RU2825102C2 - Recombinant proteins with ccn domains and fusion proteins - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to recombinant proteins of thrombospondin type I representative of family of proteins CCN and can be used in medicine for treating cancer or fibrosis. Disclosed is a monomeric fusion protein for inhibiting the effect or activity of 4-domain CCN protein, comprising a polypeptide corresponding to at least a portion of a homology domain to repeats of thrombospondin type 1 (TSP-1) of a CCN family protein and a monomer fusion partner fused therewith at the N- or C-terminus, which contains at least 6 amino acids and increases the serum half-life of said fused protein.

EFFECT: invention provides the production of fused proteins based on truncated fragments of domain III of the CCN family protein, which reproduce or have the biological activity of CCN5, are able to antagonize or inhibit effects of 4-domain CCN proteins and are resistant to proteolytic degradation.

21 cl, 16 dwg, 1 tbl, 23 ex

Description

AREA OF TECHNOLOGY

The present invention relates to recombinant proteins having an amino acid sequence corresponding to or related to the thrombospondin type I repeat homology domain (domain III) of a member of the CCN protein family and their use, including in particular such proteins that are truncated and/or contain certain amino acid modifications. Furthermore, the present invention relates to fusion proteins comprising an amino acid sequence corresponding to or related to the thrombospondin type I repeat homology domain of a member of the CCN protein family, combined with a fusion partner, optionally via a linker peptide. In particular, the fusion partner is a monomeric protein, and said fusion proteins are monomeric.

Also disclosed in the present application are novel protease-resistant Fc fragments.

LEVEL OF TECHNOLOGY

The CCN proteins are a family of secreted glycoproteins. The initial acronym chosen for CCN was derived from the first three identified members of this gene family; Cyr61, CTGF, and NOV. However, the acronym has recently been adapted to stand for "Cellular Communication Network" as ratified by the HUGO Gene Nomenclature Committee (Perbal B, Tweedie S and Bruford E, J Cell Commun Signal. 2019 Sep; 13(3):435). These proteins are often classified as extracellular matrix (ECM)-associated proteins. CCN proteins do not function as scaffolds that organize cells into tissues but are considered signaling proteins and may function as independent autocrine or paracrine signaling factors, or as modifiers of other extracellular signaling proteins. Along with a group of three Wnt-inducible signaling pathway proteins (WISP1/CCN4, WISP2/CCN5, and WISP3/CCN6), they include a family of six homologous cysteine-rich proteins in mammals that have been renamed CCN1-6.

The original members of the CCN family share a common modular structure, with an N-terminal secretion signal peptide followed by four conserved domains. The first domain shows sequence homology to insulin-like growth factor binding proteins (IGFBPs) and is thus known as the IGF-binding protein homology domain, although it has negligible affinity for IGF. The second domain is known as the von Willebrand factor type C repeat homology (VWC) domain, which is commonly found in extracellular matrix (ECM) proteins. The third domain is known as the thrombospondin type I repeat homology (TSP-1) domain, which may be involved in the attachment of CCN proteins to integrins. The fourth domain is a cysteine-rich C-terminal repeat or cystine knot homology domain, a domain that has been reported to bind heparin. The fifth member of the CCN protein family, WISP2 (Wnt1-induced signaling pathway protein 2), also known as CCN5, is atypical in that it lacks 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 source below). The four-domain CCN proteins contain 38 conserved cysteine residues along the length of the primary sequence, with the exception of CCN6, where the four cysteines in the VWC homology domain are not conserved relative to other family members. Also, in CCN5, which lacks a carboxy-terminal cystine knot homology domain, all cysteines in the IGFBP, VWC, and TSP-1 homology domains are conserved relative to other CCN family members.

A non-conserved protease-sensitive central region, often referred to as the hinge region, bisects the proteins. Expression of CCN proteins is regulated at the transcriptional, post-transcriptional, and translational levels in response to changing environmental stimuli.

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.

At the cellular level, CCN proteins can play a variety of regulatory roles at the extracellular matrix–cell surface interface. CCN proteins can regulate cell adhesion, migration, proliferation, differentiation, apoptosis, survival, aging, and gene expression. By modulating one or more aspects of these cellular functions in a cell type-specific manner, CCNs coordinate complex biological processes, including cardiovascular and skeletal development during embryogenesis, as well as inflammation, wound healing, and tissue injury and repair in adults. In general, the 4-domain CCNs 1–4 and CCN6 (specifically CCN1, CCN2, and CCN4) can exhibit profibrotic activity, whereas CCN5, which contains only 3 domains I–III, has antifibrotic activity.

CCN proteins have also been implicated in a variety of pathological conditions, such as organ failure due to progressive fibrosis, such as liver fibrosis and idiopathic pulmonary fibrosis, and cancer invasion and metastasis. For more information, see Jun and Lau, 2011, Nat. Rev. Drug. Discovery, 10(12), pp. 945-963. In general, 4-domain CCN proteins, particularly CCN2, have also been shown to be involved in the mechanisms of various fibrotic diseases, while in contrast, elevated CCN5 levels have been shown to be beneficial in preclinical models of such diseases.

In the source: et al., J. Biol. Chem., 293:46, pp. 17953-17970, report that connective tissue growth factor (CTGF), also known as CCN2, is synthesized and secreted as an inactive preprotein that requires proteolytic cleavage to release biologically active CCN2, and that the homodimer of the C-terminal fragment of domains III-IV represents the biologically fully active form of CCN2, and finally that all of the major reported activities of CCN2 can be reproduced by the homodimer of the C-terminal fragments of domains III-IV. The activity assays described et al., showed that neither unprocessed full-length CCN2 nor the N-terminal fragment consisting of domains I–II were biologically active. Furthermore, it was found that proteolytic processing of full-length CCN2 by matrix metalloproteinase (MMP) activity released its latent activity. Collectively, the data obtained et al. imply that prepro-CCN2 is autoinhibited by the N-terminal domains I and II. It was also found that the C-terminal domains III and IV of CCN1 and CCN3 were sufficient to activate rapid cell signaling and stimulate physiological responses in cells. However, it is unknown to what extent endopeptidase cleavage of the hinge region of CCN1 and/or CCN3, or any other member of the CCN protein family, is required to release biological activity.

CCN2 is known to be highly expressed during development, in a variety of pathological conditions including increased fibrogenesis and tissue fibrosis, and in some cancers (Jun and Lau, 2011, supra). The fact that CCN proteins are involved in a wide range of pathological conditions, represent extracellular proteins mechanistically involved in fibrosis, and exhibit limited expression in healthy organisms makes them attractive therapeutic targets.

In the source: Jeong et al., 2016, J. American College of Cardiology, 67: 13, pp. 1557-1568, a study was reported to investigate the role of adeno-associated virus-mediated gene transfer of CCN5 in mouse hearts after experimentally induced cardiac pressure overload. The study concluded that CCN5 could reverse established cardiac fibrosis by inhibiting the formation and increasing the apoptosis of myofibroblasts in the myocardium, suggesting that CCN5 may serve as a platform for the development of therapeutics for cardiac fibrosis.

US 2008/0207489 discloses a method for treating a disorder based on smooth muscle proliferation, comprising expressing CCN5 or introducing CCN5 protein into smooth muscle cells.

EP 2556839 proposes a composition comprising a genetic carrier comprising a nucleotide sequence encoding full-length CCN5 or CCN2ACT and suggests a role in the treatment of heart failure. CCN2ACT in EP 2556839 is defined as the amino acid sequence CCN2 truncated after K251 (Uniprot numbering).

Although overexpression of CCN5 has been described in some experimental systems to result in a phenotype opposite to that observed with overexpression of CCN2 (Jeong et al. supra, Yoon et al., J Mol Cell Cardiol, 49 (2), 294-303 Aug 2010), direct antagonism of CCN5 with four-domain CCN5 proteins has not been reported to the best of our knowledge. In particular, the structural basis for CCN5/WISP2-mediated antagonism of other CCN family members was unknown prior to the study reported herein.

The present inventors have shown at an earlier stage that full-length CCN2 (FL-CCN2) is a preproprotein, an inactive precursor, and that the fragment containing domains III and IV appears to carry out all the biologically relevant activities of CCN2. It remains unknown to what extent CCN proteins, in general, are secreted as inactive preproproteins that require proteolytic activation. However, the sensitivity of full-length CCN proteins to several proteases, as demonstrated by the present inventors ( et al., J. Biol. Chem. (2018) 293(46) 17953-17970) and other authors (Butler, GS et al. Matrix Biol 59, 23-38 (2017) and Guillon-Munos, A. et al. J Biol Chem 286, 25505- 25518 (2011)), implies that unmodified full-length CCN proteins would be highly unsuitable for use as drugs for stability reasons, both during recombinant protein production and after in vivo administration. The above-mentioned unsuitability for the use of unmodified full-length CCN proteins as therapeutic proteins also applies to fusion proteins of full-length CCN proteins, such as those 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 technical field relating to CCN proteins that the sensitivity of these proteins to proteolysis is one of the reasons for the considerable difficulty in obtaining recombinant CCN proteins. Furthermore, based on new data obtained with co-authors ( et al. J Biol Chem 2018; 293(46):17953–17970), recombinant full-length CCN proteins may be less than ideal biologic drugs, as their activity may depend on prior proteolytic processing, making pharmacokinetics and pharmacodynamics unpredictable. Furthermore, in the case of Fc fusion proteins, in addition to the proteolytic sensitivity of the components, such as the peptide linker, CCN fragment, and Fc fragment, the importance of the arrangement of the components for the efficacy and potency of recombinant fusion proteins has been shown. One example supporting this is described in a paper published by the present inventors ( et al. (2018)), where variants of Fc fusion proteins containing CCN2 domains III–IV were found to have activities that varied widely and were not readily predictable in advance.

The sensitivity of the actions of CCN proteins to the antagonizing effects of high concentrations of synthetic peptides derived from the primary sequences of CCN proteins has been described. One example is the inhibition of AKT phosphorylation stimulated by recombinant CCN2 in Rat2 fibroblasts by peptides derived from domain I, the IGFBP homology domain, and to a lesser extent by peptides derived from domain III, the 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 liver fat-storing cell adhesion by a peptide derived from domain IV, the cystine knot homology domain, of CCN2 (Gao R and Brigstock DR., J Biol Chem. 2004 Mar 5; 279(10):8848-55). Furthermore, 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 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 exert some anti-angiogenic effects in in vitro assays on 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), these peptides contain only one (Leu et al. J. Biol. Chem, 2003) or two (Karagiannis EG and Popel, Int J Biochem Cell Biol 39, 2007) of the conserved cysteines that play a central role in the invention described in the present application. Cysteines in domain III of CCN proteins are known to form disulfide bridges, as demonstrated for CCN2 endogenously expressed by HUVEC cells (Lu, S et al. (2015) Nat methods 12, 329-331) and purified recombinant CCN2 ( et al., J. Biol. Chem. 2018). The disulfide bridges demonstrated for CCN2 spanning C199-C228 (Uniprot numbering) provide a complex three-dimensional structure in which the amino acid chain folds to form a fold. This implies that one cannot expect to recapitulate the complete domain III of a CCN protein with short peptides that are not structurally constrained by disulfide bridges between cysteines, as in the complete domain III of CCN proteins produced in eukaryotic systems. Furthermore, 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 field regarding whether peptides derived from a specific domain of CCN2 can confer inhibitory capacity to four-domain CCN proteins.

In the present work, the present inventors, based on the structure-function analysis of the CCN family proteins and the observation that CCN2 requires proteolytic processing to become biologically active, discovered that the biologically active part of the CCN5 protein is represented by domain III, the thrombospondin type I repeat homology domain. These new data led to the preparation of bioactive structures based on domain III of CCN5, as well as domain III of other members of the CCN protein family.

BRIEF DESCRIPTION OF THE INVENTION

The relationship between the structure and activity of CCN5 and other CCN proteins discovered by the present inventors made it possible to obtain new biologically active recombinant proteins that reproduce cellular signaling and physiological functions of cells attributed to CCN signaling, and are also capable of counteracting 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, proteins, including in the form of fusion proteins, based on domain III, the TSP-1 homology domain, of the CCN protein are provided, which reproduce or have the biological activity of CCN5, and are capable of antagonizing or inhibiting the effects of the 4-domain CCN proteins, CCN1-4 or CCN6. In particular, the proteins according to the present application have antifibrotic activity and may also exert direct anticancer activity.

As noted above, it has been unexpectedly found that domain III (the TSP-1 homology domain) of other CCN proteins, namely, the 4-domain CCN proteins, when present as a single domain in the absence of other CCN domains, is sufficient to reproduce the disclosed activities of CCN5. Accordingly, in other words, domain III of the 4-domain CCN proteins, when present as a single domain in the absence of other CCN domains (i.e., as an isolated domain), has the same activity as CCN5, or, in other words, as domain III/TSP-1 homology domain of CNN5 (i.e., an activity opposite to the activity of the full-length 4-domain CCN proteins). Accordingly, it is clear from the experiments disclosed in this application that an isolated TSP-1 homology domain of any CCN protein can exert the same activity as the TSP-1 homology domain of CCN5. With the exception of CCN5, the activities reported may be different from those carried out by the full-length CCN protein.

It has been found that monomeric fusion proteins characterized in that domain III of the CCN protein is fused to a monomeric fusion partner have particular advantages and utility in accordance with the present invention and the description herein.

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

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

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

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

wherein the polypeptide of (i) is from 40 to 60 amino acids in length and comprises an amino acid sequence selected from SEQ ID NO: 37 or 2-6, or a sequence at least 80% identical to a sequence selected from SEQ ID NO: 37 or 2-6, wherein all cysteine residues in said sequence selected from SEQ ID NO: 37 or 2-6 are retained, and the monomeric fusion partner of (ii) and the peptide linker of (iii) do not represent or do not comprise an IGF binding protein homology domain, a von Willebrand factor type C repeat homology domain, or a cystine knot domain of a CCN family protein.

As described in more detail below, SEQ ID NOs: 37 and 2-6 represent truncated 44 amino acid fragments of domain III of CCN5, CCN3, CCN2, CCN1, CCN4 and CCN6, respectively, which contain 6 conserved cysteine residues of said domain. In particular, said fragments are flanked by the first and last cysteine residues of said domain. Such fragments were found to be particularly effective and resistant to proteolytic degradation.

According to an embodiment, the polypeptide according to (i) comprises or consists of:

(a) an amino acid sequence selected from SEQ ID NO: 1 or 8-12; or

(b) an amino acid sequence that is at least 80% identical to a sequence selected from SEQ ID NO: 1 or 8-12; or

(c) a part of the amino acid sequence according to (a) or (b), characterized in that said part comprises at least a sequence of 44 amino acids from SEQ ID NO: 37, 6, 2, 3, 4 or 5, respectively, or a sequence that is at least 80% identical to a sequence selected from SEQ ID NO: 37, 6, 2, 3, 4 or 5, respectively.

SEQ ID NOS: 1 and 8-12 represent slightly longer N-terminally truncated fragments of domain III of CCN5, CCN6, CCN3, CCN2, CCN1 and CCN4, respectively. These fragments comprise the corresponding sequences of SEQ ID NOS: 37, 6, 2, 3, 4 and 5, respectively, with any additional C-terminal sequence from the corresponding domain III.

According to a further embodiment, the polypeptide according to (i) comprises an alanine residue at a position corresponding to position 2 of said sequence selected from SEQ ID NO: 37 or 2-6, or SEQ ID NO: 1 or 8-12. According to some embodiments, the amino acid sequence according to (i) comprises an amino acid sequence selected from SEQ ID NO: 38 or 42-46, or an amino acid sequence at least 80% identical thereto. According to another embodiment, the amino acid sequence according to (i) comprises an amino acid sequence selected from SEQ ID NO: 7 or 47-51, or an amino acid sequence at least 80% identical thereto. In this regard, it has been found that substitution of said residue at position 2 is useful for increasing protein stability.

According to a further aspect of the present invention, the monomeric fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 84, 85, 88, 89, 97, 98, 102, 103, 106, 107, 111, or an amino acid sequence 80% identical thereto.

In embodiments of the above aspects, the monomeric fusion partner is selected from serum albumin, transferrin, and monomeric Fc fragments, in particular monomeric Fc fragments of IgG, more particularly human IgG.

As noted above, the substitution of position 2 in the domain III fragments of the present invention improves the stability of the fragment, in particular resistance to protease degradation. It is believed and suggested in the present application that such sequence-modified variants of domain III fragments are useful proteins in their own right, without binding to a fusion partner.

Accordingly, according to another aspect of the present invention, there is also provided a protein of 40 to 60 amino acids in length, which comprises a sequence, or a protein, which consists of the amino acid sequence presented in SEQ ID NO: 7, 38, 42-46, or 47-51, or a sequence at least 80% identical thereto, wherein said protein comprises an alanine residue at a position corresponding to position 2 of said sequence from SEQ ID NO: 7, 38, 42-16, 47-51, and all cysteine residues in said sequence are preserved.

Other proteins and fusion proteins are also provided as further aspects of the present inventions, as described in detail below.

According to a further aspect of the present invention, there is provided a recombinant protein consisting of formula (I)

Where

A is a peptide of formula II

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

B is a peptide of formula III

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-B3 is absent; and

C is a peptide of formula IV

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

D is a peptide of formula V

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

E is a peptide of formula VI

characterized in that 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 a sequence of amino acids up to about 13 amino acids in length,

where the recombinant protein contains from 40 to 60 amino acids.

According to one embodiment of the above-described aspect, a recombinant protein of formula (I) is proposed, characterized in that

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

B1 is a series (S); B2 is an amino acid selected from the group consisting of T, K, and R; B3 is an amino acid selected from the group consisting of T and S; and

C1 is an 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

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

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

where the recombinant protein contains a total of 44 to 57 amino acids.

According to another embodiment of the above-described aspect, there is provided a recombinant protein of formula I, wherein F is completely absent, partially absent, or is a peptide of approximately 13 amino acids in length comprising the amino acid sequence PPSRGRSPQNSAF.

More specifically, a recombinant protein is proposed, characterized in that said 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 fragments or variants thereof with more than 50% sequence identity to the amino acid sequences 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.

According to another aspect of the present invention, there are provided recombinant proteins as defined above, characterized in that said protein is PEGylated.

According to another aspect of the present invention, there is provided a fusion protein comprising

(i) the thrombospondin type 1 repeat homology domain (TSP-1) of the CCN family protein;

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

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

According to an embodiment of the above-described aspect, fusion proteins comprising a recombinant protein according to the present invention as described above are provided as a further aspect of the present invention.

The fusion partner of the fusion protein according to the present invention corresponds to one of the embodiments selected from the group consisting of the Fc fragment of IgG1, IgG2 or IgG4, serum albumin and transferrin.

According to a further embodiment, a fusion protein is provided, characterized in that the fusion partner (ii) is an Fc fragment of IgG1, IgG2 or IgG4 containing a stabilizing disulfide bridge. Such mutations can increase the thermal stability of the protein. Stabilizing mutations are known and have been described in the art.

According to yet another additional embodiment, a fusion protein is provided, characterized in that 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 (relates to IgG4), E233P (relates to IgG1 and IgG4), F234A (relates to IgG4), L234A (relates to IgG1), L234V (relates to IgG1), F234V (relates to IgG4), L235A (relates to IgG1 and IgG4), AG236 (relates to IgG1 and IgG4) and ΔK447 (relates to IgG1, IgG2 and IgG4).

According to another embodiment, the fusion protein may comprise an Fc region 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.

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.

According to one embodiment, said linker comprises the amino acid sequence (EAAAK)n, where n is at least 4, preferably n is 8.

According to another embodiment, said 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.

According to another embodiment, said fusion partner according to (ii) is serum albumin.

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

Also according to the present invention, in accordance with a further additional aspect, there is provided a nucleic acid molecule (eg, DNA) encoding a recombinant protein, protein or fusion protein according to the present invention.

According to one embodiment of this aspect, there is provided a DNA sequence comprising a nucleic acid sequence as presented 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 nucleic acid sequences that are approximately 80% identical to 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.

Furthermore, according to another aspect of the present invention, there is provided an expression vector comprising a DNA sequence according to the present invention. There is also provided a host cell comprising an expression vector according to the present invention.

Finally, a thrombospondin type 1 (TSP-1) repeat homology domain of a CCN family protein, protein and fusion protein as defined above is provided for use as a medicament for the treatment or prevention of disorders by inhibiting or antagonizing cell signaling and physiological functions of the cell attributed to four-domain proteins of the CCN family.

According to one aspect, there is provided a fusion protein or a protein as defined herein for use in therapy.

Said fusion protein or protein may be intended for use in the treatment of a condition associated with CCN 4-domain protein activity, in particular, undesirable or aberrant CCN 4-domain protein activity. Said activity may be associated with a fibrotic effect. Said activity may be a pro-fibrotic activity.

According to another aspect, there is provided a fusion protein or protein as defined herein for use in the treatment or prevention of fibrosis, or any condition in which fibrosis occurs (i.e., a fibrotic condition or disease). According to a further aspect, there is provided a fusion protein or protein as defined herein for use in the treatment of cancer. There is also provided a fusion protein or protein as defined herein for use in the treatment of inflammatory or autoimmune diseases, or metabolic diseases.

Also according to such aspects of the present invention there is 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 condition or disease as defined or described herein.

Such aspects also include a composition (e.g., a pharmaceutical composition) comprising a protein or fusion protein as defined herein for use in treating or preventing a condition or disease as defined or described herein.

Such aspects also include a method of treating or preventing a condition or disease as defined or described herein, characterized in that said method comprises 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.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 shows the cellular physiological processes and cellular signaling of CCN5(dIII)-Fcv2 (an embodiment of the present invention as defined in SEQ ID NO: 28).

A) The CCN5(dIII)-Fcv2 fusion protein of SEQ ID NO: 28 was shown to cause concentration-dependent inhibition of AKT (serine 473) phosphorylation in A549 lung cancer cells.

B) The CCN5(dIII)-Fcv2 fusion protein of SEQ ID. NO: 28 was shown to inhibit proliferation of the human lung fibroblast cell line, IMR90.

C) The CCN5(dIII)-Fcv2 fusion protein of SEQ ID NO: 28 was shown to inhibit the sphere-forming ability (substrate-independent growth) of the estrogen receptor-positive breast cancer cell line MCF-7 and the triple-negative breast cancer cell line MDA-MB-231.

D) The CCN5(dIII)-Fcv2 fusion protein of SEQ ID NO: 28 was shown to dose-dependently inhibit TGF-β-induced SMAD reporter activity (SMAD proteins are canonical TGF-β-regulated transcription factors).

All error bars represent SD. Statistical significance was calculated using one-way ANOVA with Dunnett's post hoc test (p<0.05 marked with *).

Fig. 2 shows the effect of different Fc hinge region variants on the protease sensitivity of embodiments of the present invention wherein CCN5(dIII) is fused to an IgG Fc region, wherein the fusion protein tested comprises the sequence shown in SEQ ID NO: 28; SEQ ID NO: 29 and SEQ ID NO: 30, respectively, see Example 6 below.

Fig. 3 shows the aggregation propensity of an embodiment of the present invention depending on the structure of the peptide linker connecting CCN5(dIII) to the Fc region of IgG, when the test fusion protein comprises the sequence presented in SEQ ID NO: 30 and SEQ ID NO: 31.

Fig. 4 shows a fusion protein according to the present invention comprising a TSP-1 repeat homology domain that is linked at the C-terminus to a peptide linker and linked via an Fc hinge to an Fc fragment.

Fig. 5 shows reduced sensitivity to endopeptidase cleavage when an embodiment of the present invention comprises a proline-195 mutation of the TSP-1 CCN5 repeat homology domain shown in SEQ ID NO: 7 (Fc-HLn8-CCN5(dIII)-P195A, SEQ ID NO: 41) relative to the wild-type P195 variant of the TSP-1 CCN5 repeat homology domain (Fc-HLn8-CCN5(dIII), SEQ ID NO: 40).

Fig. 6 shows the preparation of the protein corresponding to SEQ ID NO: 58 purified by protein A capture chromatography. It can be seen that in the absence of the reducing agent beta-mercaptoethanol, a dimer is present ((-) lane). However, in the presence of beta-mercaptoethanol ((+) lane), it can be seen that the primary product is a fragment after cleavage consisting of only the Fc fragment, and not an intact fusion protein containing all parts encoded by SEQ ID NO: 58 (the TSP-1 homology domain fragment, the peptide linker and the Fc fragment).

Fig. 7 shows the preparation of the protein corresponding to SEQ ID NO: 27, having a truncated C-terminal portion, purified by protein A capture chromatography. It is seen that said protein is significantly more resistant to protease degradation than the protein corresponding to SEQ ID NO: 58, which includes the C-terminal portion.

Fig. 8 shows the production of the protein corresponding to SEQ ID NO: 73, similar to the protein corresponding to SEQ ID NO: 27, purified by protein A capture chromatography. In this case, too, it can be seen that in the presence of beta-mercaptoethanol ((+) lane), said protein is more resistant to protease degradation than the protein corresponding to SEQ ID NO: 58.

Fig. 9 shows the results of an assay for measuring phospho-AKT (Ser-473) levels in A549 lung cancer cells following administration of varying concentrations of the protein corresponding to SEQ ID NO: 41 produced in stably transfected cells. It is seen that this protein does not show inhibition of AKT phosphorylation.

Fig. 10 shows the results of an assay for measuring phospho-AKT (Ser-473) levels in A549 lung cancer cells following administration of varying concentrations of the protein corresponding to SEQ ID NO: 80 produced in stably transfected cells. It is seen that said protein does not show significant inhibition of AKT phosphorylation and, in fact, may even lead to an increase in phospho-AKT levels at higher concentrations.

Fig. 11 shows the results of an assay for measuring phospho-AKT (Ser-473) levels in A549 lung cancer cells after administration of varying concentrations of the protein corresponding to SEQ ID NO: 80 produced in transiently transfected cells. It can be seen that when produced in transiently transfected cells, said protein is characterized by a concentration-dependent inhibitory activity with respect to AKT phosphorylation.

Fig. 12 shows the results of an assay for measuring phospho-AKT (Ser-473) levels in A549 lung cancer cells after administration of varying concentrations of proteins corresponding to SEQ ID NOs: 84, 94, and 106. It is evident that each of these proteins is characterized by a concentration-dependent inhibitory activity on AKT phosphorylation.

Fig. 13 shows the results of an assay for measuring phospho-AKT (Ser-473) levels in A549 lung cancer cells after administration of varying concentrations of the protein corresponding to SEQ ID NO: 88. It is evident that this protein is capable of inhibiting AKT phosphorylation at concentrations above 10 μg/ml.

Fig. 14 shows the results of an assay for measuring phospho-AKT (Ser-473) levels in A549 lung cancer cells after administration of varying concentrations of proteins corresponding to SEQ ID NOs: 102 and 97. It is evident that both proteins are characterized by a concentration-dependent inhibitory activity on AKT phosphorylation.

Fig. 15 shows the results of an assay for measuring phospho-AKT levels in A549 lung cancer cells after administration of varying concentrations of the protein corresponding to SEQ ID NO: 110. It is seen that the protein has a concentration-dependent inhibitory activity against AKT phosphorylation.

Fig. 16 shows the results of a series of experiments involving the protein corresponding to SEQ ID NO: 106.

A) shows that the protein inhibits human lung fibroblast migration induced by both TGF-beta and CCN2.

B) shows that the protein inhibits scratch closure induced by both TGF-beta and CCN2.

C) shows that the protein in question provides partial inhibition of TGF-beta induction of COL1A1 gene expression, which is known to be profibrotic.

D) shows that the protein in question provides partial inhibition of TGF-beta induction of FN1 gene expression, which is known to be profibrotic.

E) shows that the protein in question provides partial inhibition of TGF-beta induction of ACTA2 gene expression, which is known to be profibrotic.

F) shows that the protein in question provides partial inhibition of TGF-beta induction of CCN2 gene expression, which is known to be profibrotic.

DETAILED DESCRIPTION OF THE INVENTION

The present invention, as already mentioned, is based on the unexpected discovery that the thrombospondin type 1 repeat homology (TSP-1) domain of CCN5 is a fully active structure that mediates the CCN5/WISP2 cell signaling functions. Based on these new findings regarding the activity of the TSP-1 repeat homology domain of CCN5, the present inventors provide proteins, recombinant proteins and fusion proteins according to the present invention that can be used to inhibit or counteract the cell signaling and physiological functions of the cell attributed to the four-domain CCN proteins, i.e., CCN1, CCN2, CCN3, CCN4 and CCN6. These new data are believed to be critical to the development of a stable, homogeneous CCN5-based drug-like molecule, since it has not been previously established what portion of full-length CCN5 is required to reproduce CCN5 activity, and since full-length CCN molecules are highly susceptible to proteolysis and difficult to obtain in an active, homogeneous form. Furthermore, these data, by implying that a specific portion of CCN5 may be sufficient to reproduce the activity observed, for example, upon transient overexpression of the full-length protein, also contradict the prevailing view in the field 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 various other ECM proteins and cell surface receptors, thereby modulating their activity, rather than acting as direct modulators of cell signaling. New information on the activity of the TSP-1 repeat homology domain of CCN5, as well as information on the structural similarity to other members of the CCN protein family, suggests that the TSP-1 repeat homology domains of other CCN family proteins may also be used to inhibit the cell signaling functions of four-domain CCN family proteins. In one aspect, the recombinant proteins and fusion proteins of the present invention inhibit phosphorylation of AKT (Ser473) in A549 cells.

Inhibition of this cellular signaling has implications for the treatment of various disorders. CCN2, for example, has been implicated in a number of diseases, particularly those in which increased fibrogenesis and tissue fibrosis are a characteristic pathophysiological feature.

For example, overexpression of CCN2 by itself has been shown to be sufficient to induce fibrosis in the lung (see Sonnylal et al., Arthritis Rheum 62, 1523–1532 (2010)). CCN2 has also been found to be required 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 resulting from loss of PTEN (phosphatase and tensin homolog) expression (Parapuram, S. K. et al. Matrix Biol 43, 35-41 (2015)). Furthermore, in the absence of other stimulatory agents, CCN2 has been found to induce pulmonary fibrosis when expressed and secreted by pulmonary Clara cells (Wu, S. et al. Am J Respir Cell Mol Biol 42, 552-563 (2010)), alveolar epithelial cells type II (Chen, S. et al. Am J Physiol Lung Cell Mol Physiol 300, L330-340), when expressed 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)). Accordingly, all available reports support the conclusion that CCN2 is not only sufficient to promote fibrosis in skin or lung, but is also required for the full 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.H. et al., COPD 14, 228-237 (2017)) and systemic sclerosis. In fact, pulmonary fibrosis has been reported as a primary 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 shown to be involved in the pathophysiology of IPF (Konigshoff, M. et al., J Clin Invest 119, 772-787 (2009)) and COPD (Jang et al, supra) in human patients.

Another example is provided by neoplastic disorders. For example, in the setting of breast cancer, CCN2 has been shown to contribute to bone metastasis in a triple-negative breast cancer model (MDA-MB-231) (Kang, Y. et al., Cancer Cell 3, 537-549 (2003)). Furthermore, knockdown of CCN2 in triple-negative breast cancer cells (MDA-MB-231), a cell line that expresses high levels of CCN2 (Chen, P.S. et al., J Cell Sci 120, 2053–2065 (2007)), reduced the migratory capacity of these cells, whereas overexpression of CCN2 in hormone receptor-positive breast cancer cells MCF-7, a cell line with low endogenous CCN2 expression (Chen et al., supra), increased the migratory capacity of the latter (Chen et al., supra, Chien, W. et al., Int J Oncol 38, 1741–1747 (2011)). A later report also described that CCN2 overexpression in MCF-7 cells increased chemoresistance, whereas CCN2 knockdown in MDA-MB-231 cells decreased chemoresistance (Wang, M.Y. et al., Cancer Res 69, 3482-3491 (2009)). Increased CCN2-induced chemoresistance has also been reported in other breast cancer cells (Lai, D et al., Cancer Res 71, 2728-2738 (2011)). Furthermore, CCN2 has also been shown to contribute to epithelial-mesenchymal transition (EMT) and enhance the substrate-independent growth capacity (mammosphere formation) of breast cancer cells using overexpression or knockdown studies (Chen et al., supra, Zhu, X. et al., Oncotarget 6, 25320–25338 (2015)). The finding of both increased chemoresistance and increased EMT induced by CCN2 is consistent with the association established between EMT and chemoresistance in other cancer types as well (Fischer, K.R. et al., Nature 527, 472–476 (2015), Zheng, X. et al., Nature 527, 525–530 (2015)).

According to a particular aspect of the present invention, there is provided a monomeric fusion protein as defined above, comprising a polypeptide corresponding to at least a portion of the thrombospondin type 1 repeat homology domain (TSP-1) of a CCN family protein, wherein the sequence of said TSP-1 homology domain may be truncated and/or modified, however, the cysteine residues of said domain are retained. Said polypeptide may be referred to herein for convenience as a "TSP-1 polypeptide", and said term should accordingly be understood as not stipulating or implying any limitation to only a particular natural sequence of the TSP-1 homology domain. The term "TSP-1 polypeptide" may be used synonymously or interchangeably with "TSP-1 domain protein" or "TSP-1 domain sequence".

As demonstrated in the examples below, it has been surprisingly found that monomeric fusion partners are preferred for producing active and stable proteins compared to dimeric fusion partners, such as Fc fragments derived from IgG proteins, which form dimeric fusion proteins. The monomeric fusion proteins retain the activity of the TSP-1 domain polypeptide that they contain. In addition, these proteins are stable, including with respect to proteolytic degradation. As described in more detail below, resistance to proteolytic degradation can be improved by introducing modifications to the amino acid sequence of the TSP-1 polypeptide, including, in particular, the Ala substitution mentioned above.

Accordingly, the polypeptide of component (i) of the fusion protein may comprise insertions, deletions, substitutions, mutations, or any combination thereof relative to said sequence of SEQ ID NO: 37 or 2-6, or a sequence selected from SEQ ID NO: 1 or 8-12, provided that the polypeptide retains at least 80% sequence identity of said sequence and all cysteine residues in said sequence are retained.

According to another aspect of the present invention, there is provided a protein (e.g., a recombinant protein) that consists of or comprises a polypeptide corresponding to at least a portion of the thrombospondin type 1 repeat homology domain (TSP-1) of a CCN family protein, but not in the context of a fusion 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 2-6, or SEQ ID NO: 1 or 8-12, but wherein the cysteine residues of said domain are retained. In other words, the TSP-1 domain protein may be provided without or independently of another component, such as a fusion partner. Accordingly, the TSP-1 domain protein is not fused or linked to another protein domain or component, or another functional or structural protein sequence. For convenience, such proteins may be referred to as "Ala-substitution proteins."

In the present application, the term "conserved" means that a residue in a certain sequence is not deleted or substituted. In other words, the term "conserved" is used synonymously (and interchangeably) with the term "conserved". It simply means that cysteine residues are not deleted from the sequence. Accordingly, in the above-described context, it means that cysteine residues in a sequence selected from SEQ ID NO: 37 or 2-6 or 1 or 8-12 are not deleted or removed. It is noted that the insertion of additional residues between conserved residues (e.g., between conserved cysteine residues) or the deletion of non-conserved residues (e.g., residues other than cysteine) may change the position of the conserved residues in the polypeptide sequence relative to their position in the original reference sequence (e.g., a sequence selected from SEQ ID NO: 37 or 2-6). However, such residues are still considered "conserved" as defined in this application. Accordingly, the term "conserved" does not imply any narrowing or limitation of the position (or, more specifically, the position number) of the cysteine residues.

According to some embodiments, the polypeptide of (i) comprises or consists of:

(a) an amino acid sequence selected from SEQ ID NO: 1 or 8-12; or

b) an amino acid sequence at least 80% identical to a sequence selected from SEQ ID NO: 1 or 8-12; or

c) a part of the amino acid sequence according to (a) or (b), characterized in that said part comprises at least a sequence of 44 amino acids from SEQ ID NO: 37, 6, 2, 3, 4 or 5, respectively, or a sequence that is at least 80% identical to a sequence selected from SEQ ID NO: 37, 6, 2, 3, 4 or 5, respectively.

As noted above, the monomeric fusion partner of (ii) and the peptide linker of (iii) do not constitute or comprise an IGF binding protein homology domain, a von Willebrand factor type C repeat homology domain, or a CCN family protein cystine knot domain. In other words, the only CCN family protein domain that may be present in the fusion protein of the present invention is the TSP-1 homology domain.

Similarly and analogously, in the context of Ala-substituted proteins that are not fusion proteins, the protein does not contain any other CCN domains (other than the TSP-1 domain protein).

In some embodiments, the polypeptide of (i) or the Ala substitution protein may comprise only a portion of the TSP-1 homology domain as defined above. The present inventors have found that the minimal required fragment of the TSP-1 domain is represented by the 44 amino acid sequence of SEQ ID NO: 37, 6, 2, 3, 4 or 5. Accordingly, in some embodiments, the polypeptide of (i) is at least 44 amino acids long. In some embodiments, the polypeptide of (i) is 44 to 57 amino acids long. However, as noted above, the minimal fragment of 44 amino acids may include one or more amino acid deletions that are located between cysteine residues. Accordingly, in some embodiments, the TSP-1 polypeptide may be less than 44 residues long, i.e., 40 to 43 residues.

In some embodiments, the polypeptide of (i) consists of an amino acid sequence selected from SEQ ID NO: 37 or 2-6, or a sequence that is at least 80% identical to a sequence selected from SEQ ID NO: 37 or 2-6.

As described above, the proteins of the present invention, including fusion proteins, exhibit (or in other words, display or have) a CCN5 activity, more particularly a CCN5 biological activity. According to an embodiment, said proteins may retain, or display, or have the activity of a TSP-1 homology domain of CCN5. Alternatively, said proteins may be defined as exhibiting (or displaying, or having) an activity, in particular the biological activity of an isolated TSP-1 homology domain of a CCN protein. The above may refer to any activity of said domain, as well as to specific activities reflecting the anti-fibrotic effect of said TSP-1 homology domain. Such activity may be assayed (or tested, or detected) using any convenient assay or method based on any specific biological effect of said domain.

Note that the activity of a particular protein can conveniently be assessed by assaying the effect of the protein on AKT phosphorylation. In particular, a particular protein can be assayed for its ability to inhibit AKT (Ser-473) phosphorylation in A549 human lung cancer cells, as described in Example 2. It will be appreciated by those skilled in the art that other similar assays can be developed to assess this same activity or to assess other related antifibrotic activities.

As noted above, other aspects of the present invention provide recombinant proteins that inhibit or counteract cell signaling and cellular physiological functions attributed to four-domain CCN proteins comprising an amino acid sequence according to formula I above.

where A, B, C, D, E and F correspond to the definition above and in the attached claims.

Formula I is an alignment of the TSP-1 repeat homology domain of structurally related CCN family proteins (CCN 1 - CCN6), all of which contain 6 conserved cysteine residues, with the understanding that amino acids can be substituted without affecting the activity of the protein (conservative substitutions, as discussed in more detail below). The position of the first conserved cysteine residue of the TSP-1 repeat homology domain of the other CCN proteins is defined as position #1 of the recombinant protein of Formula I.

The five segments between the conserved cysteines are designated A, B, C, D, and E, respectively.

The first segment A is determined by the formula where A1-A9 correspond to the definition above. The amino acid at position 7 (A7) of segment A is tryptophan (W) in all members of the CCN protein family and is considered conserved.

The second segment B is defined by the formula B1-B2-B3, where B1-B3 corresponds to the definition above. According to one embodiment, B1 and B3 are either serine or threonine.

The third segment C is determined by the formula wherein amino acids C1-C14 are as defined above. According to one embodiment, amino acids C1 and C3 are glycine (G).

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

The fourth segment D is determined by the formula wherein amino acids D1-D8 are as defined above. According to one embodiment, D7 is arginine (R).

The fifth segment E is determined by the formula wherein amino acids E1-E4 are as defined above. According to one embodiment of the present invention, E4 is proline.

Following the last cysteine residue is a carboxyl-terminal peptide segment of variable length (F), containing from 0 to 13 amino acids.

F may be deleted or truncated relative to the amino acid sequences of the TSP-1 repeat homology domain of the CCN protein family. According to one embodiment, F is absent. According to another embodiment, F consists of a peptide selected from the group consisting of PPSRGRSPQNSAF, GQPVYSSL, EADLEEN, EQEPEQPTD, DVDIHTLI and DSNILKTIKIP. According to one aspect of this embodiment, the recombinant proteins may take the form of an amino acid sequence as set forth in SEQ ID NO: 8-12.

According to another aspect of the present invention, there are provided recombinant proteins 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 or variants thereof with at least 50% sequence identity to the amino acid sequences 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.

According to one aspect, a recombinant protein is provided, consisting 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 fragments or variants thereof with more than 50% sequence identity to the amino acid sequences 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.

"Recombinant proteins" in the present application are proteins encoded by recombinant nucleic acids. They are expressed from recombinant nucleic acids in a host cell as described in more detail below.

"Recombinant nucleic acid" as used herein describes a nucleic acid molecule that, by origin or manipulation, is not linked to all or part of a polynucleotide to which it is linked in nature and/or is linked to a different polynucleotide than to which it is linked in nature, as described in more detail below.

It will be understood by the skilled person that modification of the amino acid sequence of the recombinant proteins and fusion proteins according to the present invention can be introduced without changing the activity of said protein. Amino acids are generally classified as hydrophobic or hydrophilic and/or having polar or non-polar side chains. Substitutions of one amino acid for another having the same biochemical characteristics are commonly known as conservative substitutions.

Conservative amino acid substitutions include mutual substitutions of amino acids within the following groups:

• MILV

• FYW

• KRH

• AG

• ST

• QN

• ED

Generally, a "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made, and thus rarely changes the protein structure, so that biological activity is also not significantly changed.

It will be clear to the skilled person that the biological activity of a protein can also be retained if one or more amino acids are deleted, inserted or added to the amino acid sequence, provided that the structural and physicochemical properties are retained.

The symbol "Δ" in the present application before an amino acid refers to a deletion of the said amino acid, for example, ΔK447 should be understood as a protein in which K447 is missing. Also in the present application, a deletion of a specific amino acid is alternatively denoted by the symbol "-", for example, K447- should also be understood as a protein in which K447 is missing.

Accordingly, it is to be understood that the present invention encompasses recombinant proteins and fusion proteins as described in the appended claims, into which such modifications as described above (substitutions, deletions, insertions and additions of amino acids) can be introduced without substantially changing their biological activity, i.e. the ability to inhibit or counteract cellular signaling and physiological functions of the cell, attributed to the four-domain proteins of the CCN family; CCN1, CCN2, CCN3, CCN4 and CCN6.

Reference is made to amino acid sequences throughout the present specification. Reference is sometimes made to amino acid sequences in the present specification when referring to a modification of the amino acid sequence or protein in question by reference to "Uniprot numbering" or Eu numbering. Uniprot numbering refers to the numbering used in the Uniprot database (Uniprot Consortium, Nucleic Acids Res. 2019 Jan 8;47(D1):D506-D515). Uniprot numbering is used to number the amino acids of CCN proteins. Eu numbering refers to the numbering of the Eu antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85) and is used for amino acids in the Fc portions of human IgG subclasses, with or without mutations, or chimeras other than the wild type. The numbering system is available, for example, in the international information system iMMunoGeneTics (IMGT) of the IMGT Scientific chart resource. The IMGT system is described in the source: Lefranc M-R, Biomolecules. 2014 Dec; 4(4): 1102- 1139.

In the context of the present description, when speaking of "sequence identity", a sequence with at least x% identity to a second sequence means that x% corresponds to the number of amino acids in the first sequence that are identical to the corresponding amino acids of the second sequence when performing an optimal sequence alignment by global alignment over the entire length of the second amino acid sequence. An optimal alignment of both sequences is achieved if x is maximal. The alignment and determination of the percent identity can be performed manually or automatically.

Alignment for the purpose of determining percent amino acid identity can be achieved in a variety of ways within the skill of the art, for example using open source software such as ClustalOmega (Sievers F, Higgins DG (2018) Protein Sci 27:135-145), Protein BLAST (from the National Center for Biotechnology Information (NCBI), USA), or commercial software such as Megalign (DNASTAR) software. Those skilled in the art can determine suitable metrics for measuring alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared. NCBI BLAST is another example of software for determining amino acid sequence identity (MacWilliam et al., Nucleic Acids Res. 2013 Jul; 41(Web Server issue): W597-W600).

According to one aspect of the present invention, there is provided 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, 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 with at least 50% sequence identity to the amino acid sequences 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.

According to another aspect, a recombinant protein is provided, comprising an amino acid sequence with at least 60%, 70%, 80%, 90%, or 95% sequence identity to 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.

Biologically active proteins and peptides play an important role in the clinical treatment of human diseases. However, many proteins and peptides are hampered by their less than ideal pharmacokinetic properties, as they are eliminated by renal filtration due to their small size and/or proteolytic metabolism. Such factors may impose limitations or lead to difficulties in administering the drug to the subject requiring treatment, such as the need for continuous infusions or frequent subcutaneous administration to maintain circulating concentrations of the protein or peptide at an effective therapeutic level. The need for continuous or very frequent administration of a drug is clinically undesirable due to the obvious problems and inconvenience to both the patient and the treating physician.

One strategy to increase the half-life of a biologically active peptide or protein is to link a polyethyleneglycol (PEG) group to the peptide or protein of interest through a process called PEGylation (see, e.g., Dozie et al. (2015) Int. J. Mot Sci, 16(10) 25831–25864). A general strategy for protein PEGylation is to react a functional group on the protein with a complementary group on a PEG molecule to form a protein-PEG conjugate. The PEG moiety offers a number of advantages for increasing protein stability and circulating half-life due to its flexibility, hydrophilicity, variable size, and low toxicity.

According to one embodiment of the present invention, accordingly, there is provided a recombinant protein according to the description above, characterized in that said said protein is PEGylated. Fusion proteins according to the present invention can also be PEGylated.

Fused proteins

Another way to avoid the difficulties associated with the medical use of peptides and proteins is to extend the half-life of the bioactive protein or peptide by producing fusion proteins (see, for example, Valeria et al. (2017), “A New Approach to Drag 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 through genetic recombination, the molecular weight of the protein of interest can be increased to approximately 60-70 kDa, which is within the threshold for renal filtration.

The present invention provides a fusion protein comprising

(i) The thrombospondin type 1 repeat homology domain (TSP-1) of the CCN family protein;

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

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

In the present description, the TSP-I repeat homology domain may also be referred to as domain III and refers to domain III, i.e., the third domain of the CCN family of proteins.

In one preferred aspect, the fusion partner is a monomeric fusion partner and results in a monomeric fusion protein. Such fusion proteins, and in particular their TSP-1 domains, are defined above and described in more detail below.

However, the present invention also includes other embodiments, both with respect to the TSP-1 domain protein component and to the fusion partner component.

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

The TSP-1 repeat homology domain according to another embodiment is a recombinant protein having an amino acid sequence as defined in any of the sequences presented in SEQ ID NOs: 1-12, 37 and 38, or a recombinant protein of formula I as defined above.

According to one embodiment, the TSP-1 repeat homology domain is a recombinant protein comprising an amino acid sequence with at least 50%, 60%, 70%, 80%, 90% or 95% sequence identity to 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.

Proteins are inherently susceptible to degradation by proteases. To prevent protease degradation of recombinant proteins and fusion proteins according to the present invention, modifications can be introduced into the amino acid sequence, for example, by site-directed mutagenesis, to obtain protease-resistant recombinant proteins and fusion proteins. For example, a point mutation can be introduced into the thrombospondin type 1 repeat homology domain (TSP-1) of a CCN family protein as defined in SEQ ID NOs: 1-12, 37 or 38, or more specifically, into a protein as defined in any of SEQ ID NOs: 1-6, 8-12 or 37, a fusion protein or a recombinant protein. According to one embodiment, a point mutation is introduced that reduces sensitivity 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 the introduction of a point mutation corresponding to a proline to alanine substitution at position 195 (P195A) of domain III of CCN5, such as shown in SEQ ID NO: 7. A similar mutation can also be introduced into amino acid sequences derived from domain III of other CCN family members. SEQ ID NO: 38 corresponds to a truncated 44-amino acid sequence of the TSP-1 CCN5 domain containing an Ala substitution. SEQ ID NOs: 42-46 correspond to truncated 44-amino acid sequences of the TSP-1 homology domains of CCN1, 2, 3, 4 and 6, respectively, containing an Ala substitution. SEQ ID NOs: 47-51 correspond to longer sequences of homology to TSP-1 CCN1, 2, 3, 4 and 6, respectively, containing the Ala substitution. Any such sequence, or a sequence at least 80% identical thereto, can be used according to the present invention.

As noted above, according to a preferred embodiment, said fusion partner (ii) of the fusion protein according to the present invention is monomeric. Any monomeric fusion partner may be used. Accordingly, the fusion partner may be any protein or part thereof (e.g., a protein domain) that is formed and remains in a monomeric form when fused with a component protein with a TSP-1 homology domain. Accordingly, the fusion protein comprising a monomeric fusion partner and a protein with a TSP-1 homology domain remains monomeric. This means that it does not dimerize and does not form higher order multimers with itself.

Various proteins are known to be suitable as possible fusion partners, and may include natural proteins or fragments or variants thereof with modified amino acid sequences, as well as synthetic proteins or amino acid homopolymers. Such proteins include, in particular, Fc fragments of IgG, serum albumin or transferrin.

A fusion partner in the present application is broadly defined as a second polypeptide (or second amino acid sequence) that is not present in combination (e.g., not adjacent to, or not linked, directly or indirectly) with a first CCN polypeptide with homology to TSP-1 in nature, and is linked to the first CCN polypeptide with homology to TSP-1 in a synthetic or artificial combination. Accordingly, a fusion protein comprises a non-naturally occurring combination of at least two amino acid sequences or polypeptides linked or fused together.

The fusion partner may 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 provides a function or property of the fusion protein, such as stabilizing the fusion protein (making the first polypeptide more stable) or increasing its serum half-life. Accordingly, the fusion partner may be a structural protein or have a structural function, or it may provide an activity or property of the fusion protein, such as binding activity (e.g., the fusion partner may be a member of a binding pair, or may be an affinity binding partner, etc.). In representative examples, the fusion partner may be an albumin (particularly serum albumin), fibrinogen, glutathione-S-transferase, transferrin, streptavidin or streptavidin-like protein, or an immunoglobulin or a portion thereof, particularly the Fc portion of an immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4), or a portion or modified version 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 act to improve the pharmacokinetic properties of the fusion protein, such as synthetic polypeptides such as an amino acid homopolymer, a proline-alanine-serine polymer, or an elastin-like peptide, such as those described in Strohl, 2015, BioDrugs 29, 215-239. Any fusion partner known in the art for use with therapeutic proteins can be used.

According to an embodiment, the fusion partner (ii) of the fusion protein according to the present invention may be the Fc fragment of IgG (of any subclass or a chimera of any subclasses), serum albumin or transferrin.

The fusion partner may be fused at the N- or C-terminus to a component protein with a TSP-1 homology domain of the fusion protein, such as a TSP-1 repeat homology domain of CCN5 or any other CCN proteins as defined herein. It may be attached directly or indirectly via a linker, as described in more detail below.

Fc fragments tend to form dimers, and when used in fusion proteins, the fusion protein design tends to include 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.

Accordingly, if the fusion partner is an Fc fragment, it is preferably a monomeric Fc fragment, such as a monomeric Fc fragment of human IgG of any class. Chimeric Fc fragments containing portions of Fc regions from different classes, as well as Fc fragments with modified sequences, are contemplated.

Fc fusion proteins are a growing class of protein therapeutics based on chimeric proteins consisting of an effector domain linked to the Fc region of an IgG isotype. A typical example of a biopharmaceutical product is etanercept (a TNF-α receptor linked to an Fc region), used in the treatment of, for example, rheumatoid arthritis. Another example of a biopharmaceutical Fc fusion protein is aflibercept. Aflibercept, a fusion protein of the VEGF receptor Fc protein, is used in the treatment of wet macular degeneration and metastatic colorectal cancer. The primary rationale for producing Fc fusion proteins is to prolong half-life by increasing molecular weight sufficiently to eliminate renal excretion and enhance renal proximal tubular reabsorption via the neonatal Fc receptor. Also, pH-dependent binding of Fc fusion proteins to the neonatal Fc receptor (FcRn) on endothelial cells allows Fc-based fusion proteins, which would otherwise be endocytosed and subsequently lysosomally degraded, to be recycled and released back into the circulation.

According to one embodiment, there is provided a fusion protein, characterized in that the fusion partner (ii) is an Fc region from human IgG (immunoglobulin G, also known as immunoglobulin γ), including any subclasses of human IgG. According to another embodiment of the present invention, there is provided a fusion protein, characterized in that the fusion partner is an Fc region of IgG1, IgG2 or IgG4. Preferably, the Fc region of human IgG is of the IgG4 (SEQ ID. NO 13) or IgG2 (SEQ ID. NO 14) subclass.

IgG1, IgG2 and IgG4 are often preferred over IgG3 due to their longer half-life of approximately 3 weeks. One skilled in the art will appreciate that the choice of a particular subclass of IgG isotype as an Fc fusion partner depends on the desired prolongation of half-life and the level of cytotoxic activity of the resulting compound. Therapeutic antibodies intended for the treatment of cancer or autoimmune diseases belong, for the most part, to the IgG1 subclass due to their high affinity for Fc receptors and their potent ability to stimulate immune effector functions. IgG2 and IgG4, on the other hand, are the preferred IgG subclasses for use as the backbone of a candidate therapeutic agent when the absence of immune effector functions is desired, since immune effector functions may be the cause of undesirable effects. The propensity of an Fc fragment to activate immune effector functions depends on the Ig isotype and subclass and varies for different immune effector functions. In addition to selecting an Fc fragment of an appropriate IgG subclass, the amino acid sequence of a given IgG subclass can be modified, for example by site-directed mutagenesis, to reduce the ability of Fc fragments to activate immune effector functions.

Fc fragments that form monomers, or more precisely, that retain or have a monomeric form, or can be modified by introducing mutations that allow or facilitate the formation of a monomeric structure, can be selected. Such mutations are referred to in this application as "mutations that provide for the formation of monomers." Examples of Fc fragments that contain mutations that provide for the formation of monomers are provided by SEQ ID NOs: 54 and 55. A person skilled in the art knows how to introduce such mutations and select monomeric Fc mutants.

Evasion of immune effector activation function by Fc fragments

For example, the biopharmaceutical fusion protein dulaglutide (Trulicity™), a GLP-1 agonist and Fc-region fusion protein used once weekly to treat type 2 diabetes, introduces well-characterized F234A and L235A mutations into the hinge region of the Fc-region of IgG4 to reduce its ability to activate immune effector functions.

According to one embodiment of the present invention, the Fc fusion partner is an IgG4 Fc fragment, characterized in that said IgG4 Fc fragment is modified so as to avoid immune effector functions, for example, it contains the above-mentioned F234A and L235A mutations.

Protease-resistant Fc fragments

Another factor that can reduce both the manufacturing yield and the biological half-life is the cleavage of the fusion protein by endopeptidase. To reduce or eliminate the risk of proteolytic degradation, modifications can be introduced into the amino acid sequence of the Fc fragment, in particular by introducing a mutation into sites susceptible to proteolytic cleavage. In the EP patent application EP2654780 B1, the Fc domain of the constant region of IgG1 was modified by replacing E233-L234-L235-G236 with P233-V234-A235 (deletion of G236) (EU numbering) to render the resulting modified Fc-containing protein resistant to proteolytic degradation.

Incorporation of the amino acid modification disclosed in EP2654780 B1 into the Fc region of IgG4 coupled to domain III of CCN5 was found to be incapable of providing sufficient resistance to endopeptidases. However, improvement in resistance to proteases was achieved by additional modifications of the IgG subtype used as a fusion partner according to the present invention.

More specifically, fusion proteins containing the complete hinge region of IgG2 and constant heavy chains 2 and 3 of IgG4 were found to exhibit excellent proteolytic stability.

In the source: 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, eculizumab, which is used in the treatment of paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome, was shown to be suitable for use, for example, to abolish the ability of IgG4 to activate the FcγR-dependent immune effector response. In addition, it was shown that constant domains 2 and 3 of IgG4 of such a chimeric Fc fragment also abolish the ability of IgG2 to activate complement-dependent immune effector functions. Information on this topic is reported by Rother et al., (2007), see Nat. Biotechnol., 25(11), pp. 1256-1264 and Mueller JP et al., above.

Also in the source: Borrok et al. (2017), J. Pharm. Sci. 106; 1008-1017, the introduction of modifications into the Fc fragment to study its effect on the immune effector functions of antibodies (FQQ-YTE mutations) is described. WO 2017158426A1 discloses modifications of antibodies by introducing mutations into the Fc fragment to extend the half-life of antibodies. In particular, modifications are disclosed at one or more positions 311, 434, 428, 438 and 435 in the Fc region of an immunoglobulin.

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

According to one embodiment, the Fc region of the fusion protein according to the present invention consists of an Fc region of the IgG4 subclass comprising the following mutations: 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 Asn297 mutation resulted in an aglycosylated Fc fragment, which further resulted in the absence of IgG effector functions. Jacobsen also found that some variants (N297G) resulted in antibodies with better stability and development suitability compared to other variants (N297Q or N297A). They also introduced additional modifications (disulfide bridges), which resulted in better stability than the original IgG1.

According to the present invention, when the fusion partner is an Fc fragment, it may be aglycosylated, without a stabilizing disulfide bridge or with a stabilizing disulfide bridge, such as in SEQ ID NO: 16.

To the best of the knowledge of the present inventors, an Fc fragment consisting of the complete hinge region of IgG2 and constant heavy chains 2 and 3 of IgG4 has not previously been used to produce fusion proteins by linking said Fc fragment to an effector protein.

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

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

According to one preferred embodiment, said Fc fragment is a chimera of the hinge region of IgG2 (216 ERKCCVECPPCPAPPVA-GP 238, Eu numbering) and any other IgG subclass. Most preferably, the Fc fragment is a chimera of the hinge region of IgG2 and the constant domains of the heavy chain 2 and 3 of IgG4 with a deletion of the carboxy-terminal K477 (Eu numbering), as shown in SEQ ID. NO: 19. Said embodiment of the present invention has been shown to have improved protease resistance characteristics (see Example 6).

According to one embodiment, the fusion partner of the monomeric fusion protein of the present invention is an IgG1 Fc fragment stabilized by a disulfide bridge (R292C, V302C), aglycosylated (N297G) and containing mutations that allow the formation of monomers (C220Q, C226Q, C229Q, T366R, L368H, P395K, K409T, M428L, Eu numbering), as shown in SEQ ID NO: 54.

According to a further embodiment, the fusion partner of the monomeric fusion protein of the present invention is an Fc fragment which is a chimera of the hinge region of IgG2 and the constant domains of the heavy chain 2 and 3 of IgG4 with a deletion of the carboxy-terminal K477- and with mutations allowing the formation of monomers (C219Q, C220Q, C226Q, C229Q, L351F, T366R, P395K, F405R, Y407E) and half-life extending mutations (M252Y, S254T, T256E) (Eu numbering), as shown in SEQ ID NO: 55.

Although the example of a fusion protein according to the present invention uses 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 is also achieved when such a chimeric Fc fragment is coupled to other effector molecules, such as, for example, VEGFR, FGF-21, or GLP1. The effector molecule is the part of the Fc fusion protein that provides the desired pharmacodynamic properties, while the Fc fragment contributes to the pharmacokinetic properties.

Serum albumin as a fusion partner

An alternative strategy for extending the half-life of peptides and proteins is to use serum albumin (SA) as a fusion partner. IgG and SA have long half-lives of approximately 19 days, compared with a few days or less for most other circulating proteins. SA also has affinity for the neonatal Fc receptor (FcRn) and avoids intracellular degradation (see Andersen et al. (2014) J Biol Chem 289(19); pp. 13492–13502).

According to one embodiment of the present invention, there is provided a fusion protein as described above, characterized in that the fusion partner is serum albumin, preferably human serum albumin.

According to one embodiment, a monomeric fusion protein as described above is provided, characterized in that said fusion protein comprises amino acids 25,606 of human serum albumin, as presented in SEQ ID NO: 101.

According to a further embodiment of the present invention, said albumin, for example human serum albumin, is modified, for example, to increase or decrease half-life by changing its affinity for FcRn, with or without pH dependence, which results in an increase or decrease in half-life.

Transferrin as a fusion partner

Another 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. FJioDrugs. 2015; 29(4): 215 239). Transferrin can be used in glycosylated or non-glycosylated form.

According to one embodiment of the present invention, there is provided a fusion protein as described above, characterized in that the fusion partner is transferrin, preferably human transferrin.

According to one embodiment, a monomeric fusion protein as described above is provided, characterized in that said fusion protein comprises amino acids 20-698 of human transferrin, as presented in SEQ ID NO: 53.

Linker

According to another embodiment, the fusion proteins 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 at the N- or C-terminus to the TSP-1 repeat homology domain of the CCN protein (TSP-1 domain protein/polypeptide).

Any peptide linker (provided that it is not a CCN protein sequence) may be used, many of which are known and described in the art. The linker may be a flexible linker sequence (which may include repeats of a flexible linker sequence motif). Typical linkers known in the art are rich in small non-polar residues (e.g., glycine) or polar residues (e.g., serine or threonine), and typically consist of stretches of glycine and serine (GS) residues, or other amino acid residues such as alanine, lysine, and/or glutamate (A, K, and/or E), or, in fact, any amino acid. A frequently used linker is the (GGGGS) linker (SEQ ID NO: 121), which can be provided as repeating units of the linker (such as (GGGGS) n, wherein the copy number n can be adjusted, such as from 1-10, 1-6, 1-4, etc.). The length of the linker can be 1-50, 1-45, 1-40, 1-30, 1-25, 1-20, 1-15, 1-12, 1-10, such as 1-8, 1-6, 1-5, or 1-4 amino acids. Various different linkers are described and used in the examples provided below, and any of them can be used in any of the fusion proteins of the present invention.

According to some embodiments, said linker comprises no more than 50 amino acids.

The properties of the peptide linker may further improve the retention of effector functions. However, peptide linkers may be susceptible to endopeptidase cleavage and elimination of the fusion protein. Peptide linkers with glycine, with or without serine residues are often used, but this design does not always result in fusion proteins with the desired activities and endopeptidase resistance. US20180273603, which discloses a neurotrophin binding protein-Fc fusion protein, proposes the use of a-helical linkers comprising repeats of the sequence A(EAAAK)A (SEQ ID NO: 14 in the reference). In addition, US2018/0127478 discloses the use of an amino acid linker consisting of one to three repeats of the sequence EAAAK in a fusion protein with Fc.

According to the present invention, a linker consisting of the peptide sequence EAAAK (SEQ ID NO: 21 in the present application) can also be inserted between the TSP-1 homology domain and the fusion partner (Fc fragment). More preferably, the linker consists of a repeating amino acid sequence EAAAK.

If a linker is included in the fusion protein according to the present invention, said 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.

In addition, the helical linker was resistant to endopeptidase cleavage following expression of the recombinant protein in CHO cell suspension. This is important for both manufacturing purposes and in vivo efficacy. Furthermore, inclusion of an α-helical linker between the Fc fragment and the effector domain in the Fc-fusion protein was shown to reduce the aggregation propensity of the Fc-fusion protein.

Although the above results were found for a fusion protein containing domain III of the CCN protein as an effector protein, it is believed that the beneficial reduced aggregation propensity and protease resistance effects are also achieved by combining other effector molecules with the Fc fragment of the α-helical linker according to the present invention.

According to the present invention, there is accordingly provided an Fc fusion protein comprising an Fc fragment which comprises a peptide linker sequence of the formula AK1-AK2-(EAAAK)n-AK3-AK4-AK5, where n≥4, between the Fc fragment and the effector molecule, and wherein the amino acids AK1, AK2, AK3, AK4, AK5 are independently absent or represent an amino acid. The linker can be located at the N-terminus or the C-terminus of the Fc fragment. According to one embodiment, n is 8. According to another embodiment, AK1 is threonine (T), AK1, AK2, AK3, AK4 and AK5 are Ala (A). According to one embodiment, the linker for the above-mentioned Fc fusion protein is selected from the group consisting of 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 according to the present invention comprising a linker consisting of an (EAAAK) repeat, i.e. such as (EAAAK)n, where n is 8, advantageously leads to less aggregation.

An alternative linker that can be used in accordance with the present invention is a linker with the amino acid sequence shown in SEQ ID NO: 20 (TEGRMD).

According to one embodiment, the present invention may accordingly provide for the inclusion of a linker peptide between the fusion partner and domain III of CCN5 (e.g., SEQ ID NO: 1 to SEQ ID NO: 12, 37, or 38). Non-limiting examples of fusion proteins comprising the linker of SEQ ID NO: 20 are provided in SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively.

In the case where the invention uses a variant of the implementation of domain III of CCN5, genetically fused at the N-terminus with a peptide linker (as in SEQ ID NO 20) and an Fc fragment of IgG of the IgG4 subtype, comprising the following mutations (S228P, F234A, L235A, K447-, Eu-numbering) (as in SEQ ID NO 15), the complete sequence corresponds to SEQ ID NO: 28, also designated CCN5(dIII)-Fcv2.

In the case where the invention uses a variant of the implementation of domain III of CCN5, genetically fused at the N-terminus with a peptide linker (as in SEQ ID NO: 20) and an Fc fragment of IgG of the IgG4 subtype, comprising the following mutations (S228P, E233P, F234V, L235A, G236-, K447-, Eu-numbering), presented in SEQ ID NO: 18, the resulting sequence corresponds to SEQ ID NO: 29, also designated CCN5(dIII)-Fcv2.1.

In the case where the invention uses a variant of the implementation of domain III of CCN5, genetically fused at the N-terminus with a peptide linker (as in SEQ ID NO: 20) and a chimeric Fc fragment of the IgG subtype IgG2/4, presented in SEQ ID NO: 19, the resulting sequence corresponds to SEQ ID NO: 30, also designated CCN5(dIII)-Fcv2.3.

In the case where the invention uses a variant of the embodiment of domain III of CCN5 (as in SEQ ID NO: 1), genetically fused at the N-terminus with a peptide linker (as in SEQ ID NO: 25) and a chimeric Fc fragment of the IgG subtype IgG2/4, presented in SEQ ID NO: 19, the resulting sequence corresponds to SEQ ID NO: 31, also designated CCN5(dIII)-HLn8-Fcv2.3.

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

1) a point mutation in domain III of a CCN family protein, in particular CCN5 (see SEQ ID NO: 7), which results in reduced proteolytic sensitivity of said domain III;

2) an engineered chimera of the Fc region of human IgG4 and human IgG2 (SEQ ID NO: 19, which has reduced proteolytic sensitivity relative to previously described Fc region-based scaffolds used in Fc fusion proteins; and

3) comprising an optimized peptide linker composition (see SEQ ID NO: 21-25) that reduces proteolytic sensitivity, enhances the biological activity of the fusion protein, and reduces the aggregation tendency of the fusion protein.

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 the group consisting of SEQ ID NO: 20-25 or 39, or an amino acid sequence that is 80% identical thereto.

Alternative linker sequences that may be used in accordance with the present invention are shown in SEQ ID NOs: 57, 63, 65, 67 and 121.

Recombinant expression

The recombinant proteins and fusion proteins according to the present invention can be obtained by culturing a host cell allowing expression of nucleotide sequences encoding said proteins. The person skilled in the art is well aware of the various available biotechnological techniques providing expression of isolated nucleic acid sequences for the production of recombinant proteins by heterologous expression in various host cell systems using publicly available genetic engineering techniques and recombinant DNA expression systems, see for example the sources: "Recombinant Gene Expression Protocols, Methods in Molecular Biology, 1997, Ed. Rocky S Tuan, Human Press (ISSN 1064-3745) or Sambrook et al., Molecular Cloning: A laboratory Manual (third edition), 2001, CSHL Press, (ISBN 978-087969577-4). For example, nucleic acid sequences encoding recombinant proteins according to the present invention can be inserted into suitable expression vectors containing all necessary transcriptional and translational regulatory sequences, specially adapted to direct the expression of the coding nucleic acid sequence of the desired protein in a suitable host cell. Suitable expression vectors are, for example, plasmids, cosmids, viruses or yeast artificial chromosomes (YAC).

DNA sequences encoding recombinant proteins according to the present invention can be synthesized using methods well known to the skilled person or synthesized by commercial suppliers well known to the skilled person, for example, Genscript, Thermo Fisher Scientific, etc.

According to one embodiment of this aspect, there is provided a DNA molecule comprising a nucleic acid sequence as presented in SEQ ID NO: 86, 87, 90, 91, 99, 100, 104, 105, 108, 109, 112 or 113, or a sequence at least 80% identical to any of the above sequences. Expression vectors comprising such DNA molecules are also provided. According to another embodiment of this aspect, host cells comprising such vectors are also provided.

DNA sequences for expression and use in the production of recombinant proteins can be inserted into vectors, commonly known as entry vectors, using the Gateway cloning system (Esposito et al, 2009, "Gateway Cloning for Protein Expression", Methods in Molecular Biology, 498, pp. 31-54). Genes cloned into an entry vector can be easily introduced into various expression vectors by recombination. For example, a synthesized sequence encoding a recombinant protein or fusion protein according to the present invention can be recombined by BP Gateway recombinase cloning to obtain an entry vector that can be used to propagate plasmids in a suitable host cell, such as E. coli cells. According to a preferred embodiment, E. coli cells mutated in such a way that they allow efficient propagation of plasmids are used, such as, for example, One Shot Top 10™ cells.

According to one embodiment of the present invention, an expression vector is provided comprising a DNA sequence encoding a recombinant protein or fusion protein of the present invention operably linked to a promoter. It will be understood by one skilled in the art that a "promoter" as used herein refers to a region of DNA located upstream (in the 5' direction) of a coding DNA sequence that controls and initiates transcription of a particular gene. A promoter controls the recognition and binding of RNA polymerase and other proteins to initiate transcription. "Operably linked" refers to a functional relationship between a promoter and a second sequence wherein the promoter sequence initiates and mediates transcription of a DNA sequence corresponding to the second sequence. In general, "operably linked" means that the linked nucleic acid sequences are contiguous.

The input vector, as well as the expression vector, such as that derived from the receiving vector mentioned below, can be isolated using standard plasmid isolation techniques well known to those skilled in the art, for example using the QIAprep™ Spin Miniprep Kit from Qiagen™ or the QIAGEN™ Plasmid Plus Maxi Kit.

If an input vector containing a DNA sequence encoding a recombinant protein or fusion protein according to the present invention is used, said input vector can be further recombined with a host vector using LR Gateway recombinase to obtain an expression vector. The expression vector can then be used to express the protein-encoding DNA sequence in a suitable host cell. A non-limiting example of a suitable host vector is, for example, pUCOE-DHFR-DEST as described in the source: et al., J. Biol. Chem, 293:46, pp.17953-17970.

The final expression vector can also be verified using standard restriction enzyme digestion and gel electrophoresis analysis.

DNA.

According to one aspect of the present invention, there is provided an expression vector comprising a nucleic acid sequence encoding a recombinant protein of formula (I). According to another aspect of the present invention, there is provided an expression vector comprising a nucleic acid sequence encoding a 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: 8-12, 37, 38, 84, 85, 88, 89, 97, 98, 102, 103, 106, 107, 110 and 111; and fragments or variants thereof with at least 50% sequence identity to the amino acid sequences 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 and SEQ ID NO: 38; and SEQ ID NO: 84, 85, 88, 89, 97, 98, 102, 103, 106, 107, 110 and 111.

According to another aspect, there is provided an expression vector encoding a recombinant protein comprising an amino acid sequence with at least 60%, 70%, 80%, 90% or 95% sequence identity to 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 NO: 84, 85, 88, 89, 97, 98, 102, 103, 106, 107, 111 and 111.

According to an embodiment of the present invention, expression vectors encoding a fusion protein according to the present invention are provided.

The skilled person is well aware of the degeneracy of the genetic code and the preferred use of specific codons in different organisms. Accordingly, depending on the choice of host cell, the nucleic acid sequence encoding the recombinant protein and fusion proteins according to the present invention can be adapted to contain codons preferred by the host cell. Accordingly, the amino acids of the proteins according to the present invention can be encoded by any combination of codons from those presented in the table below:

Preferably, said codons are further optimized for high expression levels in accordance with the selected host cell.

For the expression of proteins by recombinant DNA technology, in addition to a particular embodiment of the present invention, a DNA sequence encoding a signal peptide is preferably added to the N-terminus of the protein sequence. The signal peptide may serve to direct the localization of the fusion protein during and/or after synthesis in the host cell. It may accordingly be a sequence directing the secretion of the fusion protein. The use of such signal peptide sequences is well known in the art. The signal peptide may be in any form, for example, it may be a signal peptide of an IgGk chain, or it may be a signal peptide from human serum albumin (SEQ ID. NO 32).

In the case where the signal peptide from human serum albumin (SEQ ID NO: 32) is added to the N-terminus of SEQ ID NO: 28, the protein sequence for expression may correspond to SEQ ID NO: 33 or SEQ ID NO: 85, 89, 98, 103, 107 or 111.

In addition, for the expression of a protein using recombinant DNA technology, according to one specific embodiment of the present invention, a protein having the amino acid sequence presented in SEQ ID NO: 33 can be expressed using a nucleotide sequence presented in SEQ ID NO: 34 or SEQ ID NO: 86, 90, 99, 104, 108 or 112, wherein the 3' end of the coding sequence is supplemented with a translational stop codon.

In the case where the embodiment of the invention is the nucleotide sequence of SEQ ID NO: 34, said nucleotide sequence is preferably complemented immediately at the 5' end of the coding sequence by a KOZAK sequence, for example GCCACC, 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 allowing subcloning, such as, for example, the attB Gateway recombinase sites. However, any cloning or synthetic strategy may be used to obtain the DNA sequence and facilitate subcloning into an expression vector. In the case where the DNA sequence includes Gateway recombinase sites allowing subcloning, the nucleotide sequence may correspond to that presented in SEQ ID NO 36 or SEQ ID NO: 87, 91, 100, 105, 109 or 113.

The resulting expression vector comprising a nucleic acid sequence encoding a recombinant protein or fusion protein according to the present invention can be introduced into suitable host cells to produce the desired protein. Various commercially available or home-made host cells can be used. For example, the expression vector can be transferred to eukaryotic host cells such as CHO cells, for example, suspension culture-adapted CHO DG44 DHFR (dihydrofolate reductase ^-/- ) cells. Transfection of host cells with the expression vector can be performed using methods well known to those skilled in the art, for example, using electroporation.

Upon culturing the host cells in a suitable culture medium, the recombinant proteins or fusion proteins of the present invention encoded by the expression vector will be produced in the host cell, and the resulting protein can be collected and purified using methods well known to those skilled in the art.

The expression vector may include signal sequences, commonly known as a "signal peptide," for secretion of the expressed protein or fusion protein into the culture medium.

To isolate and purify the secreted recombinant protein from the cell culture medium, one or more pretreatment or purification steps are typically performed, primarily to remove large particles and biomass. Non-limiting examples of suitable pretreatment steps include, for example, reverse osmosis, centrifugation, filtration and diafiltration methods, or a combination thereof. The resulting protein is then typically purified using one or more of a variety of chromatographic techniques well known to those skilled in the art, such as affinity chromatography, ion exchange chromatography, mixed mode chromatography, hydrophobic interaction chromatography, size exclusion chromatography, or other chromatographic techniques, or a combination thereof.

For example, a recombinant protein or fusion protein expressed by a suitable host cell can be purified using an affinity chromatography method, such as using MabSelect™ SuRe™ media, such as on a 5 ml HiTrap MabSelect™ SuRe™ column mounted on a high performance liquid chromatography system, such as a BioRad NGC Discover™ 10 Pro system equipped with a 5 mm pathlength UV detection flow cell. After loading a sample containing the protein to be purified, the column is typically washed one or more times with one or more suitable wash buffers, after which the protein is eluted using a suitable elution buffer. The resulting protein can be further purified using one or more of the chromatographic methods listed above.

It will be appreciated that various modifications can be introduced into the nucleic acid sequences encoding the recombinant proteins of the present invention using techniques well known to those skilled in the art, for example to facilitate expression. By using site-directed mutagenesis, a modification can be introduced to adapt the coding sequence to the desired host used to express the sequence and, accordingly, produce the recombinant protein. The existence of host-specific codons is well known to those skilled in the art and that adaptation of a heterologous nucleic acid sequence to include host-specific codons increases the expression efficiency, as mentioned above. Other modifications can also be introduced, for example to facilitate isolation and purification, i.e. by adding a sequence encoding a peptide or protein suitable for such purposes. Also, nucleic acid sequences encoding a signal peptide that ensures the secretion of the desired recombinant protein from the host cell can be additionally linked to the nucleic acid sequences of the present invention.

The present invention also provides a host cell suitable for producing a recombinant protein or a fusion protein according to the present invention. Various commercially available host cells specifically adapted for producing recombinant proteins can be used, both prokaryotic host cells and eukaryotic host cells. Non-limiting examples of suitable host cells are, for example, CHO cells, HEK293 cells, Pichia pastoris cells, NS0 cells or E. coli cells.

Finally, the present invention also relates to a thrombospondin type 1 (TSP-1) repeat homology domain of a CCN family protein, and to a fusion protein comprising said TSP-1 repeat homology domain for use as a medicament for treating or preventing disorders by inhibiting or counteracting cell signaling and physiological functions of a cell attributed to CCN family proteins.

According to one aspect of the present invention, there is provided a protein, for example a fusion protein as defined herein, for use in therapy.

In some aspects, the protein, such as a fusion protein, may be for use in treating or preventing fibrosis, or any condition in which fibrosis occurs (i.e., any fibrotic condition or disorder). Fibrosis may affect any tissue or organ, including, for example, the lungs, eye, heart, skeletal muscle, peritoneum, kidney, liver, pancreas, bile ducts, skin, blood vessels, or more global systems. In particular, the condition in which fibrosis is manifested may be selected from pulmonary fibrosis, which may be of any etiology, 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, muscular 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, scars, systemic sclerosis, atherosclerosis, epidural fibrosis.

In the context of cardiac fibrosis, conditions to be treated or prevented may include cardiac hypertrophy and heart failure with or without preserved ejection fraction.

According to a further aspect of the present invention, there is provided a protein, for example a fusion protein as defined herein, for use in the treatment of an inflammatory or autoimmune disease. According to some embodiments, said inflammatory disease is selected from rheumatoid arthritis, amyotrophic lateral sclerosis (ALS), inflammatory bowel disease, ulcerative colitis, Crohn's disease.

According to a further aspect of the present invention, there is provided a protein, for example a fusion protein as defined herein, for use in the treatment of cancer. In this regard, it is known that CCN 4-domain proteins can both induce oncogenic responses in isolated cancer cells and promote metastasis, chemoresistance and resistance to immunotherapy by acting directly on cancer cells or on the tumor stroma. The activity of the proteins according to the present invention related to the inhibition of the effect or activity of a CCN 4-domain protein, respectively, is the rationale for their use in the treatment of cancer. Cancer can be any malignant or pre-malignant neoplastic condition. It can be a cancer of any tissue or any organ. In one embodiment, cancer can manifest itself as solid tumors. According to another embodiment, cancer can be a cancer of the hematopoietic system or be in the hematopoietic system. It can be a primary cancer or a secondary cancer, or metastases. The cancer may, respectively, be a pancreatic, breast, prostate, cervical, ovarian, liver, bladder, brain, blood, bone, skin, lung, or stomach cancer. According to some embodiments, the cancer is selected from pancreatic cancer, pancreatic duct 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.

According to a further aspect of the present invention, there is provided a protein, for example a fusion protein as defined herein, for use in the treatment of a metabolic disease. Said metabolic disease may be or may be associated with insulin resistance or impaired glucose tolerance. According to some embodiments, said metabolic disease is selected from type 2 diabetes and metabolic syndrome.

The fusion protein of the present invention may also be used in the above-described methods for treating conditions. Similarly, the fusion protein of the present invention may be used in methods for preparing a medicament for use in treating the conditions described above.

EXAMPLES

Example 1

Expression of a fusion protein according to the present invention

This example describes the production of a fusion protein comprising amino acids 194-246 of CCN5 (SEQ ID NO: 1) fused at the N-terminus to a peptide linker (SEQ ID NO: 20) and the Fc fragment of IgG subclass IgG4 of SEQ ID NO: 15 (S228P, F234A, L235A, K447-, Eu-numbering) (CCN5(dIII)-Fcv2) (i.e., the fusion protein according to SEQ ID NO: 28). Said fusion protein was also supplemented with an N-terminal signal sequence derived from albumin of SEQ ID NO: 32 and expressed in mammalian cells as described below.

The DNA sequence shown as SEQ ID NO: 36 was synthesized and verified by a commercial supplier. The synthesized sequence was recombined with pDonrZeo by BP Gateway recombinase cloning to generate the entry vector. Following transfection of competent E. coli mutated to allow efficient plasmid propagation (One Shot Top10™ cells), the entry vector was isolated using standard plasmid isolation techniques using the QIAprep™ Spin Miniprep Kit from Qiagen™. Following plasmid isolation, the entry vector was verified by restriction enzyme digestion followed by DNA gel electrophoresis according to standard techniques well known to those skilled in the art.

The input vector containing the sequence SEQ ID NO: 35 was then recombined with the receiving vector using LR Gateway recombinase. The receiving vector used was pUCOE-DHFR-DEST as described by Kaasb0ll et al., 2018, see above.

Following transfection of competent E. coli mutated to allow efficient propagation of the plasmids (One Shot Top 10™ cells), the expression vector was isolated using standard plasmid isolation techniques using the QIAGEN™ 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. The resulting expression vector was then transformed into suspension-adapted CHO ExpiCHO cells according to the Max Titer protocol from the manufacturer of the CHO Expifectamine™ Transfection Kit (Gibco Cat. No.: A29129) and a brief description of the et al., 2018, supra. Cells were pelleted 6 days post-transfection by centrifugation at 4750g for 20 min at 4°C, and the cell culture medium supernatant was collected. 0.1 M PMSF in 100% isopropanol was added to a concentration of 1 mM and 0.5 M EDTA to a concentration of 2 mM. Then 96% ethanol was added to a final concentration of approximately 3%. Before chromatographic purification, 1 M TrisHCl, pH 7.4, was added to a final concentration of 25 mM.

The capture step of the purification was performed by affinity chromatography on Protein A chromatography medium. The medium used in this experiment was rProtein A FF (GE Healthcare). A 5 ml HiTrap™ rProtein A FF column (GE Healthcare) was used to purify the expressed recombinant protein from 60 ml of cell culture medium collected and supplemented as described above. The HiTrap™ rProtein A FF column was installed on a fast high-performance liquid chromatography system (BioRad NGC Discover™ 10 Pro System) equipped with a 5 mm pathlength UV detection flow cell and equilibrated with a buffer containing 25 mM TrisHCl, pH 7.4, 25 mM NaCl, and 3% ethanol. The harvested cell culture medium containing the recombinant protein was loaded using a sample pump at 2.5 ml/min, followed by washing with 6 column volumes of wash buffer (25 mM TrisHCl pH 7.4, 25 mM NaCl and 3% ethanol) before elution with 0.1 M Na citrate, pH 3.0, in 3% ethanol. The eluate with a UV absorbance at 280 nm greater than 100 mOEDP was collected in 3 ml fractions into low protein binding tubes pre-filled with 1 ml of 1 M TrisHCl, pH 9.0. The fraction containing the UV absorbance peak was concentrated to 500 µl using a Vivaspin® 20 ml concentrator with a nominal molecular weight cut-off of 30 kDa. After concentration, the sample was loaded into the sample loading loop of a fast FPLC system (BioRad NGC Discover™ 10 Pro System). The FPLC system was equipped with a Superdex® 200 Increase 10/300 GL column (GE Healthcare) equilibrated with 50 mM NaCl, 20 mM HEPES, pH 7.0. The sample was injected and the column was perfused with pre-equilibration buffer (50 mM NaCl, 20 mM HEPES, pH 7.0) at a flow rate of 0.25 mL/min. The major UV absorbance peak at 280 nm was found to contain the purified recombinant protein (CCN5(dIII)-Fcv2, SEQ ID NO: 28). Samples of 10 µl of pooled fractions were subjected to SDS-PAGE using Mini-PROTEAN® TGX Stain-Free™ precast gels and isolated recombinant proteins were visualized using the ChemiDoc™ imaging system (BioRad).

It is well known to those skilled in the art that recombinant proteins can be produced in a variety of expression systems and purified using a variety of chromatographic methods with similar results.

Example 2

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

The resulting protein was tested for its ability to inhibit pro-survival signaling (phosphorylation of serine-473 in AKT) in human lung cancer A549 cells (Fig. 1A). Tissue culture-treated 96-well Corning Incorporated Costar® sterile polystyrene plates were coated with fibronectin (Sigma, Cat. #F1141, diluted to 10 μg/mL in BioWhittaker® Dulbecco's Phosphate Buffered Saline (Lonza, Cat. #17-512F, hereafter referred to as PBS)). The coating solution containing fibronectin was dispensed into wells at a volume of 100 μl/well, incubated for 1 hour at room temperature, then the coating solution was decanted, 100 μl of PBS was dispensed into fibronectin-coated wells, and also decanted. A549 cells, subcultured to maintain a density of 80% maximum confluency, were split by enzyme treatment (Accutase®, Cat# L0950-100 from Biowest®) diluted in Dulbecco's modified Eagle's medium high glucose (Gibco Cat# 41965-039), 10% heat-inactivated fetal bovine serum (FBS) (500 mL FBS flasks (Cat# 16000-044 from Gibco), equilibrated to room temperature, incubated in 60°C water with shaking for 30 minutes) and 50 μg/mL gensumycin (Sanofi)) to a concentration of 110,000 cells/mL, and 100 μL of the cell solution were dispensed into fibronectin-coated wells. All cell incubations were performed in cell culture incubators maintained at 37°C, in a humidified atmosphere with room air and 5% CO2. After overnight incubation, A549 cells were washed twice with PBS and 90 µl Dulbecco's modified Eagle's medium high glucose (DMEM, Gibco cat. # 41965-039), and 50 µg/ml gensumycin (Sanofi) without FBS were dispensed into the wells. After 18 h of incubation in the medium without FBS, the cells were stimulated with 10 µl of the recombinant protein in question. After stimulation for 60 min, the medium was decanted and the cells were harvested by adding 50 μl of lysis buffer with blocking reagent according to the Cisbio phospho-AKT kit (Ser473) (Cisbio Inc, Cat#: 64AKSPEG). After adding the lysis buffer with blocking reagent, the 96-well plate was incubated for 60 min on a PST-60HL Plus plate shaker (ThermoFisher) at 500 rpm. After mixing, the lysed samples were ground and 16 μl from each well was transferred into white 96-well low-volume HTRF plates (Cisbio Inc., Cat#: 66PL96025). To analyze the amount of phosphorylated AKT (Ser473), 4 μl of a mixture of labeled antibodies (50/50 v/v mixture of antibodies, AB to phospho-AKT d2 and cryptate-labeled AB to phospho-AKT from Cisbio bic, cat. no.: 64AKSPEG) were added to each well (only cryptate-labeled antibody was added to negative control wells), the plates were sealed with adhesive polymer film and incubated at 4°C overnight, after which they were read on a PolarStar Omega plate reader (BMG Labtech, Germany) equipped with a TR-FRET recorder and an emission filter at 337 nm and excitation filters at 615 nm and 665 nm. The ratio between the recorded excitation values at 665 nm and 615 nm was corrected for blanks, and the values for recombinant protein from stimulated wells were expressed as a percentage relative to vehicle-stimulated wells.

Example 3

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

The resulting protein was tested for its ability to inhibit pro-fibrotic TGF-β-stimulated transcription (from SMAD2/3 binding cis-elements) in IMR90 human lung fibroblasts (Fig. 1D). The assay was technically performed as described by Kaasbell et al. (2018) above, except for using 2500 IMR90 lung fibroblasts/well instead of Rat2 cells. The proteins used for stimulation were as described in Fig. 1D. IMR90 cells were subcultured prior to use as described for A549 cells, see above. IMR90 cells were used up to passage 20, i.e., until replicative senescence was reached.

Example 4

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

The resulting protein was tested for its ability to inhibit proliferation of the human lung fibroblast cell line IMR90 (Fig. 1B). IMR90 cells were subcultured as described for A549 cells before use, above. IMR90 cells were used until passage 20, i.e., until replicative senescence was reached. For experiments, IMR90 cells were harvested as described for A549 cells, above, washed with PBS, diluted with DMEM with 1% FBS and gensumycin as described for Experiment 2, above, and seeded into xCELLigence impedance plates at a density of 12,000 cells/well. After 2 h, cells were stimulated with 10 µl of the recombinant protein in question or FBS and incubated for an additional 72 h, after which data were collected using the CellTiter-Glo® (Promega Inc.) as described in et al., (2018), supra.

Example 5

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

The resulting protein was tested for its ability to inhibit the sphere-forming ability (substrate-independent growth) of estrogen receptor-positive breast cancer cell line MCF-7 and triple-negative breast cancer cell line MDA-MB-231 (Fig. 1C) as described et al., supra. MDA-MB-231 cells were treated as described for the MCF-7 cell line and co-authors, above. MCF-7 and MDA-MB-231 cell lines were subcultured as described for the A549 cell line, above.

Example 6

DNA sequences encoding a fusion protein comprising amino acids 194,246 of CCN5 (SEQ ID NO: 1) fused at the N-terminus to a peptide linker (SEQ ID NO: 20) and either the IgG subclass IgG4 Fc fragment of SEQ ID NO: 15 (S228P, F234A, L235A, K447-, Eu numbering) (CCN5(dIII)-Fcv2) or the IgG subclass IgG4 Fc fragment of SEQ ID NO: 18 (S228P, E233P, F234V, L235A, G236-, K447-, Eu numbering) (CCN5(dIII)-Fcv2.1) or a chimeric IgG2/4 subclass Fc fragment (SEQ ID NO: 19) (CCN5(dIII)-Fcv2.3) was expressed to produce recombinant protein according to SEQ ID NO: 28 (CCN5(dIII)-Fcv2), SEQ ID NO: 29 (CCN5(dIII)-Fcv2.1) and SEQ ID NO: 30 (CCN5(dIII)-Fcv2.3).

In particular, the expression vectors for the 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 suspension culture-adapted CHO ExpiCHO cells according to the Max Titer protocol from the manufacturer of the CHO Expifectamine™ transfection kit (Gibco, Cat. No.: A29129) and a brief description of the et al., supra. Cells were pelleted 6 days post-transfection by centrifugation at 13,000 rpm in a Heraeus Biofuge Pico benchtop centrifuge for 5 min, and cell culture medium supernatant was collected. Samples of the collected cell culture medium supernatants were separated by SDS-PAGE using Mini-PROTEAN® TGX Stain-Free™ precast gels, and recombinant proteins were visualized using the ChemiDoc™ Imaging System (BioRad). Separated proteins were then transferred to PVDF membranes using the Trans-Blot Turbo Semi-Dry Blotting System (Bio-Rad) for Western blot analysis. The blot was probed with horseradish peroxidase-conjugated anti-human IgG4 antibody (Invitrogen, Cat. #: A10654), which was used in combination with SuperSignal™ West Femto Maximum Sensitivity Substrate (ThermoFisherScientific) and the ChemiDoc™ Imaging System (BioRad) for visualization.

Fig. 2 shows data demonstrating improved protease resistance of the Fc-based backbone consisting of the IgG2/4 chimera (shown in SEQ ID NO: 19).

CCN5/WISP2 (domain III) fused to the IgG4 Fc fragment either via the IgG4 hinge with silencing of immune effector functions (as defined in SEQ ID NO: 28); CCN5 (domain III)-Fcv2, where the same IgG4 backbone includes IgG2-based mutations (as defined in SEQ ID NO: 29); CCN5 (domain III)-Fcv2.1, or the same IgG4 backbone with the complete IgG2 hinge region (as defined in SEQ ID NO: 30); CCN5 (domain III)-Fcv2.3 were expressed in the ExpiCHO system and the conditioned medium (CM) was collected after 6 days. Western blotting and total protein staining of SDS-PAGE gels showed that the CCN5 (domain III)-Fcv2.3 variant is the least sensitive to proteases present during cultivation. Note that the immunoreactivity of the anti-IgG4 antibody to the Fc fragment is partially lost when replaced by IgG2 sequences, and, accordingly, the assessment of the level of this protein based on total protein staining is underestimated.

Example 7

DNA sequences encoding a fusion protein comprising amino acids 194,246 of CCN5 (SEQ ID NO: 1) fused at the N-terminus to the peptide linker described in SEQ ID NO: 20 and the chimeric Fc fragment of the IgG2/4 subclasses (SEQ ID NO: 19) (CCN5(dIII)-Fcv2.3), or to the peptide linker described in SEQ ID NO: 25 and the chimeric Fc fragment of the IgG2/4 subclasses (SEQ ID NO: 19) (CCN5(dIII)-HLn8-Fcv2.3), were expressed to produce a recombinant protein according to SEQ ID NO: 30 (CCN5(dIII)-Fcv2.3) and SEQ ID NO: 31 (CCN5(dIII)-HLn8-Fcv2.3).

In particular, the expression vectors for expression of SEQ ID NO: 30 (CCN5(dIII)-Fcv2.3) and SEQ ID NO: 31 (CCN5(dIII)-HLn8-Fcv2.3) were transfected into suspension culture-adapted CHO ExpiCHO cells according to the Max Titer protocol from the manufacturer of the CHO Expifectamine™ transfection kit (Gibco, Cat. No.: A29129) and a brief description of the et al., supra. Cells were pelleted 4 days post-transfection by centrifugation at 13,000 rpm in a Heraeus Biofuge Pico tabletop centrifuge for 5 min, and the cell culture medium supernatant was collected. Samples of the collected cell culture medium supernatant were separated by SDS-PAGE using Mini-PROTEAN® TGX Stain-Free™ precast gels. Separated proteins were transferred to PVDF membranes using the Trans-Blot Turbo Semi-Dry Blotting System (Bio-Rad) for Western blot analysis. The blot was probed with horseradish peroxidase-conjugated anti-human IgG4 antibody (Invitrogen, Cat.#: A10654), which was used for visualization in combination with SuperSignal™ West Femto Maximum Sensitivity Substrate (ThermoFisherScientific) and the ChemiDoc™ Imaging System (BioRad).

Fig. 3 shows data reflecting a reduced tendency to aggregation in cases where an embodiment of the present invention includes a peptide linker as shown in SEQ ID NO: 25.

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 different peptide linkers. Western blotting shows that the fusion protein with the amino acid sequence shown in SEQ ID NO: 31; (dIII)-HLn8-Fcv2.3 has a lower propensity to aggregate than the fusion protein of the present invention having the amino acid sequence shown in SEQ ID NO: 30; CCN5 (domain III)-Fcv2.3. This result demonstrates that the peptide linker as defined in SEQ ID NO: 25 provides a lower propensity for aggregation of the fusion protein compared to a fusion protein containing the peptide linker as defined in SEQ ID NO: 20.

Example 8

DNA sequences encoding a fusion protein comprising either amino acids 194-246 of CCN5 (SEQ ID. NO: 1) or amino acids 194-246 of CCN5 (SEQ ID. NO: 7) where the amino acid at position 195 (proline) is replaced by alanine, fused at the C-terminus to a peptide linker (SEQ ID NO: 39) and the Fc fragment of IgG subtype IgG4 of SEQ ID NO: 15 (S228P, F234A, L235A, K447-, Eu-numbering) were expressed to produce the recombinant protein according to SEQ ID NO: 40 (Fc-HLn8-CCN5(dIII)) or SEQ ID NO: 41 (Fc-HLn8-CCN5(dIII)-P195A).

In particular, the expression vectors for the expression of SEQ ID NO: 40 (Fc-HLn8-CCN5(dIII)) and SEQ ID NO: 41 (Fc-HLn8-CCN5(dIII)-P195A) were transfected into suspension culture-adapted CHO ExpiCHO cells in accordance with the Max Titer protocol from the manufacturer of the CHO Expifectamine™ transfection kit (Gibco, Cat. No.: A29129) and a brief description et al., supra. Cells were pelleted 3 days post-transfection by centrifugation at 13,000 rpm in a Heraeus Biofuge Pico tabletop centrifuge for 5 min, and the cell culture medium supernatant was collected. Samples of the collected cell culture medium supernatant were separated by SDS-PAGE using Mini-PROTEAN® TGX Stain-Free™ precast gels. Separated proteins were transferred to PVDF membranes using the Trans-Blot Turbo semi-dry blotting system (Bio-Rad) for Western blotting analysis. The blot was probed with horseradish peroxidase-conjugated anti-human IgG4 antibody (Invitrogen, Cat. #: A10654), which was used in combination with SuperSignal™ West Femto Maximum Sensitivity Substrate (ThermoFisherScientific) and the ChemiDoc™ Imaging System (BioRad) for visualization.

Fig. 5 shows data reflecting reduced sensitivity to endopeptidase cleavage when an embodiment of the present invention includes the proline-195 mutation in the TSP-1 repeat homology domain of CCN5 shown in SEQ ID NO: 7.

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

Example 9

A fusion protein comprising amino acids 194-250 of human CCN5 (SEQ ID NO: 56) fused at the N-terminus to a peptide linker (SEQ ID NO: 57) and the Fc portion of human IgG, subclass IgG4, of SEQ ID NO: 15 (S228P, F234A, L235A, K447-, Eu-numbering) to produce a protein sequence according to SEQ ID NO: 58 (CCN5(dIII)-SL-Fcv0) is disclosed. An N-terminal secretion signal sequence derived from albumin of SEQ ID NO: 32 was further added to said fusion protein to produce a fusion protein according to SEQ ID NO: 59, and it was expressed in mammalian cells as described below.

The DNA sequence encoding the fusion protein of SEQ ID NO: 59 was codon-optimized for protein expression in hamster cells (using a commercial supplier's algorithm), a KOZAK sequence was added for translation at the 5' end, and a stop codon was introduced at the 3' end, yielding the DNA sequence of SEQ ID NO: 60. This DNA sequence was also complemented at both ends with Gateway attB sites, yielding the 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 into pDonrZeo by BP Gateway recombinase cloning to generate the entry vector. Following transfection of competent E. coli mutated to allow efficient propagation of the plasmids (One Shot Top10™ cells), the input vector was isolated using standard plasmid isolation techniques using the QIAprep™ Spin Miniprep Kit from Qiagen™. Following plasmid isolation, the input vector was verified by restriction enzyme digestion followed by DNA gel electrophoresis according to standard techniques well known to those skilled in the art.

The input vector containing the sequence SEQ ID NO: 60 was further recombined with the receiving vector using LR Gateway recombinase. The receiving vector pUCOE-DHFR-DEST was used as described in the source: et al., 2018, J. Biol. Chem, 293:46, pp. 17953-17970.

Following transfection of competent E. coli mutated to allow efficient propagation of the plasmids (One Shot Top10™ cells), the expression vector was isolated using standard plasmid isolation techniques using the QIAGEN™ 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. The resulting expression vector was then transfected into suspension-adapted ExpiCHO cells according to the Max Titer protocol from the manufacturer of the Expifectamine™ CHO Transfection Kit (Gibco, Cat. No.: A29129) and a brief description of the et al., 2018, supra. Cells were pelleted 4 days post-transfection by centrifugation at 4750xg for 20 min at 4°C and the cell culture medium supernatant was collected. ODM PMSF in 100% isopropanol was added to a concentration of 1 mM and 0.5 M EDTA was added to a concentration of 2 mM. Then 96% ethanol was added to a final concentration of approximately 3%. 1 M TrisHCl pH 7.4 was added to a final concentration of 25 mM before chromatographic purification.

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™ rProtein A FF column (GE Healthcare) was used to purify the expressed recombinant protein from 120 ml of cell culture medium collected and supplemented as described above. The HiTrap™ rProtein A FF column was mounted on a fast high-performance liquid chromatography system (BioRad NGC Discover™ 10 Pro System) equipped with a 5 mm pathlength UV detection flow cell and equilibrated with a buffer containing 25 mM TrisHCl pH 7.4, 25 mM NaCl and 3% ethanol. The collected cell culture medium containing the recombinant protein was loaded using a sample pump at 2.5 ml/min, followed by washing with 10 column volumes of wash buffer (25 mM TrisHCl pH 7.4, 25 mM NaCl and 3% ethanol) before elution with ODM Na citrate, pH 3.0, in 3% ethanol. Eluted fractions of 3 ml were collected in low protein binding tubes pre-filled with 1 ml of 1 M TrisHCl, pH 9.0. Protein elution was monitored by UV absorbance at 280 nm, and 10 µl samples of pooled fractions containing a UV absorbance peak at 280 nm were subjected to SDS-PAGE using Mini-PROTEAN® TGX Stain-Free™ precast gels in the presence or absence of the reducing agent β-mercaptoethanol, and the isolated recombinant proteins were visualized using the ChemiDoc™ imaging system (BioRad).

It is well known to those skilled in the art that recombinant proteins can be produced in various expression systems and purified by various chromatographic methods to obtain similar results.

Fig. 6 shows that expression and purification of the protein corresponding to SEQ ID NO: 58 does result in protein that migrates above the expected level in the absence of the reducing agent β-mercaptoethanol, thus indicating dimer formation. However, as can be seen in the lane containing purified protein, in the presence of the reducing agent β-mercaptoethanol, expression and purification of the protein corresponding to SEQ ID NO: 58 primarily results in cleaved fragments rather than intact protein.

Example 10

Several variants of the sequence of SEQ ID NO: 58 were prepared in order to increase the proteolytic stability of the protein corresponding to SEQ ID NO: 58. The DNA sequences were synthesized and verified by a commercial supplier before subcloning to produce plasmids as described in Example 9, and the proteins were expressed as described in Example 9. These variants include proteins with the modifications shown below:

1) an N-terminal signal sequence derived from albumin of SEQ ID NO: 32, from the amino terminus of the CCN5 fragment consisting of amino acids 194-249, including the mutation (P245L), corresponding to SEQ ID NO: 62, in combination with a truncated peptide linker corresponding to SEQ ID NO: 63 and the Fc fragment of SEQ ID NO: 15, to obtain a sequence corresponding to SEQ ID NO: 64,

2) an N-terminal signal sequence derived from the albumin of SEQ ID NO: 32, at the amino terminus of the CCN5 fragment consisting of amino acids 194-246, corresponding to SEQ ID NO: 1, in combination with a peptide linker variant corresponding to SEQ ID NO: 65 and the Fc fragment of SEQ ID NO: 15, to obtain a sequence corresponding to SEQ ID NO: 66,

3) an N-terminal signal sequence derived from the albumin of SEQ ID NO: 32, at the amino terminus of the CCN5 fragment consisting of amino acids 194-246, corresponding to SEQ ID NO: 1, in combination with a peptide linker variant corresponding to SEQ ID NO: 67 and the Fc fragment of SEQ ID NO: 15, to obtain a sequence corresponding to SEQ ID NO: 68,

4) an N-terminal signal sequence derived from the albumin of SEQ ID NO: 32, from the amino terminus of the CCN5 fragment consisting of amino acids 194-246, corresponding to SEQ ID NO: 1, in combination with a peptide linker variant corresponding to SEQ ID NO: 65 and the Fc fragment of SEQ ID NO: 19, to obtain a sequence corresponding to SEQ ID NO: 69.

The versions (1-4, above) of the protein disclosed in Example 12 did show some increase in resistance to proteolytic cleavage when expressed in the ExpiCHO system as described in Example 9. However, when expressing proteins corresponding to SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68 and SEQ ID 69, it was found that the degree of proteolytic resistance was still insufficient to ensure the production of intact purified proteins.

Example 11

A fusion protein was obtained containing amino acids 194-237 of CCN5, where the amino acid at position 195 (proline) was replaced with alanine (SEQ ID NO: 38), fused at the N-terminus with a peptide linker (SEQ ID NO: 21) and a chimeric Fc fragment of IgG subtype IgG2/4 with a deletion of the carboxy-terminal K477- (Eu numbering) (SEQ ID NO: 19), to obtain SEQ ID NO: 27. An N-terminal secretion signal sequence derived from albumin SEQ ID NO: 32 was additionally added to the fusion protein, to obtain 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. (using a commercial supplier's algorithm), a KOZAK sequence for translation was added at the 5' end and a stop codon was introduced at the 3' end, yielding the DNA sequence of SEQ ID NO: 71. This DNA sequence was also complemented at both ends with attB Gateway sites, yielding the DNA sequence of SEQ ID NO: 72.

The DNA sequences were synthesized and verified by a commercial supplier before subcloning to produce plasmids as described in Example 9, and the protein corresponding to SEQ ID NO: 70 was expressed by transient transfection of ExpiCHO cells as described in Example 9.

Cells were pelleted 6 days post-transfection by centrifugation at 4750 x g for 20 min at 4°C and the cell culture medium supernatant was collected. 0.1 M PMSF in 100% isopropanol was added to a concentration of 0.1 mM. 1 M Na citrate, pH 5.5, was added to a final concentration of 30 mM before chromatographic purification.

The protein was purified by tandem chromatography consisting of a capture step on a 1 ml HiTrap™ MabSelectSuRe™ column (GE Healthcare) immediately followed by salting out on a 10 ml BioScale™ Mini Bio-Gel® P-6 column (BioRad). The columns were installed on an FPLC system (BioRad NGC Discover™ 10 Pro system) equipped with a 5 mm pathlength UV detection flow cell. The MabSelectSuRe™ column was installed on the first column switching valve and equilibrated with a buffer consisting of 30 mM Na citrate, pH 5.5, and the Bio-Gel® column was installed on the second column switching valve and equilibrated with buffer A2 (100 mM NaH ₂ PO4/Na ₂ HPO4 pH 6.5). With the second column switching valve containing the Bio-Gel® column set to the bypass position, 140 mL of harvested cell culture medium containing the recombinant protein was loaded onto the MabSelectSuRe™ column using the sample pump at a flow rate of 2.0 mL/min, followed by a wash with 5 column volumes of Wash Buffer A1 (30 mM Na citrate, pH 5.5), then 5 column volumes of Wash Buffer A3 (30 mM Na citrate, 0.5 M NaCl, pH 5.5), and then 3 column volumes of Wash Buffer A1. Before elution with the elution buffer (30 mM citric acid, pH 3.4), the Bio-Gel® column set on the second column switching valve was set to the flow channel position. After elution with 2 ml of elution buffer, the MabSelectSuRe™ column was disconnected from the flow channel and the purified protein was eluted from the Bio-Gel® column with buffer A2. Protein elution was monitored by UV absorbance at 280 nm and collection was initiated when the absorbance was greater than 100 mOOP. The collected fractions were pooled and a 10 µl sample was subjected to SDS-PAGE using Mini-PROTEAN® TGX Stain-Free™ precast gels in the presence of the reducing agent β-mercaptoethanol and the isolated recombinant proteins were visualized using the ChemiDoc™ Imaging System (BioRad).

Fig. 7 shows that the expression and purification of the protein corresponding to SEQ ID NO: 27, where the carboxy-terminal fragment of CCN5 is truncated, is substantially more proteolytically stable than variants where all carboxy-terminal amino acids of CCN5 are present (as in SEQ ID NOs: 58, 64, 66, 68 and 69), even though the cell culture medium was harvested 2 days later after subculture compared to Example 9 (Fig. 6).

Example 12

A fusion protein was prepared comprising amino acids 206-249 of CCN3, where the amino acid at position 207 (isoleucine) was replaced with alanine (SEQ ID NO: 44), fused at the N-terminus to a peptide linker (SEQ ID NO: 21) and a chimeric Fc fragment of IgG subtype IgG2/4 with a deletion of the carboxy-terminal K477- (Eu numbering) (SEQ ID NO: 19), to obtain the fusion protein of SEQ ID NO: 73. An N-terminal secretion signal sequence derived from albumin of SEQ ID NO: 32 was further added to the fusion protein, to obtain the 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 (using a commercial supplier's algorithm), a KOZAK sequence for translation was added at the 5' end and a stop codon was introduced at the 3' end, yielding the DNA sequence of SEQ ID NO: 75. The DNA sequence was also complemented at both ends with attB Gateway sites, yielding the DNA sequence of SEQ ID NO: 76.

The DNA sequences were synthesized and verified by a commercial supplier before subcloning to produce plasmids as described in Example 9, and the protein corresponding to SEQ ID NO: 74 was expressed by transient transfection of ExpiCHO cells as described in Example 9.

Cells were pelleted 5 days post-transfection by centrifugation at 4750xg for 20 min at 4°C and the cell culture medium supernatant was collected. 0.1 M PMSF in 100% isopropanol was added to a concentration of 0.1 mM. 1 M Na citrate, pH 5.5, was added to a final concentration of 30 mM before chromatographic purification.

The protein was purified by tandem chromatography consisting of a capture step on a 5 ml HiTrap™ MabSelectSuRe™ column (GE Healthcare) immediately followed by salting out on a 53 ml HiPrep™ 26/10 salting out column (GE Healthcare). The columns were mounted on an FPLC system (BioRad NGC Discover™ 10 Pro system) equipped with a 5 mm pathlength UV flow cell. The MabSelectSuRe™ column was mounted on the first switching valve and equilibrated with 30 mM Na citrate, pH 5.5 buffer, and the HiPrep™ column was mounted on the second switching valve and equilibrated with Buffer A2 (100 mM NaH ₂ PO4/Na ₂ HPO4 pH 6.5). With the second column switching valve containing the HiPrep™ column set to the bypass position, 260 mL of harvested cell culture medium containing the recombinant protein was loaded onto the MabSelectSuRe™ column using the sample pump at a flow rate of 3.5 mL/min, followed by a wash with 5 column volumes of Wash Buffer A1 (30 mM Na citrate, pH 5.5), then 5 column volumes of Wash Buffer A3 (30 mM Na citrate, 0.5 M NaCl, pH 5.5), and then 2 column volumes of Wash Buffer A1. Prior to elution with the elution buffer (30 mM citric acid, pH 3.4), the HiPrep™ column, mounted on the second column switching valve, was set to the flow channel position. After elution with 10 ml of elution buffer, the MabSelectSuRe™ column was disconnected from the flow channel and the purified protein was eluted from the HiPrep™ column with buffer A2. Protein elution was monitored by UV absorbance at 280 nm and collection was initiated when the absorbance was greater than 100 mODU. The collected fractions were pooled and a 10 µl sample was subjected to SDS-PAGE using Mini-PROTEAN® TGX Stain-Free™ precast gels in the presence or absence of the reducing agent β-mercaptoethanol and the isolated recombinant proteins were visualized using the ChemiDoc™ Imaging System (BioRad).

In Fig. 8, it can be seen that the fusion protein comprising amino acids derived from CCN3/NOv (domain III/TSP-1 homology domain) as described in SEQ ID NO: 73, similar to the fusion protein comprising amino acids derived from homologous CCN5 (domain III/TSP-1 homology domain) as described in SEQ ID NO: 27, has a similar or better resistance to proteolysis than the fusion protein comprising amino acids derived from CCN5 as described in Example 11 and in Fig. 7.

Example 13

A fusion protein comprising amino acids 194-246 of CCN5, wherein the amino acid at position 195 (proline) is replaced by alanine (SEQ ID NO: 7), fused at the C-terminus with a peptide linker (SEQ ID NO: 39) and the Fc fragment of IgG subclass IgG4 from SEQ ID NO: 15 (S228P, F234A, L235A, K447-, Eu-numbering), to yield the protein sequence corresponding to SEQ ID NO: 41, was complemented with the N-terminal signal sequence for secretion derived from albumin from SEQ ID NO: 32, to yield the 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 (using a commercial supplier's algorithm), a KOZAK sequence for translation was added at the 5' end and a stop codon was introduced at the 3' end, yielding the DNA sequence of SEQ ID NO: 78. The DNA sequence was also complemented at both ends with attB Gateway sites, yielding the DNA sequence of SEQ ID NO: 79.

The sequence of SEQ ID NO: 79 was synthesized and verified by a commercial supplier. The synthesized sequence was recombined into pDonrZeo by BP Gateway recombinase cloning to generate the entry vector. Following transfection of competent E. coli mutated to allow efficient plasmid propagation (One Shot Top10™ cells), the entry vector was isolated using standard plasmid isolation techniques using the QIAprep™ Spin Miniprep Kit from Qiagen™. Following plasmid isolation, the entry vector was verified by restriction enzyme digestion followed by DNA gel electrophoresis according to standard techniques well known to those skilled in the art.

The input vector containing the sequence SEQ ID NO: 78 was further recombined with the receiving vector using LR Gateway recombinase. The receiving vector used was pUCOE-DHFR-DEST, as described in the source: et al., 2018, J. Biol. Chem, 293:46, pp. 17953 - 17970.

Following transfection of competent E. coli mutated to allow efficient propagation of the plasmids (One Shot Top10™ cells), the expression vector was isolated using standard plasmid isolation techniques using the QIAGEN™ Plasmid Plus Maxi Kit. The resulting expression vector was verified using standard restriction enzyme digestion and DNA gel electrophoresis according to standard techniques well known to those skilled in the art. The resulting expression vector was then transferred into suspension-adapted CHO DG44 cells by electroporation using the Neon Transfection System (ThermoFisherScientilic).

Cells were maintained in ventilated Erlenmeyer flasks in cell culture incubators at 37°C with 8% _CO2 on a shaking platform (as described by Kaasbøll et al., above). Transfected cells were maintained overnight in DG44 CD medium (Gibco, cat. #12610-010) before being transferred to HyClone™ ActiPro™ medium (hypoxanthine- and thymidine-free, GE Healthcare) and subcultured to 80% viability, at which point 0.1 μM methotrexate was added to the medium. After addition of 0.1 μM methotrexate, cells were subcultured until 80% viability was again reached, at which point 1 μM methotrexate was added to the medium. The cells were again subcultured until viability exceeded 98% and the doubling time decreased to less than 26 h, at which point the cell pool was considered stably transfected. Once a stable cell pool was obtained, the cell culture volume was expanded to ensure seeding with stably transfected cells for production at a density of 1 x 10 ⁶ cells/mL. Cell cultures were supplemented with 4/0.4% (v/v) HyClone™ Cell Boost™ 7a/7b daily starting on day 3 after subculture. After 10 days, cells were pelleted by centrifugation at 4750 x g for 20 min at 4°C and the cell culture medium supernatant was collected. 0.1 M PMSF in 100% isopropanol was added to a concentration of 0.1 mM. 1 M Na citrate, pH 5.5, was added to a final concentration of 30 mM before chromatographic purification.

The protein was purified by tandem chromatography consisting of a capture step on a HiTrap™ MabSelectSuRe™ 5 ml column (GE Healthcare) immediately followed by salting out on a HiPrep™ 26/10 53 ml salting out column (GE Healthcare), as described in Example 15. The purified protein composition (with no observable signs of proteolytic processing) was then tested for the ability to inhibit pro-survival signaling (AKT serine 473 phosphorylation) in A549 human lung cancer cells as described in Example 2. As can be seen in Fig. 9, the purified protein corresponding to SEQ ID NO: 41, produced by the stably transfected pool of CHO suspension cells, unexpectedly showed no signs of the ability to inhibit AKT phosphorylation (serine 473).

Example 14

A fusion protein was prepared containing amino acids 206-249 of CCN3 (SEQ ID NO: 44), where the amino acid at position 207 (isoleucine) was replaced with alanine, fused at the N-terminus to a peptide linker (SEQ ID NO: 22) and a chimeric Fc fragment of IgG subtype IgG2/4 (SEQ ID NO: 19), to yield the protein sequence corresponding to SEQ ID NO: 80, which was complemented with the N-terminal signal sequence for secretion derived from albumin from SEQ ID NO: 32, to yield the fusion protein corresponding to SEQ ID NO: 81. The DNA sequence encoding the fusion protein of SEQ ID NO: 81 was codon-optimized for expression of the protein in hamster cells (using a commercial supplier's algorithm), the KOZAK sequence for translation was added at the 5' end, and a stop codon was introduced at the 3' end, yielding the DNA sequence of SEQ ID NO: 82. The DNA sequence was also complemented at both ends with attB Gateway sites, yielding the DNA sequence of SEQ ID NO: 83.

The sequence of SEQ ID NO: 83 was synthesized and verified by a commercial supplier. The synthesized sequence was recombined into pDonrZeo by BP Gateway recombinase cloning to generate the entry vector. Following transfection of competent E. coli mutated to allow efficient plasmid propagation (One Shot Top10™ cells), the entry vector was isolated using standard plasmid isolation techniques using the QIAprep™ Spin Miniprep Kit from Qiagen™. Following plasmid isolation, the entry vector was verified by restriction enzyme digestion followed by DNA gel electrophoresis according to standard techniques well known to those skilled in the art.

The input vector containing the sequence SEQ ID NO: 82 was further recombined with the receiving vector using LR Gateway recombinase. The receiving vector used was pUCOE-DHFR-DEST, as described in the source: et al., 2018, J. Biol. Chem, 293:46, pp.17953-17970.

Following transfection of competent E. coli mutated to allow efficient propagation of the plasmids (One Shot Top10™ cells), the expression vector was isolated using standard plasmid isolation techniques using the QIAGEN™ Plasmid Plus Maxi Kit. The resulting expression vector was verified using standard restriction enzyme digestion and DNA gel electrophoresis according to standard techniques well known to those skilled in the art. The resulting expression vector was then transformed into suspension-adapted CHO ExpiCHO cells according to the protocol “Establishment and Scale-up of a Stable Cell Line Using ExpiCHO™ Products” from the manufacturer of ExpiCHO™ Stable Production Medium (Gibco, Cat.#:A3711001). Cells were maintained in ventilated Erlenmeyer flasks in cell culture incubators at 37°C with 8% _CO2 on a shaking platform (as described by Kaasbøll et al., above). Transfected cells were maintained overnight in ExpiCHO™ Cell Expression Medium before being transferred to ExpiCHO™ Cell Expression Medium supplemented with 0.1 μM methotrexate. Cells were then subcultured until viability again reached 80%, at which point 1 μM methotrexate was added to the medium. Cells were again subcultured until viability exceeded 95% and doubling time decreased to less than 20 hours, at which point the cell pool was considered stably transfected. After obtaining a stable cell pool, the cell culture volume was expanded to ensure seeding with stably transfected production cells at a density of 1*10 ^∧ 6 cells/ml. After 5 days, the cells were pelleted by centrifugation at 4750 x g for 20 min at 4°C and the cell culture medium supernatant was collected. 0.1 M PMSF in 100% isopropanol was added to a concentration of 0.1 mM. 1 M Na 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 before chromatographic purification.

The protein was purified by tandem chromatography consisting of a capture step on a HiTrap™ MabSelect PrismA™ 5 ml column (GE Healthcare) immediately followed by a salting out step on a HiPrep™ 26/10 53 ml salting out column (GE Healthcare), using the protocol described in Example 12, except for the addition of 100 mM L-Arginine to buffers A1, A2, A3 and B1. The purified protein composition (showing no evidence of proteolytic processing) was then tested for the ability to inhibit pro-survival signaling (phosphorylation of serine-473 by AKT) in A549 human lung cancer cells as described in Example 2. As can be seen in Fig. 10, the purified protein corresponding to SEQ ID NO: 80, produced by a stably transfected pool of CHO suspension cells, unexpectedly showed no evidence of the ability to inhibit AKT (serine 473) phosphorylation, demonstrating that neither of the intact dimeric Fc fusion protein formulations containing amino acids derived from CCN5 (SEQ ID NO: 41, Example 13, Fig. 9) or amino acids derived from CCN3 (SEQ ID NO: 80) are biologically active.

Example 15

The expression plasmid described in Example 14, comprising SEQ ID NO: 82 encoding a fusion protein comprising amino acids 206-249 of CCN3 (SEQ ID NO: 44), wherein the amino acid at position 207 (isoleucine) is replaced by alanine, fused at the N-terminus to a peptide linker (SEQ ID NO: 22) and a chimeric Fc fragment of IgG of the IgG2/4 subtype (SEQ ID NO: 19) to yield a protein sequence corresponding to SEQ ID NO: 80, also complemented by the N-terminal signal sequence for secretion derived from albumin of SEQ ID NO: 32 and corresponding to SEQ ID NO: 81, was expressed by transient transfection of ExpiCHO™ cells as described in Example 9. The cells were pelleted as described in In Example 14, 6 days after transfection, the medium was supplemented as described in Example 14. The protein was purified by tandem chromatography consisting of a capture step on a HiTrap™ MabSelect PrismA™ 5 ml column (GE Healthcare) immediately followed by salting out on a HiPrep™ 26/10 53 ml salting out column (GE Healthcare), as described in Example 14.

The purified protein composition (which had been partially proteolytically processed) was then tested for the ability to inhibit pro-survival signaling (AKT serine-473 phosphorylation) in A549 human lung cancer cells as described in Example 2. As can be seen in Fig. 11, the purified protein corresponding to SEQ ID NO: 80 produced by transiently transfected ExpiCHO™ cells exhibited a concentration-dependent ability to inhibit AKT phosphorylation (series 473), demonstrating that the expression system used to produce the fusion protein corresponding to SEQ ID NO: 80, and thus the extent of proteolytic processing observed, significantly impacts the activity or inactivity of the resulting protein composition.

Example 16

A fusion protein comprising amino acids 206-249 of CCN3 (SEQ ID NO: 44), wherein the amino acid at position 207 (isoleucine) is replaced by alanine, fused at the N-terminus to a peptide linker (SEQ ID NO: 21) and an Fc fragment with monomer formation-inducing and half-life-extending mutations (SEQ ID NO: 55), to yield the protein sequence corresponding to SEQ ID NO: 84, was complemented with the N-terminal signal sequence for albumin-derived secretion from SEQ ID NO: 32, to yield the 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 (using a commercial supplier's algorithm), the KOZAK sequence for translation were added at the 5' end and a stop codon was introduced at the 3' end, yielding the DNA sequence of SEQ ID NO: 86. The DNA sequence was also complemented at both ends with attB Gateway sites, yielding the DNA sequence of SEQ ID NO: 86. The DNA sequences were synthesized and verified by a commercial supplier before being subcloned to generate plasmids as described in Example 9 and expressed by transient transfection of ExpiCHO™ cells as described in Example 9. Cells were pelleted as described in Example 14 5 days post-transfection and the medium was supplemented as described in Example 14. The protein was purified by tandem chromatography consisting of a capture step on a HiTrap™ MabSelect PrismA™ 5 ml column (GE Healthcare) immediately followed by salting out on a salting out column HiPrep™ 26/10 53 ml (GE Healthcare) as described in Example 14.

The purified protein composition, which exhibited the expected monomeric form, was then tested for the ability to inhibit pro-survival signaling (AKT serine-473 phosphorylation) in A549 human lung cancer cells as described in Example 2. As can be seen in Fig. 12, the purified protein corresponding to SEQ ID NO: 84 exhibited a concentration-dependent ability to inhibit AKT phosphorylation (series 473), demonstrating that another monomeric fusion protein containing amino acids from domain III/TSP-1 homology domain of CCN protein had the ability to inhibit AKT phosphorylation (serine-473) in A549 human lung cancer cells.

Example 17

A fusion protein comprising amino acids 206-249 of CCN3 (SEQ ID NO: 44), where the amino acid at position 207 (isoleucine) is replaced by alanine, fused at the N-terminus to a peptide linker (SEQ ID NO: 21) and an Fc fragment with monomer formation and stability-inducing mutations (SEQ ID NO: 54), to yield the protein sequence corresponding to SEQ ID NO: 88, was supplemented with the N-terminal signal sequence for secretion derived from albumin from SEQ ID NO: 32 to yield the 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 (using a commercial supplier's algorithm), the KOZAK sequence for translation was added to 5' end and a stop codon was introduced at the 3' end, yielding the DNA sequence of SEQ ID NO: 90. The DNA sequence was also complemented at both ends with attB Gateway sites, yielding the DNA sequence of SEQ ID NO: 91. The DNA sequences were synthesized and verified by a commercial supplier before being subcloned to generate plasmids as described in Example 9 and expressed by transient transfection of ExpiCHO™ cells as described in Example 9. Cells were pelleted as described in Example 14 6 days post-transfection and the medium was supplemented as described in Example 14. The protein was purified by tandem chromatography consisting of a capture step on a HiTrap™ MabSelect PrismA™ 5 ml column (GE Healthcare) immediately followed by salting out on a HiPrep™ salting out column 26/10 53 ml (GE Healthcare), as described in Example 14.

The purified protein composition, which exhibited primarily the expected monomeric form, was then tested for the ability to inhibit pro-survival signaling (AKT serine-473 phosphorylation) in A549 human lung cancer cells as described in Example 2. As can be seen in Fig. 13, the purified protein corresponding to SEQ ID NO: 88 exhibited a concentration-dependent ability to inhibit AKT phosphorylation (series 473), demonstrating that another monomeric fusion protein containing amino acids from domain III/TSP-1 homology domain of CCN protein had the ability to inhibit AKT phosphorylation (serine-473) in A549 human lung cancer cells.

Example 18

A fusion protein comprising amino acids 206-249 of CCN3 (SEQ ID NO: 44), wherein the amino acid at position 207 (isoleucine) is replaced with alanine, fused at the N-terminus to a peptide linker (SEQ ID NO: 93) and a multifunctional tag comprising a 6xHis tag, HaloTag and Sumo* elements (SEQ ID NO: 92), to yield the protein sequence corresponding to SEQ ID NO: 94, was complemented with the N-terminal signal sequence for secretion derived from albumin from SEQ ID NO: 32, to yield the 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 (using a commercial supplier's algorithm), the KOZAK sequence for translation was added at the 5' end and a stop codon was introduced at the 3' end, yielding the DNA sequence of SEQ ID NO: 95. The DNA sequence was also complemented at both ends with attB Gateway sites, yielding the DNA sequence of SEQ ID NO: 96. The DNA sequences were synthesized and verified by a commercial supplier before being subcloned to generate plasmids as described in Example 9 and expressed by transient transfection of ExpiCHO™ cells as described in Example 15. The cells were pelleted as described in Example 14 5 days after transfection. 0.1 M PMSF in 100% isopropanol was added to a final concentration of 0.1 mM, 1 M Na 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 before chromatographic purification.

The protein was purified by tandem chromatography consisting of a capture step on a HiTrap™ HisTrap™ Excel 5 ml column (GE Healthcare) immediately followed by salting out on a HiPrep™ 26/10 53 ml salting out column (GE Healthcare). The columns were installed on an FPLC chromatography system (BioRad NGC Discover™ 10 Pro system) equipped with a 5 mm path length UV detection flow cell. The HisTrap™ column was installed on the first column switching valve and equilibrated with buffer A1 consisting of 5 mM imidazole, 50 mM NaCl, 100 mM L-arginine, and the HiPrep™ column was installed on the second column switching valve and equilibrated with buffer A2 (100 mM NaH2PO4/Na2HPO4, 100 mM L-arginine, pH 6.5). With the second column switching valve containing the HiPrep™ column set to the bypass position, 250 mL of harvested cell culture medium containing the recombinant protein was loaded onto the HisTrap™ column using the sample pump at a flow rate of 3.5 mL/min, followed by a wash with 5 column volumes of Wash Buffer A1, then 5 column volumes of Wash Buffer A3 (5 mM Imidazole, 0.5 M NaCl, 100 mM L-Arginine), and then 2 column volumes of Wash Buffer A1. Prior to elution with the elution buffer (250 mM Imidazole, 50 mM NaCl, 100 mM L-Arginine), the HiPrep™ column mounted on the second column switching valve was set to the flow channel position. After elution with 10 ml of elution buffer, the HisTrap™ column was disconnected from the flow channel and the purified protein was eluted from the HiPrep™ column with buffer A2. Protein elution was monitored by UV absorbance at 280 nm and collection was initiated when the absorbance exceeded 60 mOOP.

The purified protein composition, which exhibited the expected monomeric form, was then tested for the ability to inhibit pro-survival signaling (AKT serine-473 phosphorylation) in A549 human lung cancer cells as described in Example 2. As can be seen in Fig. 12, the purified protein corresponding to SEQ ID NO: 94 exhibited a concentration-dependent ability to inhibit AKT phosphorylation (series 473), demonstrating that another monomeric fusion protein containing amino acids from domain III/TSP-1 homology domain of CCN protein had the ability to inhibit AKT phosphorylation (serine-473) in A549 human lung cancer cells.

Example 19

A fusion protein comprising amino acids 194-237 of CCN5, where the amino acid at position 195 (proline) is replaced by alanine (SEQ ID NO: 38), fused at the N-terminus to a peptide linker (SEQ ID NO: 21), and amino acids 25-609 of human serum albumin (SEQ ID NO: 52), to yield SEQ ID NO: 97, was supplemented with the N-terminal albumin-derived secretion signal sequence of SEQ ID NO: 32, to yield the fusion protein corresponding to SEQ ID NO: 98. The DNA sequence encoding the fusion protein of SEQ ID NO: 98 was codon-optimized for expression of the protein in hamster cells (using a commercial supplier's algorithm), a KOZAK sequence for translation was added at the 5' end, and a stop codon was introduced at the 3' end, yielding the DNA sequence of SEQ ID NO: 99. The DNA sequence was also complemented at both ends with attB Gateway sites, yielding the DNA sequence of SEQ ID NO: 100.

DNA sequences were synthesized and verified by a commercial supplier prior to subcloning to generate plasmids as described in Example 9, and CHO DG44 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 generated, the cell culture volume was expanded to ensure seeding with stably transfected cells for production at a density of 1* ¹⁰⁶ cells/mL. After 6 days, the cells were pelleted by centrifugation at 4750 x g for 20 min at 4°C and the cell culture medium supernatant was collected, ODM 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 Na 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 ODM before chromatographic purification.

The protein was purified by tandem chromatography consisting of a capture step on a Tricorn column (GE Healthcare) filled with 3 ml of Capture Select™ Human Albumin Affinity Matrix (ThermoFisherScientific) immediately followed by salting out on a HiPrep™ 26/10 53 ml salting out column (GE Healthcare). The columns were mounted on a FPLC system (BioRad NGC Discover™ 10 Pro system) equipped with a 5 mm pathlength UV detection flow cell. The CaptureSelect™ column _was mounted on the first column switching valve and equilibrated with buffer A1 consisting of 100 mM _{NaH2PO4 / Na2HPO4} , 100 mM L-arginine, pH _6.5 , and the HiPrep™ 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). With the second column switching valve containing the HiPrep™ column set to the bypass position, 500 mL of harvested cell culture medium containing the recombinant protein was loaded onto the CaptureSelect™ column using the sample pump at a flow rate of 2.0 mL/min, followed by a wash with 5 column volumes of Wash Buffer A1, then 5 column volumes of Wash Buffer A2 (100 mM NaH ₂ PO ₄ /Na ₂ HPO ₄ , 100 mM L-Arginine, 0.25 M NaCl, pH 6.5), and then 5 column volumes of Wash Buffer A1. Prior to elution with elution buffer (30 mM citric acid, pH 3.5 + 0.5 M L-arginine), the HiPrep™ column, mounted on the second column switching valve, was positioned for connection to the flow channel. After elution with 10 ml of elution buffer, the CaptureSelect™ column was disconnected from the flow channel and the purified protein was eluted from the HiPrep™ column with buffer A1. Protein elution was monitored by UV absorbance at 280 nm and collection was initiated when the absorbance was greater than 100 mOOP.

The purified protein composition, which exhibited the expected monomeric form, was then tested for the ability to inhibit pro-survival signaling (AKT serine-473 phosphorylation) in A549 human lung cancer cells as described in Example 2. As can be seen in Fig. 14, the purified protein corresponding to SEQ ID NO: 97 exhibited a concentration-dependent ability to inhibit AKT phosphorylation (serine-473) demonstrating that another monomeric fusion protein containing amino acids from domain III/TSP-1 homology domain of CCN protein was able to inhibit AKT phosphorylation (serine-473) in A549 human lung cancer cells.

Example 20

The human serum albumin fusion protein (amino acids 25-606, SEQ ID NO: 101) was fused at the C-terminus to a peptide linker (SEQ ID NO: 22) linked to amino acids 194-246 of human CCN5, wherein the amino acid at position 195 (proline) was replaced by alanine (SEQ ID NO: 7), to yield SEQ ID NO: 103. The fusion protein corresponding to SEQ ID NO: 102 was complemented with the N-terminal albumin-derived secretion signal sequence of SEQ ID NO: 32, to yield the 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 (using a commercial supplier's algorithm), the KOZAK sequence for translational sites were added at the 5' end and a stop codon was introduced at the 3' end, yielding the DNA sequence of SEQ ID NO: 104. The DNA sequence was also complemented at both ends with attB Gateway sites, yielding the DNA sequence of SEQ ID NO: 105.

DNA sequences were synthesized and verified by a commercial supplier prior to subcloning 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 designated protein of SEQ ID NO: 104 as described in Example 13. Once a stable pool of cells was generated, the cell culture volume was expanded to ensure seeding with stably transfected cells for production at a density of 1* ¹⁰⁶ cells/mL. After 6 days, the cells were pelleted by centrifugation at 4750xg for 20 min at 4°C and the cell culture medium supernatant was collected. 0.1 M PMSF in 100% isopropanol was added to a final concentration of 0.1 mM, 0.5 M EDTA was added to a final concentration of 2 mM, 1 M Na 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 chromatographic purification.

The protein was purified by tandem chromatography consisting of a capture step on a Tricorn column (GE Healthcare) packed with 3 mL of CaptureSelect™ Human Albumin Affinity Matrix (ThermoFisherScientific) immediately followed by salting out on a HiPrep™ 26/10 53 mL salting out column (GE Healthcare) as described in Example 19, except that the sample loading rate was 0.37 mL/min instead of 2.0 mL/min.

The purified protein composition, which exhibited the expected monomeric form, was then tested for the ability to inhibit pro-survival signaling (AKT serine-473 phosphorylation) in A549 human lung cancer cells as described in Example 2. As can be seen in Fig. 14, the purified protein corresponding to SEQ ID NO: 102 exhibited a concentration-dependent ability to inhibit AKT phosphorylation (serine-473) demonstrating that another monomeric fusion protein containing amino acids from domain III/TSP-1 homology domain of CCN protein was able to inhibit AKT phosphorylation (serine-473) in A549 human lung cancer cells.

Example 21

A fusion protein comprising amino acids 206-249 of CCN3, wherein the amino acid at position 207 (isoleucine) is replaced with alanine (SEQ ID NO: 44), fused at the N-terminus to a peptide linker (SEQ ID NO: 21) and amino acids 25,609 of human serum albumin (SEQ ID NO: 52) to yield SEQ ID NO: 106, was supplemented with the N-terminal albumin-derived secretion signal sequence of SEQ ID NO: 32 to yield the 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 (using a commercial supplier's algorithm), a KOZAK sequence for translation was added at the 5' end, and a stop codon was introduced at the 3' end, yielding the DNA sequence of SEQ ID NO: 108. The DNA sequence was also complemented at both ends with attB Gateway sites, yielding the DNA sequence of SEQ ID NO: 109.

DNA sequences were synthesized and verified by a commercial supplier before subcloning to generate plasmids as described in Example 9 and expressed by transient transfection of ExpiCHO™ cells as described in Example 9. Cells were pelleted as described in Example 14 6 days post-transfection and the medium was supplemented as described in Example 19. Protein was purified by tandem chromatography consisting of a capture step on a Tricorn column (GE Healthcare) packed with 10 ml CaptureSelect™ Human Albumin Affinity Matrix (ThermoFisherScientific) immediately followed by salting out on a HiPrep™ 26/10 53 ml salting out column (GE Healthcare). The columns were loaded onto an FPLC system (BioRad NGC Discover™ 10 Pro System) equipped with a 5 mm pathlength UV flow cell. The CaptureSelect™ column was loaded onto the first column switching valve and equilibrated with buffer A1 consisting of ₁₀₀ mM _NaH2PO4 / _Na2HPO4 , 100 mM L-arginine, pH 6.5, and the HiPrep™ column was loaded onto the second column switching valve and equilibrated _with buffer A1 (100 mM _NaH2PO4 / _{Na2HPO4 ,} 100 mM L- _arginine , pH 6.5). With the second column switching valve containing the HiPrep™ column set to the bypass position, 500 mL of harvested cell culture medium containing the recombinant protein was loaded onto the CaptureSelect™ column using the sample pump at a flow rate of 1.0 mL/min, followed by a wash with 3 column volumes of Wash Buffer A1, then 2 column volumes of Wash Buffer A2 (100 mM NaH ₂ PO ₄ /Na ₂ HPO ₄ , 100 mM L-Arginine, 0.25 M NaCl, pH 6.5), and then 3 column volumes of Wash Buffer A1. Prior to elution with elution buffer (30 mM citric acid, pH 3.5 + 0.1 M L-arginine), the HiPrep™ column, mounted on the second column switching valve, was positioned for connection to the flow channel. After elution with 15 ml of elution buffer, the Capture Select™ column was disconnected from the flow channel and the purified protein was eluted from the HiPrep™ column with buffer A1. Protein elution was monitored by UV absorbance at 280 nm and collection was initiated when the absorbance was greater than 100 mOOP.

The purified protein composition, which contained the expected monomeric form, was then tested for the ability to inhibit pro-survival signaling (AKT serine-473 phosphorylation) in A549 human lung cancer cells as described in Example 2. As can be seen in Fig. 12, the purified protein corresponding to SEQ ID NO: 106 exhibited a concentration-dependent ability to inhibit AKT phosphorylation (series 473), demonstrating that another monomeric fusion protein containing amino acids from domain III/TSP-1 homology domain of CCN protein had the ability to inhibit AKT phosphorylation (serine-473) in A549 human lung cancer cells.

Example 22

A fusion protein comprising amino acids 206-249 of CCN3, where the amino acid at position 207 (isoleucine) is replaced with alanine (SEQ ID NO: 44), fused at the N-terminus to a peptide linker (SEQ ID NO: 22) and amino acids 25-609 of human serum albumin (SEQ ID NO: 52) to yield SEQ ID NO: 110, was supplemented with the N-terminal albumin-derived secretion signal sequence of SEQ ID NO: 32 to yield the fusion protein corresponding to SEQ ID NO: 111. The DNA sequence encoding the fusion protein of SEQ ID NO: 111 was codon-optimized for expression of the protein in hamster cells (using a commercial supplier's algorithm), a KOZAK sequence for translation was added at the 5' end, and a stop codon was introduced at the 3' end, yielding the DNA sequence of SEQ ID NO: 112. The DNA sequence was also complemented at both ends with attB Gateway sites, yielding the DNA sequence of SEQ ID NO: 113.

DNA sequences were synthesized and verified by a commercial supplier before subcloning to generate plasmids as described in Example 9 and expressed by transient transfection of ExpiCHO™ cells as described in Example 9. Cells were pelleted as described in Example 14 6 days post-transfection and the medium was supplemented as described in Example 19. Protein was purified by tandem chromatography consisting of a capture step on a Tricorn column (GE Healthcare) packed with 10 ml CaptureSelect™ Human Albumin Affinity Matrix (ThermoFisherScientific) immediately followed by salting out on a HiPrep™ 26/10 53 ml salting out column (GE Healthcare). The columns were loaded onto an FPLC system (BioRad NGC Discover™ 10 Pro System) equipped with a 5 mm pathlength UV flow cell. The CaptureSelect™ column was loaded onto the first column switching valve and equilibrated with buffer A1 consisting of ₁₀₀ mM _NaH2PO4 / _Na2HPO4 , 100 mM L-arginine, pH 6.5, and the HiPrep™ column was loaded onto the second column switching valve and equilibrated _with buffer A1 (100 mM _NaH2PO4 / _{Na2HPO4 ,} 100 mM L- _arginine , pH 6.5). With the second column switching valve containing the HiPrep™ column set to the bypass position, 300 mL of harvested cell culture medium containing the recombinant protein was loaded onto the CaptureSelect™ column using the sample pump at a flow rate of 1.0 mL/min, followed by a wash with 3 column volumes of Wash Buffer A1, then 2 column volumes of Wash Buffer A2 (100 mM NaH ₂ PO ₄ /Na ₂ HPO ₄ , 100 mM L-Arginine, 0.25 M NaCl, pH 6.5), and then 3 column volumes of Wash Buffer A1. Prior to elution with elution buffer (30 mM citric acid, pH 3.5 + 0.5 M L-arginine), the HiPrep™ column, mounted on the second column switching valve, was positioned for connection to the flow channel. After elution with 15 ml of elution buffer, the CaptureSelect™ column was disconnected from the flow channel and the purified protein was eluted from the HiPrep™ column with buffer A1. Protein elution was monitored by UV absorbance at 280 nm and collection was initiated when the absorbance was greater than 100 mOOP.

The purified protein composition, which contained the expected monomeric form, was then tested for the ability to inhibit pro-survival signaling (AKT serine-473 phosphorylation) in A549 human lung cancer cells as described in Example 2. As can be seen in Fig. 15, the purified protein corresponding to SEQ ID NO: 110 exhibited a concentration-dependent ability to inhibit AKT phosphorylation (series 473), demonstrating that another monomeric fusion protein containing amino acids from domain III/TSP-1 homology domain of CCN protein had the ability to inhibit AKT phosphorylation (serine-473) in A549 human lung cancer cells.

Example 23

The expression plasmid described in Example 21, comprising SEQ ID NO: 108, encoding a fusion protein comprising amino acids 206-249 of CCN3, wherein the amino acid at position 207 (isoleucine) is replaced with alanine (SEQ ID NO: 44), fused at the N-terminus to a peptide linker (SEQ ID NO: 21), and amino acids 25-609 of human serum albumin (SEQ ID NO: 52), also supplemented with the N-terminal signal sequence for albumin-derived secretion from SEQ ID NO: 32 and corresponding to SEQ ID NO: 107, was used to generate a pool of stably transfected ExpiCHO™ cells as described in Example 14. To generate a batch of conditioned medium containing the secreted protein corresponding to SEQ ID NO: 106, the pool of stably transfected cells were expanded to ensure that 250 ml of culture medium was populated with stably transfected cells at a density of 1*10 ⁶ cells/ml. The cell cultures were supplemented with 5% (v/v) 2X EfficientFeed™ C+ (Gibco™) every other day starting on day 2 after subculturing, 3% glucose (10% w/v) on day 2 after subculturing, and 5% glucose (10% w/v) on day 6 after subculturing. After 9 days, the cells were pelleted by centrifugation at 4750 x g for 20 min at 4°C and the supernatant of the cell culture medium was collected. 0.1 M PMSF in 100% isopropanol was added to a concentration of 0.1 mM. 1 M Na citrate, pH 5.5 to a final concentration of 30 mM, and 2 M L-arginine, pH 4.0 to a final concentration of 100 mM were added before chromatographic purification.

Protein was purified by 20-D chromatography consisting of a capture step on a Tricorn column (GE Healthcare) packed with 10 mL of CaptureSelect™ Human Albumin Affinity Matrix (ThermoFisherScientific) immediately followed by size exclusion chromatography on two Superdex 200 Increase 10/300 GL columns (GE Healthcare) in series. The columns were mounted on an FPLC system (BioRad NGC Discover™ 10 Pro system) equipped with a 5 mm pathlength UV flow cell and an outlet valve connected to a 5 mL sample loop. The CaptureSelect™ column was mounted on the first switching valve and equilibrated with buffer A1 consisting of 100 mM _{NaH2PO4 / Na2HPO4 ,} 100 mM L-arginine, pH _6.5 , and the Superdex 200 Increase columns were mounted on the second switching valve and equilibrated with buffer A1 (100 mM _NaH2PO4 / _Na2HPO4 , 100 mM L-arginine, pH 6.5). With the second switching valve containing _the Superdex 200 Increase columns in the bypass position, 120 mL of harvested cell culture medium containing the recombinant protein was loaded onto the CaptureSelect™ column using a sample pump at a flow rate of 3.9 mL/min. After loading the harvested cell culture medium containing the recombinant protein onto the CaptureSelect™ column, it was washed _with 3 column volumes of buffer A1, then with 2 column volumes of buffer A2 (100 mM _NaH2PO4 / _Na2HPO4 , 100 mM L- _Arginine , 0.25 M NaCl, pH 6.5), and then with 3 column volumes of buffer A1. The CaptureSelect™ column was eluted with 15 mL of buffer B1 (30 mM citric acid, 0.5 M L-arginine, pH 3.5), with the system set to collect eluate with absorbance values greater than 1200 mODU into the sample loop. After elution of the CaptureSelect™ column connected to the first column switching valve, it was disconnected from the flow channel and the second column switching valve was set to connect the columns containing Superdex 200 Increase to the flow channel. The eluate from the CaptureSelect™ column containing the eluted protein was then loaded onto the columns containing Superdex 200 Increase with buffer A1 at a flow rate of 0.5 ml/min. Protein elution was monitored by UV absorbance at 280 nm and collection was initiated when the absorbance was greater than 200 mODU. The purified protein formulation, which contained the expected monomeric form, was then tested for its ability to inhibit TGF-β and active CCN2-induced activities of normal human lung fibroblasts (NHLF) (Lonza Bioscience, Cat.#: CC-2512). NHLF were subcultured in complete growth medium (Lonza Bioscience BulletKit (Cat.#: CC-3132) with all supplements (2% fetal bovine serum, insulin, hFGF-B, gentamicin/amphotericin-B)) to maintain density at a maximum confluency of 80% according to the instructions of the vendor (Lonza Bioscience). Active CCN2 consisted of domains 3-4 of CCN2, and was produced and purified as described by et al., 2018, supra.

To test the effect of the protein corresponding to SEQ ID NO: 106 on active CCN2 and TGF-β-induced NHLF cell migration (Transwell assay/modified Boyden chamber assay), cells were first dissociated with trypsin/EDTA, neutralized with trypsin neutralizing reagents (Lonza Bioscience, Cat. No. CC-5034) and resuspended in basal growth media (Fibroblast Basal Medium (LonzaBioscience Cat. No. CC-3131, without any additives except gensumycin (50 μg/mL))), followed by seeding of 30,000 cells in a volume of 100 μL per well on the top side of 5 μm pore size Transwell inserts (24-well plate, Corning® Transwell®, Cat. No. CLS3402-48EA from SigmaAldrich (Merck KGaA)). The lower chamber of the wells contained test substances or vehicle control dissolved in 500 μl of basal growth medium without any additives except gensumycin. After 20 h of incubation, the inserts were removed from the wells, washed twice by immersion in phosphate-buffered saline (PBS, Lonza Bioscience, Cat. No.: 17-512F), and then fixed in 4% formaldehyde (Solveco, Sweden, Cat. No.: 621092) for 15 min at 37°C. Cells were permeabilized by treatment with 0.1% Triton X-100 in PBS for 10 min, and then washed twice with PBS. Non-migrated cells on the upper side of the inserts were removed by scraping with a cotton swab and the membrane was allowed to dry. Migrated cell nuclei on the back of the inserts were stained with Hoechst 33342 20 mM (1:5000 in PBS, ThermoFisherScientific, Cat# 62249) for 15 min in the dark and then washed twice by immersion in PBS. The membrane was cut out of the Transwell insert and mounted on glass slides with the migrated cell side facing the glass coated with one drop of Pro Long™ Gold Antifade (ThermoFisherScientific, Cat# P36934), covered with a glass coverslip and 5-10 images of each well were captured using a Zeiss Axio Observer Z.1 imaging system. Images were analyzed semi-automatically using ImageJ v1.51k, Rasband, WS, ImageJ software from the US National Institutes of Health, Bethesda, MD, USA, https://imagej.nih.gov/ij/, 1997-2018). As can be seen in Fig. 16A, the protein corresponding to SEQ ID NO: 106 inhibits migration induced by both TGF-β and active CCN2.

To test the effect of the protein corresponding to SEQ ID NO: 106 on active CCN2 and TGF-β-induced scratch closure assay results, NHLF were dissociated with trypsin/EDTA, neutralized with trypsin neutralizing reagents (Lonza Bioscience, Cat.# CC-5034), and 100,000 cells were seeded in 1 ml in tissue culture-treated 12-well plates (Corning Costar®, Cat.# 3513). The day after seeding, cells were washed twice with 0.9% NaCl and complete growth medium was replaced with basal growth medium. After incubation in basal growth medium for 16-20 h, the cell monolayer was scratched with a sterile 12.5 µl pipette tip (ThermoFisherScientific, Cat# 94420053), the cells were washed once with PBS and then incubated in 1 ml basal growth medium plus test substances or vehicle. The cells were incubated for an additional 24 h before being washed three times with PBS before fixation for 15 min at 37°C in 4% formaldehyde. After fixation, the cells were washed again for 3 x 3 min in PBS with gentle shaking, permeabilized with 0.1% Triton X-100 in PBS for 10 min with gentle shaking. Cell nuclei were stained with 20 mM Hoechst 33342 (1:5000, diluted in PBS), ThermoFisherScientific, Cat#: 62249) for 15 min in the dark, followed by 3 x 5 min washes in PBS with gentle shaking. 1 drop of Pro Long™ Gold Antifade (ThermoFisherScientific, Cat#: P36934) was applied before mounting the slides and 5 images were captured centered on the remaining gap in each well using a Zeiss Axio Observer Z.1 imaging system. Images were analyzed by measuring the size of the remaining gap after the scratch was made at 3 fixed intervals along the length of the scratch. The average of all measurements across all images from each well was calculated and counted as one biological replicate. As can be seen in Fig. 16 B, the protein corresponding to SEQ ID NO: 106 inhibits scratch closure induced by both TGF-β and active CCN2.

To test the effect of the protein corresponding to SEQ ID NO: 106 on TGF-β-induced gene regulation, NHLF was cleaved with trypsin/EDTA, neutralized with trypsin neutralizing reagents (Lonza Bioscience, Cat.# CC-5034), and 100,000 cells were seeded in 1 ml in tissue culture-treated 12-well plates (Corning Costar®, Cat.# 3513). The day after seeding, cells were washed twice with 0.9% NaCl and complete growth medium was replaced with basal growth medium supplemented with 0.1% heat-inactivated fetal bovine serum (Cat.# 16000-044 from Gibco™, heat inactivated as described in Example 2). After incubation in basal growth medium with 0.1% fetal bovine serum for 6 h, test substances or vehicle control were added to the wells. After 96 h, the wells were washed twice in PBS and RNA was extracted using the Qiagen RNeasy RNA Extraction Kit (Cat. No. 74106) according to the manufacturer's protocol. RNA concentrations were quantified using a NanoDrop® ND-1000 spectrophotometer (NanoDrop Technologies, USA), diluted with nuclease-free water to a final RNA concentration of 50 ng/µl before using 200 ng RNA from each replicate to prepare cDNA using the TaqMan™ Reverse Transcription Kit (Cat. No. N8080234) according to the manufacturer's protocol. Differential gene expression was analyzed in the resulting cDNA samples using appropriate TaqMan™ assays and the TaqMan Fast Advanced Master Mix (ThermoFisherScientific Cat. #4444557). (ThermoFisherScientific Cat. #4444557). TaqMan™ real-time PCR reactions were performed to generate technical triplicates for each sample using the Applied Biosystems StepOnePlus Real Time PCR System according to the manufacturer's protocols. Relative amounts of different transcripts were calculated from a standard curve before the technical triplicate data were averaged to generate a single value for each sample. All gene expression results were compared with GAPDH mRNA levels (ThermoFisherScientific, Cat.# Hs02786624_g1) and normalized as a fold change relative to the mean of vehicle control-stimulated wells. As can be seen in Fig. 19C-F, the protein corresponding to SEQ ID NO: 107 provides partial inhibition of TGF-β-induced genes; COL1A1 ('collagen type 1 α-1', ThermoFisherScientific, Cat# Hs00164004_m1), FN1 ('fibronectin 1', ThermoFisherScientific, Cat# Hs01549976_m1), ASTA2 ('smooth muscle actin α-2', ThermoFisherScientific, Cat# Hs00426835_g1) and CCN2 (ThermoFisherScientific, Cat# Hs00170014_m1), which are generally considered pro-fibrotic genes.

Numbering of CCN proteins: according to the Uniprot database, as described in the Detailed Description of the Invention section above. Numbering of Fc fragments: according to the Ei numbering system, as described in the Detailed Description of the Invention section above.

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SEQUENCE LIST

<110> Oslo University College HF/ Oslo University College HF

<120> Recombinant CCN domain proteins and fusion proteins/

RECOMBINANT PROTEINS AND FUSION PROTEINS WITH CCN DOMAINS

<130> 27.11.145331/01

<150>EP 19163970.7

<151> 2019-03-20

<160> 121

<170> PatentIn, version 3.5

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Leu Lys Gln Thr Arg Leu Cys Met Val Arg Pro Cys

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Cys Leu Val Gln Thr Thr Glu Trp Ser Ala Cys Ser Lys Thr Cys Gly

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Glu Lys Gln Ser Arg Leu Cys Met Val Arg Pro Cys

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Cys Ile Ala Tyr Thr Ser Pro Trp Ser Pro Cys Ser Thr Ser Cys Gly

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Glu Gln Glu Ser Arg Leu Cys Asn Leu Arg Pro Cys

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Cys Leu Val Gln Ala Thr Lys Trp Thr Pro Cys Ser Arg Thr Cys Gly

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Arg Lys Glu Lys Arg Leu Cys Tyr Ile Gln Pro Cys

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<223> Modified CCN5 sequence

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Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys

225

<210> 14

<211> 228

<212> PRT/Protein

<213> Homo sapiens

<400> 14

Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val

1 5 10 15

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

20 25 30

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

35 40 45

His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

50 55 60

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

65 70 75 80

Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn

85 90 95

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro

100 105 110

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln

115 120 125

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

130 135 140

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val

145 150 155 160

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

165 170 175

Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

180 185 190

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

195 200 205

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

210 215 220

Ser Pro Gly Lys

225

<210> 15

<211> 228

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Modified Fc fragment

<400> 15

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala

1 5 10 15

Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly

225

<210> 16

<211> 232

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Modified Fc fragment

<400> 16

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Cys Glu Glu Gln

65 70 75 80

Tyr Gly Ser Thr Tyr Arg Cys Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 17

<211> 231

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Modified Fc fragment

<400> 17

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

20 25 30

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

35 40 45

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

50 55 60

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Cys Glu Glu Gln Tyr

65 70 75 80

Gly Ser Thr Tyr Arg Cys Val Ser Val Leu Thr Val Leu His Gln Asp

85 90 95

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

100 105 110

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

115 120 125

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

130 135 140

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

145 150 155 160

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

165 170 175

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

180 185 190

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

195 200 205

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

210 215 220

Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 18

<211> 227

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Modified Fc fragment

<400> 18

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val

1 5 10 15

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

20 25 30

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

35 40 45

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

50 55 60

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

65 70 75 80

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

85 90 95

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

100 105 110

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

115 120 125

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

130 135 140

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

145 150 155 160

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

165 170 175

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr

180 185 190

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

195 200 205

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

210 215 220

Ser Leu Gly

225

<210> 19

<211> 227

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Modified Fc fragment

<400> 19

Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val

1 5 10 15

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

20 25 30

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

35 40 45

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

50 55 60

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

65 70 75 80

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

85 90 95

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

100 105 110

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

115 120 125

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

130 135 140

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

145 150 155 160

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

165 170 175

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr

180 185 190

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

195 200 205

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

210 215 220

Ser Leu Gly

225

<210> 20

<211> 6

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Linker

<400> 20

Thr Glu Gly Arg Met Asp

1 5

<210> 21

<211> 5

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Linker

<400> 21

Glu Ala Ala Ala Lys

1 5

<210> 22

<211> 40

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Linker

<400> 22

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu

1 5 10 15

Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala

20 25 30

Ala Ala Lys Glu Ala Ala Ala Lys

35 40

<210> 23

<211> 42

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Linker

<400> 23

Thr Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala

1 5 10 15

Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

20 25 30

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

35 40

<210> 24

<211> 43

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Linker

<400> 24

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu

1 5 10 15

Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala

20 25 30

Ala Ala Lys Glu Ala Ala Ala Lys Ala Ala Ala

35 40

<210> 25

<211> 45

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Linker

<400> 25

Thr Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala

1 5 10 15

Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

20 25 30

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala Ala Ala

35 40 45

<210> 26

<211> 276

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 26

Cys Pro Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys Gly

1 5 10 15

Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys Arg Leu

20 25 30

Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Glu Ala Ala Ala

35 40 45

Lys Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro

50 55 60

Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

65 70 75 80

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

85 90 95

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

100 105 110

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

115 120 125

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

130 135 140

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

145 150 155 160

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

165 170 175

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

180 185 190

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

195 200 205

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

210 215 220

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

225 230 235 240

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

245 250 255

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

260 265 270

Leu Ser Leu Gly

275

<210> 27

<211> 276

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 27

Cys Ala Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys Gly

1 5 10 15

Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys Arg Leu

20 25 30

Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Glu Ala Ala Ala

35 40 45

Lys Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro

50 55 60

Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

65 70 75 80

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

85 90 95

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

100 105 110

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

115 120 125

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

130 135 140

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

145 150 155 160

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

165 170 175

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

180 185 190

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

195 200 205

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

210 215 220

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

225 230 235 240

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

245 250 255

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

260 265 270

Leu Ser Leu Gly

275

<210> 28

<211> 287

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 28

Cys Pro Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys Gly

1 5 10 15

Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys Arg Leu

20 25 30

Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Pro Pro Ser Arg

35 40 45

Gly Arg Ser Pro Gln Thr Glu Gly Arg Met Asp Glu Ser Lys Tyr Gly

50 55 60

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

65 70 75 80

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

85 90 95

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

100 105 110

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

115 120 125

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

130 135 140

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

145 150 155 160

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

165 170 175

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

180 185 190

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

195 200 205

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

210 215 220

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

225 230 235 240

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

245 250 255

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

260 265 270

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

275 280 285

<210> 29

<211> 286

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 29

Cys Pro Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys Gly

1 5 10 15

Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys Arg Leu

20 25 30

Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Pro Pro Ser Arg

35 40 45

Gly Arg Ser Pro Gln Thr Glu Gly Arg Met Asp Glu Ser Lys Tyr Gly

50 55 60

Pro Pro Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val

65 70 75 80

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

85 90 95

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

100 105 110

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

115 120 125

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

130 135 140

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

145 150 155 160

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

165 170 175

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

180 185 190

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

195 200 205

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

210 215 220

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

225 230 235 240

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

245 250 255

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

260 265 270

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

275 280 285

<210> 30

<211> 286

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 30

Cys Pro Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys Gly

1 5 10 15

Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys Arg Leu

20 25 30

Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Pro Pro Ser Arg

35 40 45

Gly Arg Ser Pro Gln Thr Glu Gly Arg Met Asp Glu Arg Lys Cys Cys

50 55 60

Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val

65 70 75 80

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

85 90 95

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

100 105 110

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

115 120 125

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

130 135 140

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

145 150 155 160

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

165 170 175

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

180 185 190

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

195 200 205

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

210 215 220

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

225 230 235 240

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

245 250 255

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

260 265 270

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

275 280 285

<210> 31

<211> 325

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 31

Cys Pro Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys Gly

1 5 10 15

Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys Arg Leu

20 25 30

Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Pro Pro Ser Arg

35 40 45

Gly Arg Ser Pro Gln Thr Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala

50 55 60

Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

65 70 75 80

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala

85 90 95

Ala Ala Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Leu Gly

325

<210> 32

<211> 18

<212> PRT/Protein

<213> Homo sapiens

<400> 32

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser

<210> 33

<211> 305

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 33

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Cys Pro Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr

20 25 30

Cys Gly Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys

35 40 45

Arg Leu Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Pro Pro

50 55 60

Ser Arg Gly Arg Ser Pro Gln Thr Glu Gly Arg Met Asp Glu Ser Lys

65 70 75 80

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

85 90 95

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

100 105 110

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

115 120 125

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

130 135 140

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

145 150 155 160

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

165 170 175

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

180 185 190

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

195 200 205

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

210 215 220

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

225 230 235 240

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

245 250 255

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

260 265 270

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

275 280 285

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

290 295 300

Gly

305

<210> 34

<211> 915

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 34

atgaaatggg tcacctttat ctccctgctg ttcctgttct cctccgccta ctcttgccct 60

gagtggtcta cagcttgggg cccttgctct accacctgtg gactcggcat ggccaccaga 120

gtgtctaacc agaacagatt ctgccggctg gaaacccagc ggagactgtg cctgtctaga 180

ccctgtcctc ctagcagagg cagatcccct cagaccgagg gcagaatgga cgagtctaag 240

tacggccctc cttgtcctcc atgtcctgct ccagaagctg ctggcggccc ttccgtgttt 300

ctgttccctc caaagcctaa ggacaccctg atgatctctc ggacccctga agtgacctgc 360

gtggtggtgg atgtgtccca agaggatccc gaggtgcagt tcaattggta cgtggacggc 420

gtggaagtgc acaacgccaa gaccaagcct agagaggaac agttcaactc cacctacaga 480

gtggtgtccg tgctgaccgt gctgcaccag gattggctga acggcaaaga gtacaagtgc 540

aaggtgtcca acaagggcct gccttccagc atcgaaaaga ccatctccaa ggccaagggc 600

cagcctaggg aaccccaggt ttacaccctg cctccaagcc aagaggaaat gaccaagaac 660

caggtgtccc tgacctgcct ggtcaagggc ttctaccctt ccgatatcgc cgtggaatgg 720

gagagcaatg gccagcctga gaacaactac aagaccacac ctcctgtgct ggactccgac 780

ggctccttct ttctgtactc ccgcctgacc gtggacaagt ccagatggca agagggcaac 840

gtgttctcct gctccgtgat gcacgaggcc ctgcacaatc actacaccca gaagtccctg 900

tctctgtccc tgggc 915

<210> 35

<211> 924

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 35

gccaccatga aatgggtcac ctttatctcc ctgctgttcc tgttctcctc cgcctactct 60

tgccctgagt ggtctacagc ttggggccct tgctctacca cctgtggact cggcatggcc 120

accagagtgt ctaaccagaa cagattctgc cggctggaaa cccagcggag actgtgcctg 180

tctagaccct gtcctcctag cagaggcaga tcccctcaga ccgagggcag aatggacgag 240

tctaagtacg gccctccttg tcctccatgt cctgctccag aagctgctgg cggcccttcc 300

gtgtttctgt tccctccaaa gcctaaggac accctgatga tctctcggac ccctgaagtg 360

acctgcgtgg tggtggatgt gtcccaagag gatcccgagg tgcagttcaa ttggtacgtg 420

gacggcgtgg aagtgcacaa cgccaagacc aagcctagag aggaacagtt caactccacc 480

tacagagtgg tgtccgtgct gaccgtgctg caccaggatt ggctgaacgg caaagagtac 540

aagtgcaagg tgtccaacaa gggcctgcct tccagcatcg aaaagaccat ctccaaggcc 600

aagggccagc ctagggaacc ccaggtttac accctgcctc caagccaaga ggaaatgacc 660

aagaaccagg tgtccctgac ctgcctggtc aagggcttct acccttccga tatcgccgtg 720

gaatggggaga gcaatggcca gcctgagaac aactacaaga cccacctcc tgtgctggac 780

tccgacggct ccttctttct gtactcccgc ctgaccgtgg acaagtccag atggcaagag 840

ggcaacgtgt tctcctgctc cgtgatgcac gaggccctgc acaatcacta cacccagaag 900

tccctgtctc tgtccctggg ctaa 924

<210> 36

<211> 997

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 36

ggggacaagt ttgtacaaaa aagcaggcta tggtaccgcc accatgaaat gggtcacctt 60

tatctccctg ctgttcctgt tctcctccgc ctactcttgc cctgagtggt ctacagcttg 120

gggcccttgc tctaccacct gtggactcgg catggccacc agagtgtcta accagaacag 180

attctgccgg ctggaaaccc agcggagact gtgcctgtct agaccctgtc ctcctagcag 240

aggcagatcc cctcagaccg agggcagaat ggacgagtct aagtacggcc ctccttgtcc 300

tccatgtcct gctccagaag ctgctggcgg cccttccgtg tttctgttcc ctccaaagcc 360

taaggacacc ctgatgatct ctcggacccc tgaagtgacc tgcgtggtgg tggatgtgtc 420

ccaagaggat cccgaggtgc agttcaattg gtacgtggac ggcgtggaag tgcacaacgc 480

caagaccaag cctagagagg aacagttcaa ctccacctac agagtggtgt ccgtgctgac 540

cgtgctgcac caggattggc tgaacggcaa agagtacaag tgcaaggtgt ccaacaaggg 600

cctgccttcc agcatcgaaa agaccatctc caaggccaag ggccagccta gggaacccca 660

ggtttacacc ctgcctccaa gccaagagga aatgaccaag aaccaggtgt ccctgacctg 720

cctggtcaag ggcttctacc cttccgatat cgccgtggaa tggggagagca atggccagcc 780

tgagaacaac tacaagacca cacctcctgt gctggactcc gacggctcct tctttctgta 840

ctcccgcctg accgtggaca agtccagatg gcaagagggc aacgtgttct cctgctccgt 900

gatgcacgag gccctgcaca atcactacac ccagaagtcc ctgtctctgt ccctgggcta 960

atctagaaac ccagctttct tgtacaaagt ggtcccc 997

<210> 37

<211> 44

<212> PRT/Protein

<213> Homo sapiens

<400> 37

Cys Pro Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys Gly

1 5 10 15

Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys Arg Leu

20 25 30

Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys

35 40

<210> 38

<211> 44

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Modified CCN5 sequence

<400> 38

Cys Ala Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys Gly

1 5 10 15

Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys Arg Leu

20 25 30

Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys

35 40

<210> 39

<211> 44

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Linker

<400> 39

Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

1 5 10 15

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu

20 25 30

Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala Ala Ala

35 40

<210> 40

<211> 325

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 40

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala

1 5 10 15

Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu

225 230 235 240

Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala

245 250 255

Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala Ala Ala

260 265 270

Cys Pro Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys Gly

275 280 285

Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys Arg Leu

290 295 300

Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Pro Pro Ser Arg

305 310 315 320

Gly Arg Ser Pro Gln

325

<210> 41

<211> 325

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 41

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala

1 5 10 15

Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu

225 230 235 240

Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala

245 250 255

Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala Ala Ala

260 265 270

Cys Ala Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys Gly

275 280 285

Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys Arg Leu

290 295 300

Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Pro Pro Ser Arg

305 310 315 320

Gly Arg Ser Pro Gln

325

<210> 42

<211> 44

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Modified CCN1 sequence

<400> 42

Cys Ala Val Gln Thr Thr Ser Trp Ser Gln Cys Ser Lys Thr Cys Gly

1 5 10 15

Thr Gly Ile Ser Thr Arg Val Thr Asn Asp Asn Pro Glu Cys Arg Leu

20 25 30

Val Lys Glu Thr Arg Ile Cys Glu Val Arg Pro Cys

35 40

<210> 43

<211> 44

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Modified CCN2 sequence

<400> 43

Cys Ala Val Gln Thr Thr Glu Trp Ser Ala Cys Ser Lys Thr Cys Gly

1 5 10 15

Met Gly Ile Ser Thr Arg Val Thr Asn Asp Asn Ala Ser Cys Arg Leu

20 25 30

Glu Lys Gln Ser Arg Leu Cys Met Val Arg Pro Cys

35 40

<210> 44

<211> 44

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Modified CCN3 sequence

<400> 44

Cys Ala Glu Gln Thr Thr Glu Trp Thr Ala Cys Ser Lys Ser Cys Gly

1 5 10 15

Met Gly Phe Ser Thr Arg Val Thr Asn Arg Asn Arg Gln Cys Glu Met

20 25 30

Leu Lys Gln Thr Arg Leu Cys Met Val Arg Pro Cys

35 40

<210> 45

<211> 44

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Modified CCN4 sequence

<400> 45

Cys Ala Ala Tyr Thr Ser Pro Trp Ser Pro Cys Ser Thr Ser Cys Gly

1 5 10 15

Leu Gly Val Ser Thr Arg Ile Ser Asn Val Asn Ala Gln Cys Trp Pro

20 25 30

Glu Gln Glu Ser Arg Leu Cys Asn Leu Arg Pro Cys

35 40

<210> 46

<211> 44

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Modified CCN6 sequence

<400> 46

Cys Ala Val Gln Ala Thr Lys Trp Thr Pro Cys Ser Arg Thr Cys Gly

1 5 10 15

Met Gly Ile Ser Asn Arg Val Thr Asn Glu Asn Ser Asn Cys Glu Met

20 25 30

Arg Lys Glu Lys Arg Leu Cys Tyr Ile Gln Pro Cys

35 40

<210> 47

<211> 52

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Modified CCN1 sequence

<400> 47

Cys Ala Val Gln Thr Thr Ser Trp Ser Gln Cys Ser Lys Thr Cys Gly

1 5 10 15

Thr Gly Ile Ser Thr Arg Val Thr Asn Asp Asn Pro Glu Cys Arg Leu

20 25 30

Val Lys Glu Thr Arg Ile Cys Glu Val Arg Pro Cys Gly Gln Pro Val

35 40 45

Tyr Ser Ser Leu

50

<210> 48

<211> 52

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Modified CCN2 sequence

<400> 48

Cys Ala Val Gln Thr Thr Glu Trp Ser Ala Cys Ser Lys Thr Cys Gly

1 5 10 15

Met Gly Ile Ser Thr Arg Val Thr Asn Asp Asn Ala Ser Cys Arg Leu

20 25 30

Glu Lys Gln Ser Arg Leu Cys Met Val Arg Pro Cys Glu Ala Asp Leu

35 40 45

Glu Glu Asn Ile

50

<210> 49

<211> 53

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Modified CCN3 sequence

<400> 49

Cys Ala Glu Gln Thr Thr Glu Trp Thr Ala Cys Ser Lys Ser Cys Gly

1 5 10 15

Met Gly Phe Ser Thr Arg Val Thr Asn Arg Asn Arg Gln Cys Glu Met

20 25 30

Leu Lys Gln Thr Arg Leu Cys Met Val Arg Pro Cys Glu Gln Glu Pro

35 40 45

Glu Gln Pro Thr Asp

50

<210> 50

<211> 52

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Modified CCN4 sequence

<400> 50

Cys Ala Ala Tyr Thr Ser Pro Trp Ser Pro Cys Ser Thr Ser Cys Gly

1 5 10 15

Leu Gly Val Ser Thr Arg Ile Ser Asn Val Asn Ala Gln Cys Trp Pro

20 25 30

Glu Gln Glu Ser Arg Leu Cys Asn Leu Arg Pro Cys Asp Val Asp Ile

35 40 45

His Thr Leu Ile

50

<210> 51

<211> 55

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Modified CCN6 sequence

<400> 51

Cys Ala Val Gln Ala Thr Lys Trp Thr Pro Cys Ser Arg Thr Cys Gly

1 5 10 15

Met Gly Ile Ser Asn Arg Val Thr Asn Glu Asn Ser Asn Cys Glu Met

20 25 30

Arg Lys Glu Lys Arg Leu Cys Tyr Ile Gln Pro Cys Asp Ser Asn Ile

35 40 45

Leu Lys Thr Ile Lys Ile Pro

50 55

<210> 52

<211> 585

<212> PRT/Protein

<213> Homo sapiens

<400> 52

Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu

1 5 10 15

Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln

20 25 30

Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu

35 40 45

Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys

50 55 60

Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu

65 70 75 80

Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro

85 90 95

Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu

100 105 110

Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His

115 120 125

Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg

130 135 140

Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg

145 150 155 160

Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala

165 170 175

Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser

180 185 190

Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu

195 200 205

Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro

210 215 220

Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys

225 230 235 240

Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp

245 250 255

Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser

260 265 270

Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His

275 280 285

Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser

290 295 300

Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala

305 310 315 320

Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg

325 330 335

Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr

340 345 350

Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu

355 360 365

Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro

370 375 380

Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu

385 390 395 400

Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro

405 410 415

Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys

420 425 430

Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys

435 440 445

Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His

450 455 460

Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser

465 470 475 480

Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr

485 490 495

Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp

500 505 510

Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala

515 520 525

Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu

530 535 540

Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys

545 550 555 560

Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val

565 570 575

Ala Ala Ser Gln Ala Ala Leu Gly Leu

580 585

<210> 53

<211> 679

<212> PRT/Protein

<213> Homo sapiens

<400> 53

Val Pro Asp Lys Thr Val Arg Trp Cys Ala Val Ser Glu His Glu Ala

1 5 10 15

Thr Lys Cys Gln Ser Phe Arg Asp His Met Lys Ser Val Ile Pro Ser

20 25 30

Asp Gly Pro Ser Val Ala Cys Val Lys Lys Ala Ser Tyr Leu Asp Cys

35 40 45

Ile Arg Ala Ile Ala Ala Asn Glu Ala Asp Ala Val Thr Leu Asp Ala

50 55 60

Gly Leu Val Tyr Asp Ala Tyr Leu Ala Pro Asn Asn Leu Lys Pro Val

65 70 75 80

Val Ala Glu Phe Tyr Gly Ser Lys Glu Asp Pro Gln Thr Phe Tyr Tyr

85 90 95

Ala Val Ala Val Val Lys Lys Asp Ser Gly Phe Gln Met Asn Gln Leu

100 105 110

Arg Gly Lys Lys Ser Cys His Thr Gly Leu Gly Arg Ser Ala Gly Trp

115 120 125

Asn Ile Pro Ile Gly Leu Leu Tyr Cys Asp Leu Pro Glu Pro Arg Lys

130 135 140

Pro Leu Glu Lys Ala Val Ala Asn Phe Phe Ser Gly Ser Cys Ala Pro

145 150 155 160

Cys Ala Asp Gly Thr Asp Phe Pro Gln Leu Cys Gln Leu Cys Pro Gly

165 170 175

Cys Gly Cys Ser Thr Leu Asn Gln Tyr Phe Gly Tyr Ser Gly Ala Phe

180 185 190

Lys Cys Leu Lys Asp Gly Ala Gly Asp Val Ala Phe Val Lys His Ser

195 200 205

Thr Ile Phe Glu Asn Leu Ala Asn Lys Ala Asp Arg Asp Gln Tyr Glu

210 215 220

Leu Leu Cys Leu Asp Asn Thr Arg Lys Pro Val Asp Glu Tyr Lys Asp

225 230 235 240

Cys His Leu Ala Gln Val Pro Ser His Thr Val Val Ala Arg Ser Met

245 250 255

Gly Gly Lys Glu Asp Leu Ile Trp Glu Leu Leu Asn Gln Ala Gln Glu

260 265 270

His Phe Gly Lys Asp Lys Ser Lys Glu Phe Gln Leu Phe Ser Ser Pro

275 280 285

His Gly Lys Asp Leu Leu Phe Lys Asp Ser Ala His Gly Phe Leu Lys

290 295 300

Val Pro Pro Arg Met Asp Ala Lys Met Tyr Leu Gly Tyr Glu Tyr Val

305 310 315 320

Thr Ala Ile Arg Asn Leu Arg Glu Gly Thr Cys Pro Glu Ala Pro Thr

325 330 335

Asp Glu Cys Lys Pro Val Lys Trp Cys Ala Leu Ser His His Glu Arg

340 345 350

Leu Lys Cys Asp Glu Trp Ser Val Asn Ser Val Gly Lys Ile Glu Cys

355 360 365

Val Ser Ala Glu Thr Thr Glu Asp Cys Ile Ala Lys Ile Met Asn Gly

370 375 380

Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Phe Val Tyr Ile Ala Gly

385 390 395 400

Lys Cys Gly Leu Val Pro Val Leu Ala Glu Asn Tyr Asn Lys Ser Asp

405 410 415

Asn Cys Glu Asp Thr Pro Glu Ala Gly Tyr Phe Ala Ile Ala Val Val

420 425 430

Lys Lys Ser Ala Ser Asp Leu Thr Trp Asp Asn Leu Lys Gly Lys Lys

435 440 445

Ser Cys His Thr Ala Val Gly Arg Thr Ala Gly Trp Asn Ile Pro Met

450 455 460

Gly Leu Leu Tyr Asn Lys Ile Asn His Cys Arg Phe Asp Glu Phe Phe

465 470 475 480

Ser Glu Gly Cys Ala Pro Gly Ser Lys Lys Asp Ser Ser Leu Cys Lys

485 490 495

Leu Cys Met Gly Ser Gly Leu Asn Leu Cys Glu Pro Asn Asn Lys Glu

500 505 510

Gly Tyr Tyr Gly Tyr Thr Gly Ala Phe Arg Cys Leu Val Glu Lys Gly

515 520 525

Asp Val Ala Phe Val Lys His Gln Thr Val Pro Gln Asn Thr Gly Gly

530 535 540

Lys Asn Pro Asp Pro Trp Ala Lys Asn Leu Asn Glu Lys Asp Tyr Glu

545 550 555 560

Leu Leu Cys Leu Asp Gly Thr Arg Lys Pro Val Glu Glu Tyr Ala Asn

565 570 575

Cys His Leu Ala Arg Ala Pro Asn His Ala Val Val Thr Arg Lys Asp

580 585 590

Lys Glu Ala Cys Val His Lys Ile Leu Arg Gln Gln Gln His Leu Phe

595 600 605

Gly Ser Asn Val Thr Asp Cys Ser Gly Asn Phe Cys Leu Phe Arg Ser

610 615 620

Glu Thr Lys Asp Leu Leu Phe Arg Asp Asp Thr Val Cys Leu Ala Lys

625 630 635 640

Leu His Asp Arg Asn Thr Tyr Glu Lys Tyr Leu Gly Glu Glu Tyr Val

645 650 655

Lys Ala Val Gly Asn Leu Arg Lys Cys Ser Thr Ser Ser Leu Leu Glu

660 665 670

Ala Cys Thr Phe Arg Arg Pro

675

<210> 54

<211> 232

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Modified Fc fragment

<400> 54

Glu Pro Lys Ser Gln Asp Lys Thr His Thr Gln Pro Pro Gln Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Cys Glu Glu Gln

65 70 75 80

Tyr Gly Ser Thr Tyr Arg Cys Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Arg Cys His Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Lys Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Thr Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 55

<211> 227

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Modified Fc fragment

<400> 55

Glu Arg Lys Gln Gln Val Glu Gln Pro Pro Gln Pro Ala Pro Pro Val

1 5 10 15

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

20 25 30

Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser

35 40 45

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

50 55 60

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

65 70 75 80

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

85 90 95

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

100 105 110

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

115 120 125

Val Tyr Thr Phe Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

130 135 140

Ser Leu Arg Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

145 150 155 160

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Lys

165 170 175

Pro Val Leu Asp Ser Asp Gly Ser Phe Arg Leu Glu Ser Arg Leu Thr

180 185 190

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

195 200 205

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

210 215 220

Ser Leu Gly

225

<210> 56

<211> 57

<212> PRT/Protein

<213> Homo sapiens

<400> 56

Cys Pro Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys Gly

1 5 10 15

Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys Arg Leu

20 25 30

Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Pro Pro Ser Arg

35 40 45

Gly Arg Ser Pro Gln Asn Ser Ala Phe

50 55

<210> 57

<211> 6

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Linker

<400> 57

Ile Glu Gly Arg Met Asp

1 5

<210> 58

<211> 291

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 58

Cys Pro Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys Gly

1 5 10 15

Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys Arg Leu

20 25 30

Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Pro Pro Ser Arg

35 40 45

Gly Arg Ser Pro Gln Asn Ser Ala Phe Ile Glu Gly Arg Met Asp Glu

50 55 60

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

65 70 75 80

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

85 90 95

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

100 105 110

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

115 120 125

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

130 135 140

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

145 150 155 160

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

165 170 175

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

180 185 190

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

195 200 205

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

210 215 220

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

225 230 235 240

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr

245 250 255

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

260 265 270

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

275 280 285

Ser Leu Gly

290

<210> 59

<211> 309

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 59

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Cys Pro Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr

20 25 30

Cys Gly Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys

35 40 45

Arg Leu Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Pro Pro

50 55 60

Ser Arg Gly Arg Ser Pro Gln Asn Ser Ala Phe Ile Glu Gly Arg Met

65 70 75 80

Asp Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu

85 90 95

Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

100 105 110

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

115 120 125

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

130 135 140

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

145 150 155 160

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

165 170 175

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

180 185 190

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

195 200 205

Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn

210 215 220

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

225 230 235 240

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

245 250 255

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg

260 265 270

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

275 280 285

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

290 295 300

Ser Leu Ser Leu Gly

305

<210> 60

<211> 936

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 60

gccaccatga aatgggtcac ctttatctcc ctgctgttcc tgttctcctc cgcctactct 60

tgccctgagt ggtctacagc ttggggccct tgctctacca cctgtggact cggcatggcc 120

accagagtgt ctaaccagaa cagattctgc cggctggaaa cccagcggag actgtgtctg 180

tccagacctt gtcctcctag ccggggcaga tcccctcaga actctgcctt tatcgaggc 240

agaatggacg agtctaagta cggccctcct tgtccaccat gtcctgctcc agaagctgct 300

ggcggccctt ccgtgtttct gttccctcca aagcctaagg acaccctgat gatctctcgg 360

acccctgaag tgacctgcgt ggtggtggat gtgtcccaag aggatcccga ggtgcagttc 420

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagcctag agaggaacag 480

ttcaactcca cctacagagt ggtgtccgtg ctgaccgtgc tgcaccagga ttggctgaac 540

ggcaaagagt acaagtgcaa ggtgtccaac aagggcctgc cttccagcat cgaaaagacc 600

atctccaagg ccaagggcca gcctagggaa ccccaggttt acaccctgcc tccaagccaa 660

gaggaaatga ccaagaacca ggtgtccctg acctgcctgg tcaagggctt ctacccttcc 720

gatatcgccg tggaatggga gagcaatggc cagcctgaga acaactacaa gaccacacct 780

cctgtgctgg actccgacgg ctccttcttt ctgtactccc gcctgaccgt ggacaagtcc 840

agatggcaag agggcaacgt gttctcctgc tccgtgatgc acgaggccct gcacaatcac 900

tacacccaga agtccctgtc tctgtccctg ggctaa 936

<210> 61

<211> 1009

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 61

ggggacaagt ttgtacaaaa aagcaggcta tggtaccgcc accatgaaat gggtcacctt 60

tatctccctg ctgttcctgt tctcctccgc ctactcttgc cctgagtggt ctacagcttg 120

gggcccttgc tctaccacct gtggactcgg catggccacc agagtgtcta accagaacag 180

attctgccgg ctggaaaccc agcggagact gtgtctgtcc agaccttgtc ctcctagccg 240

gggcagatcc cctcagaact ctgcctttat cgagggcaga atggacgagt ctaagtacgg 300

ccctccttgt ccaccatgtc ctgctccaga agctgctggc ggcccttccg tgtttctgtt 360

ccctccaaag cctaaggaca ccctgatgat ctctcggacc cctgaagtga cctgcgtggt 420

ggtggatgtg tcccaagagg atcccgaggt gcagttcaat tggtacgtgg acggcgtgga 480

agtgcacaac gccaagacca agcctagaga ggaacagttc aactccacct acagagtggt 540

gtccgtgctg accgtgctgc accaggattg gctgaacggc aaagagtaca agtgcaaggt 600

gtccaacaag ggcctgcctt ccagcatcga aaagaccatc tccaaggcca agggccagcc 660

tagggaaccc caggtttaca ccctgcctcc aagccaagag gaaatgacca agaaccaggt 720

gtccctgacc tgcctggtca agggcttcta cccttccgat atcgccgtgg aatggggagag 780

caatggccag cctgagaaca actacaagac cacacctcct gtgctggact ccgacggctc 840

cttctttctg tactcccgcc tgaccgtgga caagtccaga tggcaagagg gcaacgtgtt 900

ctcctgctcc gtgatgcacg aggccctgca caatcactac acccagaagt ccctgtctct 960

gtccctgggc taatctagaa acccagcttt cttgtacaaa gtggtcccc 1009

<210> 62

<211> 56

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Modified CCN5 sequence

<400> 62

Cys Pro Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys Gly

1 5 10 15

Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys Arg Leu

20 25 30

Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Pro Pro Ser Arg

35 40 45

Gly Arg Ser Leu Gln Asn Ser Ala

50 55

<210> 63

<211> 4

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Linker

<400> 63

Gly Arg Met Asp

1

<210> 64

<211> 306

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 64

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Cys Pro Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr

20 25 30

Cys Gly Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys

35 40 45

Arg Leu Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Pro Pro

50 55 60

Ser Arg Gly Arg Ser Leu Gln Asn Ser Ala Gly Arg Met Asp Glu Ser

65 70 75 80

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

85 90 95

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

100 105 110

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

115 120 125

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

130 135 140

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

145 150 155 160

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

165 170 175

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

180 185 190

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

195 200 205

Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

210 215 220

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

225 230 235 240

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

245 250 255

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val

260 265 270

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

275 280 285

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

290 295 300

Leu Gly

305

<210> 65

<211> 6

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Linker

<400> 65

Thr Glu Gly Arg Met Asp

1 5

<210> 66

<211> 305

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 66

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Cys Pro Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr

20 25 30

Cys Gly Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys

35 40 45

Arg Leu Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Pro Pro

50 55 60

Ser Arg Gly Arg Ser Pro Gln Thr Glu Gly Arg Met Asp Glu Ser Lys

65 70 75 80

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

85 90 95

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

100 105 110

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

115 120 125

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

130 135 140

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

145 150 155 160

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

165 170 175

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

180 185 190

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

195 200 205

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

210 215 220

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

225 230 235 240

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

245 250 255

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

260 265 270

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

275 280 285

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

290 295 300

Gly

305

<210> 67

<211> 8

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Linker

<400> 67

Thr Ala Glu Ala Ala Ala Lys Ala

1 5

<210> 68

<211> 307

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 68

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Cys Pro Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr

20 25 30

Cys Gly Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys

35 40 45

Arg Leu Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Pro Pro

50 55 60

Ser Arg Gly Arg Ser Pro Gln Thr Ala Glu Ala Ala Ala Lys Ala Glu

65 70 75 80

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala

85 90 95

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

100 105 110

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

115 120 125

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

130 135 140

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

145 150 155 160

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

165 170 175

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

180 185 190

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

195 200 205

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

210 215 220

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

225 230 235 240

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

245 250 255

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr

260 265 270

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

275 280 285

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

290 295 300

Ser Leu Gly

305

<210> 69

<211> 304

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 69

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Cys Pro Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr

20 25 30

Cys Gly Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys

35 40 45

Arg Leu Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Pro Pro

50 55 60

Ser Arg Gly Arg Ser Pro Gln Thr Glu Gly Arg Met Asp Glu Arg Lys

65 70 75 80

Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro

85 90 95

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

100 105 110

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp

115 120 125

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

130 135 140

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val

145 150 155 160

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

165 170 175

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

180 185 190

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

195 200 205

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

210 215 220

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

225 230 235 240

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

245 250 255

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys

260 265 270

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

275 280 285

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

290 295 300

<210> 70

<211> 294

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 70

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Cys Ala Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr

20 25 30

Cys Gly Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys

35 40 45

Arg Leu Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Glu Ala

50 55 60

Ala Ala Lys Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala

65 70 75 80

Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

85 90 95

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

100 105 110

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

115 120 125

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

130 135 140

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

145 150 155 160

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

165 170 175

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

180 185 190

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

195 200 205

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

210 215 220

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

225 230 235 240

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

245 250 255

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

260 265 270

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

275 280 285

Leu Ser Leu Ser Leu Gly

290

<210> 71

<211> 891

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 71

gccaccatga aatgggtcac ctttatctcc ctgctgttcc tgttctcctc cgcctactct 60

tgcgccgagt ggtctacagc ttggggccct tgttctacca cctgtggcct cggcatggcc 120

accagagtgt ccaaccagaa cagattctgc cggctggaaa cccagcggag actgtgtttg 180

tccagacctt gcgaggccgc tgccaaagaa agaaagtgct gcgtggaatg ccctccttgt 240

cctgctcctc ctgtggctgg cccttccgtg tttctgttcc ctccaaagcc taaggacacc 300

ctgatgatct ctcggacccc tgaagtgacc tgcgtggtgg tggatgtgtc ccaagaggat 360

cccgaggtgc agttcaattg gtacgtggac ggcgtggaag tgcacaacgc caagaccaag 420

cctagagagg aacagttcaa ctccacctac agagtggtgt ccgtgctgac cgtgctgcac 480

caggattggc tgaacggcaa agagtacaag tgcaaggtgt ccaacaaggg actgccctcc 540

agcatcgaaa agaccatctc caaggccaag ggacagccca gagaacccca ggtgtacaca 600

ctgcctccaa gccaagagga aatgaccaag aaccaggtgt ccctgacctg cctggtcaag 660

ggcttctacc cttccgatat cgccgtggaa tgggagtcca atggccagcc tgagaacaac 720

tacaagacca cacctccagt gctggactcc gacggctcct tctttctgta ctcccgcctg 780

accgtggaca agtccagatg gcaagagggc aacgtgttct cctgctccgt gatgcacgag 840

gccctgcaca atcactacac ccagaagtcc ctgtctctgt ccctgggcta a 891

<210> 72

<211> 964

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 72

ggggacaagt ttgtacaaaa aagcaggcta tggtaccgcc accatgaaat gggtcacctt 60

tatctccctg ctgttcctgt tctcctccgc ctactcttgc gccgagtggt ctacagcttg 120

gggcccttgt tctaccacct gtggcctcgg catggccacc agagtgtcca accagaacag 180

attctgccgg ctggaaaccc agcggagact gtgtttgtcc agaccttgcg aggccgctgc 240

caaagaaaga aagtgctgcg tggaatgccc tccttgtcct gctcctcctg tggctggccc 300

ttccgtgttt ctgttccctc caaagcctaa ggacaccctg atgatctctc ggacccctga 360

agtgacctgc gtggtggtgg atgtgtccca agaggatccc gaggtgcagt tcaattggta 420

cgtggacggc gtggaagtgc acaacgccaa gaccaagcct agagaggaac agttcaactc 480

cacctacaga gtggtgtccg tgctgaccgt gctgcaccag gattggctga acggcaaaga 540

gtacaagtgc aaggtgtcca acaagggact gccctccagc atcgaaaaga ccatctccaa 600

ggccaaggga cagcccagag aaccccaggt gtacacactg cctccaagcc aagaggaaat 660

gaccaagaac caggtgtccc tgacctgcct ggtcaagggc ttctaccctt ccgatatcgc 720

cgtggaatgg gagtccaatg gccagcctga gaacaactac aagaccacac ctccagtgct 780

ggactccgac ggctccttct ttctgtactc ccgcctgacc gtggacaagt ccagatggca 840

agagggcaac gtgttctcct gctccgtgat gcacgaggcc ctgcacaatc actacaccca 900

gaagtccctg tctctgtccc tgggctaatc tagaaaccca gctttcttgt acaaagtggt 960

cccc 964

<210> 73

<211> 276

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 73

Cys Ala Glu Gln Thr Thr Glu Trp Thr Ala Cys Ser Lys Ser Cys Gly

1 5 10 15

Met Gly Phe Ser Thr Arg Val Thr Asn Arg Asn Arg Gln Cys Glu Met

20 25 30

Leu Lys Gln Thr Arg Leu Cys Met Val Arg Pro Cys Glu Ala Ala Ala

35 40 45

Lys Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro

50 55 60

Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

65 70 75 80

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

85 90 95

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

100 105 110

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

115 120 125

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

130 135 140

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

145 150 155 160

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

165 170 175

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

180 185 190

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

195 200 205

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

210 215 220

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

225 230 235 240

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

245 250 255

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

260 265 270

Leu Ser Leu Gly

275

<210> 74

<211> 294

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 74

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Cys Ala Glu Gln Thr Thr Glu Trp Thr Ala Cys Ser Lys Ser

20 25 30

Cys Gly Met Gly Phe Ser Thr Arg Val Thr Asn Arg Asn Arg Gln Cys

35 40 45

Glu Met Leu Lys Gln Thr Arg Leu Cys Met Val Arg Pro Cys Glu Ala

50 55 60

Ala Ala Lys Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala

65 70 75 80

Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

85 90 95

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

100 105 110

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

115 120 125

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

130 135 140

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

145 150 155 160

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

165 170 175

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

180 185 190

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

195 200 205

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

210 215 220

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

225 230 235 240

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

245 250 255

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

260 265 270

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

275 280 285

Leu Ser Leu Ser Leu Gly

290

<210> 75

<211> 891

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 75

gccaccatga aatgggtcac ctttatctcc ctgctgttcc tgttctcctc cgcctactct 60

tgtgccgagc agaccacaga gtggaccgcc tgctctaagt cttgcggcat gggcttctcc 120

accagagtga ccaaccggaa cagacagtgc gagatgctga agcagacccg gctgtgtatg 180

gttcgacctt gcgaggccgc tgccaaagaa agaaagtgct gcgtggaatg ccctccttgt 240

cctgctcctc ctgtggctgg cccttccgtg tttctgttcc ctccaaagcc taaggacacc 300

ctgatgatct ctcggacccc tgaagtgacc tgcgtggtgg tggatgtgtc ccaagaggat 360

cccgaggtgc agttcaattg gtacgtggac ggcgtggaag tgcacaacgc caagaccaag 420

cctagagagg aacagttcaa ctccacctac agagtggtgt ccgtgctgac cgtgctgcac 480

caggattggc tgaacggcaa agagtacaag tgcaaggtgt ccaacaaggg cctgccttcc 540

agcatcgaaa agaccatctc caaggccaag ggacagccca gagaacccca ggtgtacaca 600

ctgcctccaa gccaagagga aatgaccaag aaccaggtgt ccctgacctg cctggtcaag 660

ggcttctacc cttccgatat cgccgtggaa tgggagtcca atggccagcc tgagaacaac 720

tacaagacca cacctccagt gctggactcc gacggctcct tctttctgta ctcccgcctg 780

accgtggaca agtccagatg gcaagagggc aacgtgttct cctgctccgt gatgcacgag 840

gccctgcaca atcactacac ccagaagtcc ctgtctctgt ccctgggcta a 891

<210> 76

<211> 964

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 76

ggggacaagt ttgtacaaaa aagcaggcta tggtaccgcc accatgaaat gggtcacctt 60

tatctccctg ctgttcctgt tctcctccgc ctactcttgt gccgagcaga ccacagagtg 120

gaccgcctgc tctaagtctt gcggcatggg cttctccacc agagtgacca accggaacag 180

acagtgcgag atgctgaagc agacccggct gtgtatggtt cgaccttgcg aggccgctgc 240

caaagaaaga aagtgctgcg tggaatgccc tccttgtcct gctcctcctg tggctggccc 300

ttccgtgttt ctgttccctc caaagcctaa ggacaccctg atgatctctc ggacccctga 360

agtgacctgc gtggtggtgg atgtgtccca agaggatccc gaggtgcagt tcaattggta 420

cgtggacggc gtggaagtgc acaacgccaa gaccaagcct agagaggaac agttcaactc 480

cacctacaga gtggtgtccg tgctgaccgt gctgcaccag gattggctga acggcaaaga 540

gtacaagtgc aaggtgtcca acaagggcct gccttccagc atcgaaaaga ccatctccaa 600

ggccaaggga cagcccagag aaccccaggt gtacacactg cctccaagcc aagaggaaat 660

gaccaagaac caggtgtccc tgacctgcct ggtcaagggc ttctaccctt ccgatatcgc 720

cgtggaatgg gagtccaatg gccagcctga gaacaactac aagaccacac ctccagtgct 780

ggactccgac ggctccttct ttctgtactc ccgcctgacc gtggacaagt ccagatggca 840

agagggcaac gtgttctcct gctccgtgat gcacgaggcc ctgcacaatc actacaccca 900

gaagtccctg tctctgtccc tgggctaatc tagaaaccca gctttcttgt acaaagtggt 960

cccc 964

<210> 77

<211> 343

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 77

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

20 25 30

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

35 40 45

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

50 55 60

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

65 70 75 80

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

85 90 95

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

100 105 110

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

115 120 125

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

130 135 140

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

145 150 155 160

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

165 170 175

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

180 185 190

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

195 200 205

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

210 215 220

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

225 230 235 240

Leu Ser Leu Ser Leu Gly Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala

245 250 255

Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

260 265 270

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala

275 280 285

Ala Ala Cys Ala Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr

290 295 300

Cys Gly Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys

305 310 315 320

Arg Leu Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Pro Pro

325 330 335

Ser Arg Gly Arg Ser Pro Gln

340

<210> 78

<211> 1038

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 78

gccaccatga aatgggtcac ctttatctcc ctgctgttcc tgttctccag cgcctactcc 60

gagtctaagt acggccctcc ttgtcctcca tgtcctgctc cagaagctgc tggcggccct 120

tccgtgtttc tgttccctcc aaagcctaag gacaccctga tgatctctcg gacccctgaa 180

gtgacctgcg tggtggtgga tgtgtcccaa gaggatcccg aggtgcagtt caattggtac 240

gtggacggcg tggaagtgca caacgccaag accaagccta gagaggaaca gttcaactcc 300

acctacagag tggtgtccgt gctgaccgtg ctgcaccagg attggctgaa cggcaaagag 360

tacaagtgca aggtgtccaa caagggcctg ccttccagca tcgaaaagac catctccaag 420

gccaagggcc agcctaggga accccaggtt tacaccctgc ctccaagcca agaggaaatg 480

accaagaacc aggtgtccct gacctgcctg gtcaagggct tctacccttc cgatatcgcc 540

gtggaatggg agagcaatgg ccagcctgag aacaactaca agaccacacc tcctgtgctg 600

gactccgacg gctccttctt tctgtactcc cgcctgaccg tggacaagtc cagatggcaa 660

gagggcaacg tgttctcctg ctccgtgatg cacgaggccc tgcacaatca ctacacccag 720

aagtccctgt ctctgtccct gggagctgag gccgctgcta aagaagctgc cgctaaagag 780

gccgcagcca aagaggcagc cgccaaagaa gccgctgcaa aagaggctgc tgcaaaagaa 840

gcagcagcta aagaagctgc tgccaaggcc gctgcttgtg ccgaatggtc tacagcttgg 900

ggcccttgct ctaccacctg tggactcggc atggccacca gagtgtctaa ccagaacaga 960

ttctgccggc tggaaaccca gcggagactg tgcctgtcta gaccctgtcc tcctagcaga 1020

ggcagatccc ctcagtga 1038

<210> 79

<211> 1111

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 79

ggggacaagt ttgtacaaaa aagcaggcta tggtaccgcc accatgaaat gggtcacctt 60

tatctccctg ctgttcctgt tctccagcgc ctactccgag tctaagtacg gccctccttg 120

tcctccatgt cctgctccag aagctgctgg cggcccttcc gtgtttctgt tccctccaaa 180

gcctaaggac accctgatga tctctcggac ccctgaagtg acctgcgtgg tggtggatgt 240

gtcccaagag gatcccgagg tgcagttcaa ttggtacgtg gacggcgtgg aagtgcacaa 300

cgccaagacc aagcctagag aggaacagtt caactccacc tacagagtgg tgtccgtgct 360

gaccgtgctg caccaggatt ggctgaacgg caaagagtac aagtgcaagg tgtccaacaa 420

gggcctgcct tccagcatcg aaaagaccat ctccaaggcc aagggccagc ctagggaacc 480

ccaggtttac accctgcctc caagccaaga ggaaatgacc aagaaccagg tgtccctgac 540

ctgcctggtc aagggcttct acccttccga tatcgccgtg gaatgggaga gcaatggcca 600

gcctgagaac aactacaaga ccacacctcc tgtgctggac tccgacggct ccttctttct 660

gtactcccgc ctgaccgtgg acaagtccag atggcaagag ggcaacgtgt tctcctgctc 720

cgtgatgcac gaggccctgc acaatcacta cacccagaag tccctgtctc tgtccctggg 780

agctgaggcc gctgctaaag aagctgccgc taaagaggcc gcagccaaag aggcagccgc 840

caaagaagcc gctgcaaaag aggctgctgc aaaagaagca gcagctaaag aagctgctgc 900

caaggccgct gcttgtgccg aatggtctac agcttggggc ccttgctcta ccacctgtgg 960

actcggcatg gccaccagag tgtctaacca gaacagattc tgccggctgg aaacccagcg 1020

gagactgtgc ctgtctagac cctgtcctcc tagcagaggc agatcccctc agtgatctag 1080

aaacccagct ttcttgtaca aagtggtccc c 1111

<210> 80

<211> 311

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 80

Cys Ala Glu Gln Thr Thr Glu Trp Thr Ala Cys Ser Lys Ser Cys Gly

1 5 10 15

Met Gly Phe Ser Thr Arg Val Thr Asn Arg Asn Arg Gln Cys Glu Met

20 25 30

Leu Lys Gln Thr Arg Leu Cys Met Val Arg Pro Cys Glu Ala Ala Ala

35 40 45

Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

50 55 60

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu

65 70 75 80

Ala Ala Ala Lys Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro

85 90 95

Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

100 105 110

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

115 120 125

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

130 135 140

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

145 150 155 160

Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

165 170 175

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly

180 185 190

Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

195 200 205

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr

210 215 220

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

225 230 235 240

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

245 250 255

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

260 265 270

Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe

275 280 285

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

290 295 300

Ser Leu Ser Leu Ser Leu Gly

305 310

<210> 81

<211> 329

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 81

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Cys Ala Glu Gln Thr Thr Glu Trp Thr Ala Cys Ser Lys Ser

20 25 30

Cys Gly Met Gly Phe Ser Thr Arg Val Thr Asn Arg Asn Arg Gln Cys

35 40 45

Glu Met Leu Lys Gln Thr Arg Leu Cys Met Val Arg Pro Cys Glu Ala

50 55 60

Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala

65 70 75 80

Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala

85 90 95

Lys Glu Ala Ala Ala Lys Glu Arg Lys Cys Cys Val Glu Cys Pro Pro

100 105 110

Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Leu Gly

325

<210> 82

<211> 996

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 82

gccaccatga aatgggtcac ctttatctcc ctgctgttcc tgttctcctc cgcctactct 60

tgtgccgagc agaccacaga gtggaccgcc tgctctaagt cttgcggcat gggcttctcc 120

accagagtga ccaaccggaa cagacagtgc gagatgctga agcagacccg gctgtgtatg 180

gttcgacctt gcgaggccgc tgccaaagag gctgctgcta aagaagccgc cgcaaaagag 240

gcagcagcaa aagaggctgc cgccaaagag gccgcagcca aagaagcagc agctaaagag 300

gccgctgcaa aagaacggaa gtgctgcgtg gaatgccctc cttgtcctgc tcctcctgtg 360

gctggccctt ccgtgtttct gttccctcca aagcctaagg acaccctgat gatctctcgg 420

acccctgaag tgacctgcgt ggtggtggat gtgtcccaag aggatcccga ggtgcagttc 480

aattggtacg tggacggcgt ggaagtgcac aacgccaaga ccaagcctag agaggaacag 540

ttcaactcca cctacagagt ggtgtccgtg ctgaccgtgc tgcaccagga ttggctgaac 600

ggcaaagagt acaagtgcaa ggtgtccaac aagggcctgc cttccagcat cgaaaagacc 660

atctccaagg ccaagggaca gcccagagaa ccccaggtgt acacactgcc tccaagccaa 720

gaggaaatga ccaagaacca ggtgtccctg acctgcctgg tcaagggctt ctacccttcc 780

gatatcgccg tggaatggga gtccaatggc cagcctgaga acaactacaa gaccacacct 840

ccagtgctgg actccgacgg ctccttcttt ctgtactccc gcctgaccgt ggacaagtcc 900

agatggcaag agggcaacgt gttctcctgc tccgtgatgc acgaggccct gcacaatcac 960

tacacccaga agtccctgtc tctgtccctg ggctaa 996

<210> 83

<211> 1069

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 83

ggggacaagt ttgtacaaaa aagcaggcta tggtaccgcc accatgaaat gggtcacctt 60

tatctccctg ctgttcctgt tctcctccgc ctactcttgt gccgagcaga ccacagagtg 120

gaccgcctgc tctaagtctt gcggcatggg cttctccacc agagtgacca accggaacag 180

acagtgcgag atgctgaagc agacccggct gtgtatggtt cgaccttgcg aggccgctgc 240

caaagaggct gctgctaaag aagccgccgc aaaagaggca gcagcaaaag aggctgccgc 300

caaagaggcc gcagccaaag aagcagcagc taaagaggcc gctgcaaaag aacggaagtg 360

ctgcgtggaa tgccctcctt gtcctgctcc tcctgtggct ggcccttccg tgtttctgtt 420

ccctccaaag cctaaggaca ccctgatgat ctctcggacc cctgaagtga cctgcgtggt 480

ggtggatgtg tcccaagagg atcccgaggt gcagttcaat tggtacgtgg acggcgtgga 540

agtgcacaac gccaagacca agcctagaga ggaacagttc aactccacct acagagtggt 600

gtccgtgctg accgtgctgc accaggattg gctgaacggc aaagagtaca agtgcaaggt 660

gtccaacaag ggcctgcctt ccagcatcga aaagaccatc tccaaggcca agggacagcc 720

cagagaaccc caggtgtaca cactgcctcc aagccaagag gaaatgacca agaaccaggt 780

gtccctgacc tgcctggtca agggcttcta cccttccgat atcgccgtgg aatgggagtc 840

caatggccag cctgagaaca actacaagac cacacctcca gtgctggact ccgacggctc 900

cttctttctg tactcccgcc tgaccgtgga caagtccaga tggcaagagg gcaacgtgtt 960

ctcctgctcc gtgatgcacg aggccctgca caatcactac acccagaagt ccctgtctct 1020

gtccctgggc taatctagaa acccagcttt cttgtacaaa gtggtcccc 1069

<210> 84

<211> 276

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 84

Cys Ala Glu Gln Thr Thr Glu Trp Thr Ala Cys Ser Lys Ser Cys Gly

1 5 10 15

Met Gly Phe Ser Thr Arg Val Thr Asn Arg Asn Arg Gln Cys Glu Met

20 25 30

Leu Lys Gln Thr Arg Leu Cys Met Val Arg Pro Cys Glu Ala Ala Ala

35 40 45

Lys Glu Arg Lys Gln Gln Val Glu Gln Pro Pro Gln Pro Ala Pro Pro

50 55 60

Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

65 70 75 80

Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val

85 90 95

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

100 105 110

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

115 120 125

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

130 135 140

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

145 150 155 160

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

165 170 175

Gln Val Tyr Thr Phe Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

180 185 190

Val Ser Leu Arg Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

195 200 205

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

210 215 220

Lys Pro Val Leu Asp Ser Asp Gly Ser Phe Arg Leu Glu Ser Arg Leu

225 230 235 240

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

245 250 255

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

260 265 270

Leu Ser Leu Gly

275

<210> 85

<211> 294

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 85

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Cys Ala Glu Gln Thr Thr Glu Trp Thr Ala Cys Ser Lys Ser

20 25 30

Cys Gly Met Gly Phe Ser Thr Arg Val Thr Asn Arg Asn Arg Gln Cys

35 40 45

Glu Met Leu Lys Gln Thr Arg Leu Cys Met Val Arg Pro Cys Glu Ala

50 55 60

Ala Ala Lys Glu Arg Lys Gln Gln Val Glu Gln Pro Pro Gln Pro Ala

65 70 75 80

Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

85 90 95

Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val

100 105 110

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

115 120 125

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

130 135 140

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

145 150 155 160

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

165 170 175

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

180 185 190

Glu Pro Gln Val Tyr Thr Phe Pro Pro Ser Gln Glu Glu Met Thr Lys

195 200 205

Asn Gln Val Ser Leu Arg Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

210 215 220

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

225 230 235 240

Thr Thr Lys Pro Val Leu Asp Ser Asp Gly Ser Phe Arg Leu Glu Ser

245 250 255

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

260 265 270

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

275 280 285

Leu Ser Leu Ser Leu Gly

290

<210> 86

<211> 891

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 86

gccaccatga aatgggtcac ctttatctcc ctgctgttcc tgttctcctc cgcctactct 60

tgtgccgagc agaccacaga gtggaccgcc tgctctaagt cttgcggcat gggcttctcc 120

accagagtga ccaaccggaa cagacagtgc gagatgctga agcagacccg gctgtgtatg 180

gttcgacctt gcgaggccgc tgccaaagaa agaaagcagc aggtcgagca gcctcctcag 240

cctgctcctc ctgttgctgg cccttccgtg tttctgttcc ctccaaagcc taaggacacc 300

ctgtacatca cccgcgagcc tgaagtgacc tgcgtggtgg tggatgtgtc ccaagaggat 360

cccgaggtgc agttcaattg gtacgtggac ggcgtggaag tgcacaacgc caagaccaag 420

cctagagagg aacagttcaa ctccacctac agagtggtgt ccgtgctgac cgtgctgcac 480

caggattggc tgaacggcaa agagtacaag tgcaaggtgt ccaacaaggg cctgccttcc 540

agcatcgaaa agaccatctc caaggccaag ggacagccca gagaacccca ggtgtacaca 600

ttccctccat ctcaagagga aatgaccaag aaccaggtgt ccctgcggtg cctggtcaag 660

ggcttctacc cttctgatat cgccgtggaa tgggagtcca acggccagcc tgagaacaac 720

tacaagacca ccaagcctgt gctggactcc gacggctcct tccggcttga atctagactg 780

accgtggaca agtcccggtg gcaagagggc aacgtgttct cctgctctgt gatgcacgag 840

gccctgcaca accactacac ccagaagtcc ctgtctctgt ccctgggcta a 891

<210> 87

<211> 964

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 87

ggggacaagt ttgtacaaaa aagcaggcta tggtaccgcc accatgaaat gggtcacctt 60

tatctccctg ctgttcctgt tctcctccgc ctactcttgt gccgagcaga ccacagagtg 120

gaccgcctgc tctaagtctt gcggcatggg cttctccacc agagtgacca accggaacag 180

acagtgcgag atgctgaagc agacccggct gtgtatggtt cgaccttgcg aggccgctgc 240

caaagaaaga aagcagcagg tcgagcagcc tcctcagcct gctcctcctg ttgctggccc 300

ttccgtgttt ctgttccctc caaagcctaa ggacaccctg tacatcaccc gcgagcctga 360

agtgacctgc gtggtggtgg atgtgtccca agaggatccc gaggtgcagt tcaattggta 420

cgtggacggc gtggaagtgc acaacgccaa gaccaagcct agagaggaac agttcaactc 480

cacctacaga gtggtgtccg tgctgaccgt gctgcaccag gattggctga acggcaaaga 540

gtacaagtgc aaggtgtcca acaagggcct gccttccagc atcgaaaaga ccatctccaa 600

ggccaaggga cagcccagag aaccccaggt gtacacattc cctccatctc aagaggaaat 660

gaccaagaac caggtgtccc tgcggtgcct ggtcaagggc ttctaccctt ctgatatcgc 720

cgtggaatgg gagtccaacg gccagcctga gaacaactac aagaccacca agcctgtgct 780

ggactccgac ggctccttcc ggcttgaatc tagactgacc gtggacaagt cccggtggca 840

agagggcaac gtgttctcct gctctgtgat gcacgaggcc ctgcacaacc actacaccca 900

gaagtccctg tctctgtccc tgggctaatc tagaaaccca gctttcttgt acaaagtggt 960

cccc 964

<210> 88

<211> 281

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 88

Cys Ala Glu Gln Thr Thr Glu Trp Thr Ala Cys Ser Lys Ser Cys Gly

1 5 10 15

Met Gly Phe Ser Thr Arg Val Thr Asn Arg Asn Arg Gln Cys Glu Met

20 25 30

Leu Lys Gln Thr Arg Leu Cys Met Val Arg Pro Cys Glu Ala Ala Ala

35 40 45

Lys Glu Pro Lys Ser Gln Asp Lys Thr His Thr Gln Pro Pro Gln Pro

50 55 60

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

65 70 75 80

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

85 90 95

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

100 105 110

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Cys Glu Glu

115 120 125

Gln Tyr Gly Ser Thr Tyr Arg Cys Val Ser Val Leu Thr Val Leu His

130 135 140

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

145 150 155 160

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

165 170 175

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

180 185 190

Thr Lys Asn Gln Val Ser Leu Arg Cys His Val Lys Gly Phe Tyr Pro

195 200 205

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

210 215 220

Tyr Lys Thr Thr Lys Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

225 230 235 240

Tyr Ser Thr Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

245 250 255

Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln

260 265 270

Lys Ser Leu Ser Leu Ser Pro Gly Lys

275 280

<210> 89

<211> 299

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 89

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Cys Ala Glu Gln Thr Thr Glu Trp Thr Ala Cys Ser Lys Ser

20 25 30

Cys Gly Met Gly Phe Ser Thr Arg Val Thr Asn Arg Asn Arg Gln Cys

35 40 45

Glu Met Leu Lys Gln Thr Arg Leu Cys Met Val Arg Pro Cys Glu Ala

50 55 60

Ala Ala Lys Glu Pro Lys Ser Gln Asp Lys Thr His Thr Gln Pro Pro

65 70 75 80

Gln Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

85 90 95

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

100 105 110

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

115 120 125

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Cys

130 135 140

Glu Glu Gln Tyr Gly Ser Thr Tyr Arg Cys Val Ser Val Leu Thr Val

145 150 155 160

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

165 170 175

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

180 185 190

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

195 200 205

Glu Leu Thr Lys Asn Gln Val Ser Leu Arg Cys His Val Lys Gly Phe

210 215 220

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

225 230 235 240

Asn Asn Tyr Lys Thr Thr Lys Pro Val Leu Asp Ser Asp Gly Ser Phe

245 250 255

Phe Leu Tyr Ser Thr Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

260 265 270

Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr

275 280 285

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

290 295

<210> 90

<211> 906

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 90

gccaccatga aatgggtcac ctttatctcc ctgctgttcc tgttctcctc cgcctactct 60

tgtgccgagc agaccacaga gtggaccgcc tgctctaagt cttgcggcat gggcttctcc 120

accagagtga ccaaccggaa cagacagtgc gagatgctga agcagacccg gctgtgtatg 180

gttcgacctt gcgaggccgc tgccaaagag cctaagagcc aggacaagac ccacacacag 240

cctccacagc ctgctccaga attgctcgga ggcccttccg tgtttctgtt ccctccaaag 300

cctaaggaca ccctgatgat ctctcggacc cctgaagtga cctgcgtggt ggtggatgtg 360

tctcacgagg atcccgaagt gaagttcaat tggtacgtgg acggcgtgga agtgcacaac 420

gccaagacaa agccctgcga ggaacagtac ggctccacct acagatgcgt gtccgtgctg 480

acagtgctgc accaggattg gctgaacggc aaagagtaca agtgcaaggt gtccaacaag 540

gccctgcctg ctcctatcga aaagaccatc tccaaggcca agggccagcc tagagaaccc 600

caggtgtaca cactgccacc ttctagggac gagctgacca agaaccaggt gtccctgaga 660

tgccacgtga agggcttcta cccctccgat atcgccgtgg aatgggagtc taatggacag 720

cccgagaaca actacaagac caccaagcct gtgctggact ccgacggctc cttcttcctg 780

tactctaccc tgaccgtgga caagtccaga tggcagcagg gcaacgtgtt ctcctgctct 840

gtgctgcacg aggccctgca caatcactac acccagaagt ccctgtctct gtcccctggc 900

aagtga 906

<210> 91

<211> 979

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 91

ggggacaagt ttgtacaaaa aagcaggcta tggtaccgcc accatgaaat gggtcacctt 60

tatctccctg ctgttcctgt tctcctccgc ctactcttgt gccgagcaga ccacagagtg 120

gaccgcctgc tctaagtctt gcggcatggg cttctccacc agagtgacca accggaacag 180

acagtgcgag atgctgaagc agacccggct gtgtatggtt cgaccttgcg aggccgctgc 240

caaagagcct aagagccagg acaagacca cacacagcct ccacagcctg ctccagaatt 300

gctcggaggc ccttccgtgt ttctgttccc tccaaagcct aaggacaccc tgatgatctc 360

tcggacccct gaagtgacct gcgtggtggt ggatgtgtct cacgaggatc ccgaagtgaa 420

gttcaattgg tacgtggacg gcgtggaagt gcacaacgcc aagacaaagc cctgcgagga 480

acagtacggc tccacctaca gatgcgtgtc cgtgctgaca gtgctgcacc aggattggct 540

gaacggcaaa gagtacaagt gcaaggtgtc caacaaggcc ctgcctgctc ctatcgaaaa 600

gaccatctcc aaggccaagg gccagcctag agaaccccag gtgtacacac tgccaccttc 660

tagggacgag ctgaccaaga accaggtgtc cctgagatgc cacgtgaagg gcttctaccc 720

ctccgatatc gccgtggaat gggagtctaa tggacagccc gagaacaact acaagaccac 780

caagcctgtg ctggactccg acggctcctt cttcctgtac tctaccctga ccgtggacaa 840

gtccagatgg cagcagggca acgtgttctc ctgctctgtg ctgcacgagg ccctgcacaa 900

tcactacacc cagaagtccc tgtctctgtc ccctggcaag tgatctagaa acccagcttt 960

cttgtacaaa gtggtcccc 979

<210> 92

<211> 402

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Merged Label

<400> 92

Gly His His His His His Gly Ser Glu Ile Gly Thr Gly Phe Pro

1 5 10 15

Phe Asp Pro His Tyr Val Glu Val Leu Gly Glu Arg Met His Tyr Val

20 25 30

Asp Val Gly Pro Arg Asp Gly Thr Pro Val Leu Phe Leu His Gly Asn

35 40 45

Pro Thr Ser Ser Tyr Val Trp Arg Asn Ile Ile Pro His Val Ala Pro

50 55 60

Thr His Arg Cys Ile Ala Pro Asp Leu Ile Gly Met Gly Lys Ser Asp

65 70 75 80

Lys Pro Asp Leu Gly Tyr Phe Phe Asp Asp His Val Arg Phe Met Asp

85 90 95

Ala Phe Ile Glu Ala Leu Gly Leu Glu Glu Val Val Leu Val Ile His

100 105 110

Asp Trp Gly Ser Ala Leu Gly Phe His Trp Ala Lys Arg Asn Pro Glu

115 120 125

Arg Val Lys Gly Ile Ala Phe Met Glu Phe Ile Arg Pro Ile Pro Thr

130 135 140

Trp Asp Glu Trp Pro Glu Phe Ala Arg Glu Thr Phe Gln Ala Phe Arg

145 150 155 160

Thr Thr Asp Val Gly Arg Lys Leu Ile Ile Asp Gln Asn Val Phe Ile

165 170 175

Glu Gly Thr Leu Pro Met Gly Val Val Arg Pro Leu Thr Glu Val Glu

180 185 190

Met Asp His Tyr Arg Glu Pro Phe Leu Asn Pro Val Asp Arg Glu Pro

195 200 205

Leu Trp Arg Phe Pro Asn Glu Leu Pro Ile Ala Gly Glu Pro Ala Asn

210 215 220

Ile Val Ala Leu Val Glu Glu Tyr Met Asp Trp Leu His Gln Ser Pro

225 230 235 240

Val Pro Lys Leu Leu Phe Trp Gly Thr Pro Gly Val Leu Ile Pro Pro

245 250 255

Ala Glu Ala Ala Arg Leu Ala Lys Ser Leu Pro Asn Cys Lys Ala Val

260 265 270

Asp Ile Gly Pro Gly Leu Asn Leu Leu Gln Glu Asp Asn Pro Asp Leu

275 280 285

Ile Gly Ser Glu Ile Ala Arg Trp Leu Ser Thr Leu Glu Ile Ser Gly

290 295 300

Leu Gln Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro

305 310 315 320

Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser

325 330 335

Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu

340 345 350

Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Thr

355 360 365

Phe Leu Tyr Asp Gly Ile Glu Ile Gln Ala Asp Gln Thr Pro Glu Asp

370 375 380

Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile

385 390 395 400

Gly Gly

<210> 93

<211> 20

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Linker

<400> 93

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Ser Gly Gly

1 5 10 15

Gly Ser Ser Gly

20

<210> 94

<211> 466

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 94

Gly His His His His His Gly Ser Glu Ile Gly Thr Gly Phe Pro

1 5 10 15

Phe Asp Pro His Tyr Val Glu Val Leu Gly Glu Arg Met His Tyr Val

20 25 30

Asp Val Gly Pro Arg Asp Gly Thr Pro Val Leu Phe Leu His Gly Asn

35 40 45

Pro Thr Ser Ser Tyr Val Trp Arg Asn Ile Ile Pro His Val Ala Pro

50 55 60

Thr His Arg Cys Ile Ala Pro Asp Leu Ile Gly Met Gly Lys Ser Asp

65 70 75 80

Lys Pro Asp Leu Gly Tyr Phe Phe Asp Asp His Val Arg Phe Met Asp

85 90 95

Ala Phe Ile Glu Ala Leu Gly Leu Glu Glu Val Val Leu Val Ile His

100 105 110

Asp Trp Gly Ser Ala Leu Gly Phe His Trp Ala Lys Arg Asn Pro Glu

115 120 125

Arg Val Lys Gly Ile Ala Phe Met Glu Phe Ile Arg Pro Ile Pro Thr

130 135 140

Trp Asp Glu Trp Pro Glu Phe Ala Arg Glu Thr Phe Gln Ala Phe Arg

145 150 155 160

Thr Thr Asp Val Gly Arg Lys Leu Ile Ile Asp Gln Asn Val Phe Ile

165 170 175

Glu Gly Thr Leu Pro Met Gly Val Val Arg Pro Leu Thr Glu Val Glu

180 185 190

Met Asp His Tyr Arg Glu Pro Phe Leu Asn Pro Val Asp Arg Glu Pro

195 200 205

Leu Trp Arg Phe Pro Asn Glu Leu Pro Ile Ala Gly Glu Pro Ala Asn

210 215 220

Ile Val Ala Leu Val Glu Glu Tyr Met Asp Trp Leu His Gln Ser Pro

225 230 235 240

Val Pro Lys Leu Leu Phe Trp Gly Thr Pro Gly Val Leu Ile Pro Pro

245 250 255

Ala Glu Ala Ala Arg Leu Ala Lys Ser Leu Pro Asn Cys Lys Ala Val

260 265 270

Asp Ile Gly Pro Gly Leu Asn Leu Leu Gln Glu Asp Asn Pro Asp Leu

275 280 285

Ile Gly Ser Glu Ile Ala Arg Trp Leu Ser Thr Leu Glu Ile Ser Gly

290 295 300

Leu Gln Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro

305 310 315 320

Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser

325 330 335

Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu

340 345 350

Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Thr

355 360 365

Phe Leu Tyr Asp Gly Ile Glu Ile Gln Ala Asp Gln Thr Pro Glu Asp

370 375 380

Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile

385 390 395 400

Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Ser

405 410 415

Gly Gly Gly Ser Ser Gly Cys Ala Glu Gln Thr Thr Glu Trp Thr Ala

420 425 430

Cys Ser Lys Ser Cys Gly Met Gly Phe Ser Thr Arg Val Thr Asn Arg

435 440 445

Asn Arg Gln Cys Glu Met Leu Lys Gln Thr Arg Leu Cys Met Val Arg

450 455 460

Pro Cys

465

<210> 95

<211> 1461

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 95

gccaccatga aatgggtcac ctttatctcc ctgctgttcc tgttctcctc cgcctactct 60

ggccaccacc atcaccatca cggctccgag atcggaaccg gctttccttt cgaccctcac 120

tacgtggaag tgctgggcga gagaatgcac tatgtggacg tgggccccag agatggaacc 180

cctgtgctgt ttctgcacgg caaccctacc tccagctacg tgtggcggaa catcatccct 240

cacgtggccc ctacacacag atgtatcgcc cctgacctga tcggcatggg caagtctgac 300

aagcctgacc tgggctactt cttcgacgac cacgtgcggt tcatggacgc ctttatcgag 360

gctctgggcc tcgaagaggt ggtgctggtc atccatgatt ggggctctgc cctgggcttt 420

cactgggcca agagaaaccc cgagagagtg aagggaatcg ccttcatgga attcatccgg 480

cctattccta cctgggacga gtggcctgag ttcgccagag agacattcca ggccttcaga 540

accaccgacg tgggcagaaa gctgatcatc gaccagaacg tgttcatcga gggcaccctg 600

cctatgggag tcgtcagacc tctgaccgag gtggaaatgg accactacag agagcccttt 660

ctgaaccccg tggaccggga acctctttgg agattcccta acgagctgcc tatcgctggc 720

gagcctgcca atattgtggc cctggtggaa gagtacatgg actggctgca tcagagcccc 780

gtgcctaagc tgctgttttg gggaacaccc ggcgtgctga ttcctcctgc tgaagctgct 840

agactggcca agagcctgcc taactgcaag gccgtggata tcggccctgg cctgaatctg 900

ctgcaagagg acaaccccga tctgatcgga tctgagatcg cccggtggct gagcaccctg 960

gaaatcagtg gactgcagga ctccgaagtg aatcaagagg ccaagcctga agtgaagccc 1020

gaagtcaagc ctgagacaca catcaacctg aaggtgtccg acggctccag cgagatcttc 1080

ttcaagatca agaaaaccac acctctgcgg cggctgatgg aagcctttgc caagagacag 1140

ggcaaagaga tggactccct gaccttcctg tacgacggca tcgagatcca ggccgatcag 1200

acccctgagg acctggacat ggaagataac gacatcattg aggcccacag agagcagatc 1260

ggcggctctg gtggtagcgg aggttctggt ggatctggtg gttcttctgg cggcggatct 1320

tctggctgtg ctgagcagac aaccgagtgg accgcctgct ctaagtcttg tggcatgggc 1380

ttctccacca gagtgaccaa ccggaacaga cagtgcgaga tgctgaagca gacccggctg 1440

tgtatggtcc gaccttgcta a 1461

<210> 96

<211> 1552

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 96

ggggacaagt ttgtacaaaa aagcaggcta taagcttgct gccaccatga aatgggtcac 60

ctttatctcc ctgctgttcc tgttctcctc cgcctactct ggccaccacc atcaccatca 120

cggctccgag atcggaaccg gctttccttt cgaccctcac tacgtggaag tgctgggcga 180

gagaatgcac tatgtggacg tgggccccag agatggaacc cctgtgctgt ttctgcacgg 240

caaccctacc tccagctacg tgtggcggaa catcatccct cacgtggccc ctacacacag 300

atgtatcgcc cctgacctga tcggcatggg caagtctgac aagcctgacc tgggctactt 360

cttcgacgac cacgtgcggt tcatggacgc ctttatcgag gctctgggcc tcgaagaggt 420

ggtgctggtc atccatgatt ggggctctgc cctgggcttt cactgggcca agagaaaccc 480

cgagagagtg aagggaatcg ccttcatgga attcatccgg cctattccta cctgggacga 540

gtggcctgag ttcgccagag agacattcca ggccttcaga accaccgacg tgggcagaaa 600

gctgatcatc gaccagaacg tgttcatcga gggcaccctg cctatgggag tcgtcagacc 660

tctgaccgag gtggaaatgg accactacag agagcccttt ctgaaccccg tggaccggga 720

acctctttgg agattcccta acgagctgcc tatcgctggc gagcctgcca atattgtggc 780

cctggtggaa gagtacatgg actggctgca tcagagcccc gtgcctaagc tgctgttttg 840

gggaacaccc ggcgtgctga ttcctcctgc tgaagctgct agactggcca agagcctgcc 900

taactgcaag gccgtggata tcggccctgg cctgaatctg ctgcaagagg acaaccccga 960

tctgatcgga tctgagatcg cccggtggct gagcaccctg gaaatcagtg gactgcagga 1020

ctccgaagtg aatcaagagg ccaagcctga agtgaagccc gaagtcaagc ctgagacaca 1080

catcaacctg aaggtgtccg acggctccag cgagatcttc ttcaagatca agaaaaccac 1140

acctctgcgg cggctgatgg aagcctttgc caagagacag ggcaaagaga tggactccct 1200

gaccttcctg tacgacggca tcgagatcca ggccgatcag acccctgagg acctggacat 1260

ggaagataac gacatcattg aggcccacag agagcagatc ggcggctctg gtggtagcgg 1320

aggttctggt ggatctggtg gttcttctgg cggcggatct tctggctgtg ctgagcagac 1380

aaccgagtgg accgcctgct ctaagtcttg tggcatgggc ttctccacca gagtgaccaa 1440

ccggaacaga cagtgcgaga tgctgaagca gacccggctg tgtatggtcc gaccttgcta 1500

aatctagagc ggccgcggta ccaacccagc tttcttgtac aaagtggtcc cc 1552

<210> 97

<211> 634

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 97

Cys Ala Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys Gly

1 5 10 15

Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys Arg Leu

20 25 30

Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Glu Ala Ala Ala

35 40 45

Lys Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly

50 55 60

Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu

65 70 75 80

Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr

85 90 95

Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp

100 105 110

Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr

115 120 125

Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu

130 135 140

Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn

145 150 155 160

Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe

165 170 175

His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala

180 185 190

Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys

195 200 205

Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala

210 215 220

Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala

225 230 235 240

Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly

245 250 255

Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe

260 265 270

Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr

275 280 285

Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp

290 295 300

Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile

305 310 315 320

Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser

325 330 335

His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro

340 345 350

Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr

355 360 365

Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala

370 375 380

Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys

385 390 395 400

Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His

405 410 415

Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu

420 425 430

Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly

435 440 445

Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val

450 455 460

Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly

465 470 475 480

Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro

485 490 495

Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu

500 505 510

His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu

515 520 525

Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu

530 535 540

Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala

545 550 555 560

Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr

565 570 575

Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln

580 585 590

Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys

595 600 605

Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu

610 615 620

Val Ala Ala Ser Gln Ala Ala Leu Gly Leu

625 630

<210> 98

<211> 652

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 98

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Cys Ala Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr

20 25 30

Cys Gly Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys

35 40 45

Arg Leu Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Glu Ala

50 55 60

Ala Ala Lys Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp

65 70 75 80

Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln

85 90 95

Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu

100 105 110

Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn

115 120 125

Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val

130 135 140

Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys

145 150 155 160

Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn

165 170 175

Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr

180 185 190

Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu

195 200 205

Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe

210 215 220

Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp

225 230 235 240

Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly

245 250 255

Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys

260 265 270

Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln

275 280 285

Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp

290 295 300

Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys

305 310 315 320

Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp

325 330 335

Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu

340 345 350

Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp

355 360 365

Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys

370 375 380

Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu

385 390 395 400

Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu

405 410 415

Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp

420 425 430

Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val

435 440 445

Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln

450 455 460

Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys

465 470 475 480

Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn

485 490 495

Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg

500 505 510

Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys

515 520 525

Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys

530 535 540

Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val

545 550 555 560

Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe

565 570 575

His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys

580 585 590

Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys

595 600 605

Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys

610 615 620

Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys

625 630 635 640

Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu

645 650

<210> 99

<211> 1965

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 99

gccaccatga aatgggtcac ctttatctcc ctgctgttcc tgttctcctc cgcctactct 60

tgcgccgagt ggtctacagc ttggggccct tgttctacca cctgtggcct cggcatggcc 120

accagagtgt ccaaccagaa cagattctgc cggctggaaa cccagcggag actgtgcttg 180

tctagacctt gcgaggccgc tgccaaggac gctcataagt ctgaggtggc ccaccggttc 240

aaggacctgg gcgaagagaa cttcaaggcc ctggtgctga tcgccttcgc tcagtacttg 300

cagcagtgcc ccttcgagga ccacgtgaag ctggtcaacg aagtgaccga gttcgccaag 360

acctgcgtgg ccgatgagtc tgccgagaac tgcgacaagt ctctgcacac cctgttcggc 420

gacaagctgt gtaccgtggc taccctgaga gaaacctacg gcgagatggc cgactgctgc 480

gctaagcaag agcccgagag aaacgagtgc ttcctgcagc acaaggacga caaccctaac 540

ctgcctagac tcgtgcggcc tgaggtggac gtgatgtgta ccgccttcca cgacaacgag 600

gaaaccttcc tgaagaagta cctgtacgag atcgccagac ggcaccccta cttttacgcc 660

cctgagctgc tgttcttcgc caagcggtac aaggccgcct tcaccgagtg ttgtcaggcc 720

gctgataagg ccgcttgcct gctgcctaaa ctggacgagc tgagagatga aggcaaggcc 780

tccagcgcca agcagagact gaagtgtgcc agcctgcaga agttcggcga gagagccttt 840

aaggcctggg ccgtcgctag actgtcccag agatttccca aggccgagtt tgccgaggtg 900

tccaagctgg ttaccgacct gaccaaggtg cacaccgaat gctgtcacgg cgacctgctg 960

gaatgcgccg atgatagagc cgatctggcc aagtacatct gcgagaacca ggactccatc 1020

tcctccaagc tgaaagagtg ctgcgagaag cctctgctgg aaaagtccca ctgtatcgcc 1080

gaggtggaaa acgacgagat gcctgccgat ctgccttctc tggccgccga cttcgtggaa 1140

tctaaggacg tgtgcaagaa ctacgccgag gctaaggatg tgttcctggg catgtttctg 1200

tacgagtacg ctcggcggca ccccgactat tctgttgtgc tgctgctgag actggctaag 1260

acctacgaga caaccctcga gaagtgctgt gccgccgctg atcctcacga gtgttacgcc 1320

aaggtgttcg acgagttcaa gccactggtg gaagaacccc agaacctgat caagcagaat 1380

tgcgagctgt tcgagcagct gggcgagtac aagttccaga acgccctgct cgtgcggtac 1440

accaagaaag tgccccaggt gtccacacct acactggttg aggtgtcccg gaacctgggc 1500

aaagtgggct ctaagtgctg caagcacccc gaggccaaga gaatgccttg tgccgaggac 1560

tacctgtccg tggtgctgaa ccagctgtgc gtgctgcacg aaaagacccc tgtgtccgac 1620

agagtgacca agtgctgtac cgagagcctg gtcaacagac ggccttgctt ctctgccctg 1680

gaagtggacg agacatacgt gcccaaagag ttcaacgccg agacattcac cttccacgcc 1740

gacatctgca ccctgtccga gaaagagcgg cagatcaaga aacagaccgc tctggtggaa 1800

ctggtcaagc acaagcccaa ggccaccaaa gaacagctga aggccgtgat ggacgacttc 1860

gccgcctttg tggaaaagtg ttgcaaggcc gacgacaaag agacatgctt cgccgaagag 1920

ggcaagaaac tggtggccgc ttctcaggct gctctgggac tttaa 1965

<210> 100

<211> 2038

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 100

ggggacaagt ttgtacaaaa aagcaggcta tggtaccgcc accatgaaat gggtcacctt 60

tatctccctg ctgttcctgt tctcctccgc ctactcttgc gccgagtggt ctacagcttg 120

gggcccttgt tctaccacct gtggcctcgg catggccacc agagtgtcca accagaacag 180

attctgccgg ctggaaaccc agcggagact gtgcttgtct agaccttgcg aggccgctgc 240

caaggacgct cataagtctg aggtggccca ccggttcaag gacctgggcg aagagaactt 300

caaggccctg gtgctgatcg ccttcgctca gtacttgcag cagtgcccct tcgaggacca 360

cgtgaagctg gtcaacgaag tgaccgagtt cgccaagacc tgcgtggccg atgagtctgc 420

cgagaactgc gacaagtctc tgcacaccct gttcggcgac aagctgtgta ccgtggctac 480

cctgagagaa acctacggcg agatggccga ctgctgcgct aagcaagagc ccgagagaaa 540

cgagtgcttc ctgcagcaca aggacgacaa ccctaacctg cctagactcg tgcggcctga 600

ggtggacgtg atgtgtaccg ccttccacga caacgaggaa accttcctga agaagtacct 660

gtacgagatc gccagacggc acccctactt ttacgcccct gagctgctgt tcttcgccaa 720

gcggtacaag gccgccttca ccgagtgttg tcaggccgct gataaggccg cttgcctgct 780

gcctaaactg gacgagctga gagatgaagg caaggcctcc agcgccaagc agagactgaa 840

gtgtgccagc ctgcagaagt tcggcgagag agcctttaag gcctgggccg tcgctagact 900

gtcccagaga tttcccaagg ccgagtttgc cgaggtgtcc aagctggtta ccgacctgac 960

caaggtgcac accgaatgct gtcacggcga cctgctggaa tgcgccgatg atagagccga 1020

tctggccaag tacatctgcg agaaccagga ctccatctcc tccaagctga aagagtgctg 1080

cgagaagcct ctgctggaaa agtcccactg tatcgccgag gtggaaaacg acgagatgcc 1140

tgccgatctg ccttctctgg ccgccgactt cgtggaatct aaggacgtgt gcaagaacta 1200

cgccgaggct aaggatgtgt tcctgggcat gtttctgtac gagtacgctc ggcggcaccc 1260

cgactattct gttgtgctgc tgctgagact ggctaagacc tacgagacaa ccctcgagaa 1320

gtgctgtgcc gccgctgatc ctcacgagtg ttacgccaag gtgttcgacg agttcaagcc 1380

actggtggaa gaaccccaga acctgatcaa gcagaattgc gagctgttcg agcagctggg 1440

cgagtacaag ttccagaacg ccctgctcgt gcggtacacc aagaaagtgc cccaggtgtc 1500

cacacctaca ctggttgagg tgtcccggaa cctgggcaaa gtgggctcta agtgctgcaa 1560

gcaccccgag gccaagagaa tgccttgtgc cgaggactac ctgtccgtgg tgctgaacca 1620

gctgtgcgtg ctgcacgaaa agacccctgt gtccgacaga gtgaccaagt gctgtaccga 1680

gagcctggtc aacagacggc cttgcttctc tgccctggaa gtggacgaga catacgtgcc 1740

caaagagttc aacgccgaga cattcacctt ccacgccgac atctgcaccc tgtccgagaa 1800

agagcggcag atcaagaaac agaccgctct ggtggaactg gtcaagcaca agcccaaggc 1860

caccaaagaa cagctgaagg ccgtgatgga cgacttcgcc gcctttgtgg aaaagtgttg 1920

caaggccgac gacaaagaga catgcttcgc cgaagagggc aagaaactgg tggccgcttc 1980

tcaggctgct ctgggacttt aatctagaaa cccagctttc ttgtacaaag tggtcccc 2038

<210> 101

<211> 582

<212> PRT/Protein

<213> Homo sapiens

<400> 101

Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu

1 5 10 15

Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln

20 25 30

Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu

35 40 45

Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys

50 55 60

Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu

65 70 75 80

Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro

85 90 95

Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu

100 105 110

Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His

115 120 125

Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg

130 135 140

Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg

145 150 155 160

Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala

165 170 175

Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser

180 185 190

Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu

195 200 205

Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro

210 215 220

Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys

225 230 235 240

Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp

245 250 255

Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser

260 265 270

Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His

275 280 285

Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser

290 295 300

Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala

305 310 315 320

Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg

325 330 335

Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr

340 345 350

Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu

355 360 365

Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro

370 375 380

Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu

385 390 395 400

Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro

405 410 415

Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys

420 425 430

Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys

435 440 445

Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His

450 455 460

Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser

465 470 475 480

Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr

485 490 495

Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp

500 505 510

Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala

515 520 525

Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu

530 535 540

Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys

545 550 555 560

Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val

565 570 575

Ala Ala Ser Gln Ala Ala

580

<210> 102

<211> 675

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 102

Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu

1 5 10 15

Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln

20 25 30

Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu

35 40 45

Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys

50 55 60

Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu

65 70 75 80

Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro

85 90 95

Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu

100 105 110

Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His

115 120 125

Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg

130 135 140

Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg

145 150 155 160

Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala

165 170 175

Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser

180 185 190

Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu

195 200 205

Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro

210 215 220

Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys

225 230 235 240

Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp

245 250 255

Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser

260 265 270

Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His

275 280 285

Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser

290 295 300

Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala

305 310 315 320

Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg

325 330 335

Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr

340 345 350

Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu

355 360 365

Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro

370 375 380

Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu

385 390 395 400

Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro

405 410 415

Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys

420 425 430

Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys

435 440 445

Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His

450 455 460

Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser

465 470 475 480

Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr

485 490 495

Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp

500 505 510

Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala

515 520 525

Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu

530 535 540

Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys

545 550 555 560

Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val

565 570 575

Ala Ala Ser Gln Ala Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

580 585 590

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu

595 600 605

Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Cys Ala

610 615 620

Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys Gly Leu Gly

625 630 635 640

Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys Arg Leu Glu Thr

645 650 655

Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Pro Pro Ser Arg Gly Arg

660 665 670

Ser Pro Gln

675

<210> 103

<211> 693

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 103

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu

20 25 30

Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr

35 40 45

Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val

50 55 60

Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys

65 70 75 80

Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala

85 90 95

Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln

100 105 110

Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro

115 120 125

Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala

130 135 140

Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile

145 150 155 160

Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala

165 170 175

Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys

180 185 190

Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys

195 200 205

Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe

210 215 220

Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg

225 230 235 240

Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu

245 250 255

Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala

260 265 270

Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser

275 280 285

Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys

290 295 300

Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu

305 310 315 320

Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn

325 330 335

Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr

340 345 350

Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala

355 360 365

Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro

370 375 380

His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu

385 390 395 400

Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu

405 410 415

Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys

420 425 430

Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu

435 440 445

Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met

450 455 460

Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val

465 470 475 480

Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr

485 490 495

Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp

500 505 510

Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His

515 520 525

Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln

530 535 540

Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu

545 550 555 560

Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys

565 570 575

Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys

580 585 590

Leu Val Ala Ala Ser Gln Ala Ala Glu Ala Ala Ala Lys Glu Ala Ala

595 600 605

Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala

610 615 620

Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

625 630 635 640

Cys Ala Glu Trp Ser Thr Ala Trp Gly Pro Cys Ser Thr Thr Cys Gly

645 650 655

Leu Gly Met Ala Thr Arg Val Ser Asn Gln Asn Arg Phe Cys Arg Leu

660 665 670

Glu Thr Gln Arg Arg Leu Cys Leu Ser Arg Pro Cys Pro Pro Ser Arg

675 680 685

Gly Arg Ser Pro Gln

690

<210> 104

<211> 2088

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 104

gccaccatga aatgggtcac ctttatctcc ctgctgttcc tgttctcctc cgcctactct 60

gacgcccaca agtctgaggt ggcccacaga ttcaaggacc tgggcgaaga gaacttcaag 120

gccctggtgc tgatcgcctt cgctcagtac ttgcagcagt gccccttcga ggaccacgtg 180

aagctggtca acgaagtgac cgagttcgcc aagacctgcg tggccgatga gtctgccgag 240

aactgcgaca agtctctgca caccctgttc ggcgacaagc tgtgtaccgt ggctaccctg 300

agagaaacct acggcgagat ggccgactgc tgcgctaagc aagagcccga gagaaacgag 360

tgcttcctgc agcacaagga cgacaaccct aacctgccta gactcgtgcg gcctgaggtg 420

gacgtgatgt gtaccgcctt ccacgacaac gaggaaacct tcctgaagaa gtacctgtac 480

gagatcgcca gacggcaccc ctacttttac gcccctgagc tgctgttctt cgccaagcgg 540

tacaaggccg ccttcaccga gtgttgtcag gccgctgata aggccgcttg cctgctgcct 600

aaactggacg agctgagaga tgaaggcaag gcctccagcg ccaagcagag actgaagtgt 660

gccagcctgc agaagttcgg cgagagagcc tttaaggcct gggccgtcgc tagactgtcc 720

cagagatttc ccaaggccga gtttgccgag gtgtccaagc tggttaccga cctgaccaag 780

gtgcacaccg aatgctgtca cggcgacctg ctggaatgcg ccgatgatag agccgatctg 840

gccaagtaca tctgcgagaa ccaggactcc atctcctcca agctgaaaga gtgctgcgag 900

aagcctctgc tggaaaagtc ccactgtatc gccgaggtgg aaaacgacga gatgcctgcc 960

gatctgcctt ctctggccgc cgacttcgtg gaatctaagg acgtgtgcaa gaactacgcc 1020

gaggccaagg atgtgttcct gggcatgttt ctgtacgagt acgctcggcg gcaccccgac 1080

tattctgttg tgctgctgct gagactggct aagacctacg agacaaccct cgagaagtgc 1140

tgtgccgccg ctgatcctca cgagtgttac gccaaggtgt tcgacgagtt caagccactg 1200

gtggaagaac cccagaacct gatcaagcag aattgcgagc tgttcgagca gctgggcgag 1260

tacaagttcc agaacgccct gctcgtgcgg tacaccaaga aagtgcccca ggtgtccaca 1320

cctacactgg ttgaggtgtc ccggaacctg ggcaaagtgg gctctaagtg ctgcaagcac 1380

cctgaggcca agagaatgcc ttgcgccgag gactacctgt ccgtggtgct gaatcagctg 1440

tgcgtgctgc acgaaaagac ccctgtgtcc gacagagtga ccaagtgctg taccgagagc 1500

ctggtcaaca gacggccttg cttctctgcc ctggaagtgg acgagacata cgtgcccaaa 1560

gagttcaacg ccgagacatt caccttccac gccgacatct gcaccctgtc cgagaaagag 1620

cggcagatca agaaacagac cgctctggtg gaactggtca agcacaagcc caaggccacc 1680

aaagaacagc tgaaggccgt gatggacgac ttcgccgcct ttgtggaaaa gtgttgcaag 1740

gccgacgaca aagagacatg cttcgccgaa gagggcaaga aactggtggc cgcttctcag 1800

gctgctgagg ccgctgctaa agaggctgcc gctaaagaag ccgcagccaa agaggcagct 1860

gcaaaagaag ctgctgcaaa agaggcagcc gccaaagagg ccgctgctaa agaagcagcc 1920

gccaagtgtg ctgagtggtc tacagcttgg ggcccctgct ctacaacctg tggactcggc 1980

atggccacca gagtgtctaa ccagaacaga ttctgccggc tggaaaccca gcggagactg 2040

tgcctgtcta gaccctgtcc tcctagcaga ggcagatccc ctcagtga 2088

<210> 105

<211> 2161

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 105

ggggacaagt ttgtacaaaa aagcaggcta tggtaccgcc accatgaaat gggtcacctt 60

tatctccctg ctgttcctgt tctcctccgc ctactctgac gcccacaagt ctgaggtggc 120

ccacagattc aaggacctgg gcgaagagaa cttcaaggcc ctggtgctga tcgccttcgc 180

tcagtacttg cagcagtgcc ccttcgagga ccacgtgaag ctggtcaacg aagtgaccga 240

gttcgccaag acctgcgtgg ccgatgagtc tgccgagaac tgcgacaagt ctctgcacac 300

cctgttcggc gacaagctgt gtaccgtggc taccctgaga gaaacctacg gcgagatggc 360

cgactgctgc gctaagcaag agcccgagag aaacgagtgc ttcctgcagc acaaggacga 420

caaccctaac ctgcctagac tcgtgcggcc tgaggtggac gtgatgtgta ccgccttcca 480

cgacaacgag gaaaccttcc tgaagaagta cctgtacgag atcgccagac ggcaccccta 540

cttttacgcc cctgagctgc tgttcttcgc caagcggtac aaggccgcct tcaccgagtg 600

ttgtcaggcc gctgataagg ccgcttgcct gctgcctaaa ctggacgagc tgagagatga 660

aggcaaggcc tccagcgcca agcagagact gaagtgtgcc agcctgcaga agttcggcga 720

gagagccttt aaggcctggg ccgtcgctag actgtcccag agatttccca aggccgagtt 780

tgccgaggtg tccaagctgg ttaccgacct gaccaaggtg cacaccgaat gctgtcacgg 840

cgacctgctg gaatgcgccg atgatagagc cgatctggcc aagtacatct gcgagaacca 900

ggactccatc tcctccaagc tgaaagagtg ctgcgagaag cctctgctgg aaaagtccca 960

ctgtatcgcc gaggtggaaa acgacgagat gcctgccgat ctgccttctc tggccgccga 1020

cttcgtggaa tctaaggacg tgtgcaagaa ctacgccgag gccaaggatg tgttcctggg 1080

catgtttctg tacgagtacg ctcggcggca ccccgactat tctgttgtgc tgctgctgag 1140

actggctaag acctacgaga caaccctcga gaagtgctgt gccgccgctg atcctcacga 1200

gtgttacgcc aaggtgttcg acgagttcaa gccactggtg gaagaacccc agaacctgat 1260

caagcagaat tgcgagctgt tcgagcagct gggcgagtac aagttccaga acgccctgct 1320

cgtgcggtac accaagaaag tgccccaggt gtccacacct acactggttg aggtgtcccg 1380

gaacctgggc aaagtgggct ctaagtgctg caagcaccct gaggccaaga gaatgccttg 1440

cgccgaggac tacctgtccg tggtgctgaa tcagctgtgc gtgctgcacg aaaagacccc 1500

tgtgtccgac agagtgacca agtgctgtac cgagagcctg gtcaacagac ggccttgctt 1560

ctctgccctg gaagtggacg agacatacgt gcccaaagag ttcaacgccg agacattcac 1620

cttccacgcc gacatctgca ccctgtccga gaaagagcgg cagatcaaga aacagaccgc 1680

tctggtggaa ctggtcaagc acaagcccaa ggccaccaaa gaacagctga aggccgtgat 1740

ggacgacttc gccgcctttg tggaaaagtg ttgcaaggcc gacgacaaag agacatgctt 1800

cgccgaagag ggcaagaaac tggtggccgc ttctcaggct gctgaggccg ctgctaaaga 1860

ggctgccgct aaagaagccg cagccaaaga ggcagctgca aaagaagctg ctgcaaaaga 1920

ggcagccgcc aaagaggccg ctgctaaaga agcagccgcc aagtgtgctg agtggtctac 1980

agcttggggc ccctgctcta caacctgtgg actcggcatg gccaccagag tgtctaacca 2040

gaacagattc tgccggctgg aaacccagcg gagactgtgc ctgtctagac cctgtcctcc 2100

tagcagaggc agatcccctc agtgatctag aaacccagct ttcttgtaca aagtggtccc 2160

c 2161

<210> 106

<211> 634

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 106

Cys Ala Glu Gln Thr Thr Glu Trp Thr Ala Cys Ser Lys Ser Cys Gly

1 5 10 15

Met Gly Phe Ser Thr Arg Val Thr Asn Arg Asn Arg Gln Cys Glu Met

20 25 30

Leu Lys Gln Thr Arg Leu Cys Met Val Arg Pro Cys Glu Ala Ala Ala

35 40 45

Lys Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly

50 55 60

Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu

65 70 75 80

Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr

85 90 95

Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp

100 105 110

Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr

115 120 125

Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu

130 135 140

Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn

145 150 155 160

Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe

165 170 175

His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala

180 185 190

Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys

195 200 205

Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala

210 215 220

Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala

225 230 235 240

Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly

245 250 255

Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe

260 265 270

Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr

275 280 285

Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp

290 295 300

Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile

305 310 315 320

Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser

325 330 335

His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro

340 345 350

Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr

355 360 365

Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala

370 375 380

Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys

385 390 395 400

Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His

405 410 415

Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu

420 425 430

Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly

435 440 445

Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val

450 455 460

Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly

465 470 475 480

Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro

485 490 495

Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu

500 505 510

His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu

515 520 525

Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu

530 535 540

Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala

545 550 555 560

Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr

565 570 575

Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln

580 585 590

Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys

595 600 605

Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu

610 615 620

Val Ala Ala Ser Gln Ala Ala Leu Gly Leu

625 630

<210> 107

<211> 652

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 107

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Cys Ala Glu Gln Thr Thr Glu Trp Thr Ala Cys Ser Lys Ser

20 25 30

Cys Gly Met Gly Phe Ser Thr Arg Val Thr Asn Arg Asn Arg Gln Cys

35 40 45

Glu Met Leu Lys Gln Thr Arg Leu Cys Met Val Arg Pro Cys Glu Ala

50 55 60

Ala Ala Lys Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp

65 70 75 80

Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln

85 90 95

Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu

100 105 110

Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn

115 120 125

Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val

130 135 140

Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys

145 150 155 160

Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn

165 170 175

Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr

180 185 190

Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu

195 200 205

Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe

210 215 220

Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp

225 230 235 240

Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly

245 250 255

Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys

260 265 270

Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln

275 280 285

Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp

290 295 300

Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys

305 310 315 320

Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp

325 330 335

Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu

340 345 350

Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp

355 360 365

Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys

370 375 380

Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu

385 390 395 400

Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu

405 410 415

Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp

420 425 430

Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val

435 440 445

Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln

450 455 460

Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys

465 470 475 480

Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn

485 490 495

Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg

500 505 510

Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys

515 520 525

Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys

530 535 540

Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val

545 550 555 560

Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe

565 570 575

His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys

580 585 590

Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys

595 600 605

Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys

610 615 620

Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys

625 630 635 640

Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu

645 650

<210> 108

<211> 1965

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 108

gccaccatga aatgggtcac ctttatctcc ctgctgttcc tgttctcctc cgcctactct 60

tgtgccgagc agaccacaga gtggaccgcc tgctctaagt cttgcggcat gggcttctcc 120

accagagtga ccaaccggaa cagacagtgc gagatgctga agcagacccg gctgtgtatg 180

gttcgacctt gcgaggccgc tgccaaggat gctcataagt ctgaggtggc ccaccggttc 240

aaggacctgg gcgaagagaa cttcaaggcc ctggtgctga tcgccttcgc tcagtacttg 300

cagcagtgcc ccttcgagga ccacgtgaag ctggtcaacg aagtgaccga gttcgccaag 360

acctgcgtgg ccgatgagtc tgccgagaac tgcgacaagt ctctgcacac cctgttcggc 420

gacaagctgt gtaccgtggc taccctgaga gaaacctacg gcgagatggc cgactgctgc 480

gctaagcaag agcccgagag aaacgagtgc ttcctgcagc acaaggacga caaccctaac 540

ctgcctagac tcgtgcggcc tgaggtggac gtgatgtgta ccgccttcca cgacaacgag 600

gaaaccttcc tgaagaagta cctgtacgag atcgccagac ggcaccccta cttttacgcc 660

cctgagctgc tgttcttcgc caagcggtac aaggccgcct tcaccgagtg ttgtcaggcc 720

gctgataagg ccgcttgcct gctgcctaaa ctggacgagc tgagagatga aggcaaggcc 780

tccagcgcca agcagagact gaagtgtgcc agcctgcaga agttcggcga gagagccttt 840

aaggcctggg ccgtcgctag actgtcccag agatttccca aggccgagtt tgccgaggtg 900

tccaagctgg ttaccgacct gaccaaggtg cacaccgaat gctgtcacgg cgacctgctg 960

gaatgcgccg atgatagagc cgatctggcc aagtacatct gcgagaacca ggactccatc 1020

tcctccaagc tgaaagagtg ctgcgagaag cctctgctgg aaaagtccca ctgtatcgcc 1080

gaggtggaaa acgacgagat gcctgccgat ctgccttctc tggccgccga cttcgtggaa 1140

tctaaggacg tgtgcaagaa ctacgccgag gctaaggatg tgttcctggg catgtttctg 1200

tacgagtacg ctcggcggca ccccgattat agtgtggtgc tgctgctgag actggctaag 1260

acctacgaga caaccctcga gaagtgctgt gccgccgctg atcctcacga gtgttacgcc 1320

aaggtgttcg acgagttcaa gccactggtg gaagaacccc agaacctgat caagcagaat 1380

tgcgagctgt tcgagcagct gggcgagtac aagttccaga acgccctgct cgtgcggtac 1440

accaagaaag tgccccaggt gtccacacct acactggttg aggtgtcccg gaacctgggc 1500

aaagtgggct ctaagtgctg caagcacccc gaggccaaga gaatgccttg cgccgaggat 1560

tacctgtccg tggtgctgaa ccagctgtgc gtgctgcacg aaaagacccc tgtgtccgac 1620

cgcgtgacca agtgctgtac agagtccctg gtcaacagac ggccctgctt ctctgccctg 1680

gaagtggacg agacatacgt gcccaaagag ttcaacgccg agacattcac cttccacgcc 1740

gacatctgca ccctgtccga gaaagagcgg cagatcaaga aacagaccgc tctggtcgaa 1800

ctggtcaagc acaagcccaa ggccaccaaa gaacagctga aggccgtgat ggacgacttc 1860

gccgcctttg tggaaaagtg ttgcaaggcc gacgacaaag agacatgctt cgccgaagag 1920

ggcaagaaac tggtggccgc ttctcaggct gctctgggac tttaa 1965

<210> 109

<211> 2038

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 109

ggggacaagt ttgtacaaaa aagcaggcta tggtaccgcc accatgaaat gggtcacctt 60

tatctccctg ctgttcctgt tctcctccgc ctactcttgt gccgagcaga ccacagagtg 120

gaccgcctgc tctaagtctt gcggcatggg cttctccacc agagtgacca accggaacag 180

acagtgcgag atgctgaagc agacccggct gtgtatggtt cgaccttgcg aggccgctgc 240

caaggatgct cataagtctg aggtggccca ccggttcaag gacctgggcg aagagaactt 300

caaggccctg gtgctgatcg ccttcgctca gtacttgcag cagtgcccct tcgaggacca 360

cgtgaagctg gtcaacgaag tgaccgagtt cgccaagacc tgcgtggccg atgagtctgc 420

cgagaactgc gacaagtctc tgcacaccct gttcggcgac aagctgtgta ccgtggctac 480

cctgagagaa acctacggcg agatggccga ctgctgcgct aagcaagagc ccgagagaaa 540

cgagtgcttc ctgcagcaca aggacgacaa ccctaacctg cctagactcg tgcggcctga 600

ggtggacgtg atgtgtaccg ccttccacga caacgaggaa accttcctga agaagtacct 660

gtacgagatc gccagacggc acccctactt ttacgcccct gagctgctgt tcttcgccaa 720

gcggtacaag gccgccttca ccgagtgttg tcaggccgct gataaggccg cttgcctgct 780

gcctaaactg gacgagctga gagatgaagg caaggcctcc agcgccaagc agagactgaa 840

gtgtgccagc ctgcagaagt tcggcgagag agcctttaag gcctgggccg tcgctagact 900

gtcccagaga tttcccaagg ccgagtttgc cgaggtgtcc aagctggtta ccgacctgac 960

caaggtgcac accgaatgct gtcacggcga cctgctggaa tgcgccgatg atagagccga 1020

tctggccaag tacatctgcg agaaccagga ctccatctcc tccaagctga aagagtgctg 1080

cgagaagcct ctgctggaaa agtcccactg tatcgccgag gtggaaaacg acgagatgcc 1140

tgccgatctg ccttctctgg ccgccgactt cgtggaatct aaggacgtgt gcaagaacta 1200

cgccgaggct aaggatgtgt tcctgggcat gtttctgtac gagtacgctc ggcggcaccc 1260

cgattatagt gtggtgctgc tgctgagact ggctaagacc tacgagacaa ccctcgagaa 1320

gtgctgtgcc gccgctgatc ctcacgagtg ttacgccaag gtgttcgacg agttcaagcc 1380

actggtggaa gaaccccaga acctgatcaa gcagaattgc gagctgttcg agcagctggg 1440

cgagtacaag ttccagaacg ccctgctcgt gcggtacacc aagaaagtgc cccaggtgtc 1500

cacacctaca ctggttgagg tgtcccggaa cctgggcaaa gtgggctcta agtgctgcaa 1560

gcaccccgag gccaagagaa tgccttgcgc cgaggattac ctgtccgtgg tgctgaacca 1620

gctgtgcgtg ctgcacgaaa agacccctgt gtccgaccgc gtgaccaagt gctgtacaga 1680

gtccctggtc aacagacggc cctgcttctc tgccctggaa gtggacgaga catacgtgcc 1740

caaagagttc aacgccgaga cattcacctt ccacgccgac atctgcaccc tgtccgagaa 1800

agagcggcag atcaagaaac agaccgctct ggtcgaactg gtcaagcaca agcccaaggc 1860

caccaaagaa cagctgaagg ccgtgatgga cgacttcgcc gcctttgtgg aaaagtgttg 1920

caaggccgac gacaaagaga catgcttcgc cgaagagggc aagaaactgg tggccgcttc 1980

tcaggctgct ctgggacttt aatctagaaa cccagctttc ttgtacaaag tggtcccc 2038

<210> 110

<211> 669

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 110

Cys Ala Glu Gln Thr Thr Glu Trp Thr Ala Cys Ser Lys Ser Cys Gly

1 5 10 15

Met Gly Phe Ser Thr Arg Val Thr Asn Arg Asn Arg Gln Cys Glu Met

20 25 30

Leu Lys Gln Thr Arg Leu Cys Met Val Arg Pro Cys Glu Ala Ala Ala

35 40 45

Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

50 55 60

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu

65 70 75 80

Ala Ala Ala Lys Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys

85 90 95

Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala

100 105 110

Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn

115 120 125

Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu

130 135 140

Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr

145 150 155 160

Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala

165 170 175

Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp

180 185 190

Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys

195 200 205

Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr

210 215 220

Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe

225 230 235 240

Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala

245 250 255

Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu

260 265 270

Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln

275 280 285

Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser

290 295 300

Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr

305 310 315 320

Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu

325 330 335

Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln

340 345 350

Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu

355 360 365

Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala

370 375 380

Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys

385 390 395 400

Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr

405 410 415

Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg

420 425 430

Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala

435 440 445

Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu

450 455 460

Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu

465 470 475 480

Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr

485 490 495

Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg

500 505 510

Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys

515 520 525

Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu

530 535 540

Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys

545 550 555 560

Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu

565 570 575

Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr

580 585 590

Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys

595 600 605

Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr

610 615 620

Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu

625 630 635 640

Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly

645 650 655

Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu

660 665

<210> 111

<211> 687

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 111

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Cys Ala Glu Gln Thr Thr Glu Trp Thr Ala Cys Ser Lys Ser

20 25 30

Cys Gly Met Gly Phe Ser Thr Arg Val Thr Asn Arg Asn Arg Gln Cys

35 40 45

Glu Met Leu Lys Gln Thr Arg Leu Cys Met Val Arg Pro Cys Glu Ala

50 55 60

Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala

65 70 75 80

Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala

85 90 95

Lys Glu Ala Ala Ala Lys Asp Ala His Lys Ser Glu Val Ala His Arg

100 105 110

Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala

115 120 125

Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu

130 135 140

Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser

145 150 155 160

Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu

165 170 175

Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys

180 185 190

Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys

195 200 205

Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val

210 215 220

Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr

225 230 235 240

Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu

245 250 255

Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln

260 265 270

Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg

275 280 285

Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser

290 295 300

Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg

305 310 315 320

Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu

325 330 335

Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu

340 345 350

Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu

355 360 365

Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro

370 375 380

Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met

385 390 395 400

Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp

405 410 415

Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe

420 425 430

Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu

435 440 445

Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala

450 455 460

Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys

465 470 475 480

Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu

485 490 495

Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg

500 505 510

Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val

515 520 525

Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu

530 535 540

Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn

545 550 555 560

Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr

565 570 575

Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala

580 585 590

Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr

595 600 605

Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln

610 615 620

Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys

625 630 635 640

Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe

645 650 655

Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu

660 665 670

Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu

675 680 685

<210> 112

<211> 2070

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 112

gccaccatga aatgggtcac ctttatctcc ctgctgttcc tgttctcctc cgcctactct 60

tgtgccgagc agaccacaga gtggaccgcc tgctctaagt cttgcggcat gggcttctcc 120

accagagtga ccaaccggaa cagacagtgc gagatgctga agcagacccg gctgtgtatg 180

gttcgacctt gcgaggccgc tgccaaagag gctgctgcta aagaagccgc cgcaaaagag 240

gcagcagcaa aagaggctgc cgccaaagag gccgcagcca aagaagcagc agctaaagag 300

gccgctgcta aggacgccca caagtctgaa gtggcccacc ggtttaagga cctgggcgaa 360

gagaacttca aggccctggt gctgatcgcc ttcgctcagt acttgcagca gtgccccttc 420

gaggaccacg tgaagctggt caacgaagtg accgagttcg ccaagacctg cgtggccgat 480

gagtctgccg agaactgcga caagtctctg cacaccctgt tcggcgacaa gctgtgtacc 540

gtggctaccc tgagagaaac ctacggcgag atggccgact gctgcgctaa gcaagagccc 600

gagagaaacg agtgcttcct gcagcacaag gacgacaacc ctaacctgcc tagactcgtg 660

cggcctgagg tggacgtgat gtgtaccgcc ttccacgaca acgaggaaac cttcctgaag 720

aagtacctgt acgagatcgc cagacggcac ccctactttt acgcccctga gctgctgttt 780

ttcgccaagc ggtacaaggc cgccttcacc gagtgttgtc aggccgccga taaggccgct 840

tgtctgctgc ctaaactgga cgagctgcgc gacgaaggca aggcctcttc tgctaagcag 900

cggctgaagt gcgccagcct gcagaagttt ggcgagagag ccttcaaggc ttgggccgtc 960

gctagactgt cccagagatt tcccaaggcc gagtttgccg aggtgtccaa gctggttacc 1020

gacctgacca aggtgcacac cgaatgctgt cacggcgacc tgctggaatg cgccgatgat 1080

agagccgatc tggccaagta catctgcgag aaccaggact ccatctcctc caagctgaaa 1140

gagtgctgcg agaagcctct gctggaaaag tcccactgta tcgccgaggt ggaaaacgac 1200

gagatgcctg ccgatctgcc ttctctggcc gccgacttcg tggaatctaa ggacgtgtgc 1260

aagaactacg ccgaggccaa ggatgtgttc ctgggcatgt ttctgtacga gtacgctcgg 1320

cggcaccccg attatagtgt ggtgctgctg ctgagactgg ctaagaccta cgagacaacc 1380

ctcgagaagt gctgtgccgc cgctgatcct cacgagtgtt acgccaaggt gttcgacgag 1440

ttcaagccac tggtggaaga accccagaac ctgatcaagc agaattgcga gctgttcgag 1500

cagctgggcg agtacaagtt ccagaacgcc ctgctcgtgc ggtacaccaa gaaagtgccc 1560

caggtgtcca cacctacact ggttgaggtg tcccggaacc tgggcaaagt gggctctaag 1620

tgctgcaagc accctgaggc caagagaatg ccttgcgccg aggactacct gtccgtggtg 1680

ctgaatcagc tgtgcgtgct gcacgaaaag acccctgtgt ccgaccgcgt gaccaagtgc 1740

tgtacagagt ccctggtcaa cagacggccc tgcttctctg ccctggaagt ggacgagaca 1800

tacgtgccca aagagttcaa cgccgagaca ttcaccttcc acgccgacat ctgcaccctg 1860

tccgagaaag agcggcagat caagaaacag accgctctgg tcgagctggt taagcacaag 1920

cccaaggcca ccaaagaaca gctgaaggcc gtgatggacg acttcgccgc ctttgtggaa 1980

aagtgttgca aggccgacga caaagagaca tgcttcgccg aagagggcaa gaaactggtg 2040

gccgcttctc aggctgctct gggactttaa 2070

<210> 113

<211> 2143

<212> DNA

<213> Artificial sequence

<220>

<223> Fused protein

<400> 113

ggggacaagt ttgtacaaaa aagcaggcta tggtaccgcc accatgaaat gggtcacctt 60

tatctccctg ctgttcctgt tctcctccgc ctactcttgt gccgagcaga ccacagagtg 120

gaccgcctgc tctaagtctt gcggcatggg cttctccacc agagtgacca accggaacag 180

acagtgcgag atgctgaagc agacccggct gtgtatggtt cgaccttgcg aggccgctgc 240

caaagaggct gctgctaaag aagccgccgc aaaagaggca gcagcaaaag aggctgccgc 300

caaagaggcc gcagccaaag aagcagcagc taaagaggcc gctgctaagg acgcccacaa 360

gtctgaagtg gcccaccggt ttaaggacct gggcgaagag aacttcaagg ccctggtgct 420

gatcgccttc gctcagtact tgcagcagtg ccccttcgag gaccacgtga agctggtcaa 480

cgaagtgacc gagttcgcca agacctgcgt ggccgatgag tctgccgaga actgcgacaa 540

gtctctgcac accctgttcg gcgacaagct gtgtaccgtg gctaccctga gagaaaccta 600

cggcgagatg gccgactgct gcgctaagca agagcccgag agaaacgagt gcttcctgca 660

gcacaaggac gacaacccta acctgcctag actcgtgcgg cctgaggtgg acgtgatgtg 720

taccgccttc cacgacaacg aggaaacctt cctgaagaag tacctgtacg agatcgccag 780

acggcacccc tacttttacg cccctgagct gctgtttttc gccaagcggt acaaggccgc 840

cttcaccgag tgttgtcagg ccgccgataa ggccgcttgt ctgctgccta aactggacga 900

gctgcgcgac gaaggcaagg cctcttctgc taagcagcgg ctgaagtgcg ccagcctgca 960

gaagtttggc gagagagcct tcaaggcttg ggccgtcgct agactgtccc agagatttcc 1020

caaggccgag tttgccgagg tgtccaagct ggttaccgac ctgaccaagg tgcacaccga 1080

atgctgtcac ggcgacctgc tggaatgcgc cgatgataga gccgatctgg ccaagtacat 1140

ctgcgagaac caggactcca tctcctccaa gctgaaagag tgctgcgaga agcctctgct 1200

ggaaaagtcc cactgtatcg ccgaggtgga aaacgacgag atgcctgccg atctgccttc 1260

tctggccgcc gacttcgtgg aatctaagga cgtgtgcaag aactacgccg aggccaagga 1320

tgtgttcctg ggcatgtttc tgtacgagta cgctcggcgg caccccgatt atagtgtggt 1380

gctgctgctg agactggcta agacctacga gacaaccctc gagaagtgct gtgccgccgc 1440

tgatcctcac gagtgttacg ccaaggtgtt cgacgagttc aagccactgg tggaagaacc 1500

ccagaacctg atcaagcaga attgcgagct gttcgagcag ctgggcgagt acaagttcca 1560

gaacgccctg ctcgtgcggt acaccaagaa agtgccccag gtgtccacac ctacactggt 1620

tgaggtgtcc cggaacctgg gcaaagtggg ctctaagtgc tgcaagcacc ctgaggccaa 1680

gagaatgcct tgcgccgagg actacctgtc cgtggtgctg aatcagctgt gcgtgctgca 1740

cgaaaagacc cctgtgtccg accgcgtgac caagtgctgt acagagtccc tggtcaacag 1800

acggccctgc ttctctgccc tggaagtgga cgagacatac gtgcccaaag agttcaacgc 1860

cgagacattc accttccacg ccgacatctg caccctgtcc gagaaagagc ggcagatcaa 1920

gaaacagacc gctctggtcg agctggttaa gcacaagccc aaggccacca aagaacagct 1980

gaaggccgtg atggacgact tcgccgcctt tgtggaaaag tgttgcaagg ccgacgacaa 2040

agagacatgc ttcgccgaag agggcaagaa actggtggcc gcttctcagg ctgctctggg 2100

actttaatct agaaacccag ctttcttgta caaagtggtc ccc 2143

<210> 114

<211> 484

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Fused protein

<400> 114

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Gly His His His His His Gly Ser Glu Ile Gly Thr Gly

20 25 30

Phe Pro Phe Asp Pro His Tyr Val Glu Val Leu Gly Glu Arg Met His

35 40 45

Tyr Val Asp Val Gly Pro Arg Asp Gly Thr Pro Val Leu Phe Leu His

50 55 60

Gly Asn Pro Thr Ser Ser Tyr Val Trp Arg Asn Ile Ile Pro His Val

65 70 75 80

Ala Pro Thr His Arg Cys Ile Ala Pro Asp Leu Ile Gly Met Gly Lys

85 90 95

Ser Asp Lys Pro Asp Leu Gly Tyr Phe Phe Asp Asp His Val Arg Phe

100 105 110

Met Asp Ala Phe Ile Glu Ala Leu Gly Leu Glu Glu Val Val Leu Val

115 120 125

Ile His Asp Trp Gly Ser Ala Leu Gly Phe His Trp Ala Lys Arg Asn

130 135 140

Pro Glu Arg Val Lys Gly Ile Ala Phe Met Glu Phe Ile Arg Pro Ile

145 150 155 160

Pro Thr Trp Asp Glu Trp Pro Glu Phe Ala Arg Glu Thr Phe Gln Ala

165 170 175

Phe Arg Thr Thr Asp Val Gly Arg Lys Leu Ile Ile Asp Gln Asn Val

180 185 190

Phe Ile Glu Gly Thr Leu Pro Met Gly Val Val Arg Pro Leu Thr Glu

195 200 205

Val Glu Met Asp His Tyr Arg Glu Pro Phe Leu Asn Pro Val Asp Arg

210 215 220

Glu Pro Leu Trp Arg Phe Pro Asn Glu Leu Pro Ile Ala Gly Glu Pro

225 230 235 240

Ala Asn Ile Val Ala Leu Val Glu Glu Tyr Met Asp Trp Leu His Gln

245 250 255

Ser Pro Val Pro Lys Leu Leu Phe Trp Gly Thr Pro Gly Val Leu Ile

260 265 270

Pro Pro Ala Glu Ala Ala Arg Leu Ala Lys Ser Leu Pro Asn Cys Lys

275 280 285

Ala Val Asp Ile Gly Pro Gly Leu Asn Leu Leu Gln Glu Asp Asn Pro

290 295 300

Asp Leu Ile Gly Ser Glu Ile Ala Arg Trp Leu Ser Thr Leu Glu Ile

305 310 315 320

Ser Gly Leu Gln Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val

325 330 335

Lys Pro Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp

340 345 350

Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg

355 360 365

Arg Leu Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser

370 375 380

Leu Thr Phe Leu Tyr Asp Gly Ile Glu Ile Gln Ala Asp Gln Thr Pro

385 390 395 400

Glu Asp Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu

405 410 415

Gln Ile Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly

420 425 430

Ser Ser Gly Gly Gly Ser Ser Gly Cys Ala Glu Gln Thr Thr Glu Trp

435 440 445

Thr Ala Cys Ser Lys Ser Cys Gly Met Gly Phe Ser Thr Arg Val Thr

450 455 460

Asn Arg Asn Arg Gln Cys Glu Met Leu Lys Gln Thr Arg Leu Cys Met

465 470 475 480

Val Arg Pro Cys

<210> 115

<211> 13

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> part of a recombinant protein

<400> 115

Pro Pro Ser Arg Gly Arg Ser Pro Gln Asn Ser Ala Phe

1 5 10

<210> 116

<211> 8

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> part of a recombinant protein

<400> 116

Gly Gln Pro Val Tyr Ser Ser Leu

1 5

<210> 117

<211> 7

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> part of a recombinant protein

<400> 117

Glu Ala Asp Leu Glu Glu Asn

1 5

<210> 118

<211> 9

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> part of a recombinant protein

<400> 118

Glu Gln Gln Pro Glu Gln Pro Thr Asp

1 5

<210> 119

<211> 8

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> part of a recombinant protein

<400> 119

Asp Val Asp Ile His Thr Leu Ile

1 5

<210> 120

<211> 11

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> part of a recombinant protein

<400> 120

Asp Ser Asn Ile Leu Lys Thr Ile Lys Ile Pro

1 5 10

<210> 121

<211> 5

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Linker

<400> 121

Gly Gly Gly Gly Ser

1 5

<---

Claims (29)

1. A monomeric fusion protein for inhibiting the effect or activity of the 4-domain CCN protein, comprising: (i) a polypeptide corresponding to at least a portion of the thrombospondin type 1 repeat homology domain (TSP-1) of a CCN family protein; (ii) a monomeric fusion partner fused at the N- or C-terminus to the amino acid sequence of (i), said monomeric fusion partner comprising at least 6 amino acids and increasing the serum half-life of said fusion protein; and (iii) optionally, a peptide linker between the polypeptide of (i) and the monomeric fusion partner of (ii), wherein said polypeptide according to (i) (a) comprises an amino acid sequence selected from SEQ ID NO: 37 or 2 and has a length of from 44 to 60 amino acids, or (b) comprises a sequence that has at least 80% identity to a sequence selected from SEQ ID NO: 37 or 2 and has a length of from 40 to 60 amino acids, wherein all cysteine residues in said sequence selected from SEQ ID NO: 37 or 2 are retained, and wherein said polypeptide according to (i) exerts the same activity as the TSP-1 homology domain of CCN5, and wherein said monomeric fusion partner of (ii) and said peptide linker of (iii) do not represent or comprise an IGF binding protein homology domain, a von Willebrand factor type C repeat homology domain, or a CCN family protein cystine knot domain. 2. The fusion protein according to claim 1, characterized in that said polypeptide according to (i) has a length of 44 to 57 amino acids. 3. A fusion protein according to claim 1 or 2, characterized in that said polypeptide according to (i) contains or consists of: (a) an amino acid sequence selected from SEQ ID NO: 1 or 9; or (b) an amino acid sequence that has at least 80% identity to a sequence selected from SEQ ID NO: 1 or 9; or (c) a portion of the amino acid sequence of (a) or (b), wherein said portion comprises at least a 44 amino acid sequence from SEQ ID NO: 37 or 2, respectively, or a sequence that has at least 80% identity to a sequence selected from SEQ ID NO: 37 or 2, respectively. 4. A fusion protein according to any one of claims 1 to 3, characterized in that said polypeptide consists of an amino acid sequence selected from SEQ ID NO: 37 or 2, or a sequence that has at least 80% identity to a sequence selected from SEQ ID NO: 37 or 2. 5. A fusion protein according to any one of claims 1 to 4, characterized in that the peptide linker according to (iii) contains no more than 50 amino acids. 6. A fusion protein according to any one of claims 1 to 5, characterized in that said polypeptide according to (i) contains an alanine residue at a position corresponding to position 2 of said sequence selected from SEQ ID NO: 37 or 2, or SEQ ID NO: 1 or 9. 7. A fusion protein according to any one of claims 1 to 6, wherein the amino acid sequence of (i) comprises an amino acid sequence selected from SEQ ID NO: 7, 38, 44 or 49, or a sequence that has at least 80% identity with said sequence, wherein said protein comprises an alanine residue at a position corresponding to position 2 of said sequence of SEQ ID NO: 7, 38, 44 or 49. 8. A fusion protein according to any one of claims 1 to 7, wherein said monomeric fusion partner is selected from the group consisting of serum albumin, transferrin, and a monomeric Fc fragment of human IgG. 9. The fusion protein according to claim 8, characterized in that said monomeric Fc fragment of human IgG is a monomeric Fc fragment of IgG1, IgG2 or IgG4. 10. The fusion protein according to claim 8 or 9, characterized in that said monomeric Fc fragment is aglycosylated. 11. A fusion protein according to any one of claims 8-10, characterized in that said monomeric Fc fragment contains a stabilizing disulfide bridge and/or a mutation that stabilizes against the action of a protease. 12. A fusion protein according to any one of claims 8–11, characterized in that said monomeric Fc fragment does not have an immune effector function. 13. The fusion protein of any one of claims 1 to 12, wherein said peptide linker between the amino acid sequence of (i) and said monomeric fusion partner has an amino acid sequence selected from the group consisting of SEQ ID NO: 20 to 25, 39, 57, 63, 65 or 67, or an amino acid sequence that has 80% identity with said sequence. 14. The fusion protein of any one of claims 1 to 8, wherein said fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 84, 85, 88, 89, 97, 98, 102, 103, 106, 107, 110 and 111, or an amino acid sequence that has 80% identity to said sequence. 15. A DNA molecule encoding a monomeric fusion protein as defined in any one of claims 1 to 14. 16. A DNA molecule according to claim 15, characterized in that said molecule additionally contains a nucleotide sequence encoding a signal sequence. 17. A DNA molecule according to claim 15 or 16, characterized in that said molecule comprises a nucleotide sequence according to SEQ ID NO: 34, 35, 36, 86, 87, 90, 91, 99, 100, 104, 105, 108, 109, 112 or 113, or a nucleotide sequence that has at least 80% identity with any of the above sequences. 18. An expression vector for providing expression in a mammalian cell, comprising a DNA molecule as defined in any of paragraphs 15-17. 19. A mammalian host cell for expressing the monomeric fusion protein of any one of claims 1-14, comprising a vector as defined in claim 18. 20. The use of a fusion protein according to any one of claims 1-14 in the treatment or prevention of fibrosis, or any condition in which fibrosis is manifested. 21. Use of a fusion protein according to any one of claims 1-14 in the treatment of cancer.