5 (domain III) -Fcv2.1; or the same IgG4 backbone with an intact hinge region from IgG2 (as defined by SEQ ID No. 30), CCN5 (domain III) -Fcv2.3 were expressed in the ExpiCHO system, and conditioned medium (CM) was harvested after 6 days. Western blotting and total protein staining of SDS-PAGE gels revealed that the CCN5 (domain III) -Fcv2.3 variant was the least susceptible to proteases present during culture. Note that with the replacement of the IgG2 sequence, the immunoreactivity of the anti-IgG4 antibody to the Fc fragment was partially lost, and thus the protein levels relative to general protein staining were underestimated.

[0316] Example 7

[0317] A DNA sequence encoding a fusion protein was expressed, the fusion protein comprising amino acids 194 to 246 of CCN5 (SEQ ID No. 1) and a chimeric Fc fragment of the IgG2 / 4 subclass (SEQ ID No. 19) (CCN5 (dIII) -Fcv2.3) fused to the N-terminus with a peptide linker shown in SEQ ID No. 20 or a chimeric Fc fragment of the IgG2 / 4 subclass (SEQ ID No. 19) (CCN5 (dIII) -HLn8-Fcv2.3) with a peptide linker shown in SEQ ID No. 25 to produce recombinant proteins according to SEQ ID NO. 30 (CCN5 (dIII) -Fcv2.3) and SEQ ID No. 31 (CCN5 (dIII) -HLn8-Fcv2.3).

[0318] Specifically, according to Expifectamine TM The "Maximum Titer" protocol provided by the manufacturer of the CHO Transfection Kit (Gibco Catalog No. A29129) and as et al., 2018 (supra), expression vectors encoding for expression of SEQ ID NO.30 (CCN5 (dIII) -Fcv2.3) and SEQ ID NO.31 (CCN5 (dIII) -HLn8-Fcv2.3) were transfected into ExpiCHO suspension-adapted CHO cells. Four days after transfection, cells were pelleted by centrifugation at 13,000 rpm for 5 minutes in a Heraeus biofuge pico benchtop centrifuge, and the supernatant cell culture medium was harvested. Stain-Free TM Precast gels were separated by SDS-PAGE to separate the harvested cell culture supernatant samples. The separated proteins were transferred to PVDF membranes for Western blot analysis using the Trans-Blot-Turbo semi-dry blotting system (Bio-Rad). The blots were probed with anti-human IgG4 antibody conjugated to horseradish peroxidase (Invitrogen catalog number: A10654) and the antibody was used with SuperSignal TM West Femto Maximum Sensitivity Substrate (ThermoFisherScientific) and ChemiDoc TM An imaging system (BioRad) was used in conjunction for visualization.

[0319] exist Figure 3 , data are provided showing that when embodiments of the present invention are incorporated into a peptide linker as shown in SEQ ID NO. 25, the aggregation tendency is reduced.

[0320] Non-reducing SDS-PAGE of CM from transiently transfected CHO suspension cells expressing CCN5 (domain III) fused to the amino terminus of a chimeric IgG2 / 4 Fc fragment via various peptide linkers. Western blotting revealed that the fusion protein (dIII)-HLn8-Fcv2.3 having the amino acid sequence shown in SEQ ID No.31 had a lower aggregation tendency than the fusion protein CCN5 (domain III)-Fcv2.3 of the present invention having the amino acid sequence shown in SEQ ID No.30. This finding indicates that the peptide linker defined in the sequence SEQ ID No.25 provides a lower aggregation tendency of the fusion protein compared to the fusion protein containing the peptide linker defined in the sequence SEQ ID No.20.

[0321] Example 8

[0322] A DNA sequence encoding a fusion protein was expressed, comprising amino acids 194 to 246 of CCN5 (SEQ ID No. 1) or amino acids 194 to 246 of CCN5 (SEQ ID No. 7) (wherein the amino acid (proline) at position 195 is replaced by alanine) with a C-terminal fusion peptide linker (SEQ ID No. 39) and an Fc fragment of the IgG subtype IgG4 of SEQ ID No. 15 (S228P, F234A, L235A, K447-, Eu numbering) to produce a recombinant protein according to SEQ ID NO.: 40 (Fc-HLn8-CCN5 (dIII)) or SEQ ID No. 41 (Fc-HLn8-CCN5 (dIII)-P195A).

[0323] Specifically, according to Expifectamine TM The "Maximum Titer" protocol provided by the manufacturer of the CHO Transfection Kit (Gibco Catalog No. A29129) and as et al., 2018 (supra), expression vectors encoding for expression of SEQ ID NO.: 40 (Fc-HLn8-CCN5 (dIII)) and SEQ ID NO.: 41 (Fc-HLn8-CCN5 (dIII)-P195A) were transfected into ExpiCHO suspension-adapted CHO cells. Three days after transfection, cells were pelleted by centrifugation at 13,000 rpm for 5 minutes in a Heraeus biofugepico benchtop centrifuge, and the supernatant cell culture medium was harvested. Stain-Free TM Precast gels were separated by SDS-PAGE to separate the harvested cell culture supernatant samples. The separated proteins were transferred to PVDF membranes for Western blot analysis using the Trans-Blot-Turbo semi-dry blotting system (Bio-Rad). The blots were probed with anti-human IgG4 antibody conjugated to horseradish peroxidase (Invitrogen catalog number: A10654) and the antibody was used with SuperSignal TM West Femto Maximum Sensitivity Substrate (ThermoFisherScientific) and ChemiDoc TM An imaging system (BioRad) was used in conjunction for visualization.

[0324] exist Figure 5 , data are provided showing that when embodiments of the present invention are incorporated into a mutation of proline 195 of the CCN5 TSP-1 repeat homology domain (as shown in SEQ ID No 7), susceptibility to endopeptidase cleavage is reduced.

[0325] Reducing SDS-PAGE of CM from transiently transfected CHO suspension cells expressing CCN5 (domain III) fused to the carboxyl terminus of an IgG4 Fc fragment as shown in SEQ ID No. 15 incorporating a P195A mutation (Fc-HLn8-CCN5(dIII)-P195A) or expressing a wild-type P195 variant of the CCN5 TSP-1 repeat homology domain (Fc-HLn8-CCN5(dIII)). This blot demonstrates that the P195A mutation confers proteolytic resistance to the TSP-1 repeat homology domain of CCN5.

[0326] Example 9

[0327] Disclosed is a fusion protein of amino acids 194 to 250 of human CCN5 (SEQ ID No. 56) comprising an N-terminal fusion peptide linker (SEQ ID No. 57) and an Fc fragment of human IgG subclass IgG4 (S228P, F234A, L235A, K447-, Eu numbering) of SEQ ID No. 15, resulting in a protein sequence corresponding to SEQ ID No. 58 (CCN5 (dIII) -SL-Fcv0). The fusion protein is also attached with an N-terminal signal sequence for secretion derived from albumin of SEQ ID No. 32 to produce a fusion protein corresponding to SEQ ID No. 59, and is expressed in mammalian cells as disclosed below.

[0328] The DNA sequence encoding the fusion protein of SEQ ID No.59 was codon-optimized for protein expression in hamster cells (by the algorithm of the commercial supplier), and a KOZAK sequence for translation was appended at the 5' end, and a stop codon was introduced at the 3' end, resulting in a DNA sequence of SEQ ID.No.60. This DNA sequence was further appended with pathway attB sites at both ends, resulting in a DNA sequence of SEQ ID No.61. The sequence of SEQ ID No.61 was synthesized and verified by the commercial supplier. The synthesized sequence was recombined with pDonrZeo by BP pathway recombinase cloning to produce an entry vector. In transfection of competent E. coli (One Shot Top10 TM Cells) were then isolated using standard plasmid isolation techniques using TM QIAprep TM The entry vector was isolated using the Spin Miniprep Kit. After plasmid isolation, the entry vector was verified by restriction enzyme digestion followed by DNA gel electrophoresis according to standard techniques well known to the skilled artisan.

[0329] The entry vector containing the sequence SEQ ID NO.60 was further recombined with the destination vector using the LR pathway recombinase. The destination vector used was pUCOE-DHFR-DEST, such as et al., 2018, J. Biol. Chem, 293: 46, pp. 17953-17970.

[0330] In transfecting competent E. coli (One Shot Top10 TM After PCR, standard plasmid isolation techniques were used using QIAGEN TM The expression vector was isolated by Plasmid Plus Maxi kit. The resulting expression vector was verified by standard restriction enzyme digestion and DNA gel electrophoresis according to standard techniques well known to technicians. TM The "Maximum Titer" protocol provided by the manufacturer of the CHO Transfection Kit (Gibco Catalog No. A29129) and as et al., 2018 (supra), the resulting expression vector was transfected into ExpiCHO cells adapted to suspension culture. Four days after transfection, cells were pelleted by centrifugation at 4750xg for 20 minutes at 4°C, and the supernatant cell culture medium was harvested. 0.1M PMSF in 100% isopropanol was added to a concentration of 1mM, and 0.5M EDTA was added to a concentration of 2mM. Then, 96% ethanol was added to a final concentration of about 3%. 1M TrisHCl pH 7.4 was added to a final concentration of 25mM, followed by chromatography.

[0331] The protein was purified by affinity chromatography using protein A chromatography medium. The chromatography medium used in this experiment was rProtein A FF (GE Healthcare). A 5 mL HiTrap TM The expressed recombinant protein was purified from 120 mL of cell culture medium harvested and supplemented as described above using an rProtein A FF column (GE Healthcare). TM The rProtein A FF column was installed in a FPLC chromatography system (BioRad NGC Discovery TM10Pro system) and equilibrated with a buffer containing 25mM TrisHCl pH 7.4, 25mM NaCl and 3% ethanol. The harvested cell culture medium containing the recombinant protein was loaded with a sample pump at a speed of 2.5ml / min, then washed with 10 column volumes of wash buffer (25mM TrisHCl pH 7.4, 25mM NaCl and 3% ethanol), and then eluted with 0.1M sodium citrate (pH 3.0) in 3% ethanol. The 3mL eluted fractions were collected in a low protein binding tube prefilled with 1mL 1M TrisHCl pH 9.0. Protein elution was monitored by 280nm UV absorbance and was measured using a 4% 4% 4% 2-mercaptoethanol precipitate. Stain-Free TM Precast gels were subjected to SDS-PAGE of 10 μL of the pooled fractions containing the UV 280 nm absorbance peak and analyzed using ChemiDoc TM The separated recombinant proteins were visualized using the Imaging System (BioRad).

[0332] It is well known to the skilled artisan that recombinant proteins can be produced in a variety of expression systems and purified by a variety of chromatographic methods with similar results.

[0333] exist Figure 6 , it is shown that in the absence of the reducing agent β-mercaptoethanol, expression and purification of the protein corresponding to SEQ ID No. 58 does result in a protein that migrates higher than expected, indicating the formation of dimers. However, as can be seen from the lanes containing the protein purified in the presence of the reducing agent β-mercaptoethanol, expression and purification of the protein corresponding to SEQ ID No. 58 results mainly in cleavage fragments rather than intact protein.

[0334] Example 10

[0335] In an attempt to improve the proteolytic resistance of the protein corresponding to SEQ ID No. 58, multiple variants of the sequence of SEQ ID No. 58 were generated. The DNA sequence was synthesized and verified by a commercial supplier and then subcloned to produce plasmids as described in Example 9 and expressed proteins as described in Example 9. The variants included proteins with the modifications listed below:

[0336] 1) a fragment of CCN5 consisting of amino acids 194 to 249 of SEQ ID NO. 62 with incorporated mutation (P245L) combined with a truncated peptide linker corresponding to SEQ ID NO. 63 and an Fc fragment of SEQ ID NO. 15, resulting in a sequence corresponding to SEQ ID NO. 64,

[0337] 2) a fragment of CCN5 consisting of amino acids 194 to 246 corresponding to SEQ ID NO.1 and an Fc fragment of SEQ ID NO.15 combined with a variant of a peptide linker corresponding to SEQ ID NO.65, resulting in a sequence corresponding to SEQ ID NO.66,

[0338] 3) a fragment of CCN5 consisting of amino acids 194 to 246 of SEQ ID NO.1 and an Fc fragment of SEQ ID NO.15 combined with a variant of a peptide linker corresponding to SEQ ID NO.67, resulting in a sequence corresponding to SEQ ID NO.68,

[0339] 4) A fragment of CCN5 consisting of amino acids 194 to 246 corresponding to SEQ ID NO. 1 and an Fc fragment of SEQ ID NO. 19 combined with a variant of a peptide linker corresponding to SEQ ID NO. 65, resulting in a sequence corresponding to SEQ ID NO. 69.

[0340] Iterations of these proteins disclosed in Example 12 (1 to 4, supra) did show some improvement in their resistance to proteolytic cleavage during expression in the ExpiCHO system, as performed as described in Example 9. However, expression of proteins corresponding to SEQ ID No. 64, SEQ ID No. 66, SEQ ID No. 68 and SEQ ID 69 revealed that the degree of proteolytic resistance was still insufficient to allow the production of intact purified proteins.

[0341] Embodiment 11

[0342] Amino acids 194 to 237 of CCN5 (wherein the amino acid at position 195 (proline) was replaced with alanine) (SEQ ID No. 38) and a chimeric Fc fragment of IgG subtype IgG2 / 4 with a carboxyl-terminal K477- (Eu numbering) deletion (SEQ ID No. 19) were generated, resulting in a fusion protein of SEQ ID No. 27. The fusion protein was also appended with an albumin-derived N-terminal signal sequence for secretion of SEQ ID No. 32 to produce a fusion protein corresponding to SEQ ID No. 70. The DNA sequence encoding the fusion protein of SEQ ID No. 70 was codon-optimized for protein expression in hamster cells (by the algorithm of the commercial supplier), and a KOZAK sequence for translation was appended at the 5' end, and a stop codon was introduced at the 3' end, resulting in a DNA sequence of SEQ ID. No. 71. The DNA sequence was further appended with access attB sites at both ends, resulting in a DNA sequence of SEQ ID No. 72.

[0343] The DNA sequence was synthesized and verified by a commercial supplier, then subcloned to produce a plasmid as described in Example 9 and a protein corresponding to SEQ ID No. 70 expressed by transient transfection of ExpiCHO cells as described in Example 9.

[0344] Six days after transfection, cells were pelleted by centrifugation at 4750 xg for 20 minutes at 4°C and the supernatant cell culture medium was harvested. 0.1 M PMSF in 100% isopropanol was added to a concentration of 0.1 mM. 1 M sodium citrate pH 5.5 was added to a final concentration of 30 mM prior to chromatography.

[0345] The protein was purified by tandem chromatography consisting of: TM MabSelectSuRe TM Column (GE Healthcare), followed by a capture step using BioScale TM Mini The column was installed in a FPLC chromatography system (BioRad NGC Discovery 500) equipped with a 5 mm UV flow cell. TM 10 Pro system). TM The column was mounted on the first column switching valve and equilibrated with a buffer consisting of 30 mM sodium citrate pH 5.5, while The column was mounted on the second column switching valve and equilibrated with buffer A2 (100 mM NaH2PO4 / Na2HPO4 pH 6.5). The second column switching valve of the column was set to bypass, and 140 mL of harvested cell culture medium containing recombinant protein was loaded into MabSelectSuRe using a sample pump at a rate of 2.0 ml / min. TM The column was then washed with 5 column volumes of wash buffer A1 (30 mM sodium citrate pH 5.5), followed by 5 column volumes of wash buffer A3 (30 mM sodium citrate, 0.5 M sodium chloride, pH 5.5), and then 3 column volumes of wash buffer A1. The column was switched to the inlet flow channel and then eluted with elution buffer (30 mM citric acid pH 3.4). After elution with 2 mL of elution buffer, MabSelectSuRe TM The column is switched out of the flow path and buffer A2 is used from The purified protein was eluted from the column. Protein elution was monitored by UV absorbance at 280 nm and collection was triggered once the absorbance exceeded 100 mAU. The collected fractions were combined and concentrated using TGX Stain-Free TM 10 μL of sample was subjected to SDS-PAGE on a precast gel and analyzed using ChemiDoc TM The separated recombinant proteins were visualized using the Imaging System (BioRad).

[0346] exist Figure 7 In the present invention, it is shown that the expression and purification of the protein corresponding to SEQ ID NO. 27 (in which the carboxyl-terminal tail of CCN5 is truncated) has significantly greater resistance to proteolysis than variants containing all the carboxyl-terminal amino acids of CCN5 (such as SEQ ID NOs. 58, 64, 66, 68 and 69), even though the cell culture medium was harvested 2 days after subculture relative to Example 9 ( Figure 6 ).

[0347] Example 12

[0348] A fusion protein of amino acids 206 to 249 of CCN3 (wherein the amino acid at position 207 (isoleucine) was replaced with alanine) (SEQ ID No. 44) comprising an N-terminal fusion peptide linker (SEQ ID No. 21) and a chimeric Fc fragment of IgG subtype IgG2 / 4 with a carboxyl-terminal K477- (Eu numbering) deletion (SEQ ID No. 19) was generated, resulting in a fusion protein of SEQ ID No. 73. The fusion protein was also appended with an albumin-derived N-terminal signal sequence for secretion of SEQ ID No. 32 to produce a fusion protein corresponding to SEQ ID No. 74. The DNA sequence encoding the fusion protein of SEQ ID No. 74 was codon-optimized for protein expression in hamster cells (by the algorithm of the commercial supplier), and a KOZAK sequence for translation was appended at the 5' end, and a stop codon was introduced at the 3' end, resulting in a DNA sequence of SEQ ID. No. 75. This DNA sequence was further appended with access attB sites at both ends, resulting in a DNA sequence of SEQ ID No. 76.

[0349] The DNA sequence was synthesized and verified by a commercial supplier, then subcloned to generate a plasmid as described in Example 9 and expressed the protein corresponding to SEQ ID No. 74 by transient transfection of ExpiCHO cells as described in Example 9.

[0350] 5 days after transfection, cells were pelleted by centrifugation at 4750 xg for 20 minutes at 4°C and supernatant cell culture medium was harvested. 0.1 M PMSF in 100% isopropanol was added to a concentration of 0.1 mM. 1 M sodium citrate pH 5.5 was added to a final concentration of 30 mM prior to chromatography.

[0351] The protein was purified by tandem chromatography consisting of: TM MabSelectSuRe TM Column (GE Healthcare), followed by a capture step with 53 mL HiPrep TM Desalting was performed using a 26 / 10 desalting column (GE Healthcare). The column was installed in an FPLC chromatography system (BioRad NGCDiscover TM 10 Pro system). TM The column was mounted on the first column switching valve and equilibrated with a buffer consisting of 30 mM sodium citrate pH 5.5, while the HiPrep TMThe column was mounted on the second column switching valve and equilibrated with buffer A2 (100 mM NaH2PO4 / Na2HPO4 pH 6.5). TM The second column switching valve of the column was set to bypass, and 260 mL of harvested cell culture medium containing recombinant protein was loaded into MabSelectSuRe using a sample pump at a rate of 3.5 ml / min. TM The column was then washed with 5 column volumes of wash buffer A1 (30 mM sodium citrate pH 5.5), followed by 5 column volumes of wash buffer A3 (30 mM sodium citrate, 0.5 M NaCl, pH 5.5), and then washed with 2 column volumes of wash buffer A1. The HiPrep TM The column was set to enter the flow channel and then eluted using elution buffer (30 mM citric acid pH 3.4). After elution with 10 mL of elution buffer, MabSelectSuRe TM The column was switched out of the flow path and buffer A2 was used from the HiPrep TM The purified protein was eluted from the column. Protein elution was monitored by UV absorbance at 280 nm and collection was triggered once the absorbance exceeded 100 mAU. The collected fractions were combined and purified using TGX Stain-Free TM 10 μL of sample was subjected to SDS-PAGE on a precast gel and analyzed using ChemiDoc TM The separated recombinant proteins were visualized using the Imaging System (BioRad).

[0352] exist Figure 8 As can be seen, the fusion protein containing amino acids derived from CCN3 / Nov (domain III / TSP-1 homology domain) as disclosed in SEQ ID No. 73 is similar to the fusion protein containing amino acids derived from homologous CCN5 (domain III / TSP-1 homology domain) as disclosed in SEQ ID No. 27, and has similar properties to those described in Example 11 and Figure 7 The fusion proteins shown in have similar or better proteolytic resistance than those containing amino acids derived from CCN5.

[0353] Embodiment 13

[0354] Amino acids 194 to 246 of CCN5 (wherein the amino acid (proline) at position 195 is replaced by alanine) (SEQ ID No. 7) and the Fc fragment of the IgG subclass IgG4 of SEQ ID NO. 15 (S228P, F234A, L235A, K447-, Eu numbering) resulting in a fusion protein corresponding to the protein sequence of SEQ ID No. 41 with the addition of the N-terminal signal sequence for secretion derived from albumin of SEQ ID No. 32, resulting in a fusion protein corresponding to SEQ ID No. 77. The DNA sequence encoding the fusion protein of SEQ ID No. 77 was codon-optimized for protein expression in hamster cells (by the algorithm of the commercial supplier), and a KOZAK sequence for translation was added at the 5' end, and a stop codon was introduced at the 3' end, resulting in a DNA sequence of SEQ ID. No. 78. This DNA sequence further had access attB sites added at both ends, resulting in a DNA sequence of SEQ ID No. 79.

[0355] The sequence of SEQ ID No. 79 was synthesized by a commercial supplier and sequence verified. The synthesized sequence was recombined with pDonrZeo by BP pathway recombinase cloning to generate an entry vector. In transfection of competent E. coli (One Shot Top10 TM Cells) were then isolated using standard plasmid isolation techniques using TM QIAprep TM The entry vector was isolated using the Spin Miniprep Kit. After plasmid isolation, the entry vector was verified by restriction enzyme digestion followed by DNA gel electrophoresis according to standard techniques well known to the skilled artisan.

[0356] The entry vector containing the sequence SEQ ID No. 78 was further recombined with the destination vector using the LR pathway recombinase. The destination vector used was pUCOE-DHFR-DEST, such as et al., 2018, J. Biol. Chem, 293: 46, pp. 17953-17970.

[0357] In transfecting competent E. coli (One Shot Top10 TM After PCR, standard plasmid isolation techniques were used using QIAGEN TMThe expression vector was isolated by Plasmid Plus Maxi kit. The resulting expression vector was verified by standard restriction enzyme digestion and DNA gel electrophoresis according to standard techniques well known to the skilled person. The resulting expression vector was then transferred into DG44 CHO cells adapted to suspension culture by electroporation using the Neon transfection system (ThermoFisherScientific).

[0358] The cells were maintained in an open Erlenmeyer flask on a shaking platform in a cell culture incubator maintained at 37°C and 8% CO2. The transfected cells were maintained overnight in CD DG44 cell culture medium (Gibco, catalog number 12610-010) and then transferred to HyClone TM ActiPro TM The cells were cultured in culture medium (without hypoxanthine and thymidine, GE Healthcare) and subcultured until the viability approached 80%, at which time 0.1 μM methotrexate was supplemented to the culture medium. After adding 0.1 μM methotrexate, the cells were subcultured until the viability approached 80% again, at which time 1 μM methotrexate was supplemented to the culture medium. The cells were subcultured again until the viability exceeded 98% and the doubling time was reduced to less than 26 hours, at which time the cell pool was considered to be stably transfected. Once a stable cell pool was established, the cell culture volume was expanded to allow stably transfected cells to be inoculated at a density of 1×10^6 cells / mL for production. The cell culture was supplemented with 4 / 0.4% v / v HyClone every day from the 3rd day after subculture. TM Cell Boost TM 7a / 7b. After 10 days, cells were pelleted by centrifugation at 4750 x g for 20 minutes at 4°C and the supernatant cell culture medium was harvested. 0.1 M PMSF in 100% isopropanol was added to a concentration of 0.1 mM. 1 M sodium citrate pH 5.5 was added to a final concentration of 30 mM prior to chromatography.

[0359] The protein was purified by tandem chromatography consisting of: 5 mL HiTrap TM MabSelectSuRe TM Column (GE Healthcare), followed by a capture step with 53 mL HiPrep TMDesalting was performed using a 26 / 10 desalting column (GE Healthcare) as described in Example 15. The purified protein preparation (which showed no signs of proteolytic processing) was then tested for its ability to inhibit pro-survival signaling (serine-473 phosphorylation of AKT) in A549 human lung cancer cells as described in Example 2. Fig. 9 As can be seen, the purified protein corresponding to SEQ ID No. 41 produced from a pool of stably transfected CHO suspension cells surprisingly showed no signs of the ability to inhibit phosphorylation of AKT (serine 473).

[0360] A fusion protein corresponding to the protein sequence of SEQ ID No. 80 was generated with amino acids 206 to 249 of CCN3 (SEQ ID No. 44) (wherein the amino acid at position 207 (isoleucine) was replaced with alanine) and a chimeric Fc fragment of IgG subtype IgG2 / 4 (SEQ ID No. 19) comprising an N-terminal fusion peptide linker (SEQ ID No. 22), which was supplemented with an N-terminal signal sequence for secretion derived from albumin of SEQ ID No. 32, resulting in a fusion protein corresponding to SEQ ID No. 81. The DNA sequence encoding the fusion protein of SEQ ID No. 81 was codon-optimized for protein expression in hamster cells (by the algorithm of the commercial supplier), and a KOZAK sequence for translation was supplemented at the 5' end and a stop codon was introduced at the 3' end, resulting in a DNA sequence of SEQ ID No. 82. This DNA sequence was further supplemented with access attB sites at both ends, resulting in a DNA sequence of SEQ ID No. 83.

[0361] The sequence of SEQ ID No. 83 was synthesized by a commercial supplier and sequence verified. The synthesized sequence was recombined with pDonrZeo by BP pathway recombinase cloning to generate the entry vector. In transfection of competent E. coli (One Shot Top10 TM Cells) were then isolated using standard plasmid isolation techniques using TM QIAprep TM The entry vector was isolated using the Spin Miniprep Kit. After plasmid isolation, the entry vector was verified by restriction enzyme digestion followed by DNA gel electrophoresis according to standard techniques well known to the skilled artisan.

[0362] The entry vector containing the sequence SEQ ID No. 82 was further recombined with the destination vector using the LR pathway recombinase. The destination vector used was pUCOE-DHFR-DEST, such as et al., 2018, J. Biol. Chem, 293: 46, pp. 17953-17970.

[0363] In transfecting competent E. coli (One Shot Top10 TM After PCR, standard plasmid isolation techniques were used using QIAGEN TM The expression vector was isolated using the Plasmid Plus Maxi kit. The resulting expression vector was verified by standard restriction enzyme digestion and DNA gel electrophoresis according to standard techniques well known to those skilled in the art. TM The manufacturer of the Stable Production Medium (Gibco Catalog No. A3711001) provided the "Use ExpiCHO TM Creation and Scale up of a Stable Cell Line Using ExpiCHO TM The expression vector was transferred into CHO cells adapted to ExpiCHO suspension culture using the "Products" protocol. The cells were maintained in an open conical flask on a shaking platform in a cell culture incubator maintained at 37°C and 8% CO2 (eg al., (supra)). The transfected cells were cultured in ExpiCHO TM The cells were maintained overnight in expression medium and then transferred to ExpiCHO medium supplemented with 0.1 μM methotrexate. TM The cells were then subcultured until the viability again approached 80%, at which point the medium was supplemented with 1 μM methotrexate. The cells were subcultured again until the viability exceeded 95% and the doubling time decreased to less than 20 hours, at which point the cell pool was considered stably transfected. Once a stable cell pool was established, the cell culture volume was expanded to allow for the expression of 1×10 6 Stably transfected cells were inoculated at a density of 10 cells / mL for production. After 5 days, cells were pelleted by centrifugation at 4750 x g for 20 minutes at 4 °C and the supernatant cell culture medium was harvested. 0.1 M PMSF in 100% isopropanol was added to a concentration of 0.1 mM. 1 M sodium citrate pH 5.5 was added to a final concentration of 30 mM, and 2 M L-arginine pH 4.0 was added to a final concentration of 100 mM, followed by chromatography.

[0364] The protein was purified by tandem chromatography using the same protocol as described in Example 12 consisting of: TM MabSelect PrismATM A capture step was performed using a 53 mL HiPrep TM Desalting was performed using a 26 / 10 desalting column (GE Healthcare) except that 100 mM L-arginine was added to buffers A1, A2, A3 and B1. The purified protein preparation (which showed no signs of proteolytic processing) was then tested for its ability to inhibit pro-survival signaling (serine-473 phosphorylation of AKT) in A549 human lung cancer cells as described in Example 2. Fig.10 As can be seen, the purified protein corresponding to SEQ ID No. 80 produced from the stable transfected CHO suspension cell pool unexpectedly showed no signs of the ability to inhibit the phosphorylation of AKT (serine 473), indicating that the protein containing the protein derived from CCN5 (SEQ ID No. 41, Example 13, Fig. 9 ) or the complete dimeric Fc fusion protein preparations containing amino acids derived from CCN3 (SEQ ID No. 80) had no biological activity.

[0365] Embodiment 15

[0366] by ExpiCHO as described in Example 9 TM Cells were transiently transfected to express the expression plasmid described in Example 14 containing SEQ ID No. 82 encoding the following fusion protein: the fusion protein comprises amino acids 206 to 249 of CCN3 (SEQ ID No. 44) (wherein the amino acid at position 207 (isoleucine) is replaced by alanine) and a chimeric Fc fragment of IgG subtype IgG2 / 4 (SEQ ID No. 19) with an N-terminal fusion peptide linker (SEQ ID No. 22) to obtain a protein sequence corresponding to SEQ ID No. 80, and the fusion protein is also supplemented with an N-terminal signal sequence for secretion derived from albumin of SEQ ID No. 32 to correspond to SEQ ID No. 81. Six days after transfection, the cells were pelleted as described in Example 14, and the culture medium was supplemented as described in Example 14. The protein was purified by tandem chromatography as described in Example 14, which consisted of: 5 mL of HiTrap TM MabSelect PrismA TM A capture step was performed using a 53 mL HiPrep TM Desalting was performed using a 26 / 10 desalting column (GE Healthcare).

[0367] The purified protein preparation (which was partially proteolytically processed) was then tested for its ability to inhibit pro-survival signaling (serine-473 phosphorylation of AKT) in A549 human lung cancer cells as described in Example 2. Fig.11 It can be seen that from the transiently transfected ExpiCHO TM The purified protein corresponding to SEQ ID No. 80 produced by the cells showed a concentration-dependent ability to inhibit AKT (serine 473) phosphorylation, indicating that the expression system used to produce the fusion protein corresponding to SEQ ID No. 80 and therefore the level of proteolytic processing observed significantly affects the activity of the resulting protein preparation, or the lack thereof.

[0368] Example 16

[0369] Amino acids 206 to 249 of CCN3 (SEQ ID. No. 44) (wherein the amino acid at position 207 (isoleucine) was replaced with alanine) comprising an N-terminal fusion peptide linker (SEQ ID No. 21) and an Fc fragment (SEQ ID No. 55) with monomer induction and half-life extension mutations to obtain a fusion protein corresponding to the protein sequence of SEQ ID No. 84. An N-terminal signal sequence for secretion derived from albumin of SEQ ID No. 32 was appended to obtain a fusion protein corresponding to SEQ ID No. 85. The DNA sequence encoding the fusion protein of SEQ ID No. 85 was codon-optimized for protein expression in hamster cells (by the algorithm of the commercial supplier) and a KOZAK sequence for translation was appended at the 5' end and a stop codon was introduced at the 3' end to obtain a DNA sequence of SEQ ID No. 86. This DNA sequence was further appended with access attB sites at both ends to obtain a DNA sequence of SEQ ID No. 86. The DNA sequence was synthesized and verified by a commercial supplier, then subcloned to generate plasmids as described in Example 9 and cloned by ExpiCHO as described in Example 9. TM 5 days after transfection, the cells were pelleted as described in Example 14 and the culture medium was supplemented as described in Example 14. The protein was purified by tandem chromatography as described in Example 14, which consisted of: 5 mL HiTrap TM MabSelect PrismA TM A capture step was performed using a 53 mL HiPrep TM Desalting was performed using a 26 / 10 desalting column (GE Healthcare).

[0370] The purified protein preparations exhibiting the expected monomeric form were then tested for their ability to inhibit pro-survival signaling (serine-473 phosphorylation of AKT) in A549 human lung cancer cells as described in Example 2. Fig.12 It can be seen that the purified protein corresponding to SEQ ID No. 84 showed a concentration-dependent ability to inhibit AKT (serine 473) phosphorylation, indicating that another monomeric fusion protein comprising amino acids from domain III / TSP-1 homology domain of CCN protein has the ability to inhibit AKT (serine-473) phosphorylation in A549 human lung cancer cells.

[0371] Embodiment 17

[0372] Amino acids 206 to 249 of CCN3 (SEQ ID. No. 44) (wherein the amino acid at position 207 (isoleucine) was replaced with alanine) comprising an N-terminal fusion peptide linker (SEQ ID No. 21) and an Fc fragment (SEQ ID No. 54) with monomer-inducing and stability-inducing mutations gave a fusion protein corresponding to the protein sequence of SEQ ID No. 88. An N-terminal signal sequence for secretion derived from albumin of SEQ ID No. 32 was appended to give a fusion protein corresponding to SEQ ID No. 89. The DNA sequence encoding the fusion protein of SEQ ID No. 89 was codon-optimized for protein expression in hamster cells (by the algorithm of the commercial supplier) and a KOZAK sequence for translation was appended at the 5' end and a stop codon was introduced at the 3' end to give a DNA sequence of SEQ ID No. 90. This DNA sequence was further appended with access attB sites at both ends to give a DNA sequence of SEQ ID No. 91. The DNA sequence was synthesized and verified by a commercial supplier, then subcloned to generate plasmids as described in Example 9 and cloned by ExpiCHO as described in Example 9. TM 6 days after transfection, the cells were pelleted as described in Example 14 and the culture medium was supplemented as described in Example 14. The protein was purified by tandem chromatography as described in Example 14, which consisted of: 5 mL HiTrap TM MabSelect PrismA TM A capture step was performed using a 53 mL HiPrep TM Desalting was performed using a 26 / 10 desalting column (GE Healthcare).

[0373] The purified protein preparations, which exhibited predominantly the expected monomeric form, were then tested for their ability to inhibit pro-survival signaling (serine-473 phosphorylation of AKT) in A549 human lung cancer cells as described in Example 2. Fig.13 It can be seen that the purified protein corresponding to SEQ ID No. 88 showed a concentration-dependent ability to inhibit AKT (serine 473) phosphorylation, indicating that another monomeric fusion protein comprising amino acids from domain III / TSP-1 homology domain of CCN protein has the ability to inhibit AKT (serine-473) phosphorylation in A549 human lung cancer cells.

[0374] Embodiment 18

[0375] The amino acids 206 to 249 of CCN3 (SEQ ID. No. 44) (wherein the amino acid at position 207 (isoleucine) was replaced with alanine) comprising an N-terminal fusion peptide linker (SEQ ID No. 93) and a multifunctional tag (SEQ ID No. 92) comprising a 6xHis tag, a Halo tag and a Sumo* element, yielded a fusion protein corresponding to the protein sequence of SEQ ID No. 94. The N-terminal signal sequence for secretion derived from albumin of SEQ ID No. 32 was appended to yield a fusion protein corresponding to SEQ ID No. 114. The DNA sequence encoding the fusion protein of SEQ ID No. 114 was codon-optimized for protein expression in hamster cells (by the algorithm of the commercial supplier), and a KOZAK sequence for translation was appended at the 5' end and a stop codon was introduced at the 3' end, yielding a DNA sequence of SEQ ID No. 95. This DNA sequence further had access attB sites appended at both ends, yielding a DNA sequence of SEQ ID No. 96. The DNA sequence was synthesized and verified by a commercial supplier, then subcloned to generate plasmids as described in Example 9 and cloned by ExpiCHO as described in Example 15. TM 5 days after transfection, the cells were pelleted as described in Example 14. 0.1 M PMSF in 100% isopropanol was added to a concentration of 0.1 mM, 1 M sodium citrate pH 5.5 was added to a final concentration of 30 mM, and 2 M L-arginine pH 4.0 was added to a final concentration of 0.1 M, and imidazole was added to a final concentration of 5 mM, followed by chromatography.

[0376] The protein was purified by tandem chromatography consisting of: TM HisTrap TMExcel column (GE Healthcare), followed by a capture step with 53 mL HiPrep TM Desalting was performed using a 26 / 10 desalting column (GE Healthcare). The column was installed in an FPLC chromatography system (BioRad NGC Discovery ELISA) equipped with a 5 mm UV flow cell. TM 10Pro system). TM The column was installed on the first column switching valve and equilibrated with A1 buffer consisting of 5 mM imidazole, 50 mM NaCl, and 100 mM L-arginine. TM The column was mounted on the second column switching valve and equilibrated with buffer A2 (100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine pH 6.5). TM The second column switching valve of the column was set to bypass, and 250 mL of harvested cell culture medium containing recombinant protein was loaded into the HisTrap using a sample pump at a rate of 3.5 ml / min. TM The column was then washed with 5 column volumes of wash buffer A1, followed by 5 column volumes of wash buffer A3 (5 mM imidazole, 0.5 M NaCl, 100 mM L-arginine), and then washed with 2 column volumes of wash buffer A1. The HiPrep TM The column was set to enter the flow channel and then eluted with elution buffer (250 mM imidazole, 50 mM NaCl, 100 mM L-arginine). After elution with 10 mL of elution buffer, HisTrap TM The column was switched out of the flow path and buffer A2 was used from the HiPrep TM The purified protein was eluted from the column. Protein elution was monitored by UV absorbance at 280 nm and collection was triggered once the absorbance exceeded 60 mAU.

[0377] The purified protein preparations exhibiting the expected monomeric form were then tested for their ability to inhibit pro-survival signaling (serine-473 phosphorylation of AKT) in A549 human lung cancer cells as described in Example 2. Fig.12 It can be seen that the purified protein corresponding to SEQ ID No. 94 showed a concentration-dependent ability to inhibit AKT (serine 473) phosphorylation, indicating that another monomeric fusion protein comprising amino acids from domain III / TSP-1 homology domain of CCN protein has the ability to inhibit AKT (serine-473) phosphorylation in A549 human lung cancer cells.

[0378] Embodiment 19

[0379] Amino acids 194 to 237 of CCN5 (wherein the amino acid at position 195 (proline) was replaced with alanine) (SEQ ID No. 38) and amino acids 25 to 609 of human serum albumin (SEQ ID No. 52) comprising an N-terminal fusion peptide linker (SEQ ID No. 21) were appended with an albumin-derived N-terminal signal sequence for secretion of SEQ ID No. 32 to obtain a fusion protein corresponding to SEQ ID No. 98. The DNA sequence encoding the fusion protein of SEQ ID No. 98 was codon-optimized for protein expression in hamster cells (by the algorithm of the commercial supplier), and a KOZAK sequence for translation was appended at the 5' end and a stop codon was introduced at the 3' end to obtain a DNA sequence of SEQ ID No. 99. This DNA sequence was further appended with access attB sites at both ends to obtain a DNA sequence of SEQ ID No. 100.

[0380] The DNA sequence was synthesized and verified by a commercial supplier, then subcloned to generate plasmids as described in Example 9, and DG44 CHO suspension cells engineered to express a constitutively active form of AKT were used to generate a stable pool of CHO suspension cells expressing the protein of SEQ ID No. 98 as described in Example 13. Once a stable pool of cells was established, the cell culture volume was expanded to allow for culture at 1×10 6 Stably transfected cells were inoculated at a density of 10 cells / mL for production. After 6 days, cells were pelleted by centrifugation at 4750 x g for 20 minutes at 4°C and the supernatant cell culture medium was harvested. 0.1 M PMSF in 100% isopropanol was added to a concentration of 0.1 mM, 0.5 M EDTA was added to a final concentration of 2 mM, 1 M sodium citrate pH 5.5 was added to a final concentration of 30 mM, and 2 M L-arginine pH 4.0 was added to a final concentration of 0.1 M before chromatography.

[0381] The protein was purified by tandem chromatography consisting of: TM The capture step was performed using a Tricorn column (GE Healthcare) with human albumin affinity matrix (Thermo Fisher Scientific), followed by 53 mL of HiPrep TM Desalting was performed using a 26 / 10 desalting column (GE Healthcare). The column was installed in an FPLC chromatography system (BioRad NGC Discovery ELISA) equipped with a 5 mm UV flow cell.TM 10Pro system). It will include CaptureSelect TM The column was installed on the first column switching valve and equilibrated with A1 buffer consisting of 100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine pH 6.5, while the HiPrep TM The column was mounted on the second column switching valve and equilibrated with buffer A1 (100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine pH 6.5). TM The second column switching valve of the column was set to bypass, and 500 mL of harvested cell culture medium containing recombinant protein was loaded into the CaptureSelect TM , followed by washing with 5 column volumes of wash buffer A1, followed by washing with 5 column volumes of wash buffer A2 (100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine, 0.25 M NaCl, pH 6.5), followed by washing with 5 column volumes of wash buffer A1. TM The column was set to enter the flow channel and then eluted with elution buffer (30 mM citric acid, pH 3.5 + 0.5 M L-arginine). After elution with 10 mL of elution buffer, the CaptureSelect TM The column was switched out of the flow path and buffer A1 was used from the HiPrep TM The purified protein was eluted from the column. Protein elution was monitored by UV absorbance at 280 nm and collection was triggered once the absorbance exceeded 100 mAU.

[0382] The purified protein preparations exhibiting the expected monomeric form were then tested for their ability to inhibit pro-survival signaling (serine-473 phosphorylation of AKT) in A549 human lung cancer cells as described in Example 2. Fig.14 It can be seen that the purified protein corresponding to SEQ ID No. 97 showed a concentration-dependent ability to inhibit AKT (serine 473) phosphorylation, indicating that another monomeric fusion protein comprising amino acids from domain III / TSP-1 homology domain of CCN protein has the ability to inhibit AKT (serine-473) phosphorylation in A549 human lung cancer cells.

[0383] Embodiment 20

[0384] A fusion protein of human serum albumin (amino acids 25 to 606, SEQ ID No. 101) with a C-terminal fusion peptide linker (SEQ ID NO. 22) connected to amino acids 194 to 246 of human CCN5 (wherein the amino acid at position 195 (proline) is replaced with alanine) (SEQ ID No. 7) to obtain SEQ ID No. 103. The fusion protein corresponding to SEQ ID No. 102 was supplemented with an albumin-derived N-terminal signal sequence for secretion of SEQ ID No. 32 to obtain a fusion protein corresponding to SEQ ID No. 103. The DNA sequence encoding the fusion protein of SEQ ID No. 103 was codon-optimized for protein expression in hamster cells (by the algorithm of the commercial supplier), and a KOZAK sequence for translation was supplemented at the 5' end and a stop codon was introduced at the 3' end to obtain a DNA sequence of SEQ ID No. 104. This DNA sequence was further supplemented with access attB sites at both ends to obtain a DNA sequence of SEQ ID No. 105.

[0385] The DNA sequence was synthesized and verified by a commercial supplier, then subcloned to generate plasmids as described in Example 9, and DG44 CHO suspension cells engineered to express a constitutively active form of AKT were used to generate a stable pool of CHO suspension cells expressing the protein of SEQ ID No. 104 as described in Example 13. Once a stable pool of cells was established, the cell culture volume was expanded to allow for culture at 1×10 6 Stably transfected cells were inoculated at a density of 10 cells / mL for production. After 6 days, cells were pelleted by centrifugation at 4750 x g for 20 minutes at 4°C and the supernatant cell culture medium was harvested. 0.1 M PMSF in 100% isopropanol was added to a concentration of 0.1 mM, 0.5 M EDTA was added to a final concentration of 2 mM, 1 M sodium citrate pH 5.5 was added to a final concentration of 30 mM, and 2 M L-arginine pH 4.0 was added to a final concentration of 0.1 M before chromatography.

[0386] The protein was purified by tandem chromatography as described in Example 19, which consisted of: TM A capture step was performed using a Tricorn column (GE Healthcare) with human albumin affinity matrix (ThermoFisherScientific), followed by 53 mL of HiPrep TM Desalting was performed using a 26 / 10 desalting column (GE Healthcare), except that the sample loading flow rate was 0.37 ml / min instead of 2.0 ml / min.

[0387] The purified protein preparations exhibiting the expected monomeric form were then tested for their ability to inhibit pro-survival signaling (serine-473 phosphorylation of AKT) in A549 human lung cancer cells as described in Example 2. Fig.14 It can be seen that the purified protein corresponding to SEQ ID No. 102 showed a concentration-dependent ability to inhibit AKT (serine 473) phosphorylation, indicating that another monomeric fusion protein comprising amino acids from domain III / TSP-1 homology domain of CCN protein has the ability to inhibit AKT (serine-473) phosphorylation in A549 human lung cancer cells.

[0388] Embodiment 21

[0389] Amino acids 206 to 249 of CCN3 (wherein the amino acid at position 207 (isoleucine) was replaced with alanine) (SEQ ID No. 44) and amino acids 25 to 609 of human serum albumin (SEQ ID No. 52) comprising an N-terminal fusion peptide linker (SEQ ID No. 21) were added with an albumin-derived N-terminal signal sequence for secretion of SEQ ID No. 32 to obtain a fusion protein corresponding to SEQ ID No. 107. The DNA sequence encoding the fusion protein of SEQ ID No. 107 was codon-optimized for protein expression in hamster cells (by the algorithm of the commercial supplier), and a KOZAK sequence for translation was added at the 5' end and a stop codon was introduced at the 3' end to obtain a DNA sequence of SEQ ID No. 108. This DNA sequence was further added with access attB sites at both ends to obtain a DNA sequence of SEQ ID No. 109.

[0390] The DNA sequence was synthesized and verified by a commercial supplier, then subcloned to generate plasmids as described in Example 9 and cloned by ExpiCHO as described in Example 9. TM Six days after transfection, cells were pelleted as described in Example 14 and the culture medium was supplemented as described in Example 19. The protein was purified by tandem chromatography consisting of: 10 mL of CaptureSelect TM A capture step was performed using a Tricorn column (GE Healthcare) with human albumin affinity matrix (ThermoFisherScientific), followed by 53 mL of HiPrep TMDesalting was performed using a 26 / 10 desalting column (GE Healthcare). The column was installed in an FPLC chromatography system (BioRad NGC Discovery ELISA) equipped with a 5 mm UV flow cell. TM 10 Pro system). It will include CaptureSelect TM The column was installed on the first column switching valve and equilibrated with A1 buffer consisting of 100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine pH 6.5, while the HiPrep TM The column was mounted on the second column switching valve and equilibrated with buffer A1 (100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine pH 6.5). TM The second column switching valve of the column was set to bypass, and 500 mL of harvested cell culture medium containing recombinant protein was loaded into the CaptureSelect TM The column was then washed with 3 column volumes of wash buffer A1, followed by 2 column volumes of wash buffer A2 (100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine, 0.25 M NaCl, pH 6.5), and then washed with 3 column volumes of wash buffer A1. The HiPrep TM The column was set to enter the flow channel and then eluted with elution buffer (30 mM citric acid, pH 3.5 + 0.1 M L-arginine). After elution with 15 mL of elution buffer, the CaptureSelect TM The column was switched out of the flow path and buffer A1 was used from the HiPrep TM The purified protein was eluted from the column. Protein elution was monitored by UV absorbance at 280 nm and collection was triggered once the absorbance exceeded 100 mAU.

[0391] Purified protein preparations containing the expected monomeric form were then tested for their ability to inhibit pro-survival signaling (serine-473 phosphorylation of AKT) in A549 human lung cancer cells as described in Example 2. Fig.12 It can be seen that the purified protein corresponding to SEQ ID No. 106 showed a concentration-dependent ability to inhibit AKT (serine 473) phosphorylation, indicating that another monomeric fusion protein comprising amino acids from domain III / TSP-1 homology domain of CCN protein has the ability to inhibit AKT (serine-473) phosphorylation in A549 human lung cancer cells.

[0392] Embodiment 22

[0393] Amino acids 206 to 249 of CCN3 (wherein the amino acid at position 207 (isoleucine) was replaced with alanine) (SEQ ID No. 44) and amino acids 25 to 609 of human serum albumin (SEQ ID No. 52) comprising an N-terminal fusion peptide linker (SEQ ID No. 22) were appended with an albumin-derived N-terminal signal sequence for secretion of SEQ ID No. 32 to obtain a fusion protein corresponding to SEQ ID No. 111. The DNA sequence encoding the fusion protein of SEQ ID No. 111 was codon-optimized for protein expression in hamster cells (by the algorithm of the commercial supplier), and a KOZAK sequence for translation was appended at the 5' end and a stop codon was introduced at the 3' end to obtain a DNA sequence of SEQ ID No. 112. This DNA sequence was further appended with access attB sites at both ends to obtain a DNA sequence of SEQ ID No. 113.

[0394] The DNA sequence was synthesized and verified by a commercial supplier, then subcloned to generate plasmids as described in Example 9 and cloned by ExpiCHO as described in Example 9. TM Six days after transfection, cells were pelleted as described in Example 14 and the culture medium was supplemented as described in Example 19. The protein was purified by tandem chromatography consisting of: 10 mL of CaptureSelect TM A capture step was performed using a Tricorn column (GE Healthcare) with human albumin affinity matrix (ThermoFisherScientific), followed by 53 mL of HiPrep TM Desalting was performed using a 26 / 10 desalting column (GE Healthcare). The column was installed in an FPLC chromatography system (BioRad NGC Discovery ELISA) equipped with a 5 mm UV flow cell. TM 10 Pro system). It will include CaptureSelect TM The column was installed on the first column switching valve and equilibrated with A1 buffer consisting of 100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine pH 6.5, while the HiPrep TM The column was mounted on the second column switching valve and equilibrated with buffer A1 (100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine pH 6.5). TMThe second column switching valve of the column was set to bypass, and 300 mL of harvested cell culture medium containing recombinant protein was loaded into the CaptureSelect TM The column was then washed with 3 column volumes of wash buffer A1, followed by 2 column volumes of wash buffer A2 (100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine, 0.25 M NaCl, pH 6.5), and then washed with 3 column volumes of wash buffer A1. The HiPrep TM The column was set to enter the flow channel and then eluted with elution buffer (30 mM citric acid, pH 3.5 + 0.5 M L-arginine). After elution with 15 mL of elution buffer, the CaptureSelect TM The column was switched out of the flow path and buffer A1 was used from the HiPrep TM The purified protein was eluted from the column. Protein elution was monitored by UV absorbance at 280 nm and collection was triggered once the absorbance exceeded 100 mAU.

[0395] Purified protein preparations containing the expected monomeric form were then tested for their ability to inhibit pro-survival signaling (serine-473 phosphorylation of AKT) in A549 human lung cancer cells as described in Example 2. Fig.15 It can be seen that the purified protein corresponding to SEQ ID No. 110 showed a concentration-dependent ability to inhibit AKT (serine 473) phosphorylation, indicating that another monomeric fusion protein comprising amino acids from domain III / TSP-1 homology domain of CCN protein has the ability to inhibit AKT (serine-473) phosphorylation in A549 human lung cancer cells.

[0396] Embodiment 23

[0397] The stably transfected ExpiCHO described in Example 14 was generated using the expression plasmid described in Example 21 containing SEQ ID No. 108 encoding the following fusion protein: TMPool of cells: The fusion protein comprises amino acids 206 to 249 of CCN3 (wherein the amino acid at position 207 (isoleucine) is replaced by alanine) (SEQ ID No. 44) and amino acids 25 to 609 of human serum albumin (SEQ ID No. 52) with an N-terminal fusion peptide linker (SEQ ID No. 21), and the fusion protein is also supplemented with an N-terminal signal sequence for secretion derived from albumin of SEQ ID No. 32 to correspond to SEQ ID No. 107. To produce a batch of conditioned medium containing the secreted protein corresponding to SEQ ID NO. 106, the pool of stably transfected cells was expanded to allow the inoculation of 250 mL of stably transfected cells at a density of 1×106 cells / mL. Starting from the second day after subculturing, the cell culture was supplemented with 5% v / v 2XEfficientFeed every other day. TM C+(Gibco TM ), and supplemented with 3% glucose (10% w / v) on the 2nd day after subculturing, and supplemented with 5% glucose (10% w / v) on the 6th day after subculturing. After 9 days, the cells were precipitated by centrifugation at 4750 x g for 20 minutes at 4°C, and the supernatant cell culture medium was harvested. 0.1M PMSF in 100% isopropanol was added to a concentration of 0.1mM, 1M sodium citrate pH 5.5 was added to a final concentration of 30mM, and 2M L-arginine pH 4.0 was added to a final concentration of 100mM, followed by chromatography.

[0398] The protein was purified by two-dimensional chromatography consisting of: TM A capture step was performed using a Tricorn column (GE Healthcare) with human albumin affinity matrix (ThermoFisherScientific), followed by size exclusion chromatography using two Superdex 200 Increase 10 / 300 GL (GE Healthcare) columns connected in series. The columns were mounted on an FPLC chromatography system (BioRad NGC Discovery 5000) equipped with a 5 mm UV flow cell and an outlet valve connected to a 5 mL sample loop. TM 10 Pro system). It will include CaptureSelect TMThe column was installed on the first column switching valve and equilibrated with A1 buffer consisting of 100mM NaH2PO4 / Na2HPO4, 100mM L-arginine pH6.5, while the Superdex 200Increase column was installed on the second column switching valve and equilibrated with buffer A1 (100mM NaH2PO4 / Na2HPO4, 100mM L-arginine pH6.5). By setting the second column switching valve containing the Superdex 200Increase column to bypass, 120mL of harvested cell culture medium containing the recombinant protein was loaded into the CaptureSelect HPLC column at a rate of 3.9ml / min using a sample pump. TM The harvested cell culture medium containing the recombinant protein is loaded onto a column containing CaptureSelect TM After loading onto the column, it was washed with 3 column volumes of buffer A1, followed by 2 column volumes of buffer A2 (100 mM NaH2PO4 / Na2HPO4, 100 mM L-arginine, 0.25 M NaCl, pH 6.5), and then 3 column volumes of buffer A1. TM The column was eluted with 15 mL of buffer B1 (30 mM citric acid, 0.5 M L-arginine, pH 3.5), during which the system was set to collect eluate with an absorbance exceeding 1200 mAU into the sample loop. TM After elution from the column, connect the CaptureSelect TM The column containing CaptureSelect was switched out of the flow path and the second column switching valve was set to switch the column containing Superdex 200Increase into the flow path. TM The eluate from the column containing the eluted protein was loaded onto a column containing Superdex 200 Increase at 0.5 ml / min with buffer A1. Protein elution was monitored by UV absorbance at 280 nm and collection was triggered once the absorbance exceeded 200 mAU.

[0399] Subsequently, the ability of the purified protein preparation containing the expected monomeric form to inhibit TGFβ-induced and active CCN2-induced activity of normal human lung fibroblasts (NHLF) (Lonza Bioscience, catalog number: CC-2512) was tested. NHLF were subcultured in complete growth medium (Lonza Bioscience bullet kit (catalog number: CC-3132) with all supplements (2% fetal bovine serum, insulin, hFGF-B, gentamicin / amphotericin B)) according to the instructions of the commercial supplier (Lonza Bioscience) to maintain a density of maximum 80% confluency. Active CCN2 consists of domains 3 to 4 of CCN2 and was produced and purified as described by Kaasb0ll et al., 2018 (supra).

[0400] To test the effect of the protein corresponding to SEQ ID No. 106 on active CCN2 and TGFβ-induced NHLF cell migration (transwell assay / modified Boyden chamber assay), cells were first detached with trypsin / EDTA, neutralized with trypsin neutralizing reagent (Lonza Bioscience, catalog number CC-5034), and resuspended in basal growth medium (fibroblast basal medium (Lonza Bioscience catalog number: CC-3131, without other supplements except gentamicin (50 μg / mL)), and then 30,000 cells were seeded in a volume of 100 μL per well on the upper side of a transwell insert with a pore size of 5 μm (24-well plate, Catalog number CLS3402-48EA from SigmaAldrich (Merck KGaA). The lower chamber of the well contained the test substance or vehicle control dissolved in 500 μL of basal growth medium (without additives other than gentamicin). After 20 hours of incubation, the insert was removed from the well, washed twice by immersion in phosphate buffered saline (PBS, Lonza Bioscience, catalog number: 17-512F), and then fixed in 4% formaldehyde (Solveco, Swe., catalog number: 621092) at 37°C for 15 minutes. The cells were permeabilized by treatment with 0.1% Triton X-100 in PBS for 10 minutes and then washed twice with PBS. The non-migrated cells located on the upper side of the insert were removed by scraping with a cotton swab, and the membrane was then dried. The nuclei of the migrating cells on the underside of the insert were stained with Hoechst 33342 20 mM (1:5000 dilution in PBS, ThermoFisherScientific, catalog number: 62249) for 15 minutes in the dark and subsequently washed twice by immersion in PBS. The membrane was cut out of the transwell insert and mounted on a glass slide (with the migrating cells facing the glass) and covered with a drop of ProLong TM Gold Antifade (ThermoFisherScientific, catalog number: P36934), coverslips were mounted, and 5 to 10 images were captured for each well on a Zeiss Axio Observer Z.1 imaging system. Images can be semi-automatically analyzed using ImageJ software v1.5lk, Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, Maryland, USA, https: / / imagej.nih.gov / ij / , 1997-2018.). Fig.16 A As can be seen, the protein corresponding to SEQ ID No. 106 inhibits migration induced by both TGFβ and active CCN2.

[0401] To test the effect of the protein corresponding to SEQ ID No. 106 on active CCN2 and TGFβ-induced scratch-wound assays, NHLF were detached with trypsin / EDTA, neutralized with trypsin neutralizing reagent (Lonza Bioscience, catalog number CC-5034), and then 100,000 cells were seeded in a volume of 1 mL in 12-well tissue culture treated plates (Corning Catalog number 3513). The next day after inoculation, the cells were washed twice with 0.9% NaCl and the complete growth medium was replaced with the basal growth medium. After incubation in the basal growth medium for 16 to 20 hours, a scratch was made on the cell monolayer with a sterile 12.5 μL pipette tip (ThermoFisherScientific, catalog number: 94420053), the cells were washed once with PBS, and then the cells were incubated in 1 mL of basal growth medium with the test substance or vehicle. The cells were incubated for another 24 hours, then washed 3 times in PBS, and then fixed in 4% formaldehyde at 37°C for 15 minutes. After fixation, the cells were washed again in PBS for 3×3 minutes with gentle shaking and permeabilized with 0.1% Triton X-100 in PBS for 10 minutes with gentle shaking. In the dark, the nuclei were stained with Hoechst 33342 20 mM (1:5000 dilution in PBS, ThermoFisherScientific, catalog number: 62249) for 15 minutes, followed by 3×5 minute washes in PBS with gentle shaking. TM GoldAntifade (ThermoFisherScientific, catalog number: P36934), then mounted, and 5 images centered on the remaining gap were captured from each well using a Zeiss AxioObserver Z.1 imaging system, and the images were analyzed by measuring the remaining gap distance along the length of the scratch after making scratches at 3 fixed intervals. The average of all measurements from all images of each well was calculated and counted as one biological replicate. Fig.16 B As can be seen, the protein corresponding to SEQ ID No. 106 inhibits the closure of scratch wounds induced by both TGFβ and active CCN2.

[0402] To test the effect of the protein corresponding to SEQ ID No. 106 on TGFβ-induced gene regulation, NHLF were detached with trypsin / EDTA, neutralized with trypsin neutralizing reagent (Lonza Bioscience, catalog number CC-5034), and then 100,000 cells were plated in a volume of 1 mL in 12-well tissue culture treated plates (Corning Catalog No. 3513). The day after seeding, cells were washed twice with 0.9% NaCl and complete growth medium was replaced with medium supplemented with 0.1% heat-inactivated fetal bovine serum (from Gibco TM, catalog number 16000-044, heat inactivated as described in Example 2). After incubation for 6 hours in basal growth medium containing 0.1% fetal bovine serum, test substances or vehicle control were added to the wells. After 96 hours, the wells were washed twice in PBS and RNA was extracted using the Qiagen RNeasy RNA extraction kit (catalog number 74106) according to the manufacturer's protocol. RNA concentration was 400 μg / mL. The RNA was quantified by spectrophotometer (NanoDrop Technologies, US), diluted with nuclease-free water to a final RNA concentration of 50 ng / μL, and then analyzed by TaqMan TM cDNA was generated using 200 ng RNA from each replicate using the Reverse Transcription Kit (Cat. No. N8080234) according to the manufacturer's protocol. TM Differential gene expression analysis was analyzed from the resulting cDNA samples using the TaqMan Fast Advanced Master Mix (ThermoFisherScientific, catalog number 4444557). The TaqMan PCR was run using the Applied Biosystems StepOnePlus Real-Time PCR System according to the manufacturer's protocol. TM Real-time PCR reaction, each sample technology is repeated three times. The relative amount of different transcripts is calculated from the standard curve, and then the average of three technical replicates is taken to generate a single value from each sample. All gene expression results are related to GAPDH (ThermoFisherScientific, catalog number Hs02786624_g1) mRNA levels and normalized to be expressed as a multiple of the average value of the wells stimulated by the vehicle control. As can be seen from Figures 19C-F, the protein corresponding to SEQ ID No. 107 provides partial inhibition of TGFβ-induced genes; COL1A1 ("Collagen type 1 alpha-1", ThermoFisherScientific, Catalog No. Hs00164004_m1), FN1 ("Fibronectin 1", ThermoFisherScientific, Catalog No. Hs01549976_m1), ACTA2 ("Smooth muscle actin alpha-2", ThermoFisherScientific, Catalog No. Hs00426835_g1) and CCN2 (ThermoFisherScientific, Catalog No. Hs00170014_m1), which are generally considered to be pro-fibrotic genes.

[0403] Summary of serial numbers mentioned in the specification and sequence listing

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416] CCN proteins are numbered according to the uniprot database, as described above in the "Detailed Description of the Invention". Fc fragments are numbered according to the Eu numbering system described above in the "Detailed Description of the Invention".

Claims

1. A monomeric fusion protein comprising: (i) a polypeptide corresponding to a portion of the thrombospondin type 1 repeat (TSP-1) homology domain of CCN3 and defined by SEQ ID NO: 44; (ii) a monomeric fusion partner fused to the amino acid sequence of (i) at the N-terminus or C-terminus, wherein the monomeric fusion partner comprises at least 6 amino acids and increases the serum half-life of the fusion protein; and (iii) optionally a peptide linker between the polypeptide of (i) and the monomeric fusion partner of (ii), wherein the monomeric fusion partner of (ii) and the peptide linker of (iii) are not or do not comprise an IGF binding protein homology domain of a CCN family protein, a von Willebrand factor C-type repeat homology domain or a cysteine ​​knot domain.

2. The fusion protein of claim 1, wherein the peptide linker of (iii) comprises no more than 50 amino acids.

3. The fusion protein of claim 1 or claim 2, wherein the monomeric fusion partner is selected from serum albumin, transferrin, and a portion of an immunoglobulin, optionally a monomeric Fc fragment of human IgG. The fusion protein of claim 3 , wherein the monomeric Fc fragment of human IgG is a monomeric Fc fragment of IgG1, IgG2 or IgG4.

5. The fusion protein of claim 3 or claim 4, wherein the monomeric Fc fragment is aglycosylated.

6. The fusion protein of any one of claims 3 to 5, wherein the monomeric Fc fragment comprises a stabilizing disulfide bridge and / or a protease stabilizing mutation.

7. The fusion protein of any one of claims 3 to 6, wherein the monomeric Fc fragment does not have immune effector function.

8. The fusion protein of any one of claims 1 to 7, wherein the peptide linker between the amino acid sequence of (i) and the monomeric fusion partner has the following: an amino acid sequence selected from SEQ ID NO: 20 to 25, 39, 57, 63, 65 or 67.

9. The fusion protein of any one of claims 1 to 3, wherein the fusion protein has the following: an amino acid sequence selected from SEQ ID NO: 84, 85, 88, 89, 106, 107, 110 and 111.

10. A DNA molecule encoding a monomeric fusion protein as defined in any one of claims 1 to 9.

11. The DNA molecule of claim 10, wherein the molecule further comprises a nucleotide sequence encoding a signal sequence.

12. The DNA molecule of claim 10 or 11, wherein the molecule comprises the nucleotide sequence shown in SEQ ID NO:86, 87, 90, 91, 108, 109, 112 or 113.

13. An expression vector comprising a DNA molecule as defined in any one of claims 10 to 12.

14. A host cell comprising the vector defined in claim 13.

15. Use of the fusion protein according to any one of claims 1 to 9 in the preparation of a medicament for treating or preventing pulmonary fibrosis.

16. Use of the fusion protein according to any one of claims 1 to 9 in the preparation of a medicament for treating breast cancer or lung cancer.

Images