Multivalent binding molecules that activate WNT signaling and their applications

Abstract

This article describes methods for influencing the binding of multivalent binding molecules to both the FZD receptor and the Wnt co-receptor on cells, wherein the binding of the multivalent binding molecule to both the FZD receptor and the co-receptor activates the Wnt signaling pathway. This article also describes multivalent binding molecules that activate the Wnt signaling pathway and methods of using them, wherein the multivalent binding molecule comprises an FZD receptor-binding domain and a Wnt co-receptor-binding domain at either end of an Fc domain.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 630,772, filed February 14, 2018, based on 35 USC119(e), the entire contents of which are incorporated herein by reference.

[0002] This application contains a sequence list that has been electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on February 12, 2019, named 115773_PA895WO_SL.txt, and is 220,360 bytes in size. Background Technology

[0003] The Wnt signaling pathway is crucial for embryonic development and tissue homeostasis in adults. Wnt ligands are secreted growth factors that regulate various cellular processes, such as proliferation, differentiation, survival, and migration. Wnt ligands are universally important for controlling the self-renewal of tissue stem cells and regulating many progenitor cell populations. The hydrophobicity and sensitive tertiary structure of Wnt proteins make their biochemical purification challenging and prevent their use in vivo and in vitro.

[0004] Nineteen Wnt ligands exist in humans, interacting with a network of ten coiled-protein cell surface receptors (FZDs) and several co-receptors that selectively guide different intracellular signaling pathways (Wodarz, A. and Nusse, R. Annu. Rev. Cell Dev. Biol. 14, 59–88 (1998); Angers, S. and Moon, RT, transduction. Nat. Rev. Mol. Cell Biol. 10, 468–477 (2009)). FZDs possess conserved structural features, including seven hydrophobic transmembrane domains and a cysteine-rich ligand-binding domain. FZDs are known to function in three distinct signaling pathways: the Wnt planar cell polarity (PCP) pathway, the typical Wnt / β-catenin pathway, and the Wnt / calcium pathway. Activation of the Wnt signaling pathway also requires the presence of Wnt co-receptors to indicate differential involvement in intracellular signaling cascades and regulate gene expression influencing cellular mechanisms underlying the aforementioned cellular processes. For example, Wnt ligands bind to members of the coilin receptor and low-density lipoprotein receptor-associated protein 5 and 6 (LRP5 / 6) co-receptor families to activate the Wnt / β-catenin pathway, or with receptor tyrosine kinase-like orphan receptors 1 and 2 (ROR1 / 2) involving receptor tyrosine kinase (RYK) or protein tyrosine kinase 7 (PTK7) co-receptors to initiate the Wnt / PCP pathway or alternative, β-catenin-independent signaling pathways. The Wnt / β-catenin pathway, sometimes referred to as the canonical pathway, peaks in the post-translational accumulation of the transcriptional effector β-catenin, which interacts with the transcription factor T-cytokine / lymphocyte enhancer factor (LEF / TCF) family to regulate the expression of specific background genes. Summary of the Invention

[0005] Wnt requires lipidation to function (Janda et al., Science. 337, 59–64 (2012); Kadowaki et al., Genes Dev. 10, 3116–3128 (1996)), and its hydrophobicity complicates biochemical manipulation; therefore, only a small number of Wnts are purified (Willert et al., Nature 423, 448–452 (2003). Furthermore, Wnt exhibits inherent cross-reactivity with multiple receptors, especially when overexpressed or applied at high doses (He et al., Science. 275, 1652–1654 (1997); Andres et al., Systematic mapping of Wnt-Frizzled interactions reveals functional selectivity by distinct Wnt-Frizzled pairs. Journal of Biological (2015)). http: / / www.jbc.org / content / early / 2015 / 01 / 20 / jbc.M114. 12648.short); Holmen et al., J. Biol. Chem. 277, 34727–34735 (2002)). Therefore, it is not possible to selectively activate the coilin receptor complex to determine its specific function in different situations or to assess its therapeutic potential for degenerative conditions. The multivalent binding molecules and methods described herein selectively activate preselected coilin receptor-co-receptor complexes. It is anticipated that the administration of the multivalent binding molecules described herein will treat degenerative conditions by activating the appropriate coilin co-receptor complex.

[0006] This article describes a method that influences the binding of peptides to both the FZD receptor and the Wnt co-receptor on cells, wherein the binding of the peptide to both the FZD receptor and the co-receptor activates the Wnt signaling pathway.

[0007] This document also describes a multivalent binding molecule that activates the Wnt signaling pathway and its usage. The multivalent binding molecule binds to both the FZD receptor and the Wnt co-receptor, thereby activating the Wnt signaling pathway. The multivalent binding molecule of this invention is also referred to herein as an "FZD agonist" or "FZDag". In a specific embodiment in which the molecule of this invention binds to FZD and LRP5 / 6, the molecule may be referred to as a "crepuscular protein and LRP5 / 6 agonist" or "FLAg". The multivalent binding molecule comprises an Fc domain or a fragment thereof containing a CH3 domain, and a first binding domain for binding the FZD receptor and a second binding domain for binding the Wnt co-receptor, wherein the FZD binding domain is attached to one end of the Fc domain, and the co-receptor binding domain is attached to the other end of the Fc domain. Therefore, the binding domain for the FZD receptor and the binding domain for the co-receptor are not directly connected, but are separated by the Fc domain, or a fragment thereof containing a CH3 domain. This conformation of the binding domains produces a surprisingly high level of Wnt signaling pathway activation. The FZD binding domain can be monovalent with a single binding site (complementary site) of the FZD receptor, or it can be polyvalent with more than one binding site of the FZD receptor. For example, the binding domain can be bivalent, trivalent, or tetravalent. The Wnt co-receptor binding domain can be monovalent with a single binding site (complementary site) of the Wnt co-receptor, or it can be polyvalent with more than one binding site of the Wnt co-receptor. For example, the binding domain can be bivalent, trivalent, or tetravalent.

[0008] The methods described herein for generating multivalent binding molecules enable the selective and potent activation of any FZD receptor complex both in vitro and in vivo. Utilizing a group of hundreds of synthetic antibodies targeting FZD and its co-receptors, we generate multivalent binding molecules for the selective and rational activation of one, two, or more FZD receptors. The multivalent binding molecules of this invention are highly stable, suitable for large-scale production and easy to purify, exhibit predictable pharmacokinetics, and are expected to demonstrate low immunogenicity.

[0009] In one embodiment of the invention, the binding domain of the multivalent binding molecule as described herein binds to one or more FZD receptors and LRPs, such as LRP5 and / or LRP6, and is alternatively referred to herein as FLAg. FLAg targeting specific FZDs and their LRP co-receptors will improve directed differentiation and cell therapy, maintain the growth of tissue organoids, mobilize endogenous stem cells in vivo, promote tissue repair after injury, and restore function after tissue degeneration.

[0010] The Fc domain of an FZD agonist may be the Fc domain of an immunoglobulin. The immunoglobulin may be IgG, such as IgG1. In one embodiment of the invention, the multivalent binding molecule is a peptide dimer, wherein the peptide dimers either intrinsically via the Fc domain or via a knock-in-hole conformation within the Fc, thereby allowing the specific assembly of two distinct peptides to produce the multivalent binding domain. A method for dimerizing peptides via a knock-in-hole conformation is described in WO2018 / 026942, by inventors Van Dyk et al., and is incorporated herein by reference.

[0011] One or both of the multivalent binding domains of the FZD agonists described herein can be bivalent and monospecific, having two binding sites for the same epitope of their respective receptors or co-receptor targets. One or both of the binding domains can be bivalent and bispecific, having two binding sites, each binding a different epitope on its respective target.

[0012] In one embodiment of the invention, the FZD-binding domain may comprise two single-chain variable fragments (scFv) for binding to the same or different epitopes on the FZD receptor. In other embodiments of the invention, the FZD-binding domain comprises one or more heavy chain variable domain (VH) fragments and / or one or more light chain variable domain (VL) fragments that bind FZD. In other embodiments of the invention, the FZD-binding domain consists of one or more single-domain antibody fragments that bind FZD. In other embodiments of the invention, the FZD-binding domain comprises an FZD ligand or a fragment thereof that binds to the FZD receptor. In one embodiment of the invention, the FZD-binding domain comprises a synthetic peptide that binds FZD, such as an affinity protein, ankylosing spondylamine, fibronectin repeat protein, fynomer, or anticalin. In one embodiment of the invention, the FZD multivalent binding domain does not comprise scFv. The FZD ligand may be, for example, a fragment of a Wnt protein or a fragment of Norrin that binds to the FZD receptor, or may be another natural or synthetic peptide with affinity matured to interact with one or more FZD receptors. Norrin is an FZD4-specific ligand that forms a complex with LRP5 and / or LRP6, which is associated with activation of typical Wnt signaling.

[0013] In one embodiment of the invention, the co-receptor binding domain may comprise two single-chain variable fragments (scFv) for binding the same or different epitopes on the co-receptor. In other embodiments of the invention, the Wnt co-receptor binding domain comprises one or more heavy chain variable domain (VH) fragments and / or one or more light chain variable domain (VL) fragments binding the Wnt co-receptor. In other embodiments of the invention, the co-receptor binding domain consists of one or more single-domain antibody fragments binding the co-receptor. In one embodiment of the invention, the Wnt co-receptor binding domain comprises a peptide binding the Wnt co-receptor, wherein the peptide is a fragment of a naturally occurring ligand binding the Wnt co-receptor or a synthetic peptide binding the Wnt co-receptor, such as an affinity compound, ankylosing repeat protein, fibronectin repeat protein, fynomer, or anticalin. In another embodiment of the invention, the co-receptor binding domain comprises a co-receptor ligand or a fragment thereof binding the co-receptor (e.g., ligand Dkk1 for co-receptor LRP5 / 6) or another natural or synthetic peptide with affinity matured to interact with one or more co-receptors.

[0014] In one embodiment of the invention, the co-receptor multivalent binding domain does not contain scFv.

[0015] In one embodiment of the invention, each binding domain of the molecule described herein may be formed by two peptides, each peptide comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL), wherein the VH and VL from one peptide pair with the VL and VH of the other peptide to form a bispecific antibody. In this conformation, the binding domain has two binding sites that bind to its target; that is, the FZD binding domain has two binding sites for the FZD receptor, and the co-receptor binding domain has two binding sites for the co-receptor. Using the mortar-and-mortar Fc conformation, peptides containing VH and VL can be engineered to be distinct but still pair to form a bispecific binding domain capable of binding to two different sites on the FZD receptor or co-receptor (see [link to 1]). Figure 3A ).

[0016] In one embodiment of the invention, one or both of the multivalent binding domains comprise two peptides forming a bispecific antibody at each end of the Fc domain. Each bispecific antibody has two binding sites for an epitope on its respective FZD receptor or coreceptor target. The bispecific antibody may be monospecific, wherein the binding sites bind to the same epitope on the FZD receptor or coreceptor, or the bispecific antibody may bispecifically bind to two different epitopes on the FZD receptor or coreceptor.

[0017] The peptides that form scFv or bisomatic antibodies can be derived from antibodies that bind to FZD receptors or from antibodies that bind to Wnt co-receptors. For the FZD-binding domain, the antibody can be one that binds to more than one FZD receptor and antagonizes Wnt signaling or inhibits Wnt binding to a given FZD receptor(s), or the antibody can be one that binds to more than one FZD receptor without inhibiting Wnt binding to FZD receptors. For the co-receptor-binding domain, the antibody can be one that binds to the co-receptor and antagonizes Wnt signaling or inhibits Wnt binding to the co-receptor, or the antibody can be one that binds to the co-receptor without inhibiting Wnt binding to the co-receptor.

[0018] The FZD-binding domain can bind to more than one member of the FZD receptor family, such as coilin receptor 1 (FZD1), coilin receptor 2 (FZD2), coilin receptor 3 (FZD3), coilin receptor 4 (FZD4), coilin receptor 5 (FZD5), coilin receptor 6 (FZD6), coilin receptor 7 (FZD7), coilin receptor 8 (FZD8), coilin receptor 9 (FZD9), or coilin receptor 10 (FZD10). The co-receptor binding domain can bind any Wnt co-receptor, such as LRP5 / 6, PTK7, ROR1 / 2, RYK, GPR124, TSPAN12, or CD133. In one embodiment of the invention, the co-receptor binding domain binds LRP5 and / or LRP6. In one embodiment of the invention, the co-receptor binding domain binds a single epitope on the co-receptor, for example, an epitope of the LRP protein that binds Wnt1 or Wnt3a. In one embodiment of the invention, the co-receptor binding domain binds two epitopes on the co-receptor, for example, an epitope on the LRP that binds to Wnt1 and an epitope that binds to Wnt3a.

[0019] One embodiment of the present invention includes a method for generating induced pluripotent stem cells (iPS), the method comprising culturing somatic cells under conditions suitable for reprogramming somatic cells in the presence of an effective amount of a multivalent binding molecule as described herein. The multivalent binding molecule can be included in an amount that accelerates iPS cell production compared to the generation of iPS cells under the same culture conditions without the multivalent binding molecule.

[0020] Another embodiment of the invention is a method for directly differentiating iPS or other pluripotent stem cells (PSCs) into various lineages by culturing these cells in the presence of an effective amount of multivalent binding molecules as described herein.

[0021] One embodiment of the present invention includes a method for generating tissue organoids, the method comprising culturing a tissue sample under conditions suitable for organoid generation in the presence of an effective amount of a multivalent binding molecule, as described herein, as part of a culture mixture. In one embodiment or of the present invention, the generation frequency of tissue organoids cultured in a medium containing multivalent binding molecules is increased compared to organoids cultured in the same medium without multivalent binding molecules. In one embodiment or of the present invention, tissue organoids are generated faster when cultured in a medium containing multivalent binding molecules compared to tissue samples cultured in the same medium without multivalent binding molecules.

[0022] One embodiment of the present invention includes a method for enhancing the maintenance of tissue organoids, the method comprising culturing organoids in the presence of an effective amount of a multivalent binding molecule, as described herein, as part of a culture mixture. As described herein, tissue organoids cultured in a medium containing a multivalent binding molecule exhibit prolonged survival compared to organoids cultured in the same medium without the multivalent binding molecule.

[0023] One aspect of the present invention is a method for preparing multivalently bound molecules as described herein. In one embodiment of the invention, the multivalently bound molecules are generated by means of the following manner:

[0024] a) Select an Fc domain with both C-terminals and N-terminals.

[0025] b) Recognize antibodies that bind to more than one FZD receptor and

[0026] c) Recognize antibodies that bind to more than one Wnt co-receptor.

[0027] d) Generate a nucleic acid molecule comprising: (i) a nucleotide sequence encoding the Fc domain of step a, (ii) a VL and / or VH of the antibody of step b, or a nucleotide sequence derived from the VL and / or VH of the antibody of step b that binds to more than one FZD, and (iii) a VL and VH of the antibody of step c, or a nucleotide sequence derived from the VL and VH of the antibody of step c that binds to more than one Wnt receptor of step c.

[0028] e) Expressing the nucleic acid molecule of (d) to produce a polypeptide, wherein the polypeptide dimerizes to form a multivalent binding molecule comprising an Fc domain, an FZD binding domain and a Wnt co-receptor binding domain, wherein the FZD binding domain comprises the VL and VH of the antibody of step b or an antibody derived from step b and is attached to one end of the Fc domain, and the Wnt co-receptor binding domain comprises the VL and VH of the antibody of step c or an antibody derived from step c and is attached to the other end of the Fc domain, thereby forming a multispecific binding molecule.

[0029] The antibody in step (b) can be an antibody or antibody fragment that binds to more than one FZD receptor and antagonizes Wnt signaling or inhibits Wnt binding to the receptor. The antibody in step (c) can be an antibody or antibody fragment that binds to more than one FZD receptor without antagonizing Wnt signaling or inhibiting Wnt binding to the receptor. The antibody in step (c) can be an antibody or antibody fragment that binds to more than one Wnt co-receptor and antagonizes Wnt signaling or inhibits Wnt binding to the co-receptor, or binds to the co-receptor without antagonizing Wnt signaling or inhibiting Wnt binding to the co-receptor. The binding domain can be linked to the Fc domain via a linker. The modular aspect of the invention allows for mixing and matching binding domains of antibodies against any given FZD receptor and co-receptor at the ends of the Fc domain to produce multivalent binding molecules that can bind multiple coilin receptor-co-receptor complexes or selectively bind a single coilin receptor-co-receptor complex to activate Wnt signaling.

[0030] Multivalent binding molecules comprise peptide dimers conformed to have an Fc domain, a binding domain that binds to one or more FZD receptors, and a second binding domain that binds to one or more Wnt co-receptors, wherein the FZD binding domain is attached to one end of the Fc domain and the co-receptor binding domain is attached to the other end of the Fc domain. Each binding domain can be monovalent or multivalent, such as divalent, trivalent, or tetravalent.

[0031] Another embodiment of the invention is a method for using multivalent binding molecules, such as for generating induced pluripotent stem (iPS) cells, for directed differentiation of pluripotent stem cells, and for generating and / or maintaining organoids, or for enhancing tissue regeneration in subjects in need.

[0032] Another embodiment of the present invention is a method for activating the Wnt signaling pathway to mobilize an endogenous stem / progenitor cell pool for regenerative medicine and for conditions or diseases associated with insufficient Wnt signaling. Attached Figure Description

[0033] Figure 1AThe binding specificity of five selected antibodies to the extracellular domain (ECD) of human LRP6 was described. LRP6-binding antibodies were selected from a synthetic antibody library by selecting antibodies that bind to the recombinant extracellular domain (ECD) of human LRP6. Binding of the antibodies to human LRP6, mouse LRP6, and mouse LRP5 was detected by ELISA. Binding to Fc peptide and bovine serum albumin (BSA) was included as negative controls.

[0034] Figure 1B The results of a luciferase reporter gene assay monitoring Wnt signal activation are described, showing that IgG 2539 and IgG2542 (100 µM) bind to different sites on the LRP6 ECD through opposite responses to stimulation of Wnt1 (transient transfection) and Wnt3a (0.5 µg / ml purified protein). An anti-MBP antibody was used as a control.

[0035] Figure 2A Describe representative bispecific IgG (Bi-IgG) and bispecific bibody antibodies (bi-diabody) that contain an FZD binding domain (5019) and an LRP6-W1 (2942, L6) at the same end of the Fc domain. 1 ) or W3 (2539, L6 3 Combined with structural domains.

[0036] Figure 2B This indicates that, as determined in the TOPFlash luciferase reporter gene assay in HEK293 cells, the bispecific IgGs (5019-2539 Bi-IgG and 5019-2542 Bi-IgG) do not activate Wnt signaling, but rather act as antagonists of Wnt signaling.

[0037] Figure 2C-2G The binding of bispecific bimeric antibodies is described, wherein the Fc domain is in a club-and-mortar conformation (K / H). Two bimeric antibodies thus obtained, 5019-2539-K / H (FZD / LRP6-W3) and 5019-2542-K / H (FZD / LRP6-W1), retain the FZD binding properties of the original IgG and the LRP6 binding activity, albeit very weakly. Figure 2C Describe the purified FZD-LRP6 bisomatic antibodies: 5019-2539-K / H and 5019-2542-K / H. Figure 2D This describes the FZD receptor binding profiles of the 5019 bisomal antibody with FZD4, FZD5, and FZD7. The 5019 FZD IgG was previously characterized to bind to FZD1, 2, 4, 5, 7, and 8. Figure 2EThe FZD receptor binding curves of the bispecific FZD / LRP6 bisomal antibody 5019-2539-K / H are described. Figure 2F The FZD receptor binding curves of the bispecific FZD / LRP6 bisomal antibody 5019-2542-K / H are described. Figure 2G This indicates that homobisomal antibodies (2539-Fc and 2542-Fc) and heterobisomal antibodies (5019-2539-Fc and 5019-2542-Fc) with binding domains at one end of the Fc domain interact with the extracellular domain of LRP6. Figure 2H This indicates that, as determined by the biolayer interference (BLI) assay, the bisomal antibodies 5019-2539-K / H and 5019-2542-K / H co-bind with FZD CRD and LRP6 ECD in solution.

[0038] Figure 2I This indicates that the FZD and LRP6 receptor bispecific antibodies forming the binding domain are located on the same side of the Fc region, and neither 5019-2539-K / H nor 5019-2542-K / H are FZD agonists that activate the Wnt-mediated pathway. The results demonstrate that, as revealed in the TOPFlash luciferase reporter gene assay in HEK293 cells, the 5019-2539-K / H bispecific antibody (selective for the Wnt3 site on LRP6) completely blocked Wnt3-mediated pathway activation at 10 nM and 50 nM, while 5019-2542-K / H was less effective.

[0039] Figure 2J A comparison of luciferase activities describing tetravalent binding molecules containing either a bispecific antibody or an scFv binding domain. Examples include bispecific antibodies containing anti-FZD scFv and anti-LRP. The molecular expression of the binding domain of ) is similar to that of anti-FZD bisomal antibodies and anti-LRP bisomal antibodies (F P+P -L6 1+3 The binding domain of the F group exhibits similar activity to that of other molecules. Conversely, it is similar to that of F... P+P -L6 1+3 In comparison, it contains anti-FZD bisomatic antibodies and anti-LRP6 scFv ( ) or containing scFv at both ends ( The molecular activity of the molecules is significantly reduced.

[0040] Figure 2K and Figure 2LThis indicates that the difference in activity between tetravalent binding molecules containing binding domains of bimeric antibodies or scFv is not due to differences in affinity, because BLI assays show that binding to LRP6 and FZD isotypes has fairly high affinity regardless of whether the complementary site is in the form of bimeric antibodies or scFv.

[0041] Figure 3A This is a schematic diagram representing a tetravalent binding molecule, which contains two FZD binding domains of homologous (recognizing the same epitope) or heterologous (recognizing different epitopes) bispecific antibodies connected to one end of the Fc domain, and two LRP6 binding domains of homologous or heterologous bispecific antibodies connected to the other end of the Fc domain.

[0042] Figure 3B Description of the multivalently bound molecule 5019-Fc-2539 (F P+P -L6 3+3 ) and 5019-Fc-2542 (F P+P -L6 1+1 Binding to FZD4, FZD5, and FZD7 ECDs. Binding to the FZD receptor was detected using the BLI assay.

[0043] Figure 3C This shows that the tetravalent binding of molecule 5019-Fc-2539 (F...) is achieved. P+P -L6 3+3 ); 5019-Fc-2542 (F P+P -L6 1 +1 ); 5019-K / H-2539-2542 (F P+P -L6 1+3 Activation of the Wnt-β-catenin signaling pathway with purified Wnt3A (0.5 μg / ml). Molecular concentrations are shown. As determined using a pBAR luciferase reporter assay, the tetravalent binding molecule is a potent agonist of the Wnt-β-catenin pathway in HEK293T cells. The 5019-Fc-2539 homobisomal antibody binds to multiple FZD receptors (5019: FZD1, 2, 4, 5, 7, 8) and to the Wnt3a site (2539) on LRP6, activating the reporter gene to levels comparable to those of purified Wnt ligands. The 5019-K / H-2539:2542 heterobisomal antibody, binding to both Wnt binding sites on LRP6, is more potent.

[0044] Figure 3DThe description involves activation of the Wnt-β catechol pathway via a multivalent binding molecule, which binds via Fc to either a monospecific LRP6 homobivalent antibody (5019-Fc-2539, 5019-Fc-2542) or a bispecific LRP6 heterobivalent antibody (5019-K / H-2539-2542, also known as 5019Ag or F). P+P -L6 1+3 FZD homologous dimeric antibody (5019) linked to either of them.

[0045] Figure 3E This study describes the activation of Wnt-β-catenin signaling via molecules containing a monovalent binding domain of either the FZD receptor or the LRP6 co-receptor. Activation of the Wnt-β-catenin pathway was detected using a pBAR luciferase reporter assay in HEK293T cells. 5019-MBP-K / H-2539-2542 contains a monovalent binding domain for FZD and still activates the Wnt pathway, but its potency is reduced by 8-fold compared to 5019Ag (which contains two FZD-binding domains that bind to the same epitope). 5019-K / H-2539-MBP, which retains only one LRP6-W3 binding domain at the C-terminus, exhibits significantly lower potency. Importantly, the lowest agonistic activity was detected in two single FZDs: single LRP6 bisomatic antibodies 5019-MBP-K / H-2539-MBP and 5019-MBP-K / H-MBP-2542, and a single LRP6-W1 site bisomatic antibody 5019-K / H-MBP-2542.

[0046] Figure 3F The description involves activation of the Wnt-β-catenin pathway via a tetravalent binding molecule, where the anti-LRP5 complement targeting the WNT3A binding site is replaced by the anti-LRP6 complement targeting the WNT1 binding site, resulting in a pathway that recruits both co-receptors and exhibits activity similar to F. P+P -L6 1+3 Similar (EC) 50 =4nM) molecules (F P+P -L5 / 6 3 ).

[0047] Figure 4AThe description refers to the activation of the Wnt-β catalin pathway via a multivalent binding molecule in cells lacking endogenous FZD4 receptor (-FZD4) or modified to express FZD4 receptor (+FZD4). This multivalent binding molecule has an FZD-binding domain specific to FZD4 on one side of the Fc domain (in this case, a homologous bisomal antibody) and a co-receptor binding domain specific to LRP6 (2539 and 2542) on the other side of the Fc domain (FZD4Ag: 5038Ag / 5038-K / H-2539-2542, 5044Ag / 5044-K / H-2539-2542, 5048Ag / 5048-K / H-2539-2542, 5063Ag / 5063-K / H-2539-2542, 5080Ag / 50180-K / H-2539-2542, 5081Ag / 5081-K / H-2539-2542). The controls are the multivalent binding molecule 5019Ag (5019-K / H-2539-2542) and the endogenous FZD4 agonist Norrin. The results indicate that replacing the 5019 FZD binding domain (recognizing FZD1, 2, 4, 5, 7, and 8) in 5019Ag / 5019-K / H-2539:2542 (pan-FZD agonist) with the selective binding domain of FZD4 enables the development of selective FZD4 agonists. HEK293T cells were transfected with plasmid pBARL (Wnt-β-catenin luciferase reporter gene) and Rluc (normalized control), which encode the listed FZD agonists and have or do not contain FZD4 and LRP6 cDNA. Norrin was used as a positive control for FZD4 activation. HEK293T cells expressed FZD4 at undetectable levels, therefore the FZD4 agonist could only activate the reporter gene in the presence of transfected FZD4 cDNA. In contrast, panFZDag 5019-K / H-2539:2542 strongly activated Wnt-β-catenin signaling by activating other endogenously expressed coiled proteins in these cells, in the absence or presence of FZD4.

[0048] Figure 4B The activation of the Wnt-β-catenin pathway was demonstrated via a multivalent binding molecule with binding domains (homodimeric antibodies) on one side of the Fc region specifically targeting FZD2 (2876, 2890), FZD2 / 7 (2886), FZD6 (2747), or FZD9 / 10 (2969, 2974), and on the other side of the Fc region specifically targeting the LRP6 heterodimeric antibody formed from antibody fragments 2539 and 2542. Activation of the Wnt-β-catenin pathway was assessed in HEK293T cells using the pBARL assay.

[0049] Figure 4C The activation of the Wnt pathway is described via a multivalent binding molecule possessing a broad-specific FZD-binding domain and derived from IgG that blocks the binding of Wnt to FZD and Wnt-β-catenin signaling. The LRP6-binding domain in these molecules is located at the C-terminus of the Fc region and consists of bimeric antibodies formed from antibodies 2539 and 2542, which have complementary sites that recognize the Wnt3 and Wnt1 binding sites on LRP6, respectively.

[0050] Figure 4D The activation of the Wnt pathway is described via a multivalent binding molecule possessing a pan-specific FZD-binding domain and derived from IgG that does not block Wnt-FZD binding and does not antagonize Wnt3-induced pathway activation. The LRP6-binding domain in these molecules is located at the C-terminus of the Fc region and consists of bimeric antibodies formed from antibodies 2539 and 2542, which have complementary sites that recognize the Wnt3 and Wnt1 binding sites on LRP6, respectively.

[0051] Figure 5 A comparison of the FZD / LRP6 binding behavior of the three tetravalent molecules described in this invention is presented. 5019-Fc-2539, 5019-Fc-2542, and 5019-Fc-2539-2542 bind tightly to FZD but show weaker LRP6 interactions (left panel) or FZD / LRP6 co-binding (middle panel). The FZD binding curve of 5019-K / H-2539-2542 (right panel) shows its recognition of FZD4, FZD5, and FZD7.

[0052] Figure 6A This is a schematic diagram of the top two propellers (E1-E2) of LRP5 / 6 that are known to mediate binding with Wnt1, and the bottom two propellers (E3-E4) of LRP5 / 6 that are close to the plasma membrane and are known to mediate interaction with Wnt3. Figure 6A It also explains the interaction of Wnt1 with LRP5 / 6 and FZD receptors, and the interaction of Wnt3 with LRP5 / 6 and FZD receptors.

[0053] Figure 6B This is a schematic diagram illustrating the potential interaction between the FZD receptor and the LRP5 / 6 receptor through the multivalent binding molecules 5019-Fc-2539, 5019-Fc-2542, and 5019-K / H-2539-2542.

[0054] Figure 6CThis indicates that, as determined using the pBAR luciferase reporter assay, the multivalent binding molecule is an agonist that strongly activates the Wnt-β catechol pathway in HEK293T cells. The 5019-Fc-2539 homobisomal antibody binds to multiple FZD receptors (5019 binds to FZD1, 2, 4, 5, 7, and 8) and also binds to the Wnt3a site (2539) on LRP6, activating the reporter gene to levels comparable to those of purified Wnt ligands. The 5019-K / H-2539:2542 heterobisomal antibody, which binds to both the Wnt3a and Wnt1 binding sites on LRP6, is even more effective.

[0055] Figure 6D The molecule 5019-K / H-2459:2460 is a tetravalent binding molecule with a club-and-mortar conformation Fc domain and a pan-FZD-specific (5019) FZD-binding domain (homodimeric antibody) and a bispecific co-receptor binding domain (2459 binding Wnt1 binding site and 2460 binding Wnt3 binding site) for two sites on LRP5, which also activates the Wnt-β catenin pathway in HEK293T cells.

[0056] Figure 7A This demonstrates that replacing the FZD-binding domain in 5019-K / H-2539:2542 (a pan-FZD agonist recognizing FZD1, 2, 4, 5, 7, and 8) with an FZD-binding domain specific to FZD5 (#2928) produces a selective FZD5 agonist. HPAF-II cell proliferation has been shown to depend on FZD5 signaling. Blocking Wnt-FZD5 signaling with the Wnt secretion inhibitor LGK974 (targeting the acyltransferase Porcupine) leads to cell cycle arrest and proliferation inhibition. Proliferation can be rescued by adding exogenous Wnt3a to conditioned medium or by adding a selective FZD5 agonist (2928-K / H-2539:2542) or a pan-FZD agonist (5019-K / H-2539:2542) as described herein. The FZD4 selective agonist 5038-K / H-2539:2542 has only moderate salvage capability.

[0057] Figure 7B The results showed that stimulation of C3H10T1 / 2 cells with FZD2-specific FLAg resulted in strong induction of the osteogenic marker alkaline phosphatase (ALPL) to levels similar to those achieved with pan-FZD FLAg, while FZD5-specific FLAg exhibited minimal activity.

[0058] Figure 8A and 8BThis demonstrates that when LGK974, a small molecule inhibitor of Porcupine (lower left figure), is used to inhibit Wnt secretion, the pan-FZDag (F...) of this invention... P+P -L6 1+3 Complete replacement of exogenous Wnt3A conditioned medium was used to rescue growth inhibition in intestinal organoids. Intestinal organoids isolated from mice were grown in the presence of recombinant R-Spondin and required the presence of Wnt ligands secreted by paneth cells. Figure 8A The image shows organoid death caused by inhibiting Wnt production with LGK974 (top right). Organoid growth was rescued by exogenous application of Wnt3A conditioned medium (bottom right) or FZDag (bottom left) in the presence of LGK974. The top left image shows organoids treated with DMSO (without LGK974) as a control. Figure 8B This indicates that, if CellTiter Glow is used... ® Assay, Promega quantified the effect by applying Wnt3A conditioned medium or FZDag (F P+P -L6 1+3 It can save organoids from death caused by LGK974 inhibiting Wnt production.

[0059] Figure 9A and 9B The description code dimerizes in a pestle-and-mortar conformation to form pan-FZDag 5019-KH-2539-2542(F P+P -L6 1+3 Examples of plasmids containing peptides. Figure 9A The description encodes a plasmid containing an Fc region with a "pickle" mutation, VH and VL of pan-FZD antibody #5019, and VL of LRP antibody #2542 and VH of LRP antibody #2539. Figure 9B The plasmids describing the peptides contain nucleic acids encoding the Fc region containing the "mortar" mutation, the VH and VL of pan-FZD antibody #5019, and the VH and VL of LRP antibody #2542 and LRP antibody #2539. The peptides encoded by these plasmids form heterodimers with a tetravalent binding domain containing a homodimer antibody generated by pairing the VH and VL of pan-specific FZD antibody #5019, and a bispecific heterodimer antibody generated by pairing the VL and VH of LRP6 antibody #2539 and the VL of LRP antibody #2542 from one peptide with the VH and VL of LRP antibody #2539 and the VL of LRP antibody #2542 from another peptide.

[0060] Figure 9C It is a heterodimer mortar conformation 5019-K / H-2539:2542 (F P+P-L6 1+3 A representative schematic diagram is shown. Using the mortar and pestle conformation within the Fc domain, the modularity of the molecule can be increased to four different binding sites. For this molecule (5019-K / H-2539:2542), a pan-FZD homobisomal antibody was engineered on one side of the Fc domain, and a heterobisomal antibody containing Wnt3(2539) and Wnt1(2542) LRP6 binding sites was engineered on the other side of the Fc domain.

[0061] Figure 10A and 10B This is a annotation of the structural domain of the nucleic acid sequence of the 5019-Chu-2539:2542 multivalent binding molecule (SEQ ID NO: 21 plus the additional 3' TGA and its complementary sequence).

[0062] Figure 11A -F describes the design and validation of a tetravalent binding molecule that binds to the FZD and LRP6 Wnt1 and Wnt3 binding sites (FLAg) as an activator of the Wnt-β catechol pathway. Figure 11A Description of anti-FZD Fab inhibitory effect (top) and specific activity (bottom). Figure 11B Describe the inhibition of Wnt1 or Wnt3A signaling by LRP6 Ab in the bisomatic antibody-Fc form. Figure 11C Describe the molecular structure of tetravalent FLAg. Figure 11D This demonstrates that serial dilution of the pan-specific FLAg protein (F...) P +P -L6 1+1 , F P+P -L6 3+3 and F P+P -L6 1+3 (x-axis) Dose-response curve of LEF / TCF reporter gene activation in HEK293T cells (y-axis). Figure 11E Describe the pan-FLAG(F) concentration at a specified level. P+P -L6 1+3 The level of β-catenin in RKO cells after 30 minutes of treatment. Figure 11F Description using 10nM generalized FLAg(F) P+P -L6 1+3 The time course of β-catenin and phosphorylated scattered protein-2 (p-Dvl2) protein levels in RKO cells treated with )

[0063] Figures 12A-12D Description of FLAG F P+P -L6 1+3 Characterization and decomposition of binding and activity. Figure 12A and 12B Display FP+P -L6 1+3 Binding dynamics with 9 out of 10 human FZD CRDs and human LRP6 ECDs. Figure 12C Indicates F P+P -L6 1+3 It behaves similarly to regular IgG and interacts with FcRn in a dose- and pH-dependent manner. Figure 12D Indicates F P+P -L6 1+3 Its interactions with other Fc effectors are similar to those of IgG. Other Fc effectors include complement (C1q), the natural killer cell marker CD16a, the B cell marker CD32a, and the monocyte and macrophage marker CD64.

[0064] Figure 13A and 13B Indicates the use of 30 nM F P+P -L6 1+3 Three days of treatment induced strong induction of the mesodermal marker BRACHYURY, and the expression of the pluripotency marker OCT4 decreased to levels comparable to those of treatment with the GSK3 inhibitor CHIR99021 at 6 μM.

[0065] Figure 14 Display carrier, C59 or pan-FLAG (F P+P -L6 1+3 Representative fluorescence images of small intestinal sections from LGR5-GFP mice treated with C59. LGR5-GFP is expressed in stem cells at the base of the crypts. Cell nuclei are counterstained with DAPI. Detailed Implementation

[0066] This document describes a multivalent binding molecule comprising an Fc domain, an FZD binding domain, and a Wnt co-receptor binding domain, wherein the binding domain is attached to opposite ends of the Fc domain. The multivalent binding molecule of this invention is an agonist of the Wnt signaling pathway, and is alternatively referred to herein as an FZD agonist or FZDag. The Wnt ligand functions by promoting the aggregation of the FZD receptor and co-receptor. Without being bound by theory, it is anticipated that multispecific molecules as described herein will simultaneously bind to both the FZD receptor and the Wnt co-receptor, thereby activating the Wnt signaling pathway.

[0067] The modularity and effectiveness of the multivalent binding molecules for activating the Wnt signaling pathway described herein contrast with prior art Wnt alternatives consisting of monovalent FZD and LRP5 / 6 binding ligands, wherein the binding ligands are not linked to opposite ends of the Fc domain. In one embodiment of the invention, the FZD binding domain comprises a binding moiety derived from an antibody or peptide that specifically binds to one or more FZD receptors, and the co-receptor binding domain comprises a binding moiety that binds to co-receptors such as LRP5 / 6, ROR1 / 2, RYK, or PTK7. In one embodiment of the invention, the antibody or peptide that specifically binds to one or more FZD receptors binds to one or more cysteine-rich domains (CRDs) of the FZD receptors.

[0068] The amino acid sequence of the FZD receptor and the nucleotide sequence encoding the FZD receptor, as well as antibodies and antibody libraries binding to FZD or Wnt co-receptors LRP5 / 6, ROR1 / 2, RYK or PTK7, are readily available or can be generated using methods well known in the art (see, for example, U.S. Publication No. 2015 / 0232554, inventors Gurney et al.; U.S. Publication No. 2016 / 0194394, inventors Sidhu et al.; U.S. Publication No. 20190040144, inventors Pan et al.; U.S. Publication No. 2017 / 0166636, inventors Wu et al.; U.S. Publication No. 2016 / 0208018, inventors Chen et al.; U.S. Publication No. 2016 / 0053022, inventors Macheda et al.; U.S. Publication No. 2015 / 031293, inventors Damelin et al.).

[0069] Methods for generating peptides or polypeptides that bind to selected targets are well known in the art, see, for example, Sidhu et al. Methods in Enzymology (2000) 328: 333-336. For example, libraries of affinity proteins for FZD or Wnt co-receptors can be obtained according to methods known in the art (see, for example, U.S. Patent No. 5,831,012 and Lofblom et al., FEBS Letters 584 (2010) 2670-2680); libraries of ankyrin repeat proteins for selecting peptides that bind to FZD or Wnt co-receptors can be obtained according to methods known in the art (see, for example, WO 02 / 020565, inventors Stumpp et al.); libraries of fibronectin repeat proteins for selecting peptides that bind to FZD or Wnt co-receptors can also be obtained according to methods known in the art (see, for example, U.S. Patent No. 9,200,273, inventors Diem and Jacobs. Peptides that bind to FZD or Wnt co-receptors can also be fynomers, small binding proteins derived from the human Fyn SH3 domain, or artificial receptor proteins “anticalins” based on human apolipoprotein D, and can be generated using methods known in the art, see, for example, Silacci et al., J. Biol. Chem (2014). 289(20):14392-8 and Vogt and Skerra, ChemBioChem (2004) 5, 191-199).

[0070] Antibodies suitable as sources of antigen-binding peptides as described herein can be isolated by screening combinatorial libraries for peptides possessing the desired activity or multiple activities. For example, various methods for generating phage display libraries and screening such libraries for antibodies possessing the desired binding properties are known in the art. Such methods have been reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further described in, for example, McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004). In some phage display methods, libraries of the VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in the phage library. Then, antigen-binding phages can be screened as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phages typically display antibody fragments, either single-chain Fv (scFv) fragments or Fab fragments. Libraries derived from immunogens can provide high-affinity antibodies against immunogens without the need for hybridoma construction. Alternatively, as described in Griffiths et al., EMBO J, 12: 725-734 (1993), a naive repertoire (e.g., from humans) can be cloned without any immunization to provide a single source of antibodies against a wide range of non-self and self antigens.Finally, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992), a natural library can also be synthesized by cloning an unrearranged V gene fragment from stem cells, encoding a highly variable CDR3 region using PCR primers containing random sequences, and performing the rearrangement in vitro. Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Publications Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360. Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0071] Therefore, those skilled in the art will readily prepare Fc domains and mix and match multivalent FZD-binding domains and Wnt co-receptor-binding domains with desired specificity at the N and C ends of the Fc domains to prepare multivalent binding molecules that bind the desired FZD receptor and co-receptor, thereby activating specific Wnt pathways. These specific agonists will serve as powerful tools for enhancing cell proliferation, differentiation, organoid survival and maintenance, and tissue regeneration in vivo. These specific agonists also serve as powerful tools for analyzing the FZD specificity involved in these processes. For example, as shown in this paper, FZD5Ag, rather than FZD4Ag, rescued growth defects in the LGK974-treated RNF43 mutant PDAC cell line, highlighting the importance of FZD5 relative to the FZD4 receptor in this process.

[0072] One embodiment of the present invention is a method for influencing the binding of a peptide to an FZD receptor and a Wnt co-receptor on a cell, wherein the binding of the peptide to the FZD receptor and the co-receptor activates the Wnt signaling pathway in the cell. The method includes selecting an Fc domain having a C-terminus and an N-terminus, or a fragment thereof containing a CH3 domain; attaching a first multivalent binding domain for binding the FZD receptor to one end of the Fc domain; and attaching a second multivalent binding domain for binding the Wnt co-receptor to the other end of the Fc domain, thereby forming a multivalent binding molecule; and then contacting the multivalent binding molecule with cells expressing the FZD receptor and the co-receptor under conditions of activated Wnt signaling pathway.

[0073] In one embodiment of the invention, the multivalent binding domain may comprise a single-chain variable fragment (ScFv) binding to one or more FZD receptors, a ligand of an FZD receptor or co-receptor, or a fragment thereof binding to an FZD receptor or co-receptor. In another embodiment, the binding domain does not comprise a single-chain variable fragment (ScFv) binding to one or more FZD receptors, a ligand of an FZD receptor or co-receptor, or a fragment thereof binding to an FZD receptor or co-receptor.

[0074] In one embodiment of the invention, at least one of the FZD or co-receptor multivalent binding domains comprises a bimeric antibody having two peptides, each peptide having a heavy chain variable domain (VH) linked to a light chain variable domain (VL), wherein the VH and VL from one peptide pair with the VL and VH of the other peptide, thereby giving the binding domain two epitope binding sites. The VH and VL domains may be the VH and VL of an antibody that binds to a Wnt binding site on an FZD receptor or co-receptor. The VH or VL derived from the antibody, i.e., the source antibody, may have 50%, 55%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the VH and VL of the source antibody, and still retains binding to the FZD receptor or co-receptor site bound by the antibody.

[0075] In one embodiment of the invention, the multivalent binding molecule of the present invention comprises the multivalent binding molecules of Table 1 (Table 1 includes Tables 1A and 1B: Table 1A shows the nucleotide and amino acid sequences of exemplary multivalent binding molecules of the present invention; Table 1B shows the nucleotide sequences of the various domains of the exemplary multivalent binding molecules). In one embodiment of the invention, the multivalent binding molecule of the present invention is substantially composed of the multivalent binding molecules of Table 1. In one embodiment of the invention, the multivalent binding molecule of the present invention is composed of the multivalent binding molecules of Table 1.

[0076] In one embodiment of the invention, the multivalent binding domain comprises one or more VL and VH domains of the molecules in Table 1. In one embodiment of the invention, the multivalent binding domain of the multivalent molecule is substantially composed of one or more VL and VH domains of the molecules in Table 1. In one embodiment of the invention, the multivalent binding domain of the multivalent molecule is composed of one or more VL and VH domains of the molecules in Table 1. In one embodiment of the invention, the binding domain of the multivalent molecule as described herein comprises VH and VL domains having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity with the VH and VL domains of the molecules listed in Table 1, and retains binding to the antigen bound by the molecules listed in Table 1.

[0077] In one embodiment of the invention, the binding domain of the multivalent molecule as described herein includes one or more complementarity-determining regions (CDRs) of the molecules listed in Table 1. In one embodiment of the invention, the binding domain of the multivalent molecule as described herein includes a CDR having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity with the CDRs of the molecules listed in Table 1, and maintains binding to the antigen bound to the molecules shown in Table 1.

[0078] The FZD receptor bound by the multivalent binding molecule of the present invention can be FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, or FZD10. The FZD receptor can be FZD1, FZD2, FZD4, FZD5, FZD7, or FZD8. The multivalent binding molecule can bind only one FZD receptor, or it can panspecifically bind more than one FZD receptor. The FZD multivalent binding domain can bind, for example, FZD1, FZD2, FZD4, FZD5, FZD7, and FZD8. The FZD multivalent binding domain can specifically bind one FZD receptor, such as FZD2, FZD4, FZD5, or FZD6.

[0079] In one embodiment of the invention, the FZD-binding domain is single-specific and binds to a single epitope on the FZD receptor. In another embodiment of the invention, the FZD-binding domain is dual-specific and binds to two epitopes on the FZD receptor.

[0080] The co-receptor binding domain can bind any Wnt co-receptor, such as LRP5 / 6 or ROR1 / 2. The multivalent co-receptor binding domain can bind, for example, LRP5 / 6, PTK7, ROR1 / 2, RYK, GPR12, TSPAN12, or CD133. In one embodiment of the invention, the multivalent co-receptor binding domain binds LRP5 or LRP6.

[0081] In one embodiment of the invention, the co-receptor multivalent binding domain binds to a single epitope on the co-receptor, such as an epitope on LRP5 / 6 that binds Wnt1 or Wnt3. In another embodiment, the co-receptor multivalent binding domain binds to two epitopes within the co-receptor, such as an epitope on LRP5 / 6 that binds to Wnt1 and an epitope that binds to Wnt3. The Wnt co-receptor bound by the multivalent binding molecule of the present invention can be LRP5 or LRP6, PTK7, ROR1, ROR2, RYK, GPR124, TSPAN12, or CD133.

[0082] In one embodiment of the invention, the multivalent binding molecule includes an Fc domain, wherein the Fc domain is the Fc domain of an immunoglobulin or a fragment thereof containing a CH3 domain. In one embodiment of the invention, the immunoglobulin is IgG. In one embodiment of the invention, the IgG is IgG1.

[0083] One embodiment of the present invention is a method for activating the Wnt signaling pathway in cells, the method comprising contacting cells having FZD receptors and Wnt co-receptors with the multivalent binding molecule of the present invention in an amount that effectively activates Wnt signaling.

[0084] In one embodiment of the invention, at least one multivalent binding domain comprises an scFv that binds to an FZD receptor or co-receptor, or a ligand or fragment thereof that contains an FZD receptor or co-receptor. In one embodiment of the invention, at least one multivalent binding domain does not comprise an scFv that binds to an FZD receptor or co-receptor, and does not comprise a ligand or fragment thereof that contains an FZD receptor or co-receptor.

[0085] In one embodiment of the invention, the FZD multivalent binding domain comprises an FZD bivalent antibody, and the co-receptor multivalent binding domain comprises a co-receptor bivalent antibody, wherein the bivalent antibody comprises two polypeptides, each comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL), wherein the binding domain is formed by pairing the VH and VL from one peptide with the VL and VH from the other peptide.

[0086] The VH and VL of the FZD binding domain can originate from antibodies that bind to the FZD receptor and antagonize Wnt signaling or inhibit the binding of Wnt ligands to the FZD receptor. Conversely, the VH and VL of the FZD binding domain can originate from antibodies that bind to the FZD receptor without antagonizing or inhibiting the binding of Wnt ligands to the FZD receptor.

[0087] The VH and VL co-receptor binding domains can originate from antibodies that bind to the co-receptor and antagonize Wnt signaling or inhibit the binding of Wnt ligands to the co-receptor. Conversely, the VH and VL co-receptor binding domains can originate from antibodies that bind to the co-receptor without antagonizing Wnt signaling or inhibiting the binding of Wnt ligands to the co-receptor.

[0088] In the multivalent binding molecule of the present invention, one or both of the binding domains may be bivalent, and one or both of the bivalent binding domains may be bispecific to the FZD receptor or co-receptor. In one embodiment of the invention, both binding domains are bivalent and bispecific, each binding domain binding to two different epitopes on its respective target FZD receptor or co-receptor. For example, the binding molecule may contain an FZD binding domain that is bivalent and bispecific (binding to two different epitopes) to the FZD receptor, or the binding molecule may contain a co-receptor binding domain that is bivalent and bispecific to the co-receptor.

[0089] In one embodiment of the invention, the FZD binding domain is connected to the N-terminus of the Fc domain of the multivalent binding molecule, and the co-receptor binding domain is connected to the C-terminus of the Fc domain.

[0090] Another embodiment of the present invention is a nucleic acid molecule encoding a multivalent binding molecule as described herein, comprising an expression cassette and a vector containing the nucleic acid molecule encoding the multivalent binding molecule. The nucleic acid molecule can be inserted into the vector and expressed in a suitable host cell, and then the multivalent binding molecule can be isolated from the cell using methods well known in the art. As used herein, the term "vector" refers to a nucleic acid delivery vector or plasmid that can be engineered to contain a nucleic acid molecule, such as a nucleic acid sequence encoding a multivalent binding molecule as described herein. A vector that can express a protein when a polynucleotide is inserted is called an expression vector. The vector can be inserted into a host cell by transformation, transduction, or transfection, thereby allowing the carried genetic material to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phage particles; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages, such as λ phage or M13 phage, and animal viruses, etc. Animal viruses may include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), varicella-zoster virus, baculoviruses, papillomaviruses, and multivacuolar papillomaviruses (e.g., SV40). Vectors may contain multiple components that control the expression of multivalently binding molecules as described herein, including but not limited to promoters such as viral or eukaryotic promoters such as the CMV promoter, signal peptides such as the TRYP2 signal peptide, transcription initiation factors, enhancers, selection elements, and reporter genes. Furthermore, vectors may also contain a replication initiation site.

[0091] As used in this invention, the term "host cell" refers to a cell into which a vector can be introduced, including but not limited to prokaryotic cells such as Escherichia coli and Bacillus subtilis, fungal cells such as yeast and Aspergillus, insect cells such as S2 Drosophila cells and Sf9, or animal cells, including human cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0092] One embodiment of the invention is a pharmaceutical composition comprising an FZD agonist as described herein and a pharmaceutically acceptable excipient. The pharmaceutical composition may further comprise additional agents that activate the Wnt pathway, such as Norrin or R-Spondin. The pharmaceutical composition may consist of, or substantially consist of, a multivalent binding molecule as described herein and a pharmaceutically acceptable carrier or excipient. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th edition) ed. AR Gennaro, Mack Publishing Company, Easton, Pa. 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextran solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Other carriers include sustained-release formulations, such as a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, in the form of a shaped article, such as a membrane, liposome, or microparticle. It will be apparent to those skilled in the art that certain carriers may be preferred, depending on, for example, the route of administration and the concentration of the FZD agonist applied.

[0093] Wnt signaling is a pervasive pathway regulating cell and tissue differentiation. For example, in eye development, a specific Wnt pathway, the Norrin-FZD4 pathway, has been identified, playing a role in retinal angiogenesis. Signaling via the Norrin-FZD4 pathway is essential for the development and maintenance of the retinal vascular system. Mutations in genes affecting this pathway can lead to several pediatric vitreoretinopathy disorders, such as Norrie's disease, familial exudative vitreoretinopathy (FEVR), as well as pseudoglioma and osteoporosis syndrome. Furthermore, retinopathy of preterm birth (ROP) is associated with mutations in this pathway, and Wnt pathway mutations have been reported in Coats' disease and persistent fetal vascular system (PFV). The Norrin-FZD pathway is also involved in CNS vascular development. Genetic ablation of Norrin, FZD4, Lrp5, and the co-receptor tetraspanin-12 (Tspan-12) leads to defects in angiogenesis and barrier disruption of retinal and cerebellar vessels (Cho et al. (2017) Neuron 95, 1056-1073; Zhou et al., (2014) J Clin Invest 124:3825–3846). This paper particularly considers the FZD4 agonist of the present invention, especially FZD4 FLAg, which contains an FZD4-binding domain at one end of the Fc receptor and binding domains of LRP5 and / or LRP6 on the other side of the Fc domain, enhancing barrier function and promoting angiogenesis. For example, treatment with FZD4 FLAg promotes the development and maintenance of the retinal vascular system and / or the blood-retinal barrier (BRB) and blood-brain barrier (BBB). Therefore, one aspect of the invention is a method for promoting and / or maintaining the retinal vascular system by treating ocular tissues, such as retinal tissue, with an effective amount of FZD4 FLAg, either locally or systemically. Another aspect of the invention is a method for promoting and / or maintaining the BBB vascular system by treating BBB with an effective amount of FZD4 FLAg after systemic administration. Yet another aspect of the invention is a method for treating a subject suffering from a condition characterized by reduced retinal or cerebral angiogenesis by administering an effective amount of FZD4 FLAg to the subject, wherein the effective amount is sufficient to increase retinal or cerebral angiogenesis in the subject. The subject may be a fetus.

[0094] Pathologically low levels of Wnt signaling are associated with osteoporosis, polycystic kidney disease, and neurodegenerative diseases. Controlled activation of the Wnt pathway has been shown to promote regenerative processes such as tissue repair and wound healing. Zhao J, KimKA, and Abo A, Trends Biotechnol. 27(3):131-6 (March 2009). See also, Logan CY and Nusse R, Annu. Rev. Cell. Dev. Biol. 20:781-810 (2004); Nusse R., Cell Res.15(1):28-32 (January 2005); Clevers H, Cell 127(3):469-80 (November 3, 2006). Proof-of-concept experiments have been conducted to demonstrate the role of Wnt signaling in osteoporosis or mucositis. Furthermore, increased Wnt signaling has been proposed to be beneficial for the treatment of diabetes and other metabolic diseases. Decreased Wnt signaling is associated with metabolic diseases. LRP6 with loss of function R611CMutations lead to early coronary artery disease, metabolic syndrome, and osteoporosis in humans. Main A et al., Science 315:1278 (2007). "LRP5 loss-of-function mutation is associated with osteoporosis, impaired glucose metabolism, and hypercholesterolaemia in human." Saarinnen et al., Clin Endocrinol 72:481 (2010). In mice lacking both LRP5 and apoE, severe hypercholesterolemia, impaired lipid tolerance, and advanced atherosclerosis are present. Magoori K. et al., JBC 1 1331 (2003). LRP5 is essential for normal cholesterol metabolism and glucose-induced insulin secretion in mice. Fujino et al., PNAS 100:229 (2003). TCF7L2 variant confers risk of type 2 diabetes. Grant et al., Nat Genet 38:320 (2006); Florez et al., N Engl J Med 355:241 (2006). Increased Wnt signaling is beneficial for the treatment of metabolic diseases. Therefore, administration of the multivalent binding molecule of the present invention to subjects with metabolic diseases can be used to treat the subjects' metabolic diseases.

[0095] Inflammatory bowel disease (IBP) is a group of inflammatory conditions of the colon and small intestine. The main types of IBP are Crohn's disease and ulcerative colitis. The RSP01 protein has been shown to improve inflammatory bowel disease in animal models. (Zhao J et al., Gastroenterology 132:1331 (2007)). Therefore, administration of the multivalent binding molecule of the present invention to subjects with IBP is useful for treating IBP in subjects.

[0096] Therefore, one embodiment of the present invention is a method for treating a subject suffering from a condition associated with reduced Wnt signaling, the method comprising administering an effective amount of the FZD agonist of the present invention to the subject in need. Conditions may include, for example, osteoporosis, polycystic kidney disease, neurodegenerative diseases, mucositis, short bowel syndrome, bacterial translocation in the gastrointestinal mucosa, enterotoxin or enteropathic infectious diarrhea, celiac disease, nontropical stomatitis diarrhea, lactose intolerance and other conditions in which dietary exposure causes mucosal villus insensitization and malabsorption, atrophic gastritis and diabetes, fractures, tissue regeneration such as tissue repair and wound healing, and metabolic diseases such as diabetes, and melanoma. Examples of damaged tissues that can be treated using the method of the present invention include, but are not limited to, intestinal tissue, heart tissue, liver tissue, kidney tissue, skeletal muscle, brain tissue, bone tissue, connective tissue, and skin tissue. The multivalent binding molecule of the present invention can be administered to a subject suffering from a disease or condition characterized by low Wnt signaling. The multivalent binding molecule of the present invention is administered to the subject in an amount that effectively increases Wnt signaling and improves the subject's disease or condition.

[0097] Mucositis is a clinical complication of cancer treatment. It is caused by the cytotoxic effects of radiation or chemotherapy on rapidly proliferating cells. Mucositis consists of epithelial damage primarily affecting the intestinal and oral mucosa. Clinical symptoms include severe oral pain, nausea, diarrhea, malnutrition, and in severe cases, sepsis and death. These symptoms often limit the dosage of cancer treatments. Currently, there are no oral or gastrointestinal mucositis treatments associated with chemotherapy or radiation therapy for solid tumors.

[0098] Oral mucositis is a common and often debilitating complication of cancer treatment. 50% of patients receiving radiotherapy for head and neck cancer and 10-15% of patients receiving 5-FU treatment develop grade 3-4 oral mucositis. RSP01 has been shown to improve oral mucositis in animal models. Zhao J et al., PNAS 106:2331 (2010).

[0099] Short bowel syndrome (SBS) is caused by the loss of function or anatomy of a large segment of the small intestine, resulting in severe impairment of digestive and absorptive capacity. Each year, many people undergo long-segment resections of the small intestine for various conditions, including trauma, inflammatory bowel disease, malignancies, and mesenteric ischemia. Various non-surgical procedures, such as radiation exposure, can also lead to functional short bowel syndrome. Current treatments for short bowel syndrome include dietary approaches, total parenteral nutrition (TPN), bowel transplantation, and non-transplant abdominal surgery. While these treatments help improve the prognosis of patients with SBS, they only partially correct the underlying problems of reduced bowel function. Currently, there is no therapy that can accelerate the recovery of the remaining small intestine in patients with SBS. See Seetharam and Rodrigues, The Saudi Journal of Gastroenterology 17, 229-235 (2011).

[0100] The adult mammalian gut constitutes one of the most rapidly self-renewing tissues, with the intestinal mucosa comprising continuous structures folded into proliferative crypts and differentiated villi. In response to mucosal damage, the host initiates a healing response, leading to restoration of mucosal integrity and regeneration of mucosal structures. This process largely depends on the proliferation of intestinal stem cells. (Neal et al., Journal of Surgical Research 167, 1-8 (2010); van der Flier and Clevers, Annual Review of Physiology 71, 241-261 (2009)).

[0101] Therefore, factors regulating intestinal stem cell activity play a major role in the host's response to intestinal damage. Since Wnt protein is the most important growth factor supporting intestinal stem cell proliferation, enhancing Wnt signaling will increase the proliferation of intestinal epithelial cells. This will lead to an increase in the number of small intestinal villi and an increase in the mucosal absorptive surface area.

[0102] Therefore, in one embodiment, the multivalent binding molecule of the present invention is applied to a person suffering from short bowel syndrome. The multivalent binding molecule is applied in an amount sufficient to increase the absorptive surface area of ​​the gastrointestinal mucosa. The application of the multivalent binding molecule of the present invention has a successful prognosis when a person with acute short bowel syndrome adapts to enteral feeding, or when a person with pre-existing SBS absorbs nutrients from enteral feeding, or when a person reduces the amount of total parenteral nutrition required daily to maintain their weight.

[0103] Prevention of bacterial translocation. In one embodiment, the antibody of the present invention is administered to a person at risk of sepsis caused by intestinal bacteria. The multivalent binding molecule is administered in an amount sufficient to increase the integrity of the gastrointestinal mucosa, thereby preventing intestinal bacteria from entering the bloodstream. Decreased gastrointestinal mucosal integrity (compared to that of the normal population) is a major cause of bloodstream infections and sepsis in critically ill patients. Administration of the multivalent binding molecule has a successful prognosis when fewer cases of bacteremia and sepsis are observed in patients in the intensive care unit (ICU) than in patients who have not received the multivalent binding molecule of the present invention.

[0104] Accelerates recovery during or after enterotoxin- or enteropathic infectious diarrhea. Infectious diarrhea is a major pediatric problem. In one embodiment, the multivalent conjugate molecule of the invention is administered in an amount sufficient to shorten the time to the end of diarrhea or to normal bowel movements. The multivalent conjugate molecule of the invention may also be administered in addition to standard care including oral or parenteral rehydration and sometimes antibiotics. When observed in pediatric patients, successful outcomes have been seen in reducing hospitalizations, shortening hospital stays, or lowering the incidence of dehydration complications and electrolyte abnormalities compared to pediatric patients who did not receive the multivalent conjugate molecule of the invention.

[0105] Celiac diarrhea, nontropical stomatitis diarrhea, lactose intolerance, and other conditions in which dietary exposure causes mucosal villi desensitization and malabsorption. In one embodiment, the multivalent binding molecule of the invention is applied in an amount sufficient to increase the mucosal absorptive surface area. The multivalent binding molecule of the invention can be applied in addition to standard care, which primarily involves avoiding unpleasant foods and sometimes dietary supplements. Application of the multivalent binding molecule of the invention will yield a successful outcome when a person with celiac disease, nontropical stomatitis diarrhea, lactose intolerance, or other conditions is adapted to enteral feeding, or when a person with any of these conditions is absorbing nutrients from enteral feeding, or when a person reduces the amount of total parenteral nutrition required for their daily weight maintenance.

[0106] Atrophic gastritis, particularly a form known as environmental metaplastic atrophic gastritis, is a common condition among the elderly and is currently treated with vitamin B12 injections. Patients have an increased risk of developing carcinoid tumors and adenocarcinomas. Medical experts have observed that in the case of carcinoid tumors, the administration of multivalent binding molecules has a successful prognosis in reducing tumor incidence by decreasing gastrin production from metaplastic G cells. Multivalent binding molecules should not be administered to subjects if medical experts determine that the tumor is due to increased activation of the Wnt pathway.

[0107] The FZD agonist of the present invention can be administered, for example, by injection (e.g., subcutaneous, intravenous, intraperitoneal, etc.), topical, or oral administration. Depending on the route of administration, the active compound may be encapsulated in a material to protect it from acids and other natural conditions that can inactivate it. The multivalently bound molecules described herein can be dissolved or suspended in a pharmaceutically acceptable carrier, preferably an aqueous carrier. Furthermore, the composition may contain excipients such as buffers, binders, blasting agents, diluents, flavoring agents, lubricants, etc. A broad list of excipients that can be used in such compositions can be obtained, for example, from A. Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). The multivalently bound molecules can also be administered together with immunostimulants such as cytokines.

[0108] One embodiment of the present invention includes a method for generating induced pluripotent stem (iPS) cells, the method comprising culturing somatic cells under conditions suitable for reprogramming somatic cells, wherein said culture conditions further comprise multivalent binding molecules as described herein. Methods for generating pluripotent stem cells are well known in the art; see, for example, Takahashi and Yamanaka, (2006), Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors, Cell 126, 663–676; Takahashi et al. (2007) Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors, Cell 131, 861–872; Yu et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920; U.S. Patent Nos. 8,546,140 and 8,268,620. In one embodiment of the invention, the multivalent binding molecule of the invention is contained in the culture medium in an amount sufficient to accelerate the generation of iPS cells.

[0109] One embodiment of the present invention includes a method for directed differentiation of pluripotent or multipotent stem cells (PSCs) or induced pluripotent stem cells (iPSCs), comprising culturing cells under conditions suitable for directed differentiation, wherein said culture conditions further comprise an effective amount of a multivalent binding molecule as described herein. Studies of mouse and human PSCs have identified specific methods for adding growth factors, including Wnt, which can induce PSC differentiation into different lineages. Methods for directed differentiation of PSCs involving activation of Wnt signaling are known in the art, see, for example, Lam et al. (2014) SeminNephol 34(4); 445-461; Yucer et al. (September 6, 2017) Scientific Reports 7, article number 10741. It is contemplated that multivalent binding molecules as described herein can be used to influence the activation of the Wnt signaling pathway to direct PSC differentiation.

[0110] One embodiment of the present invention is a method for enhancing tissue regeneration in a subject by administering an effective amount of a multivalent binding peptide as described herein to activate Wnt signaling in that subject.

[0111] One embodiment of the present invention includes a method for enhancing bone healing and / or regeneration in a subject in need, such as a subject suffering from osteoporosis or a fracture, by administering an effective amount of a multivalent binding molecule as described herein. In a specific embodiment, the multivalent binding molecule of the present invention comprises a binding domain binding to FZD2 and a binding domain binding to LRP5 and / or LRP6. The binding domains may be monovalent or multivalent, such as bivalent, trivalent, or tetravalent, and may be monospecific or multispecific, such as bispecific.

[0112] Subjects can be any animal (e.g., mammals), including but not limited to humans, non-human primates, horses, cattle, dogs, cats, rodents, etc. Typically, subjects are humans.

[0113] Effective doses and schedules for administering multivalent binding molecules as described herein can be determined empirically, and such determinations are within the scope of the art. Those skilled in the art will understand that the dose of such FZD agonists that must be administered will vary depending, for example, the subject receiving the antibody, the route of administration, the specific type of FZD agonist used, and other medications being administered. Guidelines for selecting appropriate doses of FZD agonists can be found in the literature concerning the therapeutic use of antibodies, for example, Handbook of Monoclonal Antibodies, Ferrone, eds., Noges Publications, Park Ridge, NJ, (1985) ch. 22 and pp. 303-357; Smith, Antibodies in Human Diagnosis and Therapy, Haber, eds., Raven Press, New York (1977) pp. 365-389. The dose range for administering the composition is one that is sufficiently wide to produce the desired effect. The dose should not be too high to cause adverse side effects, such as adverse cross-reactions, allergic reactions, etc. Typically, the dosage will vary depending on the patient's age, condition, sex, and degree of inflammation, and can be determined by someone skilled in the art. In the absence of any contraindications, the dosage may be adjusted by the individual physician. The dosage can vary and may be administered once or multiple times daily for one or several days. Despite variations in individual needs, determining the optimal range for the effective amount of the carrier is within the scope of the art.

[0114] In recent years, methods have been developed for culturing microorganisms known as "organoids," which encapsulate the gross anatomy and cell type composition of different tissues. Notably, complete organoids can be generated from single tissue stem cells, as demonstrated by the first isolation of intestinal LGR5+ stem cells from mice. The components of the culture medium activating the Wnt-β catechol pathway are known to be essential for organoid derivation, growth, survival, and maintenance. Therefore, purified or provided R-spondin and Wnt ligands as conditioned media are universally required for the growth of organoids from various tissues. However, purified Wnt proteins typically exhibit low specific activity and cannot sustain organoid growth. Therefore, those skilled in the art rely on the addition of Wnt3A conditioned media or small molecules such as GSK3 inhibitors to generate organoids. However, the manufacture of Wnt3A conditioned media is labor-intensive, the properties of conditioned media are inconsistent, and small-molecule GSK3 inhibitors can strongly activate the pathway to toxic levels. The multivalent binding molecules described herein address these issues because they are easy to manufacture and purify, have consistent and reproducible properties, and specifically activate Wnt by selectively binding to the desired combination of FZD receptors and co-receptors.

[0115] One embodiment of the present invention includes a method for generating tissue organoids, the method comprising culturing tissue with an effective amount of multivalent binding molecules as described herein. Organoids are 3D multicellular in vitro tissue constructs that mimic their corresponding in vivo organs, and are therefore used to study various aspects of the organ in tissue culture dishes. Methods for generating organoids are well known in the art, and epithelial organoids, for example, derived from adult stem cells in various gastrointestinal organs almost always require a Wnt signaling agonist (among other signaling factors, including those embedded in matrix gel) that can both maintain cells and generate in vivo complement-like complement. Wnt signaling also enhances the development of inner ear organoids in 3D culture and has been used for the generation of kidney organoids, see, for example, Natalie de Souza (2018) Nature Methods 15(1): 23; DeJonge et al. (2016) PLosOne 11(9), e0162508; Akkerman and Defize, (2017) Bioessays39, 4, 1600244. The multivalent binding molecules of the present invention can be included in the culture medium of organoids in an amount sufficient to enhance their growth, survival, and maintenance in culture. Thus, embodiments of the present invention include a method for enhancing the culture of tissue organoids comprising a culture medium containing an effective amount of the multivalent binding molecules as described herein.

[0116] Another aspect of the present invention is a method for preparing multivalently bound molecules as described herein. In one embodiment of the invention, the multivalently bound molecules are generated in the following manner:

[0117] a) Select an Fc domain with both C-terminals and N-terminals.

[0118] b) Recognizing peptides that bind to more than one FZD receptor, or antibodies that recognize more than one FZD receptor, and

[0119] c) Recognize peptides that bind to more than one Wnt co-receptor or antibodies that bind to more than one Wnt co-receptor.

[0120] d) Generate a nucleic acid molecule comprising: (i) a nucleotide sequence encoding the Fc domain of step a, (ii) a nucleotide sequence encoding the peptide of step b that binds to one or more FZD receptors, or a nucleotide sequence encoding the VL and / or VH of an antibody of step b that binds to one or more FZD receptors, or a nucleotide sequence encoding the VL and / or VH of an antibody derived from step b that binds to one or more FZD receptors, and (iii) a nucleotide sequence encoding the peptide of step c that binds to one or more Wnt co-receptors, or a nucleotide sequence encoding the VL and / or VH of an antibody of step c that binds to one or more Wnt co-receptors, or a nucleotide sequence encoding the VL and / or VH of an antibody derived from step c that binds to one or more Wnt co-receptors.

[0121] e) Expressing the nucleic acid molecule of (d) to produce a polypeptide, wherein the polypeptide dimerizes to form a tetravalent binding molecule comprising (i) an Fc domain, (ii) an FZD binding domain and (iii) a Wnt co-receptor binding domain, wherein the FZD binding domain comprises the peptide of step b, or the VL and / or VH of step b, and is attached to one end of the Fc domain, and the Wnt co-receptor binding domain comprises the peptide of step c or the VL and / or VH of step c, and is attached to the other end of the Fc domain, thereby forming a multispecific binding molecule.

[0122] The peptide binding to more than one FZD receptor can be a synthetic polypeptide, such as a synthetic peptide, affinity peptide, ankyrin repeat protein, fibronectin repeat protein, fynomer, or anticalin, or a peptide of a naturally occurring protein that binds to the FZD receptor. Naturally occurring proteins can be, for example, Wnt, such as Wnt-1, Wnt-2, Wnt-2b, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7a / b, Wnt-7b, Wnt-8a, Wnt-8b, Wnt-9a, Wnt-9b, Wnt-10a, Wnt-10b, Wnt-11, or Wnt-16b. The peptide in step b can be multivalent, binding to more than one site on the FZD, for example, bivalent, trivalent, or tetravalent, and can be monospecific, binding to a single epitope on the FZD, or multispecific, binding to more than one epitope on the FZD.

[0123] Peptides that bind to more than one Wnt co-receptor can be synthetic peptides, such as affinity proteins, ankyrin repeats, fibronectin repeats, fynomers, or anticalins, or peptides of naturally occurring proteins that bind to Wnt co-receptors. Naturally occurring proteins can be, for example, Wnt, such as Wnt-1, Wnt-2, Wnt-2b, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7a / b, Wnt-7b, Wnt-8a, Wnt-8b, Wnt-9a, Wnt-9b, Wnt-10a, Wnt-10b, Wnt-11, or Wnt-16b, or Dickkopf-1.

[0124] The peptide in step c can be multivalent, binding to more than one epitope on the Wnt coreceptor, such as bivalent, trivalent, or tetravalent, and can be monospecific, binding to a single epitope on the Wnt coreceptor, or multispecific, binding to more than one epitope on the Wnt coreceptor.

[0125] The naturally occurring protein that binds to the FZD receptor and the naturally occurring protein that binds to the Wnt co-receptor can be the same protein.

[0126] In one embodiment, the peptide or antibody of step b may bind to FZD2, and the peptide of step c may be a peptide of Wnt5a, and the antibody of step c may be an antibody that binds to a site on a co-receptor that binds to Wnt5a.

[0127] In one embodiment, the peptide or antibody of step b may bind to FZD4, and the peptide of step c may be one or more of Norrin, Wnt1, Wnt8 or Wnt5a, and the antibody of step c may be an antibody that binds to a site on a co-receptor that binds to Norrin, Wnt1, Wnt8 or Wnt5a.

[0128] In one embodiment, the peptide or antibody of step b may bind to FZD5, and the peptide of step c may be one or more peptides selected from Wnt7a, Wnt5a, Wnt10b, or Wnt2, and the antibody of step c may be an antibody that binds to a site on a co-receptor, said site binding to one or more of Wnt7a, Wnt5a, Wnt10b, or Wnt2.

[0129] In one embodiment, the peptide or antibody of step c binds to LRP6 and / or LRP5. For example, the peptide may be a peptide of Norrin, Wnt1, and / or Wnt3a, and the antibody of step c may be an antibody that binds to a site on LRP6 / LRP5 that binds to Norrin, Wnt1, and / or Wnt3a.

[0130] In one embodiment, the peptide or antibody of step c may bind to LRP6. For example, the peptide may be a peptide of Wnt1 or Wnt3a or both, and the antibody may be an antibody that binds to a site on LRP6 that binds to Wnt1 or Wnt3a.

[0131] In one implementation, the peptide or antibody in step c binds to ROR1 and / or ROR2.

[0132] In one implementation, the peptide or antibody of step c may bind to RYK.

[0133] In one implementation, the peptide or antibody of step c may bind to PTK7.

[0134] In one embodiment, the peptide or antibody in step (b) may be a peptide or antibody that binds to more than one FZD receptor and antagonizes Wnt signaling or inhibits Wnt binding to the receptor. In one embodiment, the peptide or antibody in step (b) may be a peptide or antibody that binds to more than one FZD receptor without antagonizing Wnt signaling or inhibiting Wnt binding to the receptor. In one embodiment, the peptide or antibody in step (c) may be a peptide or antibody that binds to more than one Wnt co-receptor and antagonizes Wnt signaling or inhibits Wnt binding to the co-receptor. In one embodiment, the peptide or antibody in step (c) may be a peptide or antibody that binds to a Wnt co-receptor without antagonizing Wnt signaling or inhibiting Wnt binding to the co-receptor. The binding domain can be linked to the Fc domain via a linker. The modular aspect of the present invention allows for the mixing and matching of peptides or antibodies VH and VL that bind to any given FZD receptor and Wnt co-receptor at opposite ends of the Fc domain to produce multivalent binding molecules that can bind multiple coilin receptor-co-receptor complexes or selectively bind a single coilin receptor-co-receptor complex to activate Wnt signaling.

[0135] One embodiment of the present invention is a method for preparing a multivalent binding molecule that activates the Wnt signaling pathway, comprising:

[0136] a) Select Fc domains with both C-terminus and N-terminus, such as the Fc domains of immunoglobulins containing the CH3 domain, such as IgG, for example IgG1.

[0137] b) Identify antibodies with binding specificity to more than one FZD receptor and

[0138] c) Identify antibodies that have binding specificity to the Wnt co-receptor;

[0139] d) Producing nucleic acid molecules, which contain

[0140] (i) The nucleotide sequence encoding the selected Fc domain.

[0141] (ii) The nucleotide sequence encoding the VL and / or VH of the antibody derived from step b, and

[0142] (iii) The nucleotide sequence encoding the VL and / or VH of the antibody derived from step c.

[0143] d) Expressing the nucleic acid molecule of (d) to produce a polypeptide forming a multivalent binding molecule via dimerization of the Fc domain, the multivalent binding molecule comprising (i) an Fc domain, (ii) an FZD-binding domain and (iii) a Wnt co-receptor-binding domain, such that the FZD-binding domain is attached to one end of the Fc domain and the Wnt co-receptor-binding domain is attached to the other end of the Fc domain, thereby forming a multivalent binding molecule. In a preferred embodiment, the multivalent binding molecule is a dimer of two polypeptides encoded by the nucleic acid molecule, wherein the Fc domain is in a club-and-mortar conformation. One or both of the binding domains may be multivalent binding domains. The antibody of step b may be an antibody fragment binding to the FZD receptor. The VH and / or VL in step d)(ii) may be the same as the VH and / or VL of the antibody of step b). The antibody of step c may be an antibody fragment binding to the Wnt co-receptor. The VH and / or VL in step d)(iii) may be the same as the VH and / or VL of the antibody of step c).

[0144] The multivalent molecules of the present invention can be generated by dimerizing two polypeptides in a "mortar and pestle" conformation. The mortar and pestle conformation increases the modularity of the invention by promoting association of peptides containing binding moieties, which bind to different epitopes on FZD receptors or co-receptors or different members of the same FZD receptor or co-receptor family, see, for example... Figure 3A Methods for Fc molecule engineering using mortar and pestle design are well known in the art; see, for example, WO2018 / 026942, inventors Van Dyk et al., Carter P. (2001) J. Immunol. Methods 248, 7–15; Ridgway et al. (1996) Protein Eng. 9, 617–621; Merchant AM et al. (1998) Nat. Biotechnol. 16, 677–681 and; et al. (1997) J. Mol. Biol. 270, 26–35.

[0145] Another embodiment of the present invention is a method for promoting the interaction between an FZD receptor and a co-receptor on a cell to activate the Wnt signaling pathway in the cell, comprising: a) selecting an Fc domain having a C-terminus and an N-terminus, or a fragment thereof containing a CH3 domain; b) attaching a first multivalent binding domain for binding the FZD receptor to one end of the Fc domain and attaching a second binding domain for binding the Wnt co-receptor to the other end of the Fc domain, thereby forming a binding molecule; c) wherein, with both the FZD receptor and the co-receptor bound to the multivalent binding molecule, the multivalent binding molecule is contacted with a cell expressing the FZD receptor and the Wnt co-receptor, thereby activating the Wnt signaling pathway. One or both of the binding domains may be monovalent or multivalent, for example, bivalent, trivalent, or tetravalent. The FZD-binding domain may contain a peptide of a naturally occurring protein that binds to FZD, a synthetic peptide that binds to FZD such as an affinity peptide, ankylosing spicule, fibronectin repeat, fynomer, or anticalin, a VH and / or VL fragment that binds to FZD, a scFV that binds to FZD, or a bisomatic antibody that binds to FZD. The Wnt co-receptor-binding domain may contain a peptide of a naturally occurring protein that binds to a Wnt co-receptor, a synthetic peptide that binds to a Wnt co-receptor such as an affinity peptide, ankylosing spicule, fibronectin repeat, fynomer, or anticalin, a VH and / or VL fragment that binds to a Wnt co-receptor, a scFV that binds to a Wnt co-receptor, or a bisomatic antibody that binds to a Wnt co-receptor.

[0146] One embodiment of the present invention is a molecule comprising an Fc domain and two binding domains, the first domain binding to an FZD receptor and the second domain binding to a Wnt co-receptor, the two portions being linked together via the Fc domain or a fragment thereof containing a CH3 domain, one domain being attached to the N-terminus of the Fc receptor and the other domain being attached to the C-terminus of the Fc receptor. The binding domains can be attached to the Fc receptor directly or via peptide linkers or non-peptide linkers, such as peptide linkers. Suitable linkers are well known in the art, such as the XTEN linker (see WO2013120683, inventors Schellenberger et al.).

[0147] One embodiment of the present invention is a method for activating the Wnt signaling pathway, the method comprising contacting cells expressing the FZD receptor and its co-receptor with an effective amount of the multivalent molecule of the present invention. It is not intended to be theoretically constrained, but rather that the multivalent molecule as described herein binds to both the FZD receptor and its co-receptor, thereby forming a complex mimicking the binding of the Wnt molecule to the FZD receptor and co-receptor, thereby sequentially activating the Wnt signaling pathway.

[0148] The multivalently bound molecules of the present invention can be prepared by recombination, for example by Gibson assembly (see Gibson et al. (2009). Nature Methods. 6 (5): 343–345 and Gibson DG. (2011). Methods in Enzymology. 498: 349–361), or can be synthesized, for example, using commercial synthetic equipment, such as automated synthesizers from Applied Biosystems, Inc., Beckman, etc. By using the synthesizer, naturally occurring amino acids can be replaced by non-natural amino acids. The specific sequence and preparation method will be determined by convenience, economy, desired purity, etc. If desired, various groups can be introduced into the peptide during synthesis or expression, allowing for linkage with other molecules or surfaces.

[0149] In some embodiments, the binding domain is connected to the Fc domain via a peptide linker, such as an XTEN linker. In some embodiments, the peptide linker comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 5 1, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or at least 100 amino acids. In some embodiments, the length of the peptide linker is between 5 and 75, 5 and 50, 5 and 25, 5 and 20, 5 and 15, or 5 and 10 amino acids. The length and flexibility of the Fc domain, with or without the linker, allow multivalent binding molecules to bind to the FZD receptor and its co-receptor, thereby activating the Wnt signaling pathway. In one embodiment of the invention, the Fc domain having or not having a linker, or a fragment thereof containing a CH3 domain, is greater than 100 amino acids, greater than 125 amino acids, greater than 150 amino acids, greater than 175 amino acids, or greater than 200 amino acids.

[0150] It must be noted that, as used herein and in the appended claims, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural objects. Thus, for example, reference to “cell” includes a plurality of such cells, and reference to “peptide” includes reference to more than one peptide and its equivalents, such as polypeptides known to those skilled in the art.

[0151] "Affinity-mature" antibodies, or "antibody maturity," refer to antibodies that have one or more alterations in one or more hypervariable regions (HVRs) that result in improved antibody affinity for the antigen or other desired properties of the molecule compared to parental or source antibodies that do not have such alterations.

[0152] "Comprising" means the elements listed in the claims that are required in the composition / method / kit, but other elements may be included to form the composition / method / kit, etc., within the scope of the claims. For example, as will be readily understood in the art, a composition comprising a multivalently binding molecule is a composition that may contain other elements besides the multivalently binding molecule, such as functional parts such as peptides, small molecules, or nucleic acids that bind to the multivalently binding molecule, for example, covalently bound; agents that promote the stability of the multivalently binding molecule composition; agents that promote the solubility of the multivalently binding molecule composition; adjuvants, etc.

[0153] "Substantially composed of..." means to limit the scope of the described composition or method to specified materials or steps that do not materially affect the essential and novel features(one or more) of the invention. For example, a multivalent binding molecule "substantially composed of" the disclosed sequence has an amino acid sequence at the boundary of the sequence from which the sequence is derived, with the disclosed sequence plus or minus about 5 amino acid residues, for example, fewer than about 5, 4, 3, 2, or about 1 residue of the listed binding amino acid residues, or more than about 1, 2, 3, 4, or 5 residues of the listed binding amino acid residues.

[0154] "Composed of..." means excluding from a composition, method, or kit any element, step, or component not specified in the claims. For example, a multivalently bound molecule "composed of" the disclosed sequence consists only of the disclosed amino acid sequence.

[0155] Where a range of values ​​is provided, it should be understood that, unless the context explicitly indicates otherwise, each intermediate value between the upper and lower limits of the range, up to one-tenth of the lower limit unit, is also specifically disclosed. Every smaller range between any specified value or intermediate value within the specified range and any other specified value or intermediate value within the specified range is included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range, and each range wherein either the upper or lower limit is included, does not include, or includes both the upper and lower limits, is also included in this invention, subject to any specific exclusion from the specified range. Where the range includes one or both of the upper and lower limits, ranges excluding one or both of the included upper and lower limits are also included in this invention.

[0156] The basic antibody structural unit is known to consist of a tetramer. Each tetramer consists of two pairs of identical polypeptide chains, each pair having a "light" chain (approximately 25 kDa) and a "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids, primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region, primarily responsible for effector function. Typically, antibody molecules obtained from humans are associated with any of IgG, IgM, IgA, IgE, and IgD, differing from each other in the presence of the heavy chain in the molecule. Some classes also have subclasses, such as IgG1, IgG2, etc. Furthermore, in humans, the light chain can be either a κ chain or a λ chain.

[0157] Three highly divergent segments within each of the heavy chain variable domain VH and the light chain variable domain VL, known as complementarity-determining regions (CDRs), are inserted between more conserved flanking segments called "frame regions" or "FRs." Therefore, the term "FR" refers to the amino acid sequence naturally present between and adjacent to the CDRs of immunoglobulins. The VH domain typically has four FRs, referred to herein as VH frame region 1 (FR1), VH frame region 2 (FR2), VH frame region 3 (FR3), and VH frame region 4 (FR4). Similarly, the VL domain typically has four FRs, referred herein as VL frame region 1 (FR1), VL frame region 2 (FR2), VL frame region 3 (FR3), and VL frame region 4 (FR4). In antibody molecules, the three CDRs (CDR-L1, CDR-L2, and CDR-L3) of the VL domain and the three CDRs (CDR-H1, CDR-H2, and CDR-H3) of the VH domain are interleaved relative to each other in three-dimensional space, thereby forming antigen-binding sites within the variable region of the antibody. The surface of the antigen-binding site is complementary to the three-dimensional surface of the bound antigen. The amino acid sequences of the VL and VH domains can be numbered and their CDRs and FRs identified / defined using the Kabat numbering system (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md.) or the International Immunogenetic Information System (IMGT numbering system; Lefranc et al., 2003, Development and Comparative Immunology 27:55-77). Based on commonly used numbering systems, such as the IMGT numbering system and the Kabat numbering system, those skilled in the art will have the knowledge to number the amino acid residues in the VL and VH domains and identify the CDR and FR.

[0158] As used herein, the term "antigen-binding moiety" or "antigen-binding fragment" (or simply "antibody moiety" or "antibody fragment") refers to one or more segments, portions, or domains of an antibody that retain the ability to bind specifically to an antigen. Fragments of full-length antibodies have been shown to perform the antigen-binding function of the antibody. Examples of binding fragments covered by the term "antigen-binding moiety" of an antibody include: (i) Fab fragments, monovalent fragments consisting of VL, VH, CL1, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments consisting of two F(ab)' fragments linked by disulfide bonds in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of the antibody; (v) dAb fragments consisting of the VH domain (Ward et al. (1989) Nature 241:544-546); and (vi) separated complementarity-determining regions (CDRs). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by their respective genes, they can be linked into a continuous chain using a recombinant approach via a synthetic linker, where the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be included within the term "antigen-binding moiety" of antibodies. Other forms of single-chain antibodies are also included, such as bimeric antibodies (see, for example, Holliger et al. (1993) PNAS. USA 90:6444-6448).

[0159] "Affinities" are small, single-domain proteins engineered to mimic the binding of monoclonal antibodies to a wide range of target proteins or peptides with high affinity. They consist of a triple-helix bundle based on a scaffold of one of the IgG-binding domains of Staphylococcus aureus protein A. This scaffold domain consists of 58 amino acids, 13 of which are randomized to generate a library of affinity molecules with a large number of ligand variants. See, for example, U.S. Patent No. 5,831,012 and Lofblom et al., FEBS Letters 584 (2010) 2670-2680. The molecular weight of the affinity molecule mimicking the antibody is approximately 6 kDa.

[0160] As used herein, “bimeric antibody” refers to a dimeric antibody fragment. In each polypeptide of a bimeric antibody, a heavy chain variable domain (VH) is linked to a light chain variable domain (VL). However, unlike single-chain Fv fragments, the linker between VL and VH is too short for intramolecular pairing. Therefore, each antigen-binding site is formed by pairing the VH and VL of one polypeptide with the VH and VL of another polypeptide, see, for example... Figure 3A Bispecific antibodies therefore have two antigen-binding sites and can be monospecific or bispecific. (See, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123; Kontermann and Dubeleds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).)

[0161] As used herein, the “effective amount” of an agent, such as a multivalent binding molecule or a pharmaceutical composition containing such a molecule, refers to the amount that effectively achieves the desired result at the required dose and time period. In some embodiments, the therapeutically effective amount is the amount that reduces the incidence and / or severity of one or more symptoms of a disease, condition, and / or disease, stabilizes one or more characteristics of one or more symptoms of a disease, condition, and / or disease, and / or delays the onset of one or more symptoms of a disease, condition, and / or disease.

[0162] As used herein, the term "epitope" includes any protein determinant capable of specifically binding to immunoglobulins or fragments thereof, or T-cell receptors. Epitope determinants typically consist of chemically active surface groups of a molecule, such as amino acid or sugar side chains, and usually possess specific three-dimensional structural features and specific charge characteristics. Antibodies are considered to specifically bind to antigens when the dissociation constant is ≤10 μM, for example, ≤100 nM, preferably ≤10 nM, and more preferably ≤1 nM.

[0163] The constant region of an immunoglobulin molecule is also called the fragment crystallizable region, the "Fc region," or the "Fc domain." The Fc domain consists of two identical protein fragments, derived from the second and third constant domains of the two heavy chains of an antibody, respectively. The Fc domain of IgG has a highly conserved N-glycosylation site. Glycosylation of the Fc fragment is essential for Fc receptor-mediated activity. In one embodiment of the invention, the Fc domain of a multivalent molecule is engineered to not target cells that bind to ADCC or CDC-dependent cell death multivalent molecules. In one embodiment of the invention, the Fc domain of the multivalent binding molecule is a peptide dimer in a club-and-mortar conformation. This peptide dimer can be a heterodimer.

[0164] The terms “individual,” “subject,” “host,” and “patient” are used interchangeably in this document and refer to any mammalian subject, particularly a human, to whom a diagnosis, treatment, or therapy is intended.

[0165] As used in this article, “LRP,” “LRP protein,” and “LRP receptor” refer to members of the low-density lipoprotein receptor-associated protein family. These receptors are single-transmembrane proteins that bind and internalize ligands in receptor-mediated endocytosis. LRP proteins LRP5 (GenBank accession number NM 002335.2) and LRP6 (GenBank accession number NM 002336.2) are contained in the Wnt receptor complex, which is required for activation of the Wnt-β-catenin signaling pathway.

[0166] As used herein, the term "peptide fragment" refers to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion, but wherein the remaining amino acid sequence is identical to the corresponding position in a naturally occurring sequence, for example, deduced from a full-length cDNA sequence.

[0167] As used herein, the term "complementary site" includes an antigen-binding site in the variable region of an antibody that binds to an epitope.

[0168] As used herein, the term "treatment" (and treating, etc.) generally refers to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of the disease and / or its side effects. As used herein, "treatment" encompasses any treatment of a disease in mammals and includes: (a) preventing the disease from occurring in subjects who may be susceptible to the disease but have not yet been diagnosed with it; (b) suppressing the disease, i.e., halting its development; or (c) alleviating the disease, i.e., causing its remission. Therapeutic agents may be administered before, during, or after the onset of a disease or injury. Of particular interest is the treatment of an ongoing disease in which the treatment stabilizes or alleviates unpleasant clinical symptoms in the patient. Such treatment is intended to be administered before complete loss of function in the affected tissue. The treatment may be administered during the symptomatic phase of a disease and, in some cases, after the symptomatic phase of a disease.

[0169] The ability of the multivalent binding molecules of the present invention to activate Wnt signaling can be confirmed by a variety of assays. The multivalent binding molecules of the present invention typically elicit responses or activities similar to or identical to those elicited by the natural ligands of the FZD receptor. The multivalent binding molecules of the present invention activate Wnt signaling pathways, such as the typical Wnt-β-catenin signaling pathway. As used herein, the term "activation" refers to a measurable increase in intracellular levels of Wnt signaling pathways, such as the Wnt-β-catenin signaling pathway, compared to levels in the absence of the FZD agonist of the present invention.

[0170] Various methods for measuring Wnt-β-catenin activation levels are known in the art. These include, but are not limited to, assays measuring: Wnt-β-catenin target gene expression; LEF / TCF reporter gene expression (e.g., TopFLASH, superTopFLASH, pBAR); β-catenin stabilization; LRP5 / 6 phosphorylation; and Axin translocation from the cytoplasm to the cell membrane and binding to LRP5 / 6. The typical Wnt-β-catenin signaling pathway ultimately leads to altered gene expression via transcription factors TCF1, TCF7L1, TCF7L2, and LEF. Transcriptional responses to Wnt activation have been characterized in many cells and tissues. Thus, global transcriptional profiling using methods well-known in the art can be used to assess activation of Wnt-β-catenin signaling.

[0171] Alterations in Wnt response gene expression are typically mediated by TCF and LEF transcription factors. The TCF reporter gene assay assesses transcriptional changes in TCF / LEF-controlled genes to determine the level of Wnt-β-catenin signaling. The TCF reporter gene assay was first described by Korinek, V. et al., 1997. Also known as TOP / FOP, this method involves using three copies of the optimal TCF motif CCTTTGATC or three copies of the mutant motif CCTTTTGGCC, driven by the smallest upstream c-Fos promoter, to drive luciferase expression (pTOPFLASH and pFOPFLASH, respectively), to determine the transactivation activity of endogenous β-catenin / TCF. A higher ratio of these two reporter gene activities (TOP / FOP) indicates higher β-catenin / TCF activity. A newer and more sensitive version of the reporter gene is called pBAR, which contains 12 TCF motif repeat sequences (Biechele and Moon, Methods Mol Biol. 2008;468:99-110, PMID: 19099249).

[0172] General methods in molecular and cellular biochemistry can be found in standard textbooks such as *Molecular Cloning: A Laboratory Manual, 3rd Edition* (Sambrook et al., CSH Laboratory Press 2001); *Short Protocols in Molecular Biology*, 4th Edition (Ausubel et al., John Wiley & Sons 1999); *Protein Methods* (Bollag et al., John Wiley & Sons 1996); *Nonviral Vectors for Gene Therapy* (Wagner et al., Academic Press 1999); *Viral Vectors* (Kaplift & Loewy eds., Academic Press 1995); *Immunology Methods Manual* (I. Lefkovits ed., Academic Press 1997); and *Cell and Tissue Culture: Laboratory Procedures in Biotechnology* (Doyle & Griffiths, John Wiley & Sons 1998).

[0173] A single-chain Fv or scFv antibody fragment contains the VH and VL domains of the antibody, which are contained within a single polypeptide chain. Typically, the Fv polypeptide further includes a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired antigen-binding structure. For a review of scFv and other antibody fragments, see James D. Marks, Antibody Engineering, Chapter 2, Oxford University Press (1995) (Carl K. Borrebaeck, Ed.).

[0174] Unless otherwise defined, scientific and technical terms used in conjunction with this invention shall have the meaning commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. Generally, terms and techniques used in conjunction with cell and tissue culture, molecular biology, protein and oligonucleotide or polynucleotide chemistry and hybridization as described herein are well-known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipid transfection). Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions or as commonly performed in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well-known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The terminology used in conjunction with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry, as described herein, and the laboratory procedures and techniques described above, are well-known and commonly used in the field. Standard techniques are used for chemical synthesis, chemical analysis, drug preparation, formulation, delivery, and patient treatment.

[0175] Example I

[0176] 1. Development of multivalent FZD agonists

[0177] To prepare a multivalent binding molecule having a first binding domain containing an FZD bisomatic antibody and a second binding domain containing a co-receptor bisomatic antibody, we identified FZD-specific antibodies from a synthetic Fab phage library (Library F; see U.S. Publication No. 2016 / 0194394, inventors Sidhu et al.) by selecting those binding to the cysteine-rich domain (CRD) of the FZD receptor using conventional phage display technology. Affinity or specificity maturation was performed as needed. For example, using the FZD4 CRD as an antigen, pan-FZD binding antibody #5019 (recognizing FZD1, 2, 4, 5, 7, and 8) was matured from antibodies derived from FZD7. Our previous work has also identified several antibodies that are completely specific to FZD4 (5038, 5044, 5048, 5062, 5063, 5080, 5081) or FZD5 (2928) (see, for example, US20160194394, inventors Sidhu et al. and WO2017127933A1, inventors Pan et al.).

[0178] These FZD antibodies are used to prepare FZD-specific bimeric antibodies. Bimeric antibodies are a form of antibody similar to single-chain variable fragments (scFv), but they are dimers of two peptides, each encoding VL and VH. However, unlike scFv, the linker between VH and VL within the polypeptide is too short to allow intramolecular complementarity between the VH and VL domains. Therefore, the VH-VL fragment of one polypeptide dimers with the VH-VL fragment of another polypeptide, thereby functionally reconstructing the two antigen-binding complementary sites. Homobimeric antibodies are formed by forming dimers of polypeptides with the same VL and VH, or heterobimeric antibodies are formed by forming dimers of two polypeptides with different VL and VH, resulting in bimeric antibodies with the same or different complementary sites.

[0179] LRP6 antibodies were also selected from a synthetic antibody library by selecting those that bound the recombinant extracellular domain (ECD) of human LRP6. Five Fabs with unique CDR regions were identified. After conversion to IgG form, they all showed binding to both human and mouse LRP6. LRP5 binding was not detected by ELISA, indicating that these antibodies are LRP6 specific. Figure 1A The LRP6 ECD contains four β-propeller motifs that alternate with four epidermal growth factor (EGF)-like repeat sequences. The first two β-propeller motifs are thought to be associated with Wnt1 binding, while the latter two are thought to be associated with Wnt3 binding, thus generating two potential antibody-binding epitopes. See also Figure 6AEpitope binding results showed that the five antibodies bound to two independent sites on LRP6 and could be divided into two groups: antibodies 2538, 2542, and 2543 bound to the Wnt1 binding site on LRP6, while antibodies 2539 and 2540 bound to the Wnt3 binding site on LRP6. Typically, antibodies binding to the LRP6-Wnt1 site are expected to prevent Wnt1-induced activation of the Wnt pathway.

[0180] To prepare Fc N-terminal binding domains containing homologous dimeric antibodies specific for FZD, the VH and VL fragments of selected FZD antibodies, VH-1, VH-2, VL-1, and VL-2, were amplified and isolated from their respective phage templates by PCR. The isolated fragments (VH-1 and VL-2) were then introduced into pre-digested EcoRI / XhoI vectors containing the Fc spur region (pSCST backbone) using Gibson assembly (see Gibson et al. (2009). Nature Methods. 6 (5): 343–345 and Gibson DG. (2011) Methods in Enzymology. 498: 349–361). Gibson assembly was also used to introduce fragments (VH-2 and VL-1) into pre-digested EcoRI / XhoI vectors containing the Fc spur region. Correct assembly was verified using DNA sequencing. Then, two plasmids (a pair, Fc pestle and Fc mortar) were used to introduce a second binding domain at the C-terminus of the Fc domain.

[0181] Fc club and Fc mortise conformations are required to generate multivalent binding domains, one of which is a heterodimeric antibody. However, Fc club and Fc mortise conformations are not required to prepare binding molecules containing homodimeric antibodies at both the N and C ends of the Fc domain; therefore, for such binding molecules, VH and VL are linked to the wild-type Fc region, and only one plasmid is used to generate a VH-VL-containing polypeptide to form a homodimer. Optionally, a linker, such as a peptide linker or a non-peptide linker, can be present between the binding domain and the Fc domain.

[0182] To generate the C-terminal binding domain, LRP5 / 6 antibodies were recognized, and LRP5 / 6 bisomal antibodies were generated using the same protocol as described above for generating FZD bisomal antibodies. VH-3, VH-4, VL-3, and VL-4 fragments were amplified by PCR from the corresponding LRP antibody phage templates, and then the amplified fragments were isolated to generate the C-terminal binding domain. As described above, the VH-3 and VL-4 fragments were then introduced into the PpuMI / BamHI site of the aforementioned mortar plasmid using Gibson assembly. Other VH-4 and VL-3 fragments were inserted into the PpuMI / BamHI restriction site of the mortar plasmid using Gibson assembly.

[0183] Two plasmids (a pair, Fc pestle and Fc mortar) with different VL and VH sequences are used to generate bispecificity, i.e., the ability to bind FZD or co-receptor binding domains at two different sites. Because it is not necessary to generate a pestle-mortar conformation to produce a dimer with a single-specific binding domain, if each binding domain is single-specific, only a single plasmid containing the wild-type Fc sequence is used.

[0184] Figure 9A The description encodes a plasmid containing an Fc region with a "pickle" mutation, VH and VL of pan-FZD antibody #5019, and VL of LRP antibody #2542 and VH of LRP antibody #2539. Figure 9B The description encodes plasmids containing nucleic acids encoding the Fc region with a "mortis" mutation, the VH and VL of pan-FZD antibody #5019, and the VH and VL of LRP antibody #2542 and LRP antibody #2539. The peptides encoded by these plasmids form heterodimers having a multivalent binding site comprising a homodimeric antibody derived from pan-specific FZD antibody #5019, and a multivalent binding site comprising a bispecific heterodimeric antibody generated by pairing the VL and VH of LRP antibody #2539 from one peptide with the VH and VL of LRP antibody #2542 from the other peptide.

[0185] The resulting plasmid was then sequenced, and the sequenced-validated plasmid was prepared using the PureLink HiPure Plasmid Filter Maxiprep kit (Invitrogen) according to the manufacturer's instructions. The plasmid was then transfected into Expi293F cells (Thermo Fisher Scientific), and the FectoPRO reagent (Polyplus) was used for antibody expression according to the manufacturer's instructions. Typically, small-batch antibody production is performed using 200 ml of cells.

[0186] Typically, 80 hours post-transfection, Expi 293F cell culture medium is collected by centrifugation to precipitate cells and cell debris. The supernatant is transferred to a clean bottle and buffered with 10x PBS buffer. After incubation with an appropriate amount of protein A magnetic beads (GE Healthcare) for 1 hour, the beads are washed, and binding molecules are eluted according to the manufacturer's instructions. Finally, the buffer is replaced with PBS.

[0187] 2. Heterodimeric multivalently bound molecules

[0188] Using the above method, we also produce tetravalent heterodimer molecules containing complete bispecific bimeric antibodies, said bimeric antibodies being fused to each of the N-terminus and C-terminus of the Fc domain (mortar and pestle). Figure 2A and 3A Specifically, we generated tetravalent binding molecules having a homologous bispecific antibody derived from antibody 5019 at the N-terminus of the Fc domain that binds to FZD, and a homologous bispecific antibody derived from either LRP6-W1 antibody 2542 (5019-Fc-2542) or LRP6-W3 antibody 2539 (5019-Fc-2539) at the C-terminus of the Fc domain. Surprisingly, both tetravalent molecules activated the Wnt pathway, but 5019-Fc-2542 was much less efficient. Figure 3C Without being bound by theory, this difference could reflect the difference in the ability of LRP6-W1 and LRP6-W3 to bind and activate Wnt signaling. It has been observed that LRP6-W3 binding to Wnt is more effective in activating Wnt signaling than Wnt binding to LRP6-W1.

[0189] We also produced a tetravalent trispecific binding molecule, which has a homologous bispecific antibody binding FZD to antibody 5019 at the N-terminus derived from the Fc domain, and an LRP heterologous bispecific antibody (5019-K / H-2539-2542, named 5019Ag) to LRP6-W1 antibody 2542 and LRP6-W3 antibody 2539 at the C-terminus derived from the Fc domain. Figure 5 Compared to molecules with monospecific LRP6 homologous bisomal antibodies, 5019Ag was unexpectedly effective in activating Wnt signaling. Figure 3C The pBAR luciferase reporter gene assay determined that all three forms activated Wnt signaling in nanomolar amounts. Figure 3D This indicates that they are effective Wnt mimics. Not wanting to be bound by theory, binding between a strong Wnt3A site and a weak Wnt1 site is expected to be more efficient than binding between two strong Wnt3A sites. The two best-performing multivalent binding molecules, “FLAg”, possessing both FZD and LRP binding domains, exhibit single-digit nanomolar efficiency (EC). 50 ~5 nM, which is practically identical in efficacy to purified Wnt3A and exhibits bell-shaped dose-response characteristics. Figure 11D We interpret this as indicating that maximum stimulation requires multivalent binding of FLAg, while the decrease in efficiency at higher concentrations may be attributed to monovalent binding with FZD or LRP6. We use F... P+P -L6 1+3Treatment of RKO cells expressing low levels of β-catenin (Major et al. Science. 316, 1043–1046 (2007)) resulted in a dose- and time-dependent increase in β-catenin levels and phosphorylation of DVL2, a marker of Wnt-FZD pathway activation. Figure 11E and Figure 11F Therefore, tetravalent FLAg is a modular, engineerable human Ab form that can be used as a synthetic agonist for FZD and LRP6.

[0190] To confirm the optimal FLAg F P+P -L6 1+3 To determine its engineering affinity and specificity, we used biolayer interferometry (BLI) to determine its binding kinetics with 9 out of 10 human FZD CRDs and with human LRP6 ECD. Figure 12A and Figure 12B FLAg binds to six FZDs recognized by a bisomatic antibody derived from the complementary site of the parental pan-FZD with an affinity in the picomolar range (KD = 10-800 pM) (Pavlovic et al. 2018), but binding to the other three FZDs was not detected. Furthermore, the affinity for LRP6 is in the nanomolar range (KD = 12 nM). Figure 12B We then used BLI to evaluate the binding of FLAg to various Fc receptors.

[0191] FLAg behaves similarly to regular IgG and interacts with FcRn in a dose- and pH-dependent manner. Figure 12C Natural IgG binds to FcRn at pH 6 rather than pH 7.4, which allows for recycling during endocytosis, resulting in a long half-life in vivo. FLAg also interacts similarly with IgG with other Fc effectors, including complement (C1q), the natural killer cell marker CD16a, the B cell marker CD32a, and the monocyte and macrophage marker CD64. Figure 12D We conclude that FLAg contains a functional Fc moiety that should confer effector function and a long half-life in vivo.

[0192] Tetravalent F P+P -L6 1+3The modular design of FLAg allows us to dissect the contribution of each of the four complementary sites to intrinsic agonist activity by replacing each complementary site with an empty complementary site that binds to an unrelated antigen, maltose-binding protein (MBP). We generated “single-binding” molecules containing an Fc domain and an FZD-binding domain attached to one end of the Fc domain and an LRP-binding domain attached to the other end of the Fc domain, but unlike bispecific antibodies, which have two binding sites for FZD or LRP, the binding domains have only one or one binding site and one maltose-binding protein binding site, “MBP.” Introducing an MBP binding site into at least one binding domain of the molecule yielded five single-binding molecules. 5019-MBP-K / H-2539-2542, containing an FZD and an MBP binding site at the N-terminus, still activates the Wnt pathway, but with an 8-fold decrease in efficacy compared to 5019Ag. Figure 3E Similarly, 5019-K / H-2539-MBP, which retains only one LRP6-W3 site at the C-terminus, exhibits significantly less Wnt activation compared to 5019Ag. Figure 3E The minimum agonistic activity of two MBP-FZD / MBP-LRP6 molecules, 5019-MBP-K / H-2539-MBP and 5019-MBP-K / H-MBP-2542, and molecule 5019-K / H-MBP-2542 with an LRP6-W1 bisomatic antibody was detected. Figure 3E These β-catenin signaling assays showed that disabling one anti-FZD complement or anti-LRP6 complement at the WNT1 binding site significantly reduced maximal stimulation, while disabling the anti-LRP6 complement at the WNT3A binding site, or simultaneously disabling one anti-FZD complement and any one of multiple anti-LRP6 complements, completely eliminated maximal stimulation. We also replaced the anti-LRP5 complement targeting the WNT3A binding site with the anti-LRP6 complement targeting the WNT1 binding site to generate recruitable co-receptors and observed FZD-like signaling. P+P -L6 1+3 Active molecules (F) P+P -L5 / 6 3 ) ( Figure 3F (EC50 = 4 nM). In summary, these data indicate that optimal agonist activity is achieved using molecules capable of recruiting two FZDs via a common epitope and LRP6 via two distinct epitopes, but activity can be modulated to moderate levels by disabling either the anti-FZD or anti-LRP6 complementary site. Furthermore, by combining the two anti-FZD complementary sites with one complementary site from each of LRP5 and LRP6, molecules capable of recruiting both FZDs and two distinct co-receptors are generated.

[0193] We also explored the requirements of geometric and spatial constraints imposed by the intermolecular bimeric antibody form by replacing bimeric antibody pairs with paired, less constrained intramolecular single-chain variable fragments (scFvs). Figure 2J ). and F P+P -L6 1+3 In comparison, it includes anti-FZD scFv ( FLAg showed similar activity to those containing anti-LRP6 scFv ( ) or contain scFv at both ends ( The activity of FLAg was significantly reduced. These differences in activity were not due to differences in affinity; as shown by BLI assays, binding to LRP6 and FZD isotypes had a fairly high affinity regardless of whether the complementary site was in the form of a bimeric antibody or scFv. Figure 2K and Figure 2L In summary, these results demonstrate that the assembly of the optimal FZD / LRP6 signaling complex requires specific stoichiometry and geometry, with constraints particularly precise for LRP6, necessitating the binding of two distinct epitopes within a specific geometry specified by the bimeric antibody form. Notably, the looser constraints on FZD binding enable significant activation at a single anti-FZD complementary site. Figure 2D This opens the door to further enhancing specificity or altering signal transduction by recruiting different cell surface proteins via additional complementary sites through binding to the anti-FZD complementary site at the N-terminus of the heterodimer Fc.

[0194] 3. Other forms of bispecific antibodies

[0195] Bispecific molecules containing the FZD binding domain of antibody #5019 and the LRP6-W1 binding domain of antibody #2942 (5019 / 2942) or the LRP6-W3 binding domain of antibody #2539 (5019 / 2539) were constructed at the same end of the Fc domain, and the corresponding proteins were purified. Figure 2A The activation of Wnt signaling was determined using a pBAR luciferase reporter gene assay. These molecules failed to activate Wnt signaling. Notably, both bispecific molecules antagonized the activity of Wnt ligands. Figure 2B Without being bound by theory, the distance and flexibility between the two complementary sites of these bispecific molecules may not recruit FZD and LRP6 receptors for activation in a suitable geometry.

[0196] Bispecific molecules containing FZD and LRP bispecific antibodies with the same ends linked to the Fc domain were also generated using a mortar and pestle conformation. FZD and LRP binding and Wnt pathway activation were measured in these designated bispecific antibodies, 5019-2539-K / H (FZD / LRP-W3) and 5019-2542-K / H (FZD / LRP-W1). Both bispecific antibodies retained the FZD binding properties and LRP6 binding activity of the original antibodies. Figure 2D-2G Both molecules bind to the FZD receptor and the LRP co-receptor, respectively. As determined by the BLI assay, 5019-2542-K / H showed co-binding with both FZD and LRP in solution. Figure 2H However, no significant co-binding was observed in 5019-2539-K / H. Neither 5019-2539-K / H nor 5019-2542-K / H, as determined in the pBAR luciferase reporter assay, activated Wnt signaling, similar to results obtained from homologous bisomal antibodies that bind only to the FZD receptor (5019-Fc) or the co-receptor (2539-Fc). Figure 2I Furthermore, both 5019-2539-K / H (FZD / LRP-W3) and 5019-2542-K / H (FZD / LRP-W1) effectively inhibited Wnt3a-mediated pathway activation. Figure 2I ).

[0197] 4. Signal Transduction Analysis of the Wnt Pathway

[0198] Wnt pathway activation was measured in HEK293 cells using the pBAR luciferase reporter system, which faithfully monitors transcriptional activation of β-catenin (Biechele and Moon, Methods Mol Biol. 2008;468:99-110, PMID: 19099249). Briefly, HEK293T cells stably expressing pBARLS and pSL9 Ef1α-Renilla luciferase constructs were seeded at 1.5E4 cells / well in 96-well plates. Twenty-four hours post-seeding, cells were treated in triplicate with a specified concentration of the designated FZD agonist or a PBS carrier control. Sixteen and a half hours post-treatment, cells were lysed according to the manufacturer's protocol, and luminescence was measured using a Dual-Luciferase Reporter Assay System (Promega #E1960). Firefly luminescence was normalized to Renilla luminescence in each well to control cell number.

[0199] We examined the agonist activity of a multivalent molecule comprising an N-terminal FZD bisomatic antibody derived from an antibody fragment (antibody #5019) that recognizes several FZD receptors (FZD1, 2, 4, 5, 7, and 8) with LRP-binding domains linked to the C-terminus of the Fc domain. The C-terminal LRP-binding domain comprises a bisomatic antibody derived from one of two LRP6 antibodies, #2539 and #2542, which binds to the Wnt3 and Wnt1 sites, respectively. Figure 6B These multivalently binding molecules, represented in nanomolar amounts as 5019-Fc-2539 and 5019-Fc-2542, activate the Wnt-β-catenin pathway. Figure 6C However, when compared to 5019-Fc-2542, treatment of cells with the molecule 5019-Fc-2539, which contains an LRP6 antibody targeting the Wnt3 site, resulted in approximately 10-fold higher activation (200-fold vs. 20-fold higher than the background). Figure 6C ).

[0200] Importantly, using a pestle-and-mortar system engineered within the Fc portion, we generated a multivalent binding molecule containing a homobiomer antibody (#5019) at one end for the pan-FZD binding domain and a heterobiomer antibody at the other end for forming the LRP6 binding domain at binding sites of Wnt1 (#2542) and Wnt3 (#2539) 5019-K / H-2539:2542. Figure 6B This conformation allows for the introduction of four distinct binding sites within the molecule with varying selectivity and affinity properties: tetravalent and trispecific. When detected in a β-catenin luciferase reporter gene assay in HEK293 cells, the molecule showed activation levels approximately 2-fold higher than 5019-Fc-2539, or about 400-fold higher than background. Figure 6C ).

[0201] We also replaced the LRP6 binding site with an equivalent LRP5 binding site within the mortar and pestle system (derived from bisomatic antibodies of 2459 and 2460, both of which bind LRP5) and the same pan-FZD bisomatic antibody (5019) that binds FZD1, 2, 4, 5, 7, and 8. This molecule, 5019-K / H-2459:2460, can also activate the Wnt-β catechol pathway in HEK293T cells. Figure 6D Although its efficacy is lower than that of agonists with LRP6 bisomatic antibodies.

[0202] 5. Characterization of selective FZD agonists (agonist modularity with binding domains derived from selective FZD and co-receptor antibody fragments)

[0203] To assess the activity of our monospecific FZD agonist, we used a cell-based assay that depends on a specific FZD isoform. We prepared multivalent binding molecules that bind only to one of the ten FZD receptors. Our previous work identified several antibodies that are completely specific to FZD4 (5038, 5044, 5048, 5062, 5063, 5080, 5081) (see, for example, US20160194394, inventors Sidhu et al. and WO2017127933A1, inventors Pan et al.). Using the Fc mortar and pestle system, we generated multivalent binding molecules containing an FZD4-specific FZD-binding domain and an LRP6-binding domain from bispecific heterobimeric antibodies derived from antibodies 2539 and 2542. These molecules can activate FZD4 signaling via the β-catenin pathway, but only when co-transfected with FZD4 cDNA into HEK293 cells. These FZD4-binding molecules failed to activate FZD4 signaling or the β-catenin pathway in unmodified HEK293T cells expressing low levels of FZD4. Therefore, this experiment demonstrates the specificity of the molecules for FZD4. 5019-K / H-2539-2542 (the aforementioned pan-FZD agonist) can activate signaling in HEK293T cells even in the absence of FZD4. Figure 4A This result is not surprising, as Wnt-mediated β-catenin signaling activation in HEK293T cells occurs via the binding of FZD1, 2, and 7 (Voloshanenko et al. FASEB 2017 FASEB J. 2017 Nov;31(11):4832-4844; PMID: 28733458) and the 5919 FZD antibody to all three receptors.

[0204] Furthermore, we generated an FZD5-specific multivalent binding molecule using the binding domain of the FZD5-specific antibody 2928, which we previously characterized as binding only to FZD5 (Steinhart et al. Nat Med. 2017 Jan; 23(1):60-68, PMID: 27869803; WO2017127933A1, inventors Pan et al.). We previously demonstrated that the proliferation of several RNF43-mutant pancreatic ductal adenocarcinoma (PDAC) cell lines depends solely on FZD5 signaling (Steinhart et al. 2017, PMID:27869803). Indeed, genome-wide CRISPR essentiality / adaptation screening in three RNF43-mutant PDAC lines revealed that FZD5 is one of the most important genes for their growth, while PDAC cell lines with WT RNF43 did not exhibit this requirement for FZD5. When RNF43 mutant cells are treated with a Porcupine inhibitor (PORCNi; e.g., LGK-974) that inhibits palmitoylation and activity of Wnt ligand, the RNF43 mutant cells cease to proliferate.

[0205] Co-treatment of RNF43 mutant cells with either pan-FZDag 5019-K / H-2539-2542 or the selective FZD5 agonist 2928-K / H-2539-2542 resulted in potent rescue of cell proliferation blocked by LGK974. These results indicate that both molecules can activate FZD5 and induce Wnt signaling in these cells, thereby mimicking the role of endogenous Wnt ligands. Figure 7B Conversely, the addition of the FZD4-specific agonist 5038-K / H-2539-2542 or the FZD2-specific agonist failed to rescue LGK974-mediated proliferation inhibition.

[0206] RNAseq analysis showed that FZD2 was the major isotype in the mesenchymal stem cell line CH3H10T1 / 2 (mouse ENCODE), suggesting that FZD2 may be responsible for the defining role of Wnt protein in osteogenic differentiation of mesenchymal cells (Day et al., Dev. Cell. 8, 739–750 (2005)). Stimulation of C3H10T1 / 2 cells with FZD2-specific FLAg resulted in a strong induction of the osteogenic marker alkaline phosphatase (ALPL) to levels similar to those achieved with pan-FZD FLAg stimulation, while FZD5-specific FLAg showed minimal activity. Figure 7B ).

[0207] 6. Co-targeting with tetravalently bound molecules

[0208] In addition to mixing and matching the FZD multivalent binding domain and co-receptor binding domain with the Fc domain to achieve desired combinations, the presence of tetravalent complementary sites in the current system provides the opportunity to simultaneously target two FZD receptors and two co-receptors with a single molecule, ensuring co-localization during in vivo application. Considering the agonistic activity of 5019-MBP-K / H-2539:2542 shown above, multivalent binding molecules with selective FZD receptor binding domains can be generated by binding the binding region in a heterobiome antibody to the N-terminus of the Fc domain. For example, binding domains derived from antibodies 5038 (binding FZD4) and 2928 (binding FZD5) will produce FZD4 and FZD5 co-targeting molecules. Binding molecules with co-receptor binding domains targeting specific or multiple co-receptors can also be generated. For example, the LRP6 / LRP5 co-targeting binding domain can be generated by binding the 2459 (binding site for Wnt1 on LRP6) and 2539 (binding site for Wnt3a on LRP6) binding domains at the C-terminus of the Fc domain. Similarly, the co-receptor binding domain can include a binding site for LRP6 that binds to another co-receptor, such as ROR1 / 2, to initiate activation of both canonical and atypical Wnt signaling pathways in a single cell.

[0209] This paper also considers multivalent binding molecules with tissue-specific binding domains derived from tissue-specific antibodies, which recruit the multivalent binding molecules to the desired tissue, where they then activate Wnt signaling by binding to FZD receptors and co-receptors. This is considered particularly useful in the use of multivalent binding molecules in regenerative therapy when the desired effect may need to be limited to a specific tissue. In summary, the tetravalent mode allows for greater design flexibility to meet a variety of functional requirements.

[0210] 7. Multivalent binding molecules with FZD binding domains and co-receptor binding domains can replace Wnt ligands to maintain intestinal organoid cultures.

[0211] The effects of the FZD agonist described herein on organoid survival and maintenance were investigated as follows. An 8-week-old female C57BL / 6 mouse was sacrificed, and intestinal crypts were collected for organoid isolation (O'Rourke et al. 2016. Isolation, Culture, and Maintenance of Mouse Intestinal Stem Cells. Bio Protoc. 20:4). Organoid cultures were passed through mechanically (O'Rourke 2016) and embedded in 25 μl of growth factor reduced matrigel (Corning, 356231) in 48-well plates. Triple-plate organoids were prepared for each experimental condition. Complete organoid culture medium (O'Rourke 2016) with experimental conditions (1 μM GK-974 + / - 40% Wnt3a conditioned medium or + / - 50 nM pan-Fzd-5056 (FZDag targeting FZD1, 2, 4, 6, 7, 8 but binding epitopes that do not compete with Wnt ligands) was added to each well on the day of subculturing and changed every 2–3 days. One week later, 150 μL of Cell Titer Glo 3D (Promega) was added to 150 μL of medium in each well. Organoids were lysed on a rocking platform at RT for 30 min. The luminescence readings of 20 μL of lysate from each well were measured in duplicate. The average luminescence readings under each condition were normalized to DMSO conditions to calculate viability.

[0212] As ubiquitous stem cell niche factors, Wnt and R-spondin are required for the derivation and maintenance of three-dimensional cultured organoids from many tissues. In vitro, in the presence of R-spondin, Wnt protein secreted by Paneth cells is sufficient to support the growth of mouse small intestinal organoids. However, if PORCNi LGK974 prevents the release and activity of Wnt, the organoids will not proliferate and will eventually die. In this paper, we demonstrate that the pan-FZD multivalent binding molecule FZDag (F P+P -L6 1 +3 The molecule can rescue and maintain organoid growth in the presence of LGK974, indicating that it functionally mimics the Wnt ligand (Figure 8) and can replace the Wnt protein to support tissue organoid growth. Since the Wnt ligand is an integral component of the mediators required for the growth of many human tissue organoids, when the antibody-derived FZD agonist of this invention is included in the culture medium, it is expected to promote the derivation, survival, and maintenance of organoids from various tissues, thereby mitigating the limitations associated with using conditioned media or purified Wnt protein.

[0213] 8. Multivalently bound molecules promote bone regeneration

[0214] A rat closed femoral fracture model was used to evaluate the regenerative properties of the multivalent binding molecule of the present invention, which has a first multivalent binding domain for binding FZD2 and a co-receptor binding domain for binding LRP5 or LRP. The first multivalent binding domain may specifically bind FZD2 or may bind FZD2 and other FZD receptors.

[0215] Following a unilateral closed fracture of the femoral shaft, rats were administered a carrier or a multivalently bound molecule (see Bonnarens and Einhorn, J. Orthop. Res. 2, 97–101 (1984)). Briefly, an 18-gauge syringe needle was inserted through the condyle into the medullary cavity. A transverse fracture of the femur was then induced by a blunt impact load on the anterior (lateral) thigh. One day post-fracture, rats were subcutaneously injected with either the saline carrier or the multivalently bound molecule twice weekly for seven weeks. At termination, the intramedullary nail was removed, and the fractured femur was analyzed by microCT.

[0216] In this model, multivalently bound molecules with a multivalent domain that binds FZD2 and a second multivalent domain that binds LRP5 or LRP6 significantly increased bone regeneration compared to bone regeneration via carrier alone.

[0217] Example II - Synthetic Antibodies Targeting FZD and LRP6

[0218] We previously used phage display to obtain hundreds of synthetic antibodies using nine recombinant FZD CRDs as antigens (FZD3 CRD could not be purified) (Steinhart et al. Nat. Med. 23, 60 (2016); Pavlovic et al. MAbs (2018), doi: 10.1080 / 19420862.2018. 1515565). Systematic characterization revealed a continuous spectrum of specificity, with some Abs exhibiting broad specificity, such as the pan-FZD Ab (FP) recognizing FZD1 / 2 / 4 / 5 / 7 / 8. Figure 11A Other Abs showed more limited specificity, and some were monospecific. Figure 11B Functional characterization showed that some antibodies competed with Wnt and inhibited β-catenin signaling, while others were non-competitive and did not interfere with Wnt signaling. Figure 11BIn summary, we fully characterized 161 anti-FZD antibodies, including 47 inhibitors of Wnt signaling. Surprisingly, as discussed in this paper, regardless of whether they compete with or inhibit Wnt signaling, all the multivalent binding molecules generated by using these anti-FZD antibodies as a source of FZD binding domains to bind LRP binding domains, such as those binding to Wnt1 and / or Wnt3a binding sites on LRP5 / 6, are agonists of the Wnt pathway.

[0219] Example III - Phenotypic Effects of FLAg in Cells, Organoids, and Animals

[0220] Having established that FLAg selectively binds to FZD and LRP to activate Wnt-related signaling pathways, we explored the phenotypic effects of these signals in progenitor stem cells (PSCs), organoids, and animals. Regulation of Wnt-β-catenin signaling activity is essential for most PSC differentiation protocols (Huggins et al. Methods Mol. Biol. 1481, 161–181 (2016)). Treatment of human PSCs with WNT3A-conditioned medium or small molecule inhibitors of GSK3 activates β-catenin signaling, leading to primitive streak induction and promoting mesodermal fate specification (Davidson et al. PNAS USA 109, 4485–4490 (2012)). In this context, we evaluated the activity of FLAg and found that 30 nM FLAg... P+P -L6 1+3 Three days of treatment with human PSCs induced strong induction of the mesodermal marker BRACHYURY, while the expression of the multipotency marker OCT4 decreased to levels comparable to those induced by treatment with the GSK3 inhibitor CHIR99021 at 6 μM. Figure 13A and Figure 13B ).

[0221] F P+P -L6 1+3 We identified mouse FZD and LRP6, which contain Fc that interacts with FcRn. Fc is expected to confer a molecularly long Ab-like half-life in vivo. Therefore, we tested Fc... P+P -L6 1+3It is unclear whether Wnt can interact with endogenous receptors in mice and accumulate to levels sufficient to activate β-catenin signaling and mobilize endogenous stem cell activity. In the intestinal stem cell niche, Wnt protein secreted by mesenchymal cells induces the expression of β-catenin target genes in crypt basal stem cells, directing their self-renewal, and the target gene LGR5 is frequently used as a marker for stem cells in various tissues. Treatment of LGR5-GFP mice with LGK974 ablates Wnt production and leads to the rapid disappearance of LGR5 expression and linked GFP signaling in crypt stem cells. Notably, intraperitoneal injection with F P+P -L6 1+3 After co-treatment, GFP expression was rescued. Figure 14 (Right figure). We conclude that F P+P -L6 1+3 It has sufficient half-life and bioavailability to activate β-catenin to a level that promotes intestinal stem cell self-renewal in the absence of endogenous Wnt.

[0222] Example IV - Materials and Methods:

[0223] 1. Ab selection and filtering

[0224] As described in (Persson et al., J. Mol. Biol. 425, 803–811 (2013)), the phage-displayed synthetic library F was used to select Fab for binding to the Wnt receptor. Briefly, Fc-tagged ECD proteins (R&D Systems) were immobilized on Maxisorp immunoassay plates (ThermoFisher, catalog 12-565-135) and used for positive binding selection with a pool of phages that had been first exposed to similarly immobilized Fc proteins to deplete nonspecific binders. After four rounds of binding selection, cloned phages were prepared and evaluated by phage ELISA (Birtalan et al., J. Mol. Biol. 377, 1518–1528 (2008)). Clones exhibiting at least a 10-fold stronger signal upon binding to the antigen compared to Fc were considered to be specific binders that had undergone further characterization.

[0225] 2. Recombinant proteins and reagents

[0226] The fusion units with the Fc tag FZD1(5988-FZ-050), FZD2(1307-FZ-050), FZD4(5847-FZ-050), FZD5(1617-FZ-050), FZD7(6178-FZ-050), FZD8(6129-FZ-050), FZD9(9175-FZ-050), and FZD10(3459-FZ-050) were purchased from R&D Systems. The Fc-tagged ECD of FZD6 (residues 19-132, UniprotO60353-1) was expressed and purified from Expi293 cells using the pFUSE-hIgG1-Fc2 vector (Invivogen). Individual promoter species were isolated from the aggregated protein by size exclusion chromatography on a Superdex 200 (10 / 300) column (GE Healthcare). Human (1505-LR-025) and mouse (2960-LR-025) LRP6 and mouse LRP5 (7344-LR-025 / CF) Fc-tagged ECD fusion proteins were purchased from R&D Systems. WNT1 (SRP4754-10ug), WNT2b (3900-WN-010 / CF), WNT5a (645-WN-010 / CF), and WNT3A (5036-WN-010 / CF) were purchased from R&D Systems, and WNT3A conditioned medium was prepared as described in (PMID: 12717451). Other proteins and chemicals were purchased from the following suppliers: FcRN (R&D, 8693-FC), C1q (Sigma, C1740), CD16a (R&D, 4325-FC), CD32a (R&D, 1330-CD / CF), CD64 (R&D, 1257-FC), LGK974 (Cayman Chemicals), the porcupine inhibitor C59 (Dalriada Therapeutics), and CHIR99021 (Sigma Aldrich).

[0227] 3. Tetravalent binding molecules of FZD and LRP, "FLAg", and antibody clones

[0228] DNA fragments encoding the variable domains of antibodies (Abs) were amplified from phage DNA templates via PCR or constructed via chemical synthesis (Twist Biosciences). The DNA fragments were cloned into mammalian expression vectors (pSCSTa) designed to generate the κ light chain and human IgG1 heavy chain. The bispecific bisomal antibodies and IgGs comprised an optimized version of the "mortar and pestle" heterodimer Fc (Ridgway et al., Protein Eng. 9, 617–621 (1996)). The FLAg and bisomal antibody-Fc fusions were aligned in a VH-VL orientation, with the variable domains separated by short GGGGS linkers (e.g., amino acids 121-125 of SEQ ID NO: 2), which facilitated intermolecular association between the VH and VL domains, thus promoting bisomal antibody formation. To generate the bisomal antibody-Fc fusion construct, the bisomal antibody chain was fused with human IgG1 Fc. The FLAg protein was constructed as VH-x-VL-y-[human IgG1Fc]-z-VH-x-VL, where the linkers are x=GGGGS (e.g., amino acids 121-125 of SEQ ID NO: 2), y=LEDKTHTKVEPKSS (amino acids 232-245 of SEQ ID NO: 4), and z=SGSETPGTSESATPESGGG (amino acids 473-501 of SEQ ID NO: 4). In this form, the human IgG1 Fc or mortis IgG1 Fc fragment spans positions 234-478 (Kabat number). For the scFv-Fc fusion, the variable domains are aligned in a VL-VH orientation and linked by a long GTTAASGSSGGSSSGA (SEQ ID NO: 75) linker, which facilitates intramolecular association between the VH and VL domains, thus promoting scFv formation. For all constructs, the entire coding region was in-frame cloned along with the secretion signal peptide into a mammalian expression vector.

[0229] 4. Protein Expression and Purification

[0230] Antigens, Ab, and FLAg proteins were generated in Expi293F (ThermoFisher) cells via transient transfection. In short, cells were grown to approximately 2.5 × 10⁻⁶ cells in Expi293 expression medium (Gibco) in baffled cell culture flasks. 6Cells were transfected at a density of 1000 cells / ml using FectoPRO transfection reagent (Polyplus-transfection) and a suitable vector using a standard manufacturing protocol (ThermoFisher). Expression was incubated at 37°C and 8% CO2 with shaking at 125 rpm for 5 days. After expression, cells were removed by centrifugation, and the protein was purified from the conditioned medium using recombinant protein A agarose gel (rProtein A Sepharose) (GE Healthcare). The purified protein was buffer-exchanged in PBS or a prepared stabilization buffer (36.8 mM citrate, 63.2 mM Na2HPO4, 10% trehalose, 0.2 M L-arginine, 0.01% Tween-80, pH 6.0) for storage. Protein concentration was determined by absorbance at 280 nm, and purity was confirmed by SDS-PAGE analysis.

[0231] 5. In vitro binding assay

[0232] BLI detection was performed using an Octet HTX instrument (ForteBio). To measure binding to the antigen, an Fc-tagged fusion of the FZD receptor (FZD-Fc protein) was captured on an AHQ BLI sensor (18-5001, ForteBio) to achieve a BLI response of 0.6–1 nm. The remaining Fc binding sites were saturated with human Fc (009-000-008, Jackson ImmunoResearch). The FZD-coated or control (Fc-coated) sensors were transferred to 100 nM Ab or FLAG in detection buffer (PBS, 1% BSA, 0.05% Tween 20) and association was monitored for 300 seconds. The sensors were then transferred to the detection buffer and dissociation was monitored for an additional 300 seconds. The shaking speed was 1000 rpm, and the temperature was 25 °C. Endpoint response values ​​were acquired after 295 seconds of association. Endpoint data is analyzed by subtracting the Fc signal from the FZD-Fc signal and then normalizing the data to the highest binding signal.

[0233] To determine binding to Fc receptors, Ab or FLAg were immobilized on an AR2G sensor (18-5092, ForteBio) via amine coupling to achieve a BLI response of 0.6–3 nm, and any remaining sites were quenched with ethanolamine. The coated sensor was equilibrated in detection buffer (PBS, 1% BSA, 0.05% Tween 20) and transferred to Fc receptor solution. Association was monitored for 600 seconds, the sensor was transferred to detection buffer, and dissociation was monitored for 600 seconds. Unless otherwise specified, CD64 and all other Fc receptors were detected at 50 nM or 300 nM at pH 7.4. The shaking speed was 1000 rpm, and the temperature was 25 °C. Endpoint response values ​​were acquired at the end of the association phase and normalized to isotype controls. Steady-state FcRN binding was detected in a similar manner, except that FcRN immobilization was performed and serial dilutions of Ab or FLAg (0.1–225 nM) were evaluated in solution. The time for consolidation and dissolution is 600 or 1200 seconds, respectively.

[0234] Surface plasmon resonance (SPR) detection was performed using the ProteOn XPR36 system (Bio-Rad). FZD-Fc or LRP-Fc proteins were immobilized on the surface of the GLC sensor (176-5011) using standard amine coupling chemistry. Ab or FLAg in detection buffer (PBS, 0.05% Tween 20, 0.5% BSA) was injected at a rate of 40 μl / min, and association was monitored for 150 seconds. Then, detection buffer was injected at a rate of 100 μl / min, and dissociation was monitored for 900 seconds. Detection was performed at 25°C. Analysis was performed using a 1:1 Langmuir model, and global fitting was performed using ProteOn Manager software to determine kon and koff values. KD was calculated as the koff / kon ratio.

[0235] 6. Epitope binning

[0236] BLI epitope grouping experiments were performed using an Octet HTX instrument (ForteBio). Fc fusions containing FZD (FZD-Fc) or LRP6 (LRP6-Fc) proteins were immobilized onto AHQ (18-5001, ForteBio) or AR2G (18-5092, ForteBio) BLI sensors, respectively. The coated sensors were transferred to 100 nM Ab in detection buffer (PBS, 1% BSA, 0.05% Tween 20) for 240 seconds to saturate the binding sites. The sensors were then transferred to 100 nM competitive Ab in detection buffer for 180 seconds. The response was measured 20 seconds after exposure to the competitive Ab and normalized to a binding signal on the unblocked antigen-coated sensor. The shaking speed was 1000 rpm, and the temperature was 25°C.

[0237] 7. Cell lines

[0238] HPAF-II and HEK293T cell lines were maintained in DMEM containing 4.5 g / L D-glucose, sodium pyruvate, and L-glutamine (ThermoFisher #12430-054) supplemented with 10% FBS (ThermoFisher) and penicillin / streptomycin (ThermoFisher #15140-163). CHO cells were maintained in DMEM / F12 (ThermoFisher #11320-033) supplemented with 10% FBS and penicillin / streptomycin. Cells were maintained at 37°C and 5% CO2.

[0239] 8. Flow cytometry

[0240] As previously described, CHO cell lines were stained with 10 nM anti-FZD Fab via indirect immunofluorescence staining (Steinhart et al. 2017 Nat Med. Jan;23(1):60-68, PMID: 27869803). Alexa Fluor488 AffiniPure F(ab')2 was used as the secondary antibody (Jackson ImmunoResearch, 109-545-097). Anti-c-MycIgG1 9E10 (primary antibody, ThermoFisher, MA1-980) and Alexa Fluor 488 IgG (secondary antibody, Life Technologies, A11001) were used as expression controls. All reagents were used according to the manufacturer's instructions.

[0241] 9. Luciferase reporter gene assay

[0242] HEK293T cells were transduced with a lentivirus encoding the pBAR1 reporter gene (Biechele and Moon in WntSignaling Signal: Pathway Methods and Mammalian Models, EE Vincan, Ed. (Humana Press, Totowa, NJ, 2008), pp. 99-110) with René luciferase as a control to generate a Wnt-β-catenin signaling reporter gene cell line. Before transfection or stimulation, 1-2 x 10⁻⁶ lentiviruses were added to 120 µl of the lentivirus. 3 Cells were seeded into each well of a 96-well plate for 24 hours. The next day, either FLAg or Ab protein was added, and after 15–20 hours of stimulation, cells were lysed and luminescence was measured using an Envision plate reader (PerkinElmer) according to the dual luciferase protocol (Promega). For the FZD4-specific agonist assay, FZD4 cDNA was transfected for 6 hours before adding FLAg protein. For the Wnt inhibition assay, Wnt1 was introduced via cDNA transfection or WNT3A protein was applied for 6 hours before adding Ab protein. All assays were repeated at least three times.

[0243] 10. Western blot detection of proteins

[0244] H1 ESCs were dissolved in lysis buffer (1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 0.1% deoxycholic acid, 50 mM Tris (pH 7.4), 0.1 mM EGTA, 0.1 mM EDTA, 20 mM sodium fluoride (NaF), 1:500 protease inhibitor (Sigma), and 1 mM sodium orthovanadate (Na3VO4)). The lysis buffer was incubated at 4°C for 30 min, centrifuged at 14,000 × g for 10 min, boiled in SDS sample buffer, separated by SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and subjected to Western blotting with the specified Abs. Abs were detected using a chemiluminescence-based detection system (ECL; ThermoFisher).

[0245] 11. Crystal violet proliferation detection

[0246] HPAF-II cells were seeded at 500 cells per well, and 100 nM LGK974 was added 24 hours later, with or without the addition of 100 nM FLAg. The culture medium was changed and the drug treatment was updated every other day. After 7 days of treatment, the cells were fixed with ice-cold methanol. The cells were stained with 0.5% crystal violet solution in 25% methanol, destained with 10% acetic acid, and quantified by measuring the absorbance at 590 nm.

[0247] 12. Immunofluorescence

[0248] H1 hES cells treated with FLAg and CHIR99021 for 3 days were washed with cold PBS and fixed with 4% PFA for 20 min. The fixed cells were rinsed with PBS, infiltrated with 0.3% Triton for 10 min, and blocked with 1% BSA for 1 h. Cells were incubated with primary antibodies for BRACHYURY (R&D systems AF2085; goat; 1:100 dilution) or OCT3 / 4 (Santa Cruz sc5279; mouse; 1:100 dilution) in 1% BSA for 2 h, and then incubated with Alexa Fluor 488-labeled donkey anti-goat or Alexa Fluor 568-labeled donkey anti-mouse Ab for 1 h. Figure 13A The coverslip was mounted using Fluoromount (Sigma-Aldrich), and analysis was performed on a Zeiss LSM700 confocal microscope using a 60× oil immersion lens. Figure 13B The image was assembled using ImageJ and Photoshop CS6 (Adobe Systems, Mountain View, CA).

[0249] 13. Intestinal crypt self-renewal detection

[0250] Lgr5-EGFP-IRES-creERT2 (B6.129P2-Lgr5tm1(cre / ERT2) Cle / J) mice aged 8-10 weeks were purchased from The Jackson Laboratory (Bar Harbor, ME). All experiments were conducted according to protocols approved by the Animal Care and Use Committee of the University of Toronto and in accordance with the regulations of the Canadian Council on Animal Care and the ARRIVE guidelines (Animal Research: In vivo experimental reporting). P+P -L6 1+3 Or negative control Ab was reconstituted in 37 mM citric acid, 63 mM Na2HPO4, 10% trehalose, 0.2 M L-arginine, 0.01% polysorbate 80, pH 6.0. Porcupine inhibitor C59 was reconstituted in ddH2O with 0.1% Tween 80 in 0.5% methylcellulose. Mice (male and female) were randomly divided into three groups (n=5-7 per group): vector, control (C59 and control Ab) or FLAg (C59 and F... P+P -L61+3 On day 1, the carrier or 10 mg / kg control Ab or F was administered via intraperitoneal injection. P +P -L6 1+3 Mice were treated. Treatment was performed blindfolded to the researchers until the end of the experiment and was repeated every two days for a total of three treatments. Starting on day 2, the vector group or both experimental groups were administered the vector or 50 mg / kg C59 by gavage twice daily at 8-hour intervals for 4 days. On day 6, the mice were sacrificed. Whole intestinal tissue was collected, washed with cold PBS, dehydrated with PBS and 30% sucrose, fixed with 4% paraformaldehyde, and embedded in an optimal cutting temperature compound (OCT). 8 μm OCT frozen sections were used for immunohistochemistry. Intestinal EGFP crypts were analyzed using a confocal microscope (Zeiss LSM700). Figure 14 The text describes the use of vectors, C59, or pan-FLAG (F P+P -L6 1+3 Representative fluorescence images of small intestinal sections from LGR5-GFP mice treated with C59. LGR5-GFP is expressed in stem cells at the base of the crypts. Cell nuclei are counterstained with DAPI.

[0251] Those skilled in the art will recognize or be able to identify many equivalents of the specific processes described herein using only conventional experiments. Such equivalents are considered to be within the scope of the invention. Various substitutions, alterations, and modifications may be made to the invention without departing from its spirit and scope. Other aspects, advantages, and modifications are within the scope of the invention. All references cited in this application, published patents, and disclosed patent applications are incorporated herein by reference. Suitable components, processes, and methods from those patents, applications, and other documents may be selected for the invention and its embodiments.

[0252] Table 1A

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275] Table 1B

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298] Table 2

[0299]

[0300] Table 3

[0301]

[0302]

[0303] sequence list <110> S. Angus (Stephane) S. Sidhu (SACHDEV) Tao Yuyong (TAO, YUYONG) <120> Multivalent binding molecules that activate WNT signaling and their applications <130> 115773.PA895WO <140> <141> <150> 62 / 630,772 <151> 2018-02-14 <160> 75 <170> PatentIn version 3.5 <210> 1 <211> 2169 <212> DNA <213> Homo sapiens <400> 1 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60 tcctgtgcag cttctggctt caacatcggt tcttcttcta tccactgggt gcgtcaggcc 120 ccgggtaagg gcctggaatg ggttgcatct atttattctg cttttgcctc tacttcttat 180 gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240 ctacaaatga acagcttaag agctgaggac actgccgtct attattgtgc tcgctaccat 300 ttcccgttcg gttttgcttt ggactactgg ggtcaaggaa ccctggtcac cgtctcctcg 360 ggtggaggtg gcagtgatat ccagatgacc cagtccccga gctccctgtc cgcctctgtg 420 ggcgataggg tcaccatcac ctgccgtgcc agtcagtccg tgtccagcgc tgtagcctgg 480 tatcaacaga aaccaggaaa agctccgaag cttctgattt actcggcatc cagcctctac 540 tctggagtcc cttctcgctt ctctggtagc cgttccggga cggatttcac tctgaccatc 600 agcagtctgc agccggaaga cttcgcaact tattactgtc agcaaggtgt ttacctgttc 660 acgttcggac agggtaccaa ggtggagatc aaactcgagg acaaaactca cacaaaagtg 720 gagcccaaaa cttctgataa gacccatact tgcccaccgt gcccagcacc tgaactcctg 780 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 840 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 900 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg cgaggagcag 960 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1020 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1080 atctccaaag ccaaagggca gccccgagaa ccaatggtgt ttgacctgcc cccatcccgg 1140 gaggagatga ccaagaacca ggtcagcctg tggtgcatgg tcaagggctt ctatcccagc 1200 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1260 cccgtgctgg actccgacgg ctccttcttc ctgtacagca agctcaccgt ggacaagagc 1320 cgctggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1380 tacacgcaga agagcctctc cctgtctccg ggtaaaagcg gcagcgagac tcccgggacc 1440 tcagagtccg ccacacccga aagtggtggc ggagaggttc agctggtgga gtctggcggt 1500 ggcctggtgc agccagggg ctcactccgt ttgtcctgtg cagcttctgg cttcaacatc 1560 tcttattctt ctatccactg ggtgcgtcag gccccgggta agggcctgga atgggttgca 1620 tatatttctt cttattatgg ctatacttat tatgccgata gcgtcaaggg ccgtttcact 1680 ataagcgcag acacatccaa aaacacagcc tacctacaaa tgaacagctt aagagctgag 1740 gacactgccg tctattattg tgctcgcgct cattacttcc cgtgggctgg tgctatggac 1800 tactggggtc aaagaaccct ggtcaccgtc tcctcgggtg gaggtggcag tgatatccag 1860 atgacccagt ccccgagctc cctgtccgcc tctgtgggcg atagggtcac catcacctgc 1920 cgtgccagtc agtccgtgtc cagcgctgta gcctggtatc aacagaaacc aggaaaagct 1980 ccgaagcttc tgatttactc ggcatccagc ctctactctg gagtcccttc tcgcttctct 2040 ggtagccgtt ccgggacgga tttcactctg accatcagca gtctgcagcc ggaagacttc 2100 gcaacttatt actgtcagca atactactgg ccgatcacgt tcggacaggg taccaaggtg 2160 gagatcaaa 2169 <210> 2 <211> 723 <212> PRT <213> Homo sapiens <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Gly Ser Ser 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Tyr Ser Ala Phe Ala Ser Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr His Phe Pro Phe Gly Phe Ala Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln 115 120 125 Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val 130 135 140 Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp 145 150 155 160 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala 165 170 175 Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser 180 185 190 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 195 200 205 Ala Thr Tyr Tyr Cys Gln Gln Gly Val Tyr Leu Phe Thr Phe Gly Gln 210 215 220 Gly Thr Lys Val Glu Ile Lys Leu Glu Asp Lys Thr His Thr Lys Val 225 230 235 240 Glu Pro Lys Thr Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 245 250 255 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 260 265 270 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 275 280 285 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 290 295 300 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 305 310 315 320 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 325 330 335 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 340 345 350 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 355 360 365 Arg Glu Pro Met Val Phe Asp Leu Pro Pro Ser Arg Glu Glu Met Thr 370 375 380 Lys Asn Gln Val Ser Leu Trp Cys Met Val Lys Gly Phe Tyr Pro Ser 385 390 395 400 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 405 410 415 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 420 425 430 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 435 440 445 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 450 455 460 Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly Ser Glu Thr Pro Gly Thr 465 470 475 480 Ser Glu Ser Ala Thr Pro Glu Ser Gly Gly Gly Glu Val Gln Leu Val 485 490 495 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 500 505 510 Cys Ala Ala Ser Gly Phe Asn Ile Ser Tyr Ser Ser Ile His Trp Val 515 520 525 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Ser 530 535 540 Tyr Tyr Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 545 550 555 560 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 565 570 575 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala His Tyr 580 585 590 Phe Pro Trp Ala Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 595 600 605 Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser 610 615 620 Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 625 630 635 640 Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys 645 650 655 Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser Leu Tyr 660 665 670 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe 675 680 685 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 690 695 700 Cys Gln Gln Tyr Tyr Trp Pro Ile Thr Phe Gly Gln Gly Thr Lys Val 705 710 715 720 Glu Ile Lys <210> 3 <211> 2187 <212> DNA <213> Homo sapiens <400> 3 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60 tcctgtgcag cttctggctt caacatcggt tcttcttcta tccactgggt gcgtcaggcc 120 ccgggtaagg gcctggaatg ggttgcatct atttattctg cttttgcctc tacttcttat 180 gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240 ctacaaatga acagcttaag agctgaggac actgccgtct attattgtgc tcgctaccat 300 ttcccgttcg gttttgcttt ggactactgg ggtcaaggaa ccctggtcac cgtctcctcg 360 ggtggaggtg gcagtgatat ccagatgacc cagtccccga gctccctgtc cgcctctgtg 420 ggcgataggg tcaccatcac ctgccgtgcc agtcagtccg tgtccagcgc tgtagcctgg 480 tatcaacaga aaccaggaaa agctccgaag cttctgattt actcggcatc cagcctctac 540 tctggagtcc cttctcgctt ctctggtagc cgttccggga cggatttcac tctgaccatc 600 agcagtctgc agccggaaga cttcgcaact tattactgtc agcaaggtgt ttacctgttc 660 acgttcggac agggtaccaa ggtggagatc aaactcgagg acaaaactca cacaaaagtt 720 gagcccaaat cttctgataa gacccataat tgcccaccgt gcccagcacc tgaactcctg 780 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 840 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 900 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 960 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1020 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1080 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccaatccgg 1140 gagctgatga ccagcaacca ggtcagcctg agctgcgccg tcaaaggctt ctatcccagc 1200 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1260 cccgtgctgg actccgacgg ctccttcttc ctcgtgagca agctcaccgt ggacaagagc 1320 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1380 tacacgcaga agagcctctc cctgtctccg ggtaaaagcg gcagcgagac tcccgggacc 1440 tcagagtccg ccacacccga aagtggtggc ggagaggttc agctggtgga gtctggcggt 1500 ggcctggtgc agccaggggg ctcactccgt ttgtcctgtg cagcttctgg cttcaacatc 1560 tcttcttatt atatccactg ggtgcgtcag gccccgggta agggcctgga atgggttgca 1620 tctatttatt cttcttatgg ctatacttct tatgccgata gcgtcaaggg ccgtttcact 1680 ataagcgcag acacatccaa aaacacagcc tacctacaaa tgaacagctt aagagctgag 1740 gacactgccg tctattattg tgctcgcact gttcgtggat ccaaaaaacc gtacttctct 1800 ggttgggcta tggactactg gggtcaagga accctggtca ccgtctcctc gggtggaggt 1860 ggcagtgata tccagatgac ccagtccccg agctccctgt ccgcctctgt gggcgatagg 1920 gtcaccatca cctgccgtgc cagtcagtcc gtgtccagcg ctgtagcctg gtatcaacag 1980 aaaccaggaa aagctccgaa gcttctgatt tactcggcat ccagcctcta ctctggagtc 2040 ccttctcgct tctctggtag ccgttccggg acggatttca ctctgaccat cagcagtctg 2100 cagccggaag acttcgcaac ttattactgt cagcaatact cttggggtcc gttcacgttc 2160 ggacagggta ccaaggtgga gatcaaa 2187 <210> 4 <211> 729 <212> PRT <213> Homo sapiens <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Gly Ser Ser 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Tyr Ser Ala Phe Ala Ser Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr His Phe Pro Phe Gly Phe Ala Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln 115 120 125 Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val 130 135 140 Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp 145 150 155 160 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala 165 170 175 Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser 180 185 190 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 195 200 205 Ala Thr Tyr Tyr Cys Gln Gln Gly Val Tyr Leu Phe Thr Phe Gly Gln 210 215 220 Gly Thr Lys Val Glu Ile Lys Leu Glu Asp Lys Thr His Thr Lys Val 225 230 235 240 Glu Pro Lys Ser Ser Asp Lys Thr His Asn Cys Pro Pro Cys Pro Ala 245 250 255 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 260 265 270 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 275 280 285 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 290 295 300 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 305 310 315 320 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 325 330 335 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 340 345 350 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 355 360 365 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ile Arg Glu Leu Met Thr 370 375 380 Ser Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser 385 390 395 400 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 405 410 415 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val 420 425 430 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 435 440 445 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 450 455 460 Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly Ser Glu Thr Pro Gly Thr 465 470 475 480 Ser Glu Ser Ala Thr Pro Glu Ser Gly Gly Gly Glu Val Gln Leu Val 485 490 495 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 500 505 510 Cys Ala Ala Ser Gly Phe Asn Ile Ser Ser Tyr Tyr Ile His Trp Val 515 520 525 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile Tyr Ser 530 535 540 Ser Tyr Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 545 550 555 560 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 565 570 575 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Val Arg 580 585 590 Gly Ser Lys Lys Pro Tyr Phe Ser Gly Trp Ala Met Asp Tyr Trp Gly 595 600 605 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile 610 615 620 Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg 625 630 635 640 Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala 645 650 655 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser 660 665 670 Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg 675 680 685 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp 690 695 700 Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Trp Gly Pro Phe Thr Phe 705 710 715 720 Gly Gln Gly Thr Lys Val Glu Ile Lys 725 <210> 5 <211> 2172 <212> DNA <213> Homo sapiens <400> 5 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60 tcctgtgcag cttctggctt caacatcggt tcttcttcta tccactgggt gcgtcaggcc 120 ccgggtaagg gcctggaatg ggttgcatct atttattctg cttttgcctc tacttcttat 180 gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240 ctacaaatga acagcttaag agctgaggac actgccgtct attattgtgc tcgctaccat 300 ttcccgttcg gttttgcttt ggactactgg ggtcaaggaa ccctggtcac cgtctcctcg 360 ggtggaggtg gcagtgatat ccagatgacc cagtccccga gctccctgtc cgcctctgtg 420 ggcgataggg tcaccatcac ctgccgtgcc agtcagtccg tgtccagcgc tgtagcctgg 480 tatcaacaga aaccaggaaa agctccgaag cttctgattt actcggcatc cagcctctac 540 tctggagtcc cttctcgctt ctctggtagc cgttccggga cggatttcac tctgaccatc 600 agcagtctgc agccggaaga cttcgcaact tattactgtc agcaaggtgt ttacctgttc 660 acgttcggac agggtaccaa ggtggagatc aaactcgagg acaaaactca cacaaaagtt 720 gagcccaaat cttctgataa gacccatact tgcccaccgt gcccagcacc tgaactcctg 780 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 840 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 900 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 960 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1020 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1080 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1140 gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1200 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1260 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1320 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc acgaggctct gcacaaccac 1380 tacacgcaga agagcctctc cctgtctccg ggtaaaagcg gcagcgagac tcccgggacc 1440 tcagagtccg ccacacccga aagtggtggc ggagaggttc agctggtgga gtctggcggt 1500 ggcctggtgc agccaggggg ctcactccgt ttgtcctgtg cagcttctgg cttcaacatc 1560 tcttattctt ctatccactg ggtgcgtcag gccccgggta agggcctgga atgggttgca 1620 tatatttctt cttattatgg ctatacttat tatgccgata gcgtcaaggg ccgtttcact 1680 ataagcgcag acacatccaa aaacacagcc tacctacaaa tgaacagctt aagagctgag 1740 gacactgccg tctattattg tgctcgcgct cattacttcc cgtgggctgg tgctatggac 1800 tactggggtc aaggaaccct ggtcaccgtc tcctcgggtg gaggtggcag tgatatccag 1860 atgacccagt ccccgagctc cctgtccgcc tctgtgggcg atagggtcac catcacctgc 1920 cgtgccagtc agtccgtgtc cagcgctgta gcctggtatc aacagaaacc aggaaaagct 1980 ccgaagcttc tgatttactc ggcatccagc ctctactctg gagtcccttc tcgcttctct 2040 ggtagccgtt ccgggacgga tttcactctg accatcagca gtctgcagcc ggaagacttc 2100 gcaacttatt actgtcagca atactcttgg ggtccgttca cgttcggaca gggtaccaag 2160 gtggagatca aa 2172 <210> 6 <211> 724 <212> PRT <213> Homo sapiens <400> 6 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Gly Ser Ser 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Tyr Ser Ala Phe Ala Ser Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr His Phe Pro Phe Gly Phe Ala Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln 115 120 125 Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val 130 135 140 Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp 145 150 155 160 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala 165 170 175 Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser 180 185 190 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 195 200 205 Ala Thr Tyr Tyr Cys Gln Gln Gly Val Tyr Leu Phe Thr Phe Gly Gln 210 215 220 Gly Thr Lys Val Glu Ile Lys Leu Glu Asp Lys Thr His Thr Lys Val 225 230 235 240 Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 245 250 255 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 260 265 270 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 275 280 285 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 290 295 300 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 305 310 315 320 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 325 330 335 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 340 345 350 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 355 360 365 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 370 375 380 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 385 390 395 400 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 405 410 415 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 420 425 430 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 435 440 445 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 450 455 460 Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly Ser Glu Thr Pro Gly Thr 465 470 475 480 Ser Glu Ser Ala Thr Pro Glu Ser Gly Gly Gly Glu Val Gln Leu Val 485 490 495 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 500 505 510 Cys Ala Ala Ser Gly Phe Asn Ile Ser Tyr Ser Ser Ile His Trp Val 515 520 525 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Ser 530 535 540 Tyr Tyr Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 545 550 555 560 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 565 570 575 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala His Tyr 580 585 590 Phe Pro Trp Ala Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 595 600 605 Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser 610 615 620 Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 625 630 635 640 Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys 645 650 655 Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser Leu Tyr 660 665 670 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe 675 680 685 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr [[ID=!17]]690 695 700 Cys Gln Gln Tyr Ser Trp Gly Pro Phe Thr Phe Gly Gln Gly Thr Lys 705 710 715 720 Val Glu Ile Lys <210> 7 <211> 2184 <212> DNA[[ID=3 !0]] <213> Homo sapiens <400> 7 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60 tcctgtgcag cttctggctt caacatcggt tcttcttcta tccactgggt gcgtcaggcc 120 ccgggtaagg gcctggaatg ggttgcatct atttattctg cttttgcctc tacttcttat 180 gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240 It should be noted that there seems to be a small error in the original text where "[[ID=3!0]]" should probably be "", and " " should probably be " ". The translation is done based on the provided text as accurately as possible while keeping the tags intact. ctacaaatga acagcttaag agctgaggac actgccgtct attattgtgc tcgctaccat 300 ttcccgttcg gttttgcttt ggactactgg ggtcaaggaa ccctggtcac cgtctcctcg 360 ggtggaggtg gcagtgatat ccagatgacc cagtccccga gctccctgtc cgcctctgtg 420 ggcgataggg tcaccatcac ctgccgtgcc agtcagtccg tgtccagcgc tgtagcctgg 480 tatcaacaga aaccaggaaa agctccgaag cttctgattt actcggcatc cagcctctac 540 tctggagtcc cttctcgctt ctctggtagc cgttccggga cggatttcac tctgaccatc 600 agcagtctgc agccggaaga cttcgcaact tattactgtc agcaaggtgt ttacctgttc 660 acgttcggac agggtaccaa ggtggagatc aaactcgagg acaaaactca cacaaaagtt 720 gagcccaaat cttctgataa gacccatact tgcccaccgt gcccagcacc tgaactcctg 780 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 840 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 900 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 960 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1020 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1080 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1140 gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1200 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1260 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1320 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc acgaggctct gcacaaccac 1380 tacacgcaga agagcctctc cctgtctccg ggtaaaagcg gcagcgagac tcccgggacc 1440 tcagagtccg ccacacccga aagtggtggc ggagaggttc agctggtgga gtctggcggt 1500 ggcctggtgc agccaggggg ctcactccgt ttgtcctgtg cagcttctgg cttcaacatc 1560 tcttcttatt atatccactg ggtgcgtcag gccccgggta agggcctgga atgggttgca 1620 tctatttatt cttcttatgg ctatacttct tatgccgata gcgtcaaggg ccgtttcact 1680 ataagcgcag acacatccaa aaacacagcc tacctacaaa tgaacagctt aagagctgag 1740 ataagcgcag acacatccaa aaacacagcc tacctacaaa tgaacagctt aagagctgag 1740 gacactgccg tctattattg tgctcgcact gttcgtggat ccaaaaaacc gtacttctct gacactgccg tctattattg tgctcgcact gttcgtggat ccaaaaaacc gtacttctct 1800 ggttgggcta tggactactg gggtcaagga accctggtca ccgtctcctc gggtggaggt ggttgggcta tggactactg gggtcaagga accctggtca ccgtctcctc gggtggaggt 1860 ggcagtgata tccagatgac ccagtccccg agctccctgt ccgcctctgt gggcgatagg ggcagtgata tccagatgac ccagtccccg agctccctgt ccgcctctgt gggcgatagg 1920 gtcaccatca cctgccgtgc cagtcagtcc gtgtccagcg ctgtagcctg gtatcaacag gtcaccatca cctgccgtgc cagtcagtcc gtgtccagcg ctgtagcctg gtatcaacag 1980 aaaccaggaa aagctccgaa gcttctgatt tactcggcat ccagcctcta ctctggagtc aaaccaggaa aagctccgaa gcttctgatt tactcggcat ccagcctcta ctctggagtc 2040 ccttctcgct tctctggtag ccgttccggg acggatttca ctctgaccat cagcagtctg ccttctcgct tctctggtag ccgttccggg acggatttca ctctgaccat cagcagtctg 2100 cagccggaag acttcgcaac ttattactgt cagcaatact actggccgat cacgttcgga cagccggaag acttcgcaac ttattactgt cagcaatact actggccgat cacgttcgga 2160 cagggtacca aggtggagat caaa 2184 cagggtacca aggtggagat caaa 2184 <210> 8<210> 8 <211> 728<211> 728 <212> PRT <212> PRT <213> 智人 <213> Homo sapiens <400> 8 <400> 8 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Gly Ser Ser Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Gly Ser Ser [[ID=3-four]]20 25 30 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Tyr Ser Ala Phe Ala Ser Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr His Phe Pro Phe Gly Phe Ala Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln 115 120 125 Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val 130 135 140 Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp 145 150 155 160 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala 165 170 175 Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser 180 185 190 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 195 200 205 Ala Thr Tyr Tyr Cys Gln Gln Gly Val Tyr Leu Phe Thr Phe Gly Gln 210 215 220 Gly Thr Lys Val Glu Ile Lys Leu Glu Asp Lys Thr His Thr Lys Val 225 230 235 240 Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 245 250 255 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 260 265 270 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 275 280 285 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 290 295 300 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 305 310 315 320 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 325 330 335 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 340 345 350 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 355 360 365 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 370 375 380 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 385 390 395 400 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 405 410 415 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 420 425 430 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 435 440 445 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 450 455 460 Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly Ser Glu Thr Pro Gly Thr 465 470 475 480 Ser Glu Ser Ala Thr Pro Glu Ser Gly Gly Gly Glu Val Gln Leu Val 485 490 495 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 500 505 510 Cys Ala Ala Ser Gly Phe Asn Ile Ser Ser Tyr Tyr Ile His Trp Val 515 520 525 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile Tyr Ser 530 535 540 Ser Tyr Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 545 550 555 560 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 565 570 575 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Val Arg 580 585 590 Gly Ser Lys Lys Pro Tyr Phe Ser Gly Trp Ala Met Asp Tyr Trp Gly 595 600 605 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile 610 615 620 Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg 625 630 635 640 Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala 645 650 655 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser 660 665 670 Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg 675 680 685 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp 690 695 700 Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Trp Pro Ile Thr Phe Gly 705 710 715 720 Gln Gly Thr Lys Val Glu Ile Lys 725 <210> 9 <211> 2175 <212> DNA <213> Homo sapiens <400> 9 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60 tcctgtgcag cttctggctt caacatctct tattattata tgcactgggt gcgtcaggcc 120 ccgggtaagg gcctggaatg ggttgcatct atttattctt attatggcta tacttattat 180 gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240 ctacaaatga acagcttaag agctgaggac actgccgtct attattgtgc tcgctcttct 300 ttctcttggg ctatggacta ctggggtcaa ggaaccctgg tcaccgtctc ctcgggtgga 360 ggtggcagtg atatccagat gacccagtcc ccgagctccc tgtccgcctc tgtgggcgat 420 agggtcacca tcacctgccg tgccagtcag tccgtgtcca gcgctgtagc ctggtatcaa 480 cagaaaccag gaaaagctcc gaagcttctg atttactcgg catccagcct ctactctgga 540 gtcccttctc gcttctctgg tagccgttcc gggacggatt tcactctgac catcagcagt 600 ctgcagccgg aagacttcgc aacttattac tgtcagcaac atccgtggtc tggtggttac 660 ctgatcacgt tcggacaggg taccaaggtg gagatcaaac tcgaggacaa aactcacaca 720 aaagtggagc ccaaaacttc tgataagacc catacttgcc caccgtgccc agcacctgaa 780 ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 840 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 900 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgcgag 960 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 1020 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1080 aaaaccatct ccaaagccaa agggcagccc cgagaaccaa tggtgtttga cctgccccca 1140 tcccgggagg agatgaccaa gaaccaggtc agcctgtggt gcatggtcaa gggcttctat 1200 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1260 acgcctcccg tgctggactc cgacggctcc ttcttcctgt acagcaagct caccgtggac 1320 aagagccgct ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1380 aaccactaca cgcagaagag cctctccctg tctccgggta aaagcggcag cgagactccc 1440 gggacctcag agtccgccac acccgaaagt ggtggcggag aggttcagct ggtggagtct 1500 ggcggtggcc tggtgcagcc agggggctca ctccgtttgt cctgtgcagc ttctggcttc 1560 aacatctctt attcttctat ccactgggtg cgtcaggccc cgggtaaggg cctggaatgg 1620 gttgcatata tttcttctta ttatggctat acttattatg ccgatagcgt caagggccgt 1680 ttcactataa gcgcagacac atccaaaaac acagcctacc tacaaatgaa cagcttaaga 1740 gctgaggaca ctgccgtcta ttattgtgct cgcgctcatt acttcccgtg ggctggtgct 1800 atggactact ggggtcaagg aaccctggtc accgtctcct cgggtggagg tggcagtgat 1860 atccagatga cccagtcccc gagctccctg tccgcctctg tgggcgatag ggtcaccatc 1920 acctgccgtg ccagtcagtc cgtgtccagc gctgtagcct ggtatcaaca gaaaccagga 1980 aaagctccga agcttctgat ttactcggca tccagcctct actctggagt cccttctcgc 2040 ttctctggta gccgttccgg gacggatttc actctgacca tcagcagtct gcagccggaa 2100 gacttcgcaa cttattactg tcagcaatac tactggccga tcacgttcgg acagggtacc 2160 aaggtggaga tcaaa 2175 <210> 10 <211> 725 <212> PRT <213> Homo sapiens <400> 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Ser Tyr Tyr 20 25 30 ​​​ Ala Ser Ile Tyr Ser Tyr Tyr Gly Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Phe Ser Trp Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 115 120 125 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 130 135 140 Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln 145 150 155 160 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser 165 170 175 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr 180 185 190 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 195 200 205 Tyr Tyr Cys Gln Gln His Pro Trp Ser Gly Gly Tyr Leu Ile Thr Phe 210 215 220 Gly Gln Gly Thr Lys Val Glu Ile Lys Leu Glu Asp Lys Thr His Thr 225 230 235 240 Lys Val Glu Pro Lys Thr Ser Asp Lys Thr His Thr Cys Pro Pro Cys 245 250 255 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 260 265 270 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 275 280 285 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 290 295 300 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 305 310 315 320 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 325 330 335 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 340 345 350 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 355 360 365 Gln Pro Arg Glu Pro Met Val Phe Asp Leu Pro Pro Ser Arg Glu Glu 370 375 380 Met Thr Lys Asn Gln Val Ser Leu Trp Cys Met Val Lys Gly Phe Tyr 385 390 395 400 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 405 410 415 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 420 425 430 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 435 440 445 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 450 455 460 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly Ser Glu Thr Pro 465 470 475 480 Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Gly Gly Glu Val Gln 485 490 495 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 500 505 510 Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Ser Tyr Ser Ser Ile His 515 520 525 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile 530 535 540 Ser Ser Tyr Tyr Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg 545 550 555 560 Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met 565 570 575 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala 580 585 590 His Tyr Phe Pro Trp Ala Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr 595 600 605 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 610 615 620 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 625 630 635 640 Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln 645 650 655 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser 660 665 670 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr 675 680 685 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 690 695 700 Tyr Tyr Cys Gln Gln Tyr Tyr Trp Pro Ile Thr Phe Gly Gln Gly Thr 705 710 715 720 Lys Val Glu Ile Lys 725 <210> 11 <211> 2193 <212> DNA <213> Homo sapiens <400> 11 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60 tcctgtgcag cttctggctt caacatctct tattattata tgcactgggt gcgtcaggcc 120 ccgggtaagg gcctggaatg ggttgcatct atttattctt attatggcta tacttattat 180 gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240 ctacaaatga acagcttaag agctgaggac actgccgtct attattgtgc tcgctcttct 300 ttctcttggg ctatggacta ctggggtcaa ggaaccctgg tcaccgtctc ctcgggtgga 360 ggtggcagtg atatccagat gacccagtcc ccgagctccc tgtccgcctc tgtgggcgat 420 agggtcacca tcacctgccg tgccagtcag tccgtgtcca gcgctgtagc ctggtatcaa 480 cagaaaccag gaaaagctcc gaagcttctg atttactcgg catccagcct ctactctgga 540 gtcccttctc gcttctctgg tagccgttcc gggacggatt tcactctgac catcagcagt 600 ctgcagccgg aagacttcgc aacttattac tgtcagcaac atccgtggtc tggtggttac 660 ctgatcacgt tcggacaggg taccaaggtg gagatcaaac tcgaggacaa aactcacaca 720 aaagttgagc ccaaatcttc tgataagacc cataattgcc caccgtgccc agcacctgaa 780 ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 840 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 900 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 960 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 1020 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1080 aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1140 atccgggagc tgatgaccag caaccaggtc agcctgagct gcgccgtcaa aggcttctat 1200 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1260 acgcctcccg tgctggactc cgacggctcc ttcttcctcg tgagcaagct caccgtggac 1320 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1380 aaccactaca cgcagaagag cctctccctg tctccgggta aaagcggcag cgagactccc 1440 gggacctcag agtccgccac acccgaaagt ggtggcggag aggttcagct ggtggagtct 1500 ggcggtggcc tggtgcagcc agggggctca ctccgtttgt cctgtgcagc ttctggcttc 1560 aacatctctt cttattatat ccactgggtg cgtcaggccc cgggtaaggg cctggaatgg 1620 gttgcatcta tttattcttc ttatggctat acttcttatg ccgatagcgt caagggccgt 1680 ttcactataa gcgcagacac atccaaaaac acagcctacc tacaaatgaa cagcttaaga 1740 gctgaggaca ctgccgtcta ttattgtgct cgcactgttc gtggatccaa aaaaccgtac 1800 ttctctggtt gggctatgga ctactggggt caaggaaccc tggtcaccgt ctcctcgggt 1860 ggaggtggca gtgatatcca gatgacccag tccccgagct ccctgtccgc ctctgtgggc 1920 gatagggtca ccatcacctg ccgtgccagt cagtccgtgt ccagcgctgt agcctggtat 1980 caacagaaac caggaaaagc tccgaagctt ctgatttact cggcatccag cctctactct 2040 ggagtccctt ctcgcttctc tggtagccgt tccgggacgg atttcactct gaccatcagc 2100 agtctgcagc cggaagactt cgcaacttat tactgtcagc aatactcttg gggtccgttc 2160 acgttcggac agggtaccaa ggtggagatc aaa 2193 <210> 12 <211> 731 <212> PRT <213> Homo sapiens <400> 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Ser Tyr Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Tyr Ser Tyr Tyr Gly Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Phe Ser Trp Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 115 120 125 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 130 135 140 Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln 145 150 155 160 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser 165 170 175 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr 180 185 190 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 195 200 205 Tyr Tyr Cys Gln Gln His Pro Trp Ser Gly Gly Tyr Leu Ile Thr Phe 210 215 220 Gly Gln Gly Thr Lys Val Glu Ile Lys Leu Glu Asp Lys Thr His Thr 225 230 235 240 Lys Val Glu Pro Lys Ser Ser Asp Lys Thr His Asn Cys Pro Pro Cys 245 250 255 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 260 265 270 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 275 280 285 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 290 295 300 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 305 310 315 320 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 325 330 335 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 340 345 350 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 355 360 365 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ile Arg Glu Leu 370 375 380 Met Thr Ser Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr 385 390 395 400 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 405 410 415 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 420 425 430 Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 435 440 445 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 450 455 460 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly Ser Glu Thr Pro 465 470 475 480 Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Gly Gly Glu Val Gln 485 490 495 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 500 505 510 Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Ser Ser Tyr Tyr Ile His 515 520 525 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile 530 535 540 Tyr Ser Ser Tyr Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg 545 550 555 560 Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met 565 570 575 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr 580 585 590 Val Arg Gly Ser Lys Lys Pro Tyr Phe Ser Gly Trp Ala Met Asp Tyr 595 600 605 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 610 615 620 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 625 630 635 640 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala 645 650 655 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 660 665 670 Tyr Ser Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 675 680 685 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 690 695 700 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Trp Gly Pro Phe 705 710 715 720 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 725 730 <210> 13 <211> 2169 <212> DNA <213> Homo sapiens <400> 13 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60 tcctgtgcag cttctggctt caacctctct tcttattcta tgcactgggt gcgtcaggcc 120 ccgggtaagg gcctggaatg ggttgcatat atttcttctt attatggcta tacttattat 180 gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240 ctacaaatga acagcttaag agctgaggac actgccgtct attattgtgc tcgcccggct 300 ccgggtcatt ggggttttga ctactggggt caaggaaccc tggtcaccgt ctcctcgggt 360 ggaggtggca gtgatatcca gatgacccag tccccgagct ccctgtccgc ctctgtgggc 420 gatagggtca ccatcacctg ccgtgccagt cagtccgtgt ccagcgctgt agcctggtat 480 caacagaaac caggaaaagc tccgaagctt ctgatttact cggcatccag cctctactct 540 ggagtccctt ctcgcttctc tggtagccgt tccgggacgg atttcactct gaccatcagc 600 agtctgcagc cggaagactt cgcaacttat tactgtcagc aatggtacta cgctccgatc 660 acgttcggac agggtaccaa ggtggagatc aaactcgagg acaaaactca cacaaaagtg 720 gagcccaaaa cttctgataa gacccatact tgcccaccgt gcccagcacc tgaactcctg 780 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 840 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 900 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg cgaggagcag 960 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1020 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1080 atctccaaag ccaaagggca gccccgagaa ccaatggtgt ttgacctgcc cccatcccgg 1140 gaggagatga ccaagaacca ggtcagcctg tggtgcatgg tcaagggctt ctatcccagc 1200 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1260 cccgtgctgg actccgacgg ctccttcttc ctgtacagca agctcaccgt gcaagagc 1320 cgctggcagc agggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1380 tacacgcaga agagcctctc cctgtctccg ggtaaaagcg gcagcgagac tcccgggacc 1440 tcagagtccg ccacacccga aagtggtggc ggagaggttc agctggtgga gtctggcggt 1500 ggcctggtgc agccagggg ctcactccgt ttgtcctgtg cagcttctgg cttcaacatc 1560 tcttattctt ctatccactg ggtgcgtcag gccccgggta agggcctgga atgggttgca 1620 tatatttctt cttattatgg ctatacttat tatgccgata gcgtcaaggg ccgtttcact 1680 ataagcgcag acacatccaa aaacacagcc tacctacaaa tgaacagctt aagagctgag 1740 gacactgccg tctattattg tgctcgcgct cattacttcc cgtgggctgg tgctatggac 1800 tactggggtc aaagaaccct ggtcaccgtc tcctcgggtg gaggtggcag tgatatccag 1860 atgacccagt ccccgagctc cctgtccgcc tctgtgggcg atagggtcac catcacctgc 1920 cgtgccagtc agtccgtgtc cagcgctgta gcctggtatc aacagaaacc aggaaaagct 1980 ccgaagcttc tgatttactc ggcatccagc ctctactctg gagtcccttc tcgcttctct 2040 ggtagccgtt ccgggacgga tttcactctg accatcagca gtctgcagcc ggaagacttc 2100 gcaacttatt actgtcagca atactactgg ccgatcacgt tcggacaggg taccaaggtg 2160 gagatcaaa 2169 <210> 14 <211> 723 <212> PRT <213> Homo sapiens <400> 14 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Leu Ser Ser Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Tyr Tyr Gly Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Pro Ala Pro Gly His Trp Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met 115 120 125 Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr 130 135 140 Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr 145 150 155 160 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser 165 170 175 Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly 180 185 190 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 195 200 205 Thr Tyr Tyr Cys Gln Gln Trp Tyr Tyr Ala Pro Ile Thr Phe Gly Gln 210 215 220 Gly Thr Lys Val Glu Ile Lys Leu Glu Asp Lys Thr His Thr Lys Val 225 230 235 240 Glu Pro Lys Thr Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 245 250 255 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 260 265 270 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 275 280 285 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 290 295 300 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 305 310 315 320 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 325 330 335 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 340 345 350 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 355 360 365 Arg Glu Pro Met Val Phe Asp Leu Pro Pro Ser Arg Glu Glu Met Thr 370 375 380 Lys Asn Gln Val Ser Leu Trp Cys Met Val Lys Gly Phe Tyr Pro Ser 385 390 395 400 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 405 410 415 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 420 425 430 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 435 440 445 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 450 455 460 Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly Ser Glu Thr Pro Gly Thr 465 470 475 480 Ser Glu Ser Ala Thr Pro Glu Ser Gly Gly Gly Glu Val Gln Leu Val 485 490 495 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 500 505 510 Cys Ala Ala Ser Gly Phe Asn Ile Ser Tyr Ser Ser Ile His Trp Val 515 520 525 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Ser 530 535 540 Tyr Tyr Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 545 550 555 560 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 565 570 575 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala His Tyr 580 585 590 Phe Pro Trp Ala Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 595 600 605 Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser 610 615 620 Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 625 630 635 640 Arg Ala Ser Green Ser Val Ser Ser Ala Val Ala Trp Tyr Green Green Light 645 650 655 Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser Leu Tyr 660 665 670 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe 675 680 685 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 690 695 700 Cys Gln Gln Tyr Tyr Trp Pro Ile Thr Phe Gly Gln Gly Thr Lys Val 705 710 715 720 Glu Ile Lys <210> 15 <211> 2187 <212> DNA <213> Homo sapiens <400> 15 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60 tcctgtgcag cttctggctt caacctctct tcttattcta tgcactgggt gcgtcaggcc 120 ccgggtaagg gcctggaatg ggttgcatat atttcttctt attatggcta tacttattat 180 gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240 ctacaaatga acagcttaag agctgaggac actgccgtct attattgtgc tcgcccggct 300 ccgggtcatt ggggttttga ctactggggt caaggaaccc tggtcaccgt ctcctcgggt 360 ggaggtggca gtgatatcca gatgacccag tccccgagct ccctgtccgc ctctgtgggc 420 gatagggtca ccatcacctg ccgtgccagt cagtccgtgt ccagcgctgt agcctggtat 480 caacagaaac caggaaaagc tccgaagctt ctgatttact cggcatccag cctctactct 540 ggagtccctt ctcgcttctc tggtagccgt tccgggacgg atttcactct gaccatcagc 600 agtctgcagc cggaagactt cgcaacttat tactgtcagc aatggtacta cgctccgatc 660 acgttcggac agggtaccaa ggtggagatc aaactcgagg acaaaactca cacaaaagtt 720 gagcccaaat cttctgataa gacccataat tgcccaccgt gcccagcacc tgaactcctg 780 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 840 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 900 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 960 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1020 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1080 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccaatccgg 1140 gagctgatga ccagcaacca ggtcagcctg agctgcgccg tcaaaggctt ctatcccagc 1200 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1260 cccgtgctgg actccgacgg ctccttcttc ctcgtgagca agctcaccgt ggacaagagc 1320 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1380 tacacgcaga agagcctctc cctgtctccg ggtaaaagcg gcagcgagac tcccgggacc 1440 tcagagtccg ccacacccga aagtggtggc ggagaggttc agctggtgga gtctggcggt 1500 ggcctggtgc agccaggggg ctcactccgt ttgtcctgtg cagcttctgg cttcaacatc 1560 tcttcttatt atatccactg ggtgcgtcag gccccgggta agggcctgga atgggttgca 1620 tctatttatt cttcttatgg ctatacttct tatgccgata gcgtcaaggg ccgtttcact 1680 ataagcgcag acacatccaa aaacacagcc tacctacaaa tgaacagctt aagagctgag 1740 gacactgccg tctattattg tgctcgcact gttcgtggat ccaaaaaacc gtacttctct 1800 ggttgggcta tggactactg gggtcaagga accctggtca ccgtctcctc gggtggaggt 1860 ggcagtgata tccagatgac ccagtccccg agctccctgt ccgcctctgt gggcgatagg 1920 gtcaccatca cctgccgtgc cagtcagtcc gtgtccagcg ctgtagcctg gtatcaacag 1980 aaaccaggaa aagctccgaa gcttctgatt tactcggcat ccagcctcta ctctggagtc 2040 ccttctcgct tctctggtag ccgttccggg acggatttca ctctgaccat cagcagtctg 2100 cagccggaag acttcgcaac ttattactgt cagcaatact cttggggtcc gttcacgttc 2160 ggacagggta ccaaggtgga gatcaaa 2187 <210> 16 <211> 729 <212> PRT ]><213> Homo sapiens <400> 16 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Leu Ser Ser Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Ser Tyr Tyr Gly Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Pro Ala Pro Gly His Trp Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met 115 120 125 Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr 130 135 140 Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr 145 150 155 160 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser 165 170 175 Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly 180 185 190 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 195 200 205 Thr Tyr Tyr Cys Gln Gln Trp Tyr Tyr Ala Pro Ile Thr Phe Gly Gln 210 215 220 Gly Thr Lys Val Glu Ile Lys Leu Glu Asp Lys Thr His Thr Lys Val 225 230 235 240 Glu Pro Lys Ser Ser Asp Lys Thr His Asn Cys Pro Pro Cys Pro Ala 245 250 255 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 260 265 270 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 275 280 285 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 290 295 300 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 305 310 315 320 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 325 330 335 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 340 345 350 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 355 360 365 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ile Arg Glu Leu Met Thr 370 375 380 Ser Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser 385 390 395 400 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 405 410 415 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val 420 425 430 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 435 440 445 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 450 455 460 Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly Ser Glu Thr Pro Gly Thr 465 470 475 480 Ser Glu Ser Ala Thr Pro Glu Ser Gly Gly Gly Glu Val Gln Leu Val 485 490 495 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 500 505 510 Cys Ala Ala Ser Gly Phe Asn Ile Ser Ser Tyr Tyr Ile His Trp Val 515 520 525 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile Tyr Ser 530 535 540 Ser Tyr Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 545 550 555 560 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 565 570 575 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Val Arg 580 585 590 Gly Ser Lys Lys Pro Tyr Phe Ser Gly Trp Ala Met Asp Tyr Trp Gly 595 600 605 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile 610 615 620 Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg 625 630 635 640 Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala 645 650 655 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser 660 665 670 Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg 675 680 685 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp 690 695 700 Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Trp Gly Pro Phe Thr Phe 705 710 715 720 Gly Gln Gly Thr Lys Val Glu Ile Lys 725 <210> 17 <211> 2175 <212> DNA <213> Homo sapiens <400> 17 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60 tcctgtgcag cttctggctt caacatctct tattattata tgcactgggt gcgtcaggcc 120 ccgggtaagg gcctggaatg ggttgcatct atttcttctt attatggctc tacttattat 180 gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240 ctacaaatga acagcttaag agctgaggac actgccgtct attattgtgc tcgctcttgg 300 tgggcttggg cttttgacta ctggggtcaa ggaaccctgg tcaccgtctc ctcgggtgga 360 ggtggcagtg atatccagat gacccagtcc ccgagctccc tgtccgcctc tgtgggcgat 420 agggtcacca tcacctgccg tgccagtcag tccgtgtcca gcgctgtagc ctggtatcaa 480 cagaaaccag gaaaagctcc gaagcttctg atttactcgg catccagcct ctactctgga 540 gtcccttctc gcttctctgg tagccgttcc gggacggatt tcactctgac catcagcagt 600 ctgcagccgg aagacttcgc aacttattac tgtcagcaac attactctgt ttacgcttct 660 ctgatcacgt tcggacaggg taccaaggtg gagatcaaac tcgaggacaa aactcacaca 720 aaagtggagc ccaaaacttc tgataagacc catacttgcc caccgtgccc agcacctgaa 780 ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 840 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 900 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgcgag 960 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 1020 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1080 aaaaccatct ccaaagccaa agggcagccc cgagaaccaa tggtgtttga cctgccccca 1140 tcccgggagg agatgaccaa gaaccaggtc agcctgtggt gcatggtcaa gggcttctat 1200 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1260 acgcctcccg tgctggactc cgacggctcc ttcttcctgt acagcaagct caccgtggac 1320 aagagccgct ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1380 aaccactaca cgcagaagag cctctccctg tctccgggta aaagcggcag cgagactccc 1440 gggacctcag agtccgccac acccgaaagt ggtggcggag aggttcagct ggtggagtct 1500 ggcggtggcc tggtgcagcc agggggctca ctccgtttgt cctgtgcagc ttctggcttc 1560 aacatctctt attcttctat ccactgggtg cgtcaggccc cgggtaaggg cctggaatgg 1620 gttgcatata tttcttctta ttatggctat acttattatg ccgatagcgt caagggccgt 1680 ttcactataa gcgcagacac atccaaaaac acagcctacc tacaaatgaa cagcttaaga 1740 gctgaggaca ctgccgtcta ttattgtgct cgcgctcatt acttcccgtg ggctggtgct 1800 atggactact ggggtcaagg aaccctggtc accgtctcct cgggtggagg tggcagtgat 1860 atccagatga cccagtcccc gagctccctg tccgcctctg tgggcgatag ggtcaccatc 1920 acctgccgtg ccagtcagtc cgtgtccagc gctgtagcct ggtatcaaca gaaaccagga 1980 aaagctccga agcttctgat ttactcggca tccagcctct actctggagt cccttctcgc 2040 ttctctggta gccgttccgg gacggatttc actctgacca tcagcagtct gcagccggaa 2100 gacttcgcaa cttattactg tcagcaatac tactggccga tcacgttcgg acagggtacc 2160 aaggtggaga tcaaa 2175 <210> 18 <211> 725 <212> PRT <213> Homo sapiens <400> 18 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Ser Tyr Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Ser Tyr Tyr Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Trp Trp Ala Trp Ala Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 115 120 125 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 130 135 140 Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln 145 150 155 160 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser 165 170 175 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr 180 185 190 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 195 200 205 Tyr Tyr Cys Gln Gln His Tyr Ser Val Tyr Ala Ser Leu Ile Thr Phe 210 215 220 Gly Gln Gly Thr Lys Val Glu Ile Lys Leu Glu Asp Lys Thr His Thr 225 230 235 240 Lys Val Glu Pro Lys Thr Ser Asp Lys Thr His Thr Cys Pro Pro Cys 245 250 255 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 260 265 270 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 275 280 285 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 290 295 300 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 305 310 315 320 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 325 330 335 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 340 345 350 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 355 360 365 Gln Pro Arg Glu Pro Met Val Phe Asp Leu Pro Pro Ser Arg Glu Glu 370 375 380 Met Thr Lys Asn Gln Val Ser Leu Trp Cys Met Val Lys Gly Phe Tyr 385 390 395 400 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 405 410 415 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 420 425 430 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 435 440 445 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 450 455 460 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly Ser Glu Thr Pro 465 470 475 480 Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Gly Gly Glu Val Gln 485 490 495 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 500 505 510 Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Ser Tyr Ser Ser Ile His 515 520 525 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile 530 535 540 Ser Ser Tyr Tyr Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg 545 550 555 560 Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met 565 570 575 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala 580 585 590 His Tyr Phe Pro Trp Ala Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr 595 600 605 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 610 615 620 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 625 630 635 640 Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln 645 650 655 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser 660 665 670 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr 675 680 685 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 690 695 700 Tyr Tyr Cys Gln Gln Tyr Tyr Trp Pro Ile Thr Phe Gly Gln Gly Thr 705 710 715 720 Lys Val Glu Ile Lys 725 <210> 19 <211> 2193 <212> DNA <213> Homo sapiens <400> 19 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60 tcctgtgcag cttctggctt caacatctct tattattata tgcactgggt gcgtcaggcc 120 ccgggtaagg gcctggaatg ggttgcatct atttcttctt attatggctc tacttattat 180 gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240 ctacaaatga acagcttaag agctgaggac actgccgtct attattgtgc tcgctcttgg 300 tgggcttggg cttttgacta ctggggtcaa ggaaccctgg tcaccgtctc ctcgggtgga 360 ggtggcagtg atatccagat gacccagtcc ccgagctccc tgtccgcctc tgtgggcgat 420 agggtcacca tcacctgccg tgccagtcag tccgtgtcca gcgctgtagc ctggtatcaa 480 cagaaaccag gaaaagctcc gaagcttctg atttactcgg catccagcct ctactctgga 540 gtcccttctc gcttctctgg tagccgttcc gggacggatt tcactctgac catcagcagt 600 ctgcagccgg aagacttcgc aacttattac tgtcagcaac attactctgt ttacgcttct 660 ctgatcacgt tcggacaggg taccaaggtg gagatcaaac tcgaggacaa aactcacaca 720 aaagttgagc ccaaatcttc tgataagacc cataattgcc caccgtgccc agcacctgaa 780 ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 840 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 900 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 960 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 1020 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1080 aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1140 atccgggagc tgatgaccag caaccaggtc agcctgagct gcgccgtcaa aggcttctat 1200 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1260 acgcctcccg tgctggactc cgacggctcc ttcttcctcg tgagcaagct caccgtggac 1320 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1380 aaccactaca cgcagaagag cctctccctg tctccgggta aaagcggcag cgagactccc 1440 gggacctcag agtccgccac acccgaaagt ggtggcggag aggttcagct ggtggagtct 1500 ggcggtggcc tggtgcagcc aggggctca ctccgtttgt cctgtgcagc ttctggcttc 1560 aacatctctt cttattat ccactgggtg cgtcaggccc cgggtaagg cctggaatgg 1620 gttgcatcta tttattcttc ttatggctat acttctttatg ccgatagcgt caagggccgt 1680 ttcactataa gcgcagacac atccaaaaac acagcctacc tacaatgaa cagcttaaga 1740 gctgaggaca ctgccgtcta ttatgtgct cgcactgttc gtggatccaa aaaaccgtac 1800 ttctctggtt gggctatgga ctactggggt caaggaaccc tggtcaccgt ctcctcgggt 1860 ggaggtggca gtgatatcca gatgacccag tccccgagct ccctgtccgc ctctgtgggc 1920 gatagggtca ccatcacctg ccgtgccagt cagtccgtgt ccagcgctgt agcctggtat 1980 caaagaaac caggaaaagc tccgaagctt ctgatttact cggcatccag cctctactct 2040 ggagtccctt ctcgcttctc tggtagccgt tccgggacgg atttcactct gaccatcagc 2100 agtctgcagc cggaagaactt cgcaacttat tactgtcagc aatactcttg gggtccgttc 2160 acgttcggac agggtaccaa ggtggagatc aaa 2193 <210> 20 <211> 731 <212> PRT <213> Homo sapiens <400> 20 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Ser Tyr Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Ser Tyr Tyr Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Trp Trp Ala Trp Ala Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 115 120 125 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 130 135 140 Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln 145 150 155 160 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser 165 170 175 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr 180 185 190 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 195 200 205 Tyr Tyr Cys Gln Gln His Tyr Ser Val Tyr Ala Ser Leu Ile Thr Phe 210 215 220 Gly Gln Gly Thr Lys Val Glu Ile Lys Leu Glu Asp Lys Thr His Thr 225 230 235 240 Lys Val Glu Pro Lys Ser Ser Asp Lys Thr His Asn Cys Pro Pro Cys 245 250 255 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 260 265 270 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 275 280 285 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 290 295 300 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 305 310 315 320 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 325 330 335 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 340 345 350 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 355 360 365 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ile Arg Glu Leu 370 375 380 Met Thr Ser Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr 385 390 395 400 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 405 410 415 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 420 425 430 Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 435 440 445 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 450 455 460 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly Ser Glu Thr Pro 465 470 475 480 Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Gly Gly Glu Val Gln 485 490 495 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 500 505 510 Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Ser Ser Tyr Tyr Ile His 515 520 525 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile 530 535 540 Tyr Ser Ser Tyr Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg 545 550 555 560 Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met 565 570 575 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr 580 585 590 Val Arg Gly Ser Lys Lys Pro Tyr Phe Ser Gly Trp Ala Met Asp Tyr 595 600 605 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 610 615 620 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 625 630 635 640 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala 645 650 655 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 660 665 670 Tyr Ser Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 675 680 685 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 690 695 700 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Trp Gly Pro Phe 705 710 715 720 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 725 730 <210> 21 <211> 2175 <212> DNA <213> Homo sapiens <400> 21 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60 tcctgtgcag cttctggctt caacatctct tattattata tccactgggt gcgtcaggcc 120 ccgggtaagg gcctggaatg ggttgcatct atttattctt cttctagcta tacttattat 180 gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240 ctacaaatga acagcttaag agctgaggac actgccgtct attattgtgc tcgctcttct 300 tacgcttggg ctattgacta ctggggtcaa ggaaccctgg tcaccgtctc ctcgggtgga 360 ggtggcagtg atatccagat gacccagtcc ccgagctccc tgtccgcctc tgtgggcgat 420 agggtcacca tcacctgccg tgccagtcag tccgtgtcca gcgctgtagc ctggtatcaa 480 cagaaaccag gaaaagctcc gaagcttctg atttactcgg catccagcct ctactctgga 540 gtcccttctc gcttctctgg tagccgttcc gggacggatt tcactctgac catcagcagt 600 ctgcagccgg aagacttcgc aacttattac tgtcagcaat ctggttggtg gggtgtttct 660 ctgatcacgt tcggacaggg taccaaggtg gagatcaaac tcgaggacaa aactcacaca 720 aaagtggagc ccaaaacttc tgataagacc catacttgcc caccgtgccc agcacctgaa 780 ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 840 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 900 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgcgag 960 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 1020 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1080 aaaaccatct ccaaagccaa agggcagccc cgagaaccaa tggtgtttga cctgccccca 1140 tcccgggagg agatgaccaa gaaccaggtc agcctgtggt gcatggtcaa gggcttctat 1200 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1260 acgcctcccg tgctggactc cgacggctcc ttcttcctgt acagcaagct caccgtggac 1320 aagagccgct ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1380 aaccactaca cgcagaagag cctctccctg tctccgggta aaagcggcag cgagactccc 1440 gggacctcag agtccgccac acccgaaagt ggtggcggag aggttcagct ggtggagtct 1500 ggcggtggcc tggtgcagcc agggggctca ctccgtttgt cctgtgcagc ttctggcttc 1560 aacatctctt attcttctat ccactgggtg cgtcaggccc cgggtaaggg cctggaatgg 1620 gttgcatata tttcttctta ttatggctat acttattatg ccgatagcgt caagggccgt 1680 ttcactataa gcgcagacac atccaaaaac acagcctacc tacaaatgaa cagcttaaga 1740 gctgaggaca ctgccgtcta ttattgtgct cgcgctcatt acttcccgtg ggctggtgct 1800 atggactact ggggtcaagg aaccctggtc accgtctcct cgggtggagg tggcagtgat 1860 atccagatga cccagtcccc gagctccctg tccgcctctg tgggcgatag ggtcaccatc 1920 acctgccgtg ccagtcagtc cgtgtccagc gctgtagcct ggtatcaaca gaaaccagga 1980 aaagctccga agcttctgat ttactcggca tccagcctct actctggagt cccttctcgc 2040 ttctctggta gccgttccgg gacggatttc actctgacca tcagcagtct gcagccggaa 2100 gacttcgcaa cttattactg tcagcaatac tactggccga tcacgttcgg acagggtacc 2160 aaggtggaga tcaaa 2175 <210> 22 <211> 725 <212> PRT <213> Homo sapiens <400> 22 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Ser Tyr Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Tyr Ser Ser Ser Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Tyr Ala Trp Ala Ile Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 115 120 125 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 130 135 140 Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln 145 150 155 160 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser 165 170 175 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr 180 185 190 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 195 200 205 Tyr Tyr Cys Gln Gln Ser Gly Trp Trp Gly Val Ser Leu Ile Thr Phe 210 215 220 Gly Gln Gly Thr Lys Val Glu Ile Lys Leu Glu Asp Lys Thr His Thr 225 230 235 240 Lys Val Glu Pro Lys Thr Ser Asp Lys Thr His Thr Cys Pro Pro Cys 245 250 255 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 260 265 270 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 275 280 285 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 290 295 300 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 305 310 315 320 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 325 330 335 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 340 345 350 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 355 360 365 Gln Pro Arg Glu Pro Met Val Phe Asp Leu Pro Pro Ser Arg Glu Glu 370 375 380 Met Thr Lys Asn Gln Val Ser Leu Trp Cys Met Val Lys Gly Phe Tyr 385 390 395 400 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 405 410 415 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 420 425 430 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 435 440 445 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 450 455 460 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly Ser Glu Thr Pro 465 470 475 480 Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Gly Gly Glu Val Gln 485 490 495 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 500 505 510 Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Ser Tyr Ser Ser Ile His 515 520 525 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile 530 535 540 Ser Ser Tyr Tyr Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg 545 550 555 560 Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met 565 570 575 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala 580 585 590 His Tyr Phe Pro Trp Ala Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr 595 600 605 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 610 615 620 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 625 630 635 640 Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln 645 650 655 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser 660 665 670 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr 675 680 685 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 690 695 700 Tyr Tyr Cys Gln Gln Tyr Tyr Trp Pro Ile Thr Phe Gly Gln Gly Thr 705 710 715 720 Lys Val Glu Ile Lys 725 <210> 23 <211> 2193 <212> DNA <213> Homo sapiens <400> 23 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60 tcctgtgcag cttctggctt caacatctct tattattata tccactgggt gcgtcaggcc 120 ccgggtaagg gcctggaatg ggttgcatct atttattctt cttctagcta tacttattat 180 gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240 ctacaaatga acagcttaag agctgaggac actgccgtct attattgtgc tcgctcttct 300 tacgcttggg ctattgacta ctggggtcaa ggaaccctgg tcaccgtctc ctcgggtgga 360 ggtggcagtg atatccagat gacccagtcc ccgagctccc tgtccgcctc tgtgggcgat 420 agggtcacca tcacctgccg tgccagtcag tccgtgtcca gcgctgtagc ctggtatcaa 480 cagaaaccag gaaaagctcc gaagcttctg atttactcgg catccagcct ctactctgga 540 gtcccttctc gcttctctgg tagccgttcc gggacggatt tcactctgac catcagcagt 600 ctgcagccgg aagacttcgc aacttattac tgtcagcaat ctggttggtg gggtgtttct 660 ctgatcacgt tcggacaggg taccaaggtg gagatcaaac tcgaggacaa aactcacaca 720 aaagttgagc ccaaatcttc tgataagacc cataattgcc caccgtgccc agcacctgaa 780 ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 840 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 900 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 960 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 1020 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1080 aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1140 atccgggagc tgatgaccag caaccaggtc agcctgagct gcgccgtcaa aggcttctat 1200 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1260 acgcctcccg tgctggactc cgacggctcc ttcttcctcg tgagcaagct caccgtggac 1320 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1380 aaccactaca cgcagaagag cctctccctg tctccgggta aaagcggcag cgagactccc 1440 gggacctcag agtccgccac acccgaaagt ggtggcggag aggttcagct ggtggagtct 1500 ggcggtggcc tggtgcagcc agggggctca ctccgtttgt cctgtgcagc ttctggcttc 1560 aacatctctt cttattatat ccactgggtg cgtcaggccc cgggtaaggg cctggaatgg 1620 gttgcatcta tttattcttc ttatggctat acttcttatg ccgatagcgt caagggccgt 1680 ttcactataa gcgcagacac atccaaaaac acagcctacc tacaaatgaa cagcttaaga 1740 gctgaggaca ctgccgtcta ttattgtgct cgcactgttc gtggatccaa aaaaccgtac 1800 ttctctggtt gggctatgga ctactggggt caaggaaccc tggtcaccgt ctcctcgggt 1860 ggaggtggca gtgatatcca gatgacccag tccccgagct ccctgtccgc ctctgtgggc 1920 gatagggtca ccatcacctg ccgtgccagt cagtccgtgt ccagcgctgt agcctggtat 1980 caacagaaac caggaaaagc tccgaagctt ctgatttact cggcatccag cctctactct 2040 ggagtccctt ctcgcttctc tggtagccgt tccgggacgg atttcactct gaccatcagc 2100 agtctgcagc cggaagactt cgcaacttat tactgtcagc aatactcttg gggtccgttc 2160 acgttcggac agggtaccaa ggtggagatc aaa 2193 <210> 24 <211> 731 <212> PRT <213> Homo sapiens <400> 24 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Ser Tyr Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Tyr Ser Ser Ser Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Tyr Ala Trp Ala Ile Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 115 120 125 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 130 135 140 Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln 145 150 155 160 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser 165 170 175 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr 180 185 190 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 195 200 205 Tyr Tyr Cys Gln Gln Ser Gly Trp Trp Gly Val Ser Leu Ile Thr Phe 210 215 220 Gly Gln Gly Thr Lys Val Glu Ile Lys Leu Glu Asp Lys Thr His Thr 225 230 235 240 Lys Val Glu Pro Lys Ser Ser Asp Lys Thr His Asn Cys Pro Pro Cys 245 250 255 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 260 265 270 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 275 280 285 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 290 295 300 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 305 310 315 320 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 325 330 335 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 340 345 350 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 355 360 365 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ile Arg Glu Leu 370 375 380 Met Thr Ser Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr 385 390 395 400 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 405 410 415 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 420 425 430 Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 435 440 445 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 450 455 460 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly Ser Glu Thr Pro 465 470 475 480 Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Gly Gly Glu Val Gln 485 490 495 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 500 505 510 Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Ser Ser Tyr Tyr Ile His 515 520 525 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile 530 535 540 Tyr Ser Ser Tyr Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg 545 550 555 560 Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met 565 570 575 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr 580 585 590 Val Arg Gly Ser Lys Lys Pro Tyr Phe Ser Gly Trp Ala Met Asp Tyr 595 600 605 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 610 615 620 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 625 630 635 640 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala 645 650 655 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 660 665 670 Tyr Ser Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 675 680 685 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 690 695 700 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Trp Gly Pro Phe 705 710 715 720 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 725 730 <210> 25 <211> 2169 <212> DNA <213> Homo sapiens <400> 25 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60 tcctgtgcag cttctggctt caacatctct tattattata tccactgggt gcgtcaggcc 120 ccgggtaagg gcctggaatg ggttgcatct atttatcctt cttctggcta tacttattat 180[[ID=4'2]] gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240 ctacaaatga acagcttaag agctgaggac actgccgtct attattgtgc tcgctcttct 300 ttctactggg ctatggacta ctggggtcaa ggaaccctgg tcaccgtctc ctcgggtgga 360 ggtggcagtg atatccagat gacccagtcc ccgagctccc tgtccgcctc tgtgggcgat 420 agggtcacca tcacctgccg tgccagtcag tccgtgtcca gcgctgtagc ctggtatcaa 480 cagaaaccag gaaaagctcc gaagcttctg atttactcgg catccagcct ctactctgga 540 gtcccttctc gcttctctgg tagccgttcc gggacggatt tcactctgac catcagcagt 600 ctgcagccgg aagacttcgc aacttattac tgtcagcaat cttacgctgc ttacctgttc 660 acgttcggac agggtaccaa ggtggagatc aaactcgagg acaaaactca cacaaaagtg 720 gagcccaaaa cttctgataa gacccatact tgcccaccgt gcccagcacc tgaactcctg 780 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 840 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 900 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg cgaggagcag 960 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1020 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1080 atctccaaag ccaaagggca gccccgagaa ccaatggtgt ttgacctgcc cccatcccgg 1140 gaggagatga ccaagaacca ggtcagcctg tggtgcatgg tcaagggctt ctatcccagc 1200 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1260 cccgtgctgg actccgacgg ctccttcttc ctgtacagca agctcaccgt ggacaagagc 1320 cgctggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1380 tacacgcaga agagcctctc cctgtctccg ggtaaaagcg gcagcgagac tcccgggacc 1440 tcagagtccg ccacacccga aagtggtggc ggagaggttc agctggtgga gtctggcggt 1500 ggcctggtgc agccaggggg ctcactccgt ttgtcctgtg cagcttctgg cttcaacatc 1560 tcttattctt ctatccactg ggtgcgtcag gccccgggta agggcctgga atgggttgca 1620 tatatttctt cttattatgg ctatacttat tatgccgata gcgtcaaggg ccgtttcact 1680 ataagcgcag acacatccaa aaacacagcc tacctacaaa tgaacagctt aagagctgag 1740 gacactgccg tctattattg tgctcgcgct cattacttcc cgtgggctgg tgctatggac 1800 tactggggtc aaggaaccct ggtcaccgtc tcctcgggtg gaggtggcag tgatatccag 1860 atgacccagt ccccgagctc cctgtccgcc tctgtgggcg atagggtcac catcacctgc 1920 cgtgccagtc agtccgtgtc cagcgctgta gcctggtatc aacagaaacc aggaaaagct 1980 ccgaagcttc tgatttactc ggcatccagc ctctactctg gagtcccttc tcgcttctct 2040 ggtagccgtt ccgggacgga tttcactctg accatcagca gtctgcagcc ggaagacttc 2100 gcaacttatt actgtcagca atactactgg ccgatcacgt tcggacaggg taccaaggtg 2160 gagatcaaa 2169 <210> 26 <211> 723 <212> PRT <213> Homo sapiens <400> 26 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15<Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Ser Tyr Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Tyr Pro Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Phe Tyr Trp Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 115 120 125 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 130 135 140 Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln 145 150 155 160 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser 165 170 175 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr 180 185 190 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 195 200 205 Tyr Tyr Cys Gln Gln Ser Tyr Ala Ala Tyr Leu Phe Thr Phe Gly Gln 210 215 220 Gly Thr Lys Val Glu Ile Lys Leu Glu Asp Lys Thr His Thr Lys Val 225 230 235 240 Glu Pro Lys Thr Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 245 250 255 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 260 265 270 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 275 280 285 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 290 295 300 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 305 310 315 320 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 325 330 335 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 340 345 350 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 355 360 365 Arg Glu Pro Met Val Phe Asp Leu Pro Pro Ser Arg Glu Glu Met Thr 370 375 380 Lys Asn Gln Val Ser Leu Trp Cys Met Val Lys Gly Phe Tyr Pro Ser 385 390 395 400 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 405 410 415 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 420 425 430 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 435 440 445 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 450 455 460 Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly Ser Glu Thr Pro Gly Thr 465 470 475 480 Ser Glu Ser Ala Thr Pro Glu Ser Gly Gly Gly Glu Val Gln Leu Val 485 490 495 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 500 505 510 Cys Ala Ala Ser Gly Phe Asn Ile Ser Tyr Ser Ser Ile His Trp Val 515 520 525 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Ser 530 535 540 Tyr Tyr Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 545 550 555 560 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 565 570 575 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala His Tyr 580 585 590 Phe Pro Trp Ala Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val 595 600 605 Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser 610 615 620 Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 625 630 635 640 Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys 645 650 655 Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser Leu Tyr 660 665 670 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe 675 680 685 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 690 695 700 Cys Gln Gln Tyr Tyr Trp Pro Ile Thr Phe Gly Gln Gly Thr Lys Val 705 710 715 720 Glu Ile Lys <210> 27 <211> 2187 <212> DNA <213> Homo sapiens <400> 27 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60 tcctgtgcag cttctggctt caacatctct tattattata tccactgggt gcgtcaggcc 120 ccgggtaagg gcctggaatg ggttgcatct atttatcctt cttctggcta tacttattat 180 gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240 ctacaaatga acagcttaag agctgaggac actgccgtct attattgtgc tcgctcttct 300 ttctactggg ctatggacta ctggggtcaa ggaaccctgg tcaccgtctc ctcgggtgga 360 ggtggcagtg atatccagat gacccagtcc ccgagctccc tgtccgcctc tgtgggcgat 420 agggtcacca tcacctgccg tgccagtcag tccgtgtcca gcgctgtagc ctggtatcaa 480 cagaaaccag gaaaagctcc gaagcttctg atttactcgg catccagcct ctactctgga 540 gtcccttctc gcttctctgg tagccgttcc gggacggatt tcactctgac catcagcagt 600 ctgcagccgg aagacttcgc aacttattac tgtcagcaat cttacgctgc ttacctgttc 660 acgttcggac agggtaccaa ggtggagatc aaactcgagg acaaaactca cacaaaagtt 720 gagcccaaat cttctgataa gacccataat tgcccaccgt gcccagcacc tgaactcctg 780 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 840 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 900 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 960 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1020 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1080 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccaatccgg 1140 gagctgatga ccagcaacca ggtcagcctg agctgcgccg tcaaaggctt ctatcccagc 1200 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1260 cccgtgctgg actccgacgg ctccttcttc ctcgtgagca agctcaccgt ggacaagagc 1320 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1380 tacacgcaga agagcctctc cctgtctccg ggtaaaagcg gcagcgagac tcccgggacc 1440 tcagagtccg ccacacccga aagtggtggc ggagaggttc agctggtgga gtctggcggt 1500 ggcctggtgc agccaggggg ctcactccgt ttgtcctgtg cagcttctgg cttcaacatc 1560 tcttcttatt atatccactg ggtgcgtcag gccccgggta agggcctgga atgggttgca 1620 tctatttatt cttcttatgg ctatacttct tatgccgata gcgtcaaggg ccgtttcact 1680 ataagcgcag acacatccaa aaacacagcc tacctacaaa tgaacagctt aagagctgag 1740 gacactgccg tctattattg tgctcgcact gttcgtggat ccaaaaaacc gtacttctct 1800 ggttgggcta tggactactg gggtcaagga accctggtca ccgtctcctc gggtggaggt 1860 ggcagtgata tccagatgac ccagtccccg agctccctgt ccgcctctgt gggcgatagg 1920 gtcaccatca cctgccgtgc cagtcagtcc gtgtccagcg ctgtagcctg gtatcaacag 1980 aaaccaggaa aagctccgaa gcttctgatt tactcggcat ccagcctcta ctctggagtc 2040 ccttctcgct tctctggtag ccgttccggg acggatttca ctctgaccat cagcagtctg 2100 cagccggaag acttcgcaac ttattactgt cagcaatact cttggggtcc gttcacgttc 2160 ggacagggta ccaaggtgga gatcaaa 2187 <210> 28 <211> 729 <212> PRT <213> Homo sapiens <400> 28 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Ser Tyr Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Tyr Pro Ser Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Phe Tyr Trp Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 115 120 125 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 130 135 140 Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln 145 150 155 160 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser 165 170 175 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr 180 185 190 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 195 200 205 Tyr Tyr Cys Gln Gln Ser Tyr Ala Ala Tyr Leu Phe Thr Phe Gly Gln 210 215 220 Gly Thr Lys Val Glu Ile Lys Leu Glu Asp Lys Thr His Thr Lys Val 225 230 235 240 Glu Pro Lys Ser Ser Asp Lys Thr His Asn Cys Pro Pro Cys Pro Ala 245 250 255 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 260 265 270 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 275 280 285 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 290 295 300 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 305 310 315 320 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 325 330 335 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 340 345 350 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 355 360 365 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ile Arg Glu Leu Met Thr 370 375 380 Ser Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser 385 390 395 400 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 405 410 415 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val 420 425 430 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 435 440 445 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 450 455 460 Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly Ser Glu Thr Pro Gly Thr 465 470 475 480 Ser Glu Ser Ala Thr Pro Glu Ser Gly Gly Gly Glu Val Gln Leu Val 485 490 495 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 500 505 510 Cys Ala Ala Ser Gly Phe Asn Ile Ser Ser Tyr Tyr Ile His Trp Val 515 520 525 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile Tyr Ser 530 535 540 Ser Tyr Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 545 550 555 560 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 565 570 575 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Val Arg 580 585 590 Gly Ser Lys Lys Pro Tyr Phe Ser Gly Trp Ala Met Asp Tyr Trp Gly 595 600 605 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile 610 615 620 Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg 625 630 635 640 Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala 645 650 655 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser 660 665 670 Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg 675 680 685 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp 690 695 700 Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Trp Gly Pro Phe Thr Phe 705 710 715 720 Gly Gln Gly Thr Lys Val Glu Ile Lys 725 <210> 29 <211> 2163 <212> DNA <213> Homo sapiens <400> 29 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60 tcctgtgcag cttctggctt caacatctct tattattcta tgcactgggt gcgtcaggcc 120 ccgggtaagg gcctggaatg ggttgcatct atttcttctt attatagctc tacttcttat 180 [[ID=3,4]]gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240 ctacaaatga acagcttaag agctgaggac actgccgtct attattgtgc tcgcttctgg 300 tacccgggta tggactactg gggtcaagga accctggtca ccgtctcctc gggtggaggt 360 ggcagtgata tccagatgac ccagtccccg agctccctgt ccgcctctgt gggcgatagg 420 gtcaccatca cctgccgtgc cagtcagtcc gtgtccagcg ctgtagcctg gtatcaacag 480 aaaccaggaa aagctccgaa gcttctgatt tactcggcat ccagcctcta ctctggagtc 540 ccttctcgct tctctggtag ccgttccggg acggatttca ctctgaccat cagcagtctg 600 cagccggaag acttcgcaac ttattactgt cagcaacatt ggtcttaccc gatcacgttc 660 ggacagggta ccaaggtgga gatcaaactc gaggacaaaa ctcacacaaa agtggagccc 720 aaaacttctg ataagaccca tacttgccca ccgtgcccag cacctgaact cctgggggga 780 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 840 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 900 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgcgagga gcagtacaac 960 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 1020 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1080 aaagccaaag ggcagccccg agaaccaatg gtgtttgacc tgcccccatc ccgggaggag 1140 atgaccaaga accaggtcag cctgtggtgc atggtcaagg gcttctatcc cagcgacatc 1200 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1260 ctggactccg acggctcctt cttcctgtac agcaagctca ccgtggacaa gagccgctgg 1320 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1380 cagaagagcc tctccctgtc tccgggtaaa agcggcagcg agactcccgg gacctcagag 1440 tccgccacac ccgaaagtgg tggcggagag gttcagctgg tggagtctgg cggtggcctg 1500 gtgcagccag ggggctcact ccgtttgtcc tgtgcagctt ctggcttcaa catctcttat 1560 tcttctatcc actgggtgcg tcaggccccg ggtaagggcc tggaatgggt tgcatatatt 1620 tcttcttatt atggctatac ttattatgcc gatagcgtca agggccgttt cactataagc 1680 gcagacacat ccaaaaacac agcctaccta caaatgaaca gcttaagagc tgaggacact 1740 gccgtctatt attgtgctcg cgctcattac ttcccgtggg ctggtgctat ggactactgg 1800 ggtcaaggaa ccctggtcac cgtctcctcg ggtggaggtg gcagtgatat ccagatgacc 1860 cagtccccga gctccctgtc cgcctctgtg ggcgataggg tcaccatcac ctgccgtgcc 1920 agtcagtccg tgtccagcgc tgtagcctgg tatcaacaga aaccaggaaa agctccgaag 1980 cttctgattt actcggcatc cagcctctac tctggagtcc cttctcgctt ctctggtagc 2040 cgttccggga cggatttcac tctgaccatc agcagtctgc agccggaaga cttcgcaact 2100 tattactgtc agcaatacta ctggccgatc acgttcggac agggtaccaa ggtggagatc 2160 aaa 2163 <210> 30 <211> 721 <212> PRTs s<213> Homo sapiens <400> 30 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Ser Tyr Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Ser Tyr Tyr Ser Ser Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Trp Tyr Pro Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln 115 120 125 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 130 135 140 Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln Gln 145 150 155 160 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser Leu 165 170 175 Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp 180 185 190 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 195 200 205 Tyr Cys Gln Gln His Trp Ser Tyr Pro Ile Thr Phe Gly Gln Gly Thr 210 215 220 Lys Val Glu Ile Lys Leu Glu Asp Lys Thr His Thr Lys Val Glu Pro 225 230 235 240 Lys Thr Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 Pro Met Val Phe Asp Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 370 375 380 Gln Val Ser Leu Trp Cys Met Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu 465 470 475 480 Ser Ala Thr Pro Glu Ser Gly Gly Gly Glu Val Gln Leu Val Glu Ser 485 490 495 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 500 505 510 Ala Ser Gly Phe Asn Ile Ser Tyr Ser Ser Ile His Trp Val Arg Gln 515 520 525 Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Ser Tyr Tyr 530 535 540 Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 545 550 555 560 Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg 565 570 575 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala His Tyr Phe Pro 580 585 590 Trp Ala Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 595 600 605 Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser 610 615 620 Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala 625 630 635 640 Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly 645 650 655 Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser Leu Tyr Ser Gly 660 665 670 Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu 675 680 685 Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln 690 695 700 Gln Tyr Tyr Trp Pro Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 705 710 715 720 Lys <210> 31 <211> 2181 <212> DNA <213> Homo sapiens <400> 31 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60 tcctgtgcag cttctggctt caacatctct tattattcta tgcactgggt gcgtcaggcc 120 ccgggtaagg gcctggaatg ggttgcatct atttcttctt attatagctc tacttcttat 180 gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240 ctacaaatga acagcttaag agctgaggac actgccgtct attattgtgc tcgcttctgg 300 tacccgggta tggactactg gggtcaagga accctggtca ccgtctcctc gggtggaggt 360 ggcagtgata tccagatgac ccagtccccg agctccctgt ccgcctctgt gggcgatagg 420 gtcaccatca cctgccgtgc cagtcagtcc gtgtccagcg ctgtagcctg gtatcaacag 480 aaaccaggaa aagctccgaa gcttctgatt tactcggcat ccagcctcta ctctggagtc 540 ccttctcgct tctctggtag ccgttccggg acggatttca ctctgaccat cagcagtctg 600 cagccggaag acttcgcaac ttattactgt cagcaacatt ggtcttaccc gatcacgttc 660 ggacagggta ccaaggtgga gatcaaactc gaggacaaaa ctcacacaaa agttgagccc 720 aaatcttctg ataagaccca taattgccca ccgtgcccag cacctgaact cctgggggga 780 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 840 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 900 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 960 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 1020 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1080 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccaat ccgggagctg 1140 atgaccagca accaggtcag cctgagctgc gccgtcaaag gcttctatcc cagcgacatc 1200 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1260 ctggactccg acggctcctt cttcctcgtg agcaagctca ccgtggacaa gagcaggtgg 1320 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1380 cagaagagcc tctccctgtc tccgggtaaa agcggcagcg agactcccgg gacctcagag 1440 tccgccacac ccgaaagtgg tggcggagag gttcagctgg tggagtctgg cggtggcctg 1500 gtgcagccag ggggctcact ccgtttgtcc tgtgcagctt ctggcttcaa catctcttct 1560 tattatatcc actgggtgcg tcaggccccg ggtaagggcc tggaatgggt tgcatctatt 1620 tattcttctt atggctatac ttcttatgcc gatagcgtca agggccgttt cactataagc 1680 gcagacacat ccaaaaacac agcctaccta caaatgaaca gcttaagagc tgaggacact 1740 gccgtctatt attgtgctcg cactgttcgt ggatccaaaa aaccgtactt ctctggttgg 1800 gctatggact actggggtca aggaaccctg gtcaccgtct cctcgggtgg aggtggcagt 1860 gatatccaga tgacccagtc cccgagctcc ctgtccgcct ctgtgggcga tagggtcacc 1920 atcacctgcc gtgccagtca gtccgtgtcc agcgctgtag cctggtatca acagaaacca 1980 ggaaaagctc cgaagcttct gatttactcg gcatccagcc tctactctgg agtcccttct 2040 cgcttctctg gtagccgttc cgggacggat ttcactctga ccatcagcag tctgcagccg 2100 gaagacttcg caacttatta ctgtcagcaa tactcttggg gtccgttcac gttcggacag 2160 ggtaccaagg tggagatcaa a 2181 <210> 32 <211> 727 <212> PRT <213> Homo sapiens <400> 32 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Ser Tyr Tyr 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Ser Tyr Tyr Ser Ser Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Trp Tyr Pro Gly Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln 115 120 125 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 130 135 140 Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln Gln 145 150 155 160 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser Leu 165 170 175 Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp 180 185 190 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 195 200 205 Tyr Cys Gln Gln His Trp Ser Tyr Pro Ile Thr Phe Gly Gln Gly Thr 210 215 220 Lys Val Glu Ile Lys Leu Glu Asp Lys Thr His Thr Lys Val Glu Pro 225 230 235 240 Lys Ser Ser Asp Lys Thr His Asn Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ile Arg Glu Leu Met Thr Ser Asn 370 375 380 Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu 465 470 475 480 Ser Ala Thr Pro Glu Ser Gly Gly Gly Glu Val Gln Leu Val Glu Ser 485 490 495 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 500 505 510 Ala Ser Gly Phe Asn Ile Ser Ser Tyr Tyr Ile His Trp Val Arg Gln 515 520 525 Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile Tyr Ser Ser Tyr 530 535 540 Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 545 550 555 560 Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg 565 570 575 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Val Arg Gly Ser 580 585 590 Lys Lys Pro Tyr Phe Ser Gly Trp Ala Met Asp Tyr Trp Gly Gln Gly 595 600 605 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met 610 615 620 Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr 625 630 635 640 Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr 645 650 655 Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser 660 665 670 Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly 675 680 685 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 690 695 700 Thr Tyr Tyr Cys Gln Gln Tyr Ser Trp Gly Pro Phe Thr Phe Gly Gln 705 710 715 720 Gly Thr Lys Val Glu Ile Lys 725 <210> 33 <211> 2166 <212> DNA <213> Homo sapiens <400> 33 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60 tcctgtgcag cttctggctt caacctctct tattattata tgcactgggt gcgtcaggcc 120 ccgggtaagg gcctggaatg ggttgcatct atttattctt attctggcta tacttattat 180 gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240 ctacaaatga acagcttaag agctgaggac actgccgtct attattgtgc tcgctcttct 300 ttcgcttggg cttttgacta ctggggtcaa ggaaccctgg tcaccgtctc ctcgggtgga 360 ggtggcagtg atatccagat gacccagtcc ccgagctccc tgtccgcctc tgtgggcgat 420 agggtcacca tcacctgccg tgccagtcag tccgtgtcca gcgctgtagc ctggtatcaa 480 cagaaaccag gaaaagctcc gaagcttctg atttactcgg catccagcct ctactctgga 540 gtcccttctc gcttctctgg tagccgttcc gggacggatt tcactctgac catcagcagt 600 ctgcagccgg aagacttcgc aacttattac tgtcagcaag gtggttgggg tccgttcacg 660 ttcggacagg gtaccaaggt ggagatcaaa ctcgaggaca aaactcacac aaaagtggag 720 cccaaaactt ctgataagac ccatacttgc ccaccgtgcc cagcacctga actcctgggg 780 ggaccgtcag tcttctctctt ccccccaaaaa cccaaggaca ccctcatgat ctcccggacc 840 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcgcga ggagcagtac 960 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020 aaggagtca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaccatc 1080 tccaaagcca aagggcagcc ccgagaacca atggtgtttg acctgccccc atcccgggag 1140 gagatgacca agaaccaggt cagcctgtgg tgcatggtca agggcttcta tcccagcgac 1200 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1260 gtgctggact ccgacggctc cttcttcctg tacagcaagc tcaccgtgga caagagccgc 1320 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1380 acgcagaaga gcctctccct gtctccgggt aaaagcggca gcgagactcc cgggacctca 1440 gagtccgcca cacccgaaag tggtggcgga gaggttcagc tggtggagtc tggcggtggc 1500 ctggtgcagc cagggggctc actccgtttg tcctgtgcag cttctggctt caacatctct 1560 tattcttcta tccactgggt gcgtcaggcc ccgggtaagg gcctggaatg ggttgcatat 1620 atttcttctt attatgcta tacttattat gccgatagcg tcaagggccg tttcactata 1680 agcgcagaca catccaaaaa cacagcctac ctacaaatga acagcttaag agctgaggac 1740 actgccgtct attattgtgc tcgcgctcat tacttcccgt gggctggtgc tatggactac 1800 tggggtcaag gaaccctggt caccgtctcc tcgggtggag gtggcagtga tatccagatg 1860 acccagtccc cgagctccct gtccgcctct gtgggcgata gggtcaccat cacctgccgt 1920 gccagtcagt ccgtgtccag cgctgtagcc tggtatcaac agaaaccagg aaaagctccg 1980 aagcttctga tttactcggc atccagcctc tactctggag tcccttctcg cttctctggt 2040 agccgttccg ggacggattt cactctgacc atcagcagtc tgcagccgga agacttcgca 2100 acttattact gtcagcaata ctactggccg atcacgttcg gacagggtac caaggtggag 2160 atcaaa 2166 <210> 34 <211> 722 <212> PRT <213> Homo sapiens<0003已排版的文本148><400> 34 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Leu Ser Tyr Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Tyr Ser Tyr Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 已排版的文本>Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 7已排版的文本5 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Phe Ala Trp Ala Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 115 120 125 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 130 135 140 Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln 145 150 155 160 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser 165 170 175 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr 180 185 190 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 195 200 205 Tyr Tyr Cys Gln Gln Gly Gly Trp Gly Pro Phe Thr Phe Gly Gln Gly 210 215 220 Thr Lys Val Glu Ile Lys Leu Glu Asp Lys Thr His Thr Lys Val Glu 225 230 235 240 Pro Lys Thr Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Met Val Phe Asp Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 370 375 380 Asn Gln Val Ser Leu Trp Cys Met Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys Ser Gly Ser Glu Thr Pro Gly Thr Ser 465 470 475 480 Glu Ser Ala Thr Pro Glu Ser Gly Gly Gly Glu Val Gln Leu Val Glu 485 490 495 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 500 505 510 Ala Ala Ser Gly Phe Asn Ile Ser Tyr Ser Ser Ile His Trp Val Arg 515 520 525 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Ser Tyr 530 535 540 Tyr Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 545 550 555 560 Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu 565 570 575 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala His Tyr Phe 580 585 590 Pro Trp Ala Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 595 600 605 Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 610 615 620 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg 625 630 635 640 Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro 645 650 655 Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser Leu Tyr Ser 660 665 670 Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr 675 680 685 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 690 695 700 Gln Gln Tyr Tyr Trp Pro Ile Thr Phe Gly Gln Gly Thr Lys Val Glu 705 710 715 720 Ile Lys <210> 35 <211> 2184 <212> DNA <213> Homo sapiens <400> 35 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60 tcctgtgcag cttctggctt caacctctct tattattata tgcactgggt gcgtcaggcc 120 ccgggtaagg gcctggaatg ggttgcatct atttattctt attctggcta tacttattat 180 gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240 ctacaaatga acagcttaag agctgaggac actgccgtct attattgtgc tcgctcttct 300 ttcgcttggg cttttgacta ctggggtcaa ggaaccctgg tcaccgtctc ctcgggtgga 360 ggtggcagtg atatccagat gacccagtcc ccgagctccc tgtccgcctc tgtgggcgat 420 agggtcacca tcacctgccg tgccagtcag tccgtgtcca gcgctgtagc ctggtatcaa 480 cagaaaccag gaaaagctcc gaagcttctg atttactcgg catccagcct ctactctgga 540 gtcccttctc gcttctctgg tagccgttcc gggacggatt tcactctgac catcagcagt 600 ctgcagccgg aagacttcgc aacttattac tgtcagcaag gtggttgggg tccgttcacg 660 ttcggacagg gtaccaaggt ggagatcaaa ctcgaggaca aaactcacac aaaagttgag 720 cccaaatctt ctgataagac ccataattgc ccaccgtgcc cagcacctga actcctgggg 780 ggaccgtcag tcttctctctt ccccccaaaaa cccaaggaca ccctcatgat ctcccggacc 840 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 900 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 960 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1020 aaggagtca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaccatc 1080 tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc aatccgggag 1140 ctgatgacca gcaaccaggt cagcctgagc tgcgccgtca aaggcttcta tcccagcgac 1200 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1260 gtgctggact ccgacggctc cttcttcctc gtgagcaagc tcaccgtgga caagagcagg 1320 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1380 acgcagaaga gcctctccct gtctccgggt aaaagcggca gcgagactcc cgggacctca 1440 gagtccgcca cacccgaaag tggtggcgga gaggttcagc tggtggagtc tggcggtggc 1500 ctggtgcagc cagggggctc actccgtttg tcctgtgcag cttctggctt caacatctct 1560 tcttattata tccactgggt gcgtcaggcc ccgggtaagg gcctggaatg ggttgcatct 1620 atttattctt cttatggcta tacttcttat gccgatagcg tcaagggccg tttcactata 1680 agcgcagaca catccaaaaa cacagcctac ctacaaatga acagcttaag agctgaggac 1740 actgccgtct attattgtgc tcgcactgtt cgtggatcca aaaaaccgta cttctctggt 1800 tgggctatgg actactgggg tcaaggaacc ctggtcaccg tctcctcggg tggaggtggc 1860 agtgatatcc agatgaccca gtccccgagc tccctgtccg cctctgtggg cgatagggtc 1920 accatcacct gccgtgccag tcagtccgtg tccagcgctg tagcctggta tcaacagaaa 1980 ccaggaaaag ctccgaagct tctgatttac tcggcatcca gcctctactc tggagtccct 2040 tctcgcttct ctggtagccg ttccgggacg gatttcactc tgaccatcag cagtctgcag 2100 ccggaagact tcgcaactta ttactgtcag caatactctt ggggtccgtt cacgttcgga 2160 cagggtacca aggtggagat caaa 2184 <210> 36 <211> 728 <212> PRT <213> Homo sapiens <400> 36 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Leu Ser Tyr Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Tyr Ser Tyr Ser Gly Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Phe Ala Trp Ala Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr 115 120 125 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 130 135 140 Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln 145 150 155 160 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser 165 170 175 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr 180 185 190 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr 195 200 205 Tyr Tyr Cys Gln Gln Gly Gly Trp Gly Pro Phe Thr Phe Gly Gln Gly 210 215 220 Thr Lys Val Glu Ile Lys Leu Glu Asp Lys Thr His Thr Lys Val Glu 225 230 235 240 Pro Lys Ser Ser Asp Lys Thr His Asn Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 355 360 365 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ile Arg Glu Leu Met Thr Ser 370 375 380 Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys Ser Gly Ser Glu Thr Pro Gly Thr Ser 465 470 475 480 Glu Ser Ala Thr Pro Glu Ser Gly Gly Gly Glu Val Gln Leu Val Glu 485 490 495 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 500 505 510 Ala Ala Ser Gly Phe Asn Ile Ser Ser Tyr Tyr Ile His Trp Val Arg 515 520 525 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile Tyr Ser Ser 530 535 540 Tyr Gly Tyr Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 545 550 555 560 Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu 565 570 575 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Val Arg Gly 580 585 590 Ser Lys Lys Pro Tyr Phe Ser Gly Trp Ala Met Asp Tyr Trp Gly Gln 595 600 605 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln 610 615 620 Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val 625 630 635 640 Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp 645 650 655 Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala 660 665 670 Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser 675 680 685 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 690 695 700 Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Trp Gly Pro Phe Thr Phe Gly [[ID=3~1]]705 710 715 720 Gln Gly Thr Lys Val Glu Ile Lys 725 <210> 37 <211> 2157 <212> DNA <X13> Homo sapiens <400> 37 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60 tcctgtgcag cttctggctt caacatctct tattcttcta tccactgggt gcgtcaggcc 120 ccgggtaagg gcctggaatg ggttgcatct atttatcctt cttatagctc tacttattat 180 gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240 ctacaaatga acagcttaag agctgaggac actgccgtct attattgtgc tcgctactac 300 gctatggact actggggtca aggaaccctg gtcaccgtct cctcgggtgg aggtggcagt 360 gatatccaga tgacccagtc cccgagctcc ctgtccgcct ctgtgggcga tagggtcacc 420 atcacctgcc gtgccagtca gtccgtgtcc agcgctgtag cctggtatca acagaaacca 480 ggaaaagctc cgaagcttct gatttactcg gcatccagcc tctactctgg agtcccttct 540 cgcttctctg gtagccgttc cgggacggat ttcactctga ccatcagcag tctgcagccg 600 gaagacttcg caacttatta ctgtcagcaa gctttctact acccgatcac gttcggacag 660 ggtaccaagg tggagatcaa actcgaggac aaaactcaca caaaagtgga gcccaaaact 720 tctgataaga cccatacttg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 780 gtttctctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 840 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 900 gacggcgtgg aggtgcataa tgccaagaca aagccgcgcgc aggagcagta caacagcacg 960 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 1020 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1080 aaagggcagc cccgagaacc aatggtgttt gacctgcccc catcccggga ggagatgacc 1140 aagaaccagg tcagcctgtg gtgcatggtc aagggcttct atcccagcga catcgccgtg 1200 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1260 tccgacggct ccttcttcct gtacagcaag ctcaccgtgg acaagagccg ctggcagcag 1320 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc aaaccacta cacgcagaag 1380 agcctctccc tgtctccggg taaaagcggc agcgagactc ccgggacctc agagtccgcc 1440 acacccgaaa gtggtggcgg agaggttcag ctggtggagt ctggcggtgg cctggtgcag 1500 ccagggggct cactccgttt gtcctgtgca gcttctggct tcaacatctc ttattcttct 1560 atccactggg tgcgtcaggc cccgggtaag ggcctggaat gggttgcata tatttcttct 1620 tattatggct atacttatta tgccgatagc gtcaagggcc gtttcactat aagcgcagac 1680 acatccaaaa acacagccta cctacaaatg aacagcttaa gagctgagga cactgccgtc 1740 tattattgtg ctcgcgctca ttacttcccg tgggctggtg ctatggacta ctggggtcaa 1800 ggaaccctgg tcaccgtctc ctcgggtgga ggtggcagtg atatccagat gacccagtcc 1860 ccgagctccc tgtccgcctc tgtgggcgat agggtcacca tcacctgccg tgccagtcag 1920 tccgtgtcca gcgctgtagc ctggtatcaa cagaaaccag gaaaagctcc gaagcttctg 1980 atttactcgg catccagcct ctactctgga gtcccttctc gcttctctgg tagccgttcc 2040 gggacggatt tcactctgac catcagcagt ctgcagccgg aagacttcgc aacttattac 2100 tgtcagcaat actactggcc gatcacgttc ggacagggta ccaaggtgga gatcaaa 2157 <210> 38 <211> 719 <212> PRT <213> Homo sapiens <400> 38 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Ser Tyr Ser 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Tyr Pro Ser Tyr Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 115 120 125 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg 130 135 140 Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro 145 150 155 160 Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser Leu Tyr Ser 165 170 175 Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr 180 185 190 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 195 200 205 Gln Gln Ala Phe Tyr Tyr Pro Ile Thr Phe Gly Gln Gly Thr Lys Val 210 215 220 Glu Ile Lys Leu Glu Asp Lys Thr His Thr Lys Val Glu Pro Lys Thr 225 230 235 240 Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Met 355 360 365 Val Phe Asp Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 370 375 380 Ser Leu Trp Cys Met Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Pro Gly Lys Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala 465 470 475 480 Thr Pro Glu Ser Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly 485 490 495 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 500 505 510 Gly Phe Asn Ile Ser Tyr Ser Ser Ile His Trp Val Arg Gln Ala Pro 515 520 525 Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Ser Tyr Tyr Gly Tyr 530 535 540 Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp 545 550 555 560 Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu 565 570 575 Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala His Tyr Phe Pro Trp Ala 580 585 590 Gly Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 595 600 605 Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu 610 615 620 Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln 625 630 635 640 Ser Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala 645 650 655 Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser Leu Tyr Ser Gly Val Pro 660 665 670 Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile 675 680 685 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr 690 695 700 Tyr Trp Pro Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 705 710 715 <210> 39 <211> 2175 <212> DNA <213> Homo sapiens <400> 39 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60 tcctgtgcag cttctggctt caacatctct tattcttcta tccactgggt gcgtcaggcc 120 ccgggtaagg gcctggaatg ggttgcatct atttatcctt cttatagctc tacttattat 180 gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240 ctacaaatga acagcttaag agctgaggac actgccgtct attattgtgc tcgctactac 300 gctatggact actggggtca aggaaccctg gtcaccgtct cctcgggtgg aggtggcagt 360 gatatccaga tgacccagtc cccgagctcc ctgtccgcct ctgtgggcga tagggtcacc 420 atcacctgcc gtgccagtca gtccgtgtcc agcgctgtag cctggtatca acagaaacca 480 ggaaaagctc cgaagcttct gatttactcg gcatccagcc tctactctgg agtcccttct 540 cgcttctctg gtagccgttc cgggacggat ttcactctga ccatcagcag tctgcagccg 600 gaagacttcg caacttatta ctgtcagcaa gctttctact acccgatcac gttcggacag 660 ggtaccaagg tggagatcaa actcgaggac aaaactcaca caaaagttga gcccaaatct 720 tctgataaga cccataattg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 780 gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 840 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 900 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 960 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 1020 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1080 aaagggcagc cccgagaacc acaggtgtac accctgcccc caatccggga gctgatgacc 1140 agcaaccagg tcagcctgag ctgcgccgtc aaaggcttct atcccagcga catcgccgtg 1200 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1260 1320 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc aaaccacta cacgcagaag 1380 agcctctccc tgtctccggg taaaagcggc agcgagactc ccgggacctc agagtccgcc 1440 acacccgaaa gtggtggcgg agaggttcag ctggtggagt ctggcggtgg cctggtgcag 1500 ccaggggct cactccgttt gtcctgtgca gcttctggct tcaacatctc ttcttattat 1560 atccactggg tgcgtcaggc cccgggtaag ggcctggaat gggttgcatc tatttattct 1620 tcttatggct atacttctta tgccgatagc gtcaagggcc gtttcactat aagcgcagac 1680 acatccaaaa acacagccta cctacaaatg aacagcttaa gagctgagga cactgccgtc 1740 tattattgtg ctcgcactgt tcgtggatcc aaaaaaccgt acttctctgg ttgggctatg 1800 gactactggg gtcaaggaac cctggtcacc gtctcctcgg gtggaggtgg cagtgatatc 1860 cagatgaccc agtccccgag ctccctgtcc gcctctgtgg gcgatagggt caccatcacc 1920 tgccgtgcca gtcagtccgt gtccagcgct gtagcctggt atcaacagaa accaggaaaa 1980 gctccgaagc ttctgattta ctcggcatcc agcctctact ctggagtccc ttctcgcttc 2040 tctggtagcc gttccgggac ggatttcact ctgaccatca gcagtctgca gccggaagac 2100 ttcgcaactt attactgtca gcaatactct tggggtccgt tcacgttcgg acagggtacc 2160 aaggtggaga tcaaa 2175 <210> 40 <211> 725 <212> PRT <213> Homo sapiens [[ID=<400> 40 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Ser Tyr Ser 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Tyr Pro Ser Tyr Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 115 120 125 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg 130 135 140 Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro 145 150 155 160 Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser Leu Tyr Ser 165 170 175 Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr 180 185 190 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 195 200 205 Gln Gln Ala Phe Tyr Tyr Pro Ile Thr Phe Gly Gln Gly Thr Lys Val 210 215 220 Glu Ile Lys Leu Glu Asp Lys Thr His Thr Lys Val Glu Pro Lys Ser 225 230 235 240 Ser Asp Lys Thr His Asn Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 Val Tyr Thr Leu Pro Pro Ile Arg Glu Leu Met Thr Ser Asn Gln Val 370 375 380 Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Pro Gly Lys Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala 465 470 475 480 Thr Pro Glu Ser Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly 485 490 495 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 500 505 510 Gly Phe Asn Ile Ser Ser Tyr Tyr Ile His Trp Val Arg Gln Ala Pro 515 520 525 Gly Lys Gly Leu Glu Trp Val Ala Ser Ile Tyr Ser Ser Tyr Gly Tyr 530 535 540 Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp 545 550 555 560 Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu 565 570 575 Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Val Arg Gly Ser Lys Lys 580 585 590 Pro Tyr Phe Ser Gly Trp Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 595 600 605 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln 610 615 620 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 625 630 635 640 Cys Arg Ala Ser Gln Ser Val Ser Ser Ala Val Ala Trp Tyr Gln Gln 645 650 655 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Ser Leu 660 665 670 Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp 675 680 685 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 690 695 700 Tyr Cys Gln Gln Tyr Ser Trp Gly Pro Phe Thr Phe Gly Gln Gly Thr 705 710 715 720 Lys Val Glu Ile Lys 725 <210> 41 <211> 2196 <212> DNA <213> Homo sapiens <400> 41 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60 tcctgtgcag cttctggctt caacatcggt tcttcttcta tccactgggt gcgtcaggcc 120 ccgggtaagg gcctggaatg ggttgcatct atttattctg cttttgcctc tacttcttat 180 gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240 ctacaaatga acagcttaag agctgaggac actgccgtct attattgtgc tcgctaccat 300 ttcccgttcg gttttgcttt ggactactgg ggtcaaggaa ccctggtcac cgtctcctcg 360 ggtggaggtg gcagtgatat ccagatgacc cagtccccga gctccctgtc cgcctctgtg 420 ggcgataggg tcaccatcac ctgccgtgcc agtcagtccg tgtccagcgc tgtagcctgg 480 tatcaacaga aaccaggaaa agctccgaag cttctgattt actcggcatc cagcctctac 540 tctggagtcc cttctcgctt ctctggtagc cgttccggga cggatttcac tctgaccatc 600 agcagtctgc agccggaaga cttcgcaact tattactgtc agcaaggtgt ttacctgttc 660 acgttcggac agggtaccaa ggtggagatc aaactcgagg acaaaactca cacaaaagtg 720 gagcccaaaa cttctgataa gacccatact tgcccaccgt gcccagcacc tgaactcctg 780 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 840 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 900 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg cgaggagcag 960 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1020 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1080 atctccaaag ccaaagggca gccccgagaa ccaatggtgt ttgacctgcc cccatcccgg 1140 gaggagatga ccaagaacca ggtcagcctg tggtgcatgg tcaagggctt ctatcccagc 1200 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1260 cccgtgctgg actccgacgg ctccttcttc ctgtacagca agctcaccgt ggacaagagc 1320 cgctggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1380 tacacgcaga agagcctctc cctgtctccg ggtaaaagcg gcagcgagac tcccgggacc 1440 tcagagtccg ccacacccga aagtggtggc ggagaggttc agctggtgga gtctggcggt 1500 ggcctggtgc agccaggggg ctcactccgt ttgtcctgtg cagcttctgg cttcaacttt 1560 tcttcttctt ctatacactg ggtgcgtcag gccccgggta agggcctgga atgggttgca 1620 tctatttctt cttcttatgg ctatacttat tatgccgata gcgtcaaggg ccgtttcact 1680 ataagcgcag acacatccaa aaacacagcc tacctacaaa tgaacagctt aagagctgag 1740 gacactgccg tctattattg tgctcgcggt ggttctggtg tttctcatta cggttctgtt 1800 tactactctt ggtgggcttt ggactactgg ggtcaaggaa ccctggtcac cgtctcctcg 1860 ggtggaggtg gcagtgatat ccagatgacc cagtccccga gctccctgtc cgcctctgtg 1920 ggcgataggg tcaccatcac ctgccgtgcc agtcagtccg tgtccagcgc tgtagcctgg 1980 tatcaacaga aaccaggaaa agctccgaag cttctgattt actcggcatc cagcctctac 2040 tctggagtcc cttctcgctt ctctggtagc cgttccggga cggatttcac tctgaccatc 2100 agcagtctgc agccggaaga cttcgcaact tattactgtc agcaagcttc ttacgctccg 2160 atcacgttcg gacagggtac caaggtggag atcaaa 2196 <210> 42 <211> 732 <212> PRT [[ID=2二十二]]<213> Homo sapiens <4 hundred> 42 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Gly Ser Ser 20 25 30 It should be noted that there may be some inaccuracies in the above translation due to the complexity and potential ambiguity of the original text,...

Claims

1. An in vitro method for selectively activating the Wnt signaling pathway in cells, the method comprising contacting cells having a coiled protein (FZD) receptor and a Wnt co-receptor with a modular tetravalent binding molecule, wherein the modular tetravalent binding molecule simultaneously binds two epitopes of the FZD receptor and the Wnt co-receptor LRP5 and / or LRP6, and the modular tetravalent binding molecule comprises: (a) An Fc domain having C-terminus and N-terminus, or a fragment thereof containing a CH3 domain. (b) An FZD-binding domain having at least two binding sites, wherein at least one binding site binds to an FZD receptor, and said FZD receptor is FZD1, FZD2, FZD4, FZD5, FZD7, or FZD8, and wherein said FZD-binding domain comprises: (i) A bimeric antibody that binds to the FZD receptor, the bimeric antibody comprising two peptides, each peptide comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL), wherein the VH and VL from one peptide pair with the VL and VH of the other peptide to form a bimeric antibody, or (ii) the scFv of the FZD receptor containing the VL and VH regions, and The FZD binding domain comprises one or more combinations of VH and VL selected from the group consisting of: VH being a combination of SEQ ID NO.45 and VL being a combination of SEQ ID NO.47, VH being a combination of SEQ ID NO.54 and VL being a combination of SEQ ID NO.55, VH being a combination of SEQ ID NO.57 and VL being a combination of SEQ ID NO.58, VH being a combination of SEQ ID NO.59 and VL being a combination of SEQ ID NO.60, VH being a combination of SEQ ID NO.61 and VL being a combination of SEQ ID NO.62, VH being a combination of SEQ ID NO.63 and VL being a combination of SEQ ID NO.64, VH being a combination of SEQ ID NO.65 and VL being a combination of SEQ ID NO.66, VH being a combination of SEQ ID NO.67 and VL being a combination of SEQ ID NO.68, and VH being a combination of SEQ ID NO.69 and VL being a combination of SEQ ID NO.70; (c) A Wnt co-receptor binding domain having at least two binding sites, wherein the Wnt co-receptor binding domain binds to both Wnt1 and Wnt3 binding sites on Wnt co-receptors LRP5 and / or LRP6; and wherein the Wnt co-receptor binding domain comprises a bisomatic antibody binding to the Wnt co-receptor, the bisomatic antibody comprising two peptides, each peptide comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL), wherein the VH and VL from one peptide pair with the VL and VH of the other peptide to form a bisomatic antibody. Wherein (1) the VH from one peptide is SEQ ID NO.49 and the VL is SEQ ID NO.50, and the VH from another peptide is SEQ ID NO.53 and the VL is SEQ ID NO.52, or (2) the VH from one peptide is SEQ ID NO.71 and the VL is SEQ ID NO.72, and the VH from another peptide is SEQ ID NO.73 and the VL is SEQ ID NO.74; The FZD binding domain is connected to one end of the Fc domain or a segment thereof, and the Wnt co-receptor binding domain is connected to the other end of the Fc domain or a segment thereof. The FZD binding domain and the Wnt co-receptor binding domain are selected based on their ability to target epitopes on the FZD receptor and the Wnt co-receptor LRP5 and / or LRP6, respectively, thereby activating specific Wnt signaling pathways.

2. The in vitro method according to claim 1, wherein the FZD binding domain binds to the Wnt ligand binding site on the FZD receptor, or the Wnt co-receptor binding domain binds to the Wnt ligand binding site on the Wnt co-receptors LRP5 and / or LRP6.

3. The in vitro method according to claim 2, wherein the Wnt co-receptor binding domain binds to Wnt3 and / or Wnt1 binding sites.

4. The in vitro method according to claim 1, wherein the FZD binding domain binds to at least two FZD receptors.

5. The in vitro method according to claim 1, wherein the Fc domain is an IgG Fc domain.

6. A modular tetravalent bonded molecule, wherein the modular tetravalent bonded molecule comprises: (a) An Fc domain having C-terminus and N-terminus, or a fragment thereof containing a CH3 domain. (b) An FZD-binding domain having at least two binding sites, wherein at least one binding site binds to an FZD receptor, and said FZD receptor is FZD1, FZD2, FZD4, FZD5, FZD7, or FZD8, and wherein said FZD-binding domain comprises: (i) A bimeric antibody that binds to the FZD receptor, the bimeric antibody comprising two peptides, each peptide comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL), wherein the VH and VL from one peptide pair with the VL and VH of the other peptide to form a bimeric antibody, or (ii) the scFv of the FZD receptor containing the VL and VH regions, and The FZD binding domain comprises one or more combinations of VH and VL selected from the group consisting of: VH being a combination of SEQ ID NO.45 and VL being a combination of SEQ ID NO.47, VH being a combination of SEQ ID NO.54 and VL being a combination of SEQ ID NO.55, VH being a combination of SEQ ID NO.57 and VL being a combination of SEQ ID NO.58, VH being a combination of SEQ ID NO.59 and VL being a combination of SEQ ID NO.60, VH being a combination of SEQ ID NO.61 and VL being a combination of SEQ ID NO.62, VH being a combination of SEQ ID NO.63 and VL being a combination of SEQ ID NO.64, VH being a combination of SEQ ID NO.65 and VL being a combination of SEQ ID NO.66, VH being a combination of SEQ ID NO.67 and VL being a combination of SEQ ID NO.68, and VH being a combination of SEQ ID NO.69 and VL being a combination of SEQ ID NO.70; (c) A Wnt co-receptor binding domain having at least two binding sites, wherein the Wnt co-receptor binding domain binds to both Wnt1 and Wnt3 binding sites on Wnt co-receptors LRP5 and / or LRP6; and wherein the Wnt co-receptor binding domain comprises a bisomatic antibody binding to the Wnt co-receptor, the bisomatic antibody comprising two peptides, each peptide comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL), wherein the VH and VL from one peptide pair with the VL and VH of the other peptide to form a bisomatic antibody. Wherein (1) the VH from one peptide is SEQ ID NO.49 and the VL is SEQ ID NO.50, and the VH from another peptide is SEQ ID NO.53 and the VL is SEQ ID NO.52, or (2) the VH from one peptide is SEQ ID NO.71 and the VL is SEQ ID NO.72, and the VH from another peptide is SEQ ID NO.73 and the VL is SEQ ID NO.74; The FZD binding domain is connected to one end of the Fc domain, and the Wnt co-receptor binding domain is connected to the other end of the Fc domain. The FZD binding domain and the Wnt co-receptor binding domain are selected based on their ability to target epitopes on the FZD receptor and the Wnt co-receptor LRP5 and / or LRP6, respectively, thereby activating specific Wnt signaling pathways.

7. The tetravalent binding molecule according to claim 6, wherein at least one of the binding domains is bispecific.

8. The tetravalent binding molecule of claim 6, wherein the VH and VL are derived from antibodies that bind to the FZD receptor and optionally inhibit the binding of the Wnt ligand to the FZD receptor, or the VH and VL are derived from antibodies that bind to the Wnt co-receptor and optionally inhibit the binding of the Wnt ligand to the co-receptor.

9. A pharmaceutical composition comprising a tetravalent binding molecule according to any one of claims 6 to 8 and a pharmaceutically acceptable carrier.

10. Use of the tetravalent binding molecule according to any one of claims 6 to 8 in the preparation of a medicament for enhancing tissue regeneration of bone in a subject in need by means of administering to the subject an amount of the tetravalent binding molecule according to any one of claims 6 to 8 sufficient to enhance tissue regeneration.

11. An in vitro method for promoting the interaction between FZD receptors and Wnt co-receptors on cells to activate the Wnt signaling pathway in said cells, comprising: a) Select an Fc domain with C-terminus and N-terminus or a fragment containing a CH3 domain; b) A bivalent FZD receptor-binding domain is attached to one end of the Fc domain, and a bivalent Wnt co-receptor-binding domain is attached to the other end of the Fc domain, thereby forming a tetravalent binding molecule; wherein the FZD receptor is FZD1, FZD2, FZD4, FZD5, FZD7, or FZD8, and wherein the FZD binding domain comprises: (i) A bimeric antibody that binds to the FZD receptor, the bimeric antibody comprising two peptides, each peptide comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL), wherein the VH and VL from one peptide pair with the VL and VH of the other peptide to form a bimeric antibody, or (ii) the scFv of the FZD receptor containing the VL and VH regions, and The FZD receptor binding domain comprises one or more combinations of VH and VL selected from the group consisting of: VH of SEQ ID NO.45 and VL of SEQ ID NO.47, VH of SEQ ID NO.54 and VL of SEQ ID NO.55, VH of SEQ ID NO.57 and VL of SEQ ID NO.58, VH of SEQ ID NO.59 and VL of SEQ ID NO.60, VH of SEQ ID NO.61 and VL of SEQ ID NO.62, VH of SEQ ID NO.63 and VL of SEQ ID NO.64, VH of SEQ ID NO.65 and VL of SEQ ID NO.66, VH of SEQ ID NO.67 and VL of SEQ ID NO.68, and VH of SEQ ID NO.69 and VL of SEQ ID NO.70; The Wnt co-receptor binding domain comprises a bimeric antibody that binds to the Wnt co-receptor. The bimeric antibody comprises two peptides, each containing a heavy chain variable domain (VH) linked to a light chain variable domain (VL). The VH and VL from one peptide pair with the VL and VH from the other peptide to form the bimeric antibody. Wherein (1) the VH from one peptide is SEQ ID NO.49 and the VL is SEQ ID NO.50, and the VH from another peptide is SEQ ID NO.53 and the VL is SEQ ID NO.52, or (2) the VH from one peptide is SEQ ID NO.71 and the VL is SEQ ID NO.72, and the VH from another peptide is SEQ ID NO.73 and the VL is SEQ ID NO.74; c) Under the condition that the tetravalent binding molecule is bound to the FZD receptor and the Wnt co-receptor, the tetravalent binding molecule is brought into contact with cells expressing the FZD receptor and the Wnt co-receptor, thereby activating the Wnt signaling pathway, wherein the Wnt co-receptor binding domain binds to both the Wnt1 and Wnt3 binding sites on the Wnt co-receptor LRP5 and / or LRP6.

12. The in vitro method of claim 11, wherein the bivalent FZD receptor-binding domain comprises a bisomatic antibody that binds to the FZD receptor, and the bivalent Wnt receptor-binding domain comprises a bisomatic antibody that binds to the Wnt co-receptor.

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