Antibody that binds to LRP5 protein and method of use

Abstract

Provided herein are antibodies that specifically bind to LRP5, and methods of use thereof. TIFF2022544308000035.tif74165

Description

[Technical Field]

[0001] Description of research funded by the federal government. none.

[0002] Cross-reference of related applications This application claims the benefit of the priority date of U.S. Provisional Application No. 62 / 886,913, filed on 14 August 2019, which is incorporated herein by reference in its entirety.

[0003] Parties in a joint research agreement none. [Background technology]

[0004] background Wnt signaling plays a crucial role in regulating various cellular processes, including cell fate determination, proliferation, survival, polarity, and migration. 1 Disruptions resulting from altered expression or mutations in the Wnt signaling pathway are associated with defects in embryonic development, as well as various pathological conditions such as cancer and osteoporosis. 2~4 Wnt signaling leads to the activation of classical and non-classical signaling pathways. 1、5The non-classical pathway activates signaling molecules that activate cytoplasmic signals that regulate the cytoskeleton and calcium storage, rather than involving the nucleus or transcription. This pathway primarily plays a role in regulating cell polarity or migration. The classical pathway primarily regulates transcriptional activity by modulating the cytoplasmic level of β-catenin. In the unstimulated state, β-catenin associates with a degradation complex composed of Axin, APC, CK1, and GSK3b, which leads to phosphorylation, ubiquitination, and proteasomal degradation of β-catenin. Wnt signaling destabilizes this complex, resulting in the accumulation of "free" β-catenin in the cytosol, which translocates to the nucleus and acts as a co-activator for TCF / LEF-mediated transcription. Wnt binds to the frizzled family of seven-transmembrane domain receptors and either LRP5 or LRP6, initiating the classical signaling pathway. 6~8 .

[0005] LRP5 and LRP6 are functionally redundant single-pass transmembrane receptors that share approximately 70% homology. Binding of Wnt ligands to Fz and LRP5 / 6 triggers the recruitment of degradation complexes and Dishevelled (Dsh / Dvl), as well as the PPPSPxS motif located in the intracellular domain. (SEQ ID NO: 1) The above results in phosphorylation of LRP5 / 6.9 This phosphorylation is mediated by GSK3b and CK1, which reduces GSK3b activity, inhibits β-catenin phosphorylation and subsequent proteasomal degradation, and enhances TCF / LEF-mediated transcriptional activity. LRP5 is widely expressed during embryonic development and in adult tissues. Mutations in LRP5 are associated with bone density disorders, and several mouse models with LRP5 knockout or mutations exhibit altered bone development. 10 .

[0006] LRP5 expression has also been shown to be elevated in human malignant tissues and human cancer cell lines, such as osteosarcoma, and Wnt signaling in such cell lines has been shown to be decreased with overexpression of dominant-negative LRP5. 11~13Furthermore, LRP5 also appears to have an important role in regulating cell invasion ability and cell motility. 14~16 In tests, LRP6 has been shown to be more potent than LRP5 in transmitting the Wnt signal. However, recent genetic experiments have shown that some Wnt ligands require the presence of both receptors to generate the classical signal (17). Due to the importance of LRPs in the regulation of Wnt signaling and their established role in several human diseases, LRPs are becoming increasingly important targets for therapeutic drug development. There is important biology surrounding LRP5, as well as its role in Wnt signaling and the etiology of various still-undiscovered diseases. A profound toolbox of synthetic antibodies can help to systematically clarify these roles and provide additional targeted therapeutics.

[0007] Wnt signaling leads to the activation of classical and non-classical signaling pathways. The non-classical pathway activates signaling molecules that activate cytoplasmic signals that do not involve the nucleus or transcription, but rather regulate the cytoskeleton and calcium levels. This pathway mainly plays a role in regulating cell polarity or migration.

[0008] The classical pathway mainly controls transcriptional activity by regulating the cytoplasmic level of β-catenin. In the unstimulated state, β-catenin associates with a degradation complex composed of Axin, APC, CD1, and GSKβ, which leads to phosphorylation, ubiquitination, and proteasomal degradation of β-catenin. Wnt signaling is active when Wnt binds to Frizzled (FDZ), a seven-pass transmembrane receptor, and a co-receptor, either low density lipoprotein receptor-related protein (LRP5 or LRP6). This signaling destabilizes the complex by attracting Disheveled (Dsh / Dvl) to the plasma membrane, resulting in the accumulation of β-catenin, which then translocates to the nucleus and activates TCF / LEF-mediated transcription.

[0009] The present invention described below utilizes prior art antibody phage display libraries and techniques to identify a series of novel synthetic antibodies that target extracellular epitopes of LRP5.

Summary of the Invention

[0010] Summary In one embodiment, an antibody that specifically binds to LRP5, comprising a light chain variable region and / or a heavy chain variable region, is provided herein, wherein the heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of the above CDR is the CDR sequence of the anti-LRP5 antibody. Set: Includes or consists of sequences selected from LRP5-A7, LRP5-A9, LRP5-C5, LRP5-C12, LRP5-D9, LRP5-E5, LRP5-G2, LRP5-G9, LRP5-G10, LRP5-G11, LRP5-H3, LRP5-H5, LRP5-H9, LRP5-R3O_D3, LRP5-R3_E8, LRP5-R3O_G6. In one embodiment, the amino acid sequence of the above CDR includes or consists of sequences selected from the sequences described below, and CDR-H1 is, LSYYYM (SEQ ID NO: 7) , ISYSYI (SEQ ID NO: 5) LSYSSM (SEQ ID NO: 2) ISSYSI (SEQ ID NO: 3) , ISYSYI (SEQ ID NO: 5) IYSYSI (SEQ ID NO: 6) , LSYYYM (SEQ ID NO: 7) FSSSSI (SEQ ID NO: 4) ,LYYYYI (SEQ ID NO: 8) LSYSSI (SEQ ID NO: 9) IYSYYI (SEQ ID NO: 10) LLYYSSM (SEQ ID NO: 122) , and FSSSSI (SEQ ID NO: 4) Selected from the group consisting of, CDR-H2 is, SIYPYYGYTY (SEQ ID NO: 11) SSSYYGYTY (SEQ ID NO: 12) SISSSYGYTY (SEQ ID NO: 13) , SIYSSYGSTS (SEQ ID NO: 14) , SIYSSYGYTY (SEQ ID NO: 15) , SIYPYSSYTS (SEQ ID NO: 16) , SIYSSYGYTY (SEQ ID NO: 15) , SIYPSYGYTY (SEQ ID NO: 17) SISPYYGYTS (SEQ ID NO: 18) SISSSYGSTS (SEQ ID NO: 19) , SIYSYYGYTY (SEQ ID NO: 20) SISSSYGYTY(SEQ ID NO: 13) SISSSYGYTY (SEQ ID NO: 13) SISSYYGYTS (SEQ ID NO: 53) , and YISPYYGYTS (SEQ ID NO: 56) Selected from the group consisting of, CDR-H3 is, HGAM (SEQ ID NO: 21) TVRGSKKPYFSGWAM (SEQ ID NO: 22) SSYYSSVSSSVYAL (SEQ ID NO: 23) TVRGSKKPYFSGWAM (SEQ ID NO: 22) HYSYOFFYAM (SEQ ID NO: 24) ,YAVYFPGYYWGM (SEQ ID NO: 25) WSHVSGHYSGM (SEQ ID NO: 26) WGAYHSSGYGM (SEQ ID NO: 27 ), GGSGVSHYGSVYYSWWAL (SEQ ID NO: 28) ,AAPYYGYYYGam (SEQ ID NO: 29) SGYGWYAM (SEQ ID NO: 30) GYWAI (SEQ ID NO: 31) SYPAM (SEQ ID NO: 32) SWAM (SEQ ID NO: 33) YWAL (SEQ ID NO: 54) GWGSPASAGYYGL (SEQ ID NO: 57) SSYYSSVSSSVYAL (SEQ ID NO: 23) TVRGSKKPYFSGWAM (SEQ ID NO: 22) , and TVRGSKKPYFSGWAM( SEQ ID NO: 22) Selected from the group consisting of, CDR-L1 is SVSSA (SEQ ID NO: 34) Therefore, CDR-L2 is SASSLYS (SEQ ID NO: 35) Therefore, the CDR-L3 is AWGWGLF (SEQ ID NO: 36) , VHYSPYSLI (SEQ ID NO: 37) , YQYSGLI (SEQ ID NO: 38) , FSHVSLI (SEQ ID NO: 39) ASYSPI (SEQ ID NO: 40) , YHYYYLF (SEQ ID NO: 41) ASYAPI (SEQ ID NO: 42) , SSSSPI (SEQ ID NO: 43) SSYSLI (SEQ ID NO: 44) GVSLI (SEQ ID NO: 45) YWFLI (SEQ ID NO: 46) , PVGHYGYPI (SEQ ID NO: 47) SSYSPI (SEQ ID NO: 48) YWAYYSPI (SEQ ID NO: 49)VSYYPLI (SEQ ID NO: 51) SSYSLI (SEQ ID NO: 44) , and VHYSPYSLI (SEQ ID NO: 37) The antibody is selected from the group consisting of the following: (i) a heavy chain variable region comprising: (i) a heavy chain amino acid sequence as described in Table 2; (ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the heavy chain amino acid sequence as described in Table 2, wherein the CDR sequence is one of the CDR sequence sets described in Table 1; or (iii) a conservatively substituted amino acid sequence of (i), wherein the CDR sequence is one of the CDR sequence sets described in Table 1. In another embodiment, the antibody comprises a light chain variable region including: (i) a light chain amino acid sequence as described in Table 2; (ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the light chain amino acid sequence as described in Table 2, wherein the CDR sequence is the set of CDR sequences described in Table 1; or (iii) a conservatively substituted amino acid sequence of (i), wherein the CDR sequence is the set of CDR sequences described in Table 1. In another embodiment, the CDR sequence is a complete set of CDR sequences selected from the antibodies identified in Table 1. In another embodiment, the antibody cross-reacts with LRP6. In another embodiment, the CDR sequence comprises a set of light chain CDR sequences or a set of heavy chain CDR sequences selected from the antibodies identified in Table 1. In another embodiment, the antibody specifically binds to LRP5. In another embodiment, the CDR sequence is a set of CDR sequences of antibodies selected from LRP5-A7, LRP5-A9, LRP5-C5, LRP5-C12, LRP5-D9, LRP5-E5, LRP5-G2, LRP5-G9, LRP5-G10, LRP5-G11, LRP5-H3, LRP5-H5, LRP5-H9, LRP5-R3O_D3, LRP5-R3_E8, and LRP5-R3O_G6. In another embodiment, the antibody blocks the binding of a Wnt ligand to the Wnt3a binding site of LRP5. In another embodiment, the antibody blocks the binding of a Wnt ligand to the non-Wnt3a binding site of LRP5. In another embodiment, the antibody is a monoclonal antibody.In another embodiment, the antibody is a humanized antibody. In another embodiment, the antibody is a single-chain antibody. In another embodiment, the antibody is an antibody-binding fragment selected from Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimer, nanobody, and minibody, and in another embodiment, the antibody is a bispecific antibody that further binds to the FZD receptor. In another embodiment, the antibody contains a non-natural glycosylation pattern. In another embodiment, the antibody contains, for example, a cysteine ​​substitution or addition to a constant region or framework region.

[0011] In another aspect, an immunoconjugate comprising an antibody provided herein and a detectable label or cytotoxic agent is provided herein. In another aspect, the immunoconjugate comprises a cytotoxic agent selected from meitansinoids, auristatin, drastatin, tubulysin, cryptophycin, pyrrolobenzodiazepine (PBD) dimers, indolinobenzodiazepine dimers, α-amanitin, trichothene, SN-38, duocalmycin, CC1065, calicheamincin, engine antibiotics, taxanes, doxorubicin derivatives, anthracyclines, and their stereoisomers, azanofide, isosteres, analogs, or derivatives.

[0012] In another aspect, nucleic acid molecules encoding antibodies provided herein are provided herein. In one embodiment, one or more of the CDR sequences are encoded by the nucleic acids in Table 2. In another embodiment, the antibody comprises a heavy chain variable region encoded by a nucleic acid, including: (i) a heavy chain nucleic acid sequence as described in Table 2; (ii) a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to the heavy chain nucleic acid sequences as described in Table 2, wherein the CDR sequence is one of the CDR sequence sets described in Table 1; or (iii) a codon degenerate nucleic acid sequence of (i), wherein the CDR sequence is one of the CDR sequence sets described in Table 1. In another embodiment, the antibody comprises a light chain variable region encoded by a nucleic acid including: (i) a light chain nucleic acid sequence as described in Table 2; (ii) a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to the light chain nucleic acid sequence as described in Table 2, wherein the CDR sequence is one of the CDR sequence sets described in Table 1; or (iii) a codon-degenerate nucleic acid sequence of (i) wherein the CDR sequence is one of the CDR sequence sets described in Table 1.

[0013] In another context, vectors comprising expression regulatory sequences functionally linked to nucleic acids provided herein are also provided herein.

[0014] In another aspect, a host cell comprising a recombinant nucleic acid molecule containing an expression regulatory sequence functionally linked to the nucleic acid provided herein is provided herein. In another aspect, the host cell is a Chinese hamster ovary (CHO) cell.

[0015] In another context, host cells containing the vectors provided herein are provided herein.

[0016] In another aspect, a method for producing an anti-LRP5 antibody is provided herein, comprising the step of culturing host cells provided herein.

[0017] In another aspect, compositions comprising antibodies, immunoconjugates, nucleic acid molecules, vectors, or host cells, provided herein, along with an optional suitable diluent, are provided herein. In one embodiment, the composition comprises one or more antibodies or immunoconjugates, and optionally, the composition is a pharmaceutical composition.

[0018] In another context, kits comprising antibodies, immunoconjugates, nucleic acid molecules, vectors, or host cells provided herein are also provided herein.

[0019] In another aspect, a method for detecting LRP5 expression is provided herein, comprising the steps of contacting a sample containing one or more cells with one or more antibodies or immunoconjugates provided herein, under conditions that allow the formation of antibody-cell complexes, and detecting the presence of any antibody complex. In one embodiment, detection is by immunofluorescence. In another embodiment, detection is by flow cytometry. In another embodiment, the method is for detecting LRP4 expression, and the antibody or immunoconjugate comprises a set of CDR sequences corresponding to an antibody selected from LRP5-A7, LRP5-A9, LRP5-C5, LRP5-C12, LRP5-D9, LRP5-E5, LRP5-G2, LRP5-G9, LRP5-G10, LRP5-G11, LRP5-H3, LRP5-H5, LRP5-H9, LRP5-R3O_D3, LRP5-R3_E8, and LRP5-R3O_G6.

[0020] In another aspect, methods are provided herein for inhibiting Wnt ligand binding to the LRP5 receptor, interfering with the Wnt signaling pathway, inhibiting Wnt-induced transcriptional activity, inhibiting disheveled activation, promoting the preservation of the β-catenin degradation complex, promoting the accumulation of β-catenin, or inhibiting cell proliferation, the methods comprising contacting cells expressing the LRP5 receptor with an antibody or immunoconjugate provided herein. In yet another aspect, antibodies or immunoconjugates provided herein are provided for use in inhibiting Wnt ligand binding to the LRP5 receptor, interfering with the Wnt signaling pathway, inhibiting Wnt-induced transcriptional activity, inhibiting disheveled activation, promoting the preservation of the β-catenin degradation complex, promoting the accumulation of β-catenin, or inhibiting cell proliferation. In a further aspect, the use of antibodies or immunoconjugates provided herein for inhibiting Wnt ligand binding to the LRP5 receptor, interfering with the Wnt signaling pathway, inhibiting Wnt-induced transcriptional activity, inhibiting disheveled activation, promoting the preservation of the β-catenin degradation complex, promoting β-catenin accumulation, or inhibiting cell proliferation is provided. In yet another aspect, the use of antibodies or immunoconjugates provided herein in the manufacture of pharmaceuticals for inhibiting Wnt ligand binding to the LRP5 receptor, interfering with the Wnt signaling pathway, inhibiting Wnt-induced transcriptional activity, inhibiting disheveled activation, promoting the preservation of the β-catenin degradation complex, promoting β-catenin accumulation, or inhibiting cell proliferation is provided. In one embodiment, the antibody or immunoconjugate blocks the binding of the Wnt ligand to the Wnt3a binding site of LRP5. In another embodiment, the antibody or immunoconjugate blocks the binding of a Wnt ligand to the non-Wnt3a binding site of LRP5.In another embodiment, the antibody or immunoconjugate comprises a set of CDR sequences corresponding to an antibody selected from LRP5-A7, LRP5-A9, LRP5-C5, LRP5-C12, LRP5-D9, LRP5-E5, LRP5-G2, LRP5-G9, LRP5-G10, LRP5-G11, LRP5-H3, LRP5-H5, LRP5-H9, LRP5-R3O_D3, LRP5-R3_E8, and LRP5-R3O_G6.

[0021] In another embodiment, a method for treating a cancer of a target in need is provided herein, comprising the step of administering an effective amount of a pharmaceutical composition comprising an antibody or immunoconjugate provided herein to the target. In another embodiment, a pharmaceutical composition comprising an antibody or immunoconjugate provided herein for use in treating a cancer of a target in need. In yet another embodiment, the use of a pharmaceutical composition comprising an antibody or immunoconjugate provided herein for treating a cancer of a target in need. In yet another embodiment, the use of a pharmaceutical composition comprising an antibody or immunoconjugate provided herein in the manufacture of a pharmaceutical for treating a cancer of a target in need. In one embodiment, the cancer is selected from cancer cells of colon cancer, lung cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, gastric cancer, liver cancer, adrenocortical carcinoma, and osteoblastoma. In another embodiment, the cancer is selected from acute myeloid leukemia, prostate cancer, glioblastoma, bladder cancer, and cervical cancer. In another embodiment, the method comprises the step of administering a first and a second antibody or antibody conjugate provided herein, the first of which blocks the binding of a Wnt ligand to the Wnt3a binding site of LRP5, and the second of which blocks the binding of a Wnt ligand to the non-Wnt3a binding site of LRP5. In another embodiment, the first antibody or immunoconjugate comprises a set of CDR sequences selected from antibodies of epitope group 2. In another embodiment, the antibody or immunoconjugate specifically binds to LRP5 in at least one assay and inhibits Wnt3a-induced signaling in at least one assay, and optionally, the antibody or immunoconjugate is an antibody or immunoconjugate provided herein. In another embodiment, the antibody or immunoconjugate comprises a set of CDR sequences corresponding to an antibody selected from LRP5-A7, LRP5-A9, LRP5-C5, LRP5-C12, LRP5-D9, LRP5-E5, LRP5-G2, LRP5-G9, LRP5-G10, LRP5-G11, LRP5-H3, LRP5-H5, LRP5-H9, LRP5-R3O_D3, LRP5-R3_E8, and LRP5-R3O_G6.

[0022] In another aspect, a method for enhancing the signaling activity of a Wnt ligand that binds to the Wnt3a binding site of LRP5 is provided herein, comprising the step of contacting cells expressing LRP5 with an antibody that blocks the binding of a Wnt ligand to a non-Wnt3a binding site of LRP5. In one embodiment, the method is performed in vitro. In another embodiment, the method is performed in vivo.

[0023] In another aspect, a method for enhancing the signaling activity of a Wnt ligand that binds to a non-Wnt3a binding site of LRP5 is provided herein, by contacting cells expressing LRP5 with an antibody that blocks the binding of a Wnt ligand to the Wnt3a binding site of LRP5. In one embodiment, the method is performed in vitro. In another embodiment, the method is performed in vivo.

[0024] The accompanying drawings incorporated herein and forming part thereof illustrate exemplary embodiments and, together with the description, further enable those skilled in the art to fabricate and use these embodiments and others that would be apparent to them. The present invention is described in further detail in conjunction with the following drawings. [Brief explanation of the drawing]

[0025]

Figure 1

Figure 2

[0026] Detailed explanation I. Definition Unless otherwise defined, scientific and technical terms used in connection with this disclosure shall have meanings generally understood by those skilled in the art. Furthermore, unless contextually required, singular terms shall include plural forms, and plural terms shall include singular forms. For example, the term “cell” includes a single cell and a group of cells or a population of cells. In general, the nomenclature and techniques used in connection with the cell and tissue cultures, molecular biology, and the chemistry and hybridization of proteins and oligonucleotides or polynucleotides described herein are well known and commonly used in the art (see, for example, Green and Sambrook, 2012).

[0027] As used herein, the term “polypeptide” refers to a molecule having a sequence of natural and / or non-natural amino acids linked via peptide bonds. The term “peptide” typically refers to a short polypeptide of 30 amino acids or less in length. The amino acid sequence of a polypeptide is called its “primary structure.” The term “protein” refers to a polypeptide having secondary, tertiary, and / or quaternary structures, such as structures stabilized by hydrogen bonds, and relationships between the secondary structure and structures formed from multiple proteins. Proteins can be further modified by other binding sites, such as carbohydrates (glycoproteins), lipids (lipoproteins), and phosphate groups (phosphoproteins).

[0028] As used herein, an amino acid sequence "consists" of only the amino acids within that sequence.

[0029] As used herein, if the first amino acid sequence (1) comprises a second amino acid sequence and (2) is 1, 2, or 3 times longer than the second amino acid sequence, then the first amino acid sequence "essentially consists of" the second amino acid sequence.

[0030] As used herein, if the second amino acid sequence includes the first amino acid sequence, the first amino acid sequence is a "fragment" of the second amino acid sequence. In certain embodiments, the first amino acid sequence, which is a fragment of the second amino acid sequence, may have one, two, three, four, five, six, seven, eight, nine, or ten fewer amino acids than the second amino acid sequence.

[0031] As used herein, a “functional equivalent” of a reference amino acid sequence is a sequence that is not identical to the reference sequence but contains minor changes, such as the insertion, deletion, or substitution of one or more amino acids. A functionally equivalent sequence retains the function (e.g., immunogenicity) of the reference sequence to which it is equivalent. If a functionally equivalent amino acid sequence contains one or more amino acid substitutions relative to the reference sequence, these are generally conservative amino acid substitutions.

[0032] As used herein, a “conservative amino acid substitution” is a substitution in which one amino acid residue is replaced by another amino acid residue without losing the desired properties of the protein. Suitable conservative amino acid substitutions can be made by substituting amino acids with similar hydrophobicity, polarity, and R-chain length. See, for example, Watson, et al., “Molecular Biology of the Gene,” 4th Edition, 1987, The Benjamin / Cummings Pub. Co., Menlo Park, CA, p. 224. Examples of conservative amino acid substitutions include (note that some categories are not mutually exclusive):

[0033] TIFF0007840842000001.tif65133

[0034] As used herein, the term “substantially identical” means the identity between the first and second amino acid sequences such that the first and second amino acid sequences contain a sufficient or minimum number of amino acid residues that are either (i) identical to aligned amino acid residues in the second amino acid sequence, or (ii) conserved substitutions of aligned amino acid residues in the second amino acid sequence, so that the first and second amino acid sequences have a common structural domain and / or common functional activity and / or common immunogenicity. For example, amino acid sequences containing a common structural or antigenic domain having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity are referred to as sufficiently or substantially identical. In the context of nucleotide sequences, the term “substantially identical” is used herein to refer to a first nucleic acid sequence containing a sufficient or minimum number of nucleotides that are identical to the aligned nucleotides in the second nucleic acid sequence, such that the first and second nucleotide sequences encode polypeptides having common functional activity, or encoding common structural polypeptide domains or common functional polypeptide activity, or encoding polypeptides having the same immunogenic properties.

[0035] As used herein, the terms “antigen,” “immunogen,” and “antibody target” refer to a molecule, compound, or complex that is recognized by an antibody, i.e., that can be bound by an antibody. The terms may refer to any molecule that can be recognized by an antibody, such as a polypeptide, polynucleotide, carbohydrate, lipid, chemical moiety, or combination thereof (e.g., phosphorylated polypeptide or glycosylated polypeptide). Those skilled in the art will understand that the terms merely indicate that a molecule can be targeted by an antibody, rather than indicating that the molecule is immunogenic in all circumstances.

[0036] As used herein, the term “epitope” refers to a site on an antigen that is recognized and bound by an antibody. An epitope may consist of several amino acids, or a portion of several amino acids, e.g., five or six, or more, e.g., twenty or more amino acids, or a portion of those amino acids. In some cases, an epitope may consist of non-protein components, e.g., carbohydrates, nucleic acids, or lipids. In some cases, an epitope is a three-dimensional portion. Thus, for example, if the target is a protein, the epitope may consist of consecutive amino acids, or amino acids from different parts of the protein that are adjacent by protein folding (e.g., a discontinuous epitope).

[0037] As used herein, the term “antibody” refers to an immunoglobulin that recognizes and specifically binds to one or more target antigens, such as proteins, polypeptides, peptides, carbohydrates, polynucleotides, lipids, or combinations thereof. This binding occurs at one or more epitopes on the antigen via at least one antigen-recognition site within the variable region of the immunoglobulin. The variable region is most important for binding specificity and affinity. As used herein, the term “antibody” includes intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments, single-chain Fv(scFv) variants, multispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, hybrid antibodies, fusion proteins, and any other immunoglobulin molecules, including antigen-recognition sites, as long as the antibody exhibits the desired biological activity. Antibodies may be (i) any of the five major classes of immunoglobulins based on the identity of their heavy chain constant domains—α (IgA), δ (IgD), ε (IgE), γ (IgG), and μ (IgM)—or (ii) a subclass (isotype) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The light chain may be either λ or κ. Antibodies may be naked or conjugated with other molecules, such as toxins, drugs, radioisotopes, chemotherapeutic agents, etc.

[0038] In one aspect, an "intact antibody" includes a tetramer composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50 - 70 kD). The heavy and light chains are linked via various covalent and non-covalent bonds (e.g., disulfide bonds) that vary in number and amount between different immunoglobulin classes. In one aspect, each chain includes a variable region and a constant region. The antigen recognition site of the variable region is composed of hypervariable regions or complementarity determining regions (CDRs) and framework regions. The framework regions typically do not contact the antigen but provide structural support for the CDRs. The constant region interacts with other immune cells in the body. Between the constant region and the variable region (only IgG, IgD, IgA, not IgM or IgE), there is a hinge region in the center between the two heavy chains that provides flexibility for linking antigen binding.

[0039] The following is a non-exhaustive list of different antibody forms that all retain antigen binding activity: (1) Whole immunoglobulin (also called an "intact" antibody) (two light chains and two heavy chains, e.g., a tetramer). (2) Immunoglobulin polypeptide (light chain or heavy chain). (3) Antibody fragments, e.g., Fv (monovalent or bivalent variable region fragments) that are variable regions (e.g., V L and / or V H ), Fab (V L C L V H C H ), F(ab’)2, Fv (V L V H ), scFv (single-chain Fv) (a polypeptide containing V L and V H linked by a linker, e.g., a peptide linker), (scFv)2, sc(Fv)2, bispecific sc(Fv)2, bispecific (scFv)2, minibody (sc(Fv)2 fused to a CH3 domain), and can only include tribody which is a trivalent sc(Fv)3 or trispecific sc(Fv)3. (4) Polyvalent antibodies (antibodies that contain binding regions that bind to two different epitopes or proteins, such as "scorpion" antibodies). (5) A fusion protein containing an immunoglobulin binding site fused to another amino acid sequence (such as a fluorescent protein).

[0040] As used herein, the term “antibody fragment” refers to a portion or part of an antibody or antibody chain that contains fewer amino acid residues than an intact or complete antibody or antibody chain, and that binds to an antigen or competes with the intact antibody. Fragments can be obtained by chemical or enzymatic treatment of an intact or complete antibody or antibody chain. Fragments can also be obtained by recombinant means. For example, the F(ab')2 fragment can be produced by treating an antibody with pepsin. The resulting F(ab')2 fragment can be processed to reduce the disulfide crosslinks and produce the Fab' fragment. Papain digestion can result in the formation of the Fab fragment. Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, and other fragments can also be constructed by recombinant expression techniques.

[0041] While various antibody fragments are defined in relation to the digestion products of intact antibodies, those skilled in the art will understand that such fragments may also be synthesized de novo chemically or constructed and expressed using recombinant DNA methods.

[0042] A single-stranded Fv (scFv) is a V molecule linked by a linker, such as a peptide linker. L and V HThis refers to polypeptides containing scFv. scFv can also be used to form tandem (or divalent) scFv or diabodies. The formation and characterization of tandem scFv and diabodies are described, for example, in Asano et al. (2011) J Biol. Chem. 286: 1812; Kenanova et al. (2010) Prot Eng Design Sel 23: 789; and Asano et al. (2008) Prot Eng Design Sel 21: 597.

[0043] The antibody fragment further comprises an Fd antibody (the heavy chain portion contained in the Fab fragment) and a single-domain antibody. The single-domain antibody (sdAb) is a variable domain of either the heavy chain or the light chain produced by recombinant methods.

[0044] As used herein, the term “CDR sequence set” refers to the three heavy-chain CDRs and / or three light-chain CDRs of a particular antibody described herein. “Light-chain” CDR sequence set refers to the light-chain CDR sequence. “Heavy-chain” CDR sequence set refers to the heavy-chain CDR sequence. “Complete” CDR sequence set refers to both the heavy-chain and light-chain CDR sequences. For example, in the case of antibody LRP5-A7, the complete CDR sequence set is SVSSA, as shown in Table 1. (SEQ ID NO: 34) (CDR L1), SASSLYS (SEQ ID NO: 35) (CDR L2) AWGWGLF (SEQ ID NO: 36) (CDR L3), LYSSSM (SEQ ID NO: 50) (CDR H1), SIYPYYGYTY (SEQ ID NO: 11) (CDR H2), and HGAM (SEQ ID NO: 21) (CDR H3) is included or consists of them. The CDR sequence of each CDR includes, essentially consists of, or can consist of, the CDRs in Table 1, for example. The CDRs are predicted based on IMGT sequence alignment.

[0045] As used herein, the term “monoclonal antibody” refers to a clonal preparation or composition of an antibody having a single binding specificity and affinity to a given epitope on an antigen (a “monoclonal antibody composition”). “Polyclonal antibody” refers to a preparation or composition of antibodies that arise for a single antigen but have different binding specificities and affinities (a “polyclonal antibody composition”).

[0046] As used herein, the term “chimeric antibody” refers to an antibody having an amino acid sequence derived from two or more species. In one embodiment, both the light and heavy chain variable regions correspond to the variable regions of an antibody derived from one species of mammal (e.g., mouse, rat, rabbit) having desired specificity, affinity, and ability, while the constant region is homologous to a sequence derived from another species (typically in the subject being treated, e.g., human) to avoid inducing an immune response.

[0047] As used herein, the term “humanized antibody” refers to a chimeric antibody in which CDRs obtained from the VH and VL regions of a non-human antibody having desired specificity, affinity, and competence are grafted onto a human framework sequence. In one embodiment, the framework residues of the humanized antibody are modified to improve and optimize the antibody’s specificity, affinity, and competence. Humanization, i.e., substitution of the corresponding sequence of a human antibody with a non-human CDR sequence, can be carried out according to the methods described, for example, U.S. Patent Nos. 5,545,806, 5,569,825, 5,633,425, 5,661,016, Riechmann et al., Nature 332: 323-327 (1988); Marks et al., Bio / Technology 10: 779-783 (1992); Morrison, Nature 368: 812-13 (1994); Fishwild et al., Nature Biotechnology 14: 845-51 (1996).

[0048] As used herein, the term "human antibody" refers to an antibody produced by a human being, or an antibody having a corresponding amino acid sequence prepared by any technique known in the art.

[0049] As used herein, the term “hybrid antibody” refers to an antibody in which pairs of heavy and light chains derived from antibodies having different antigenic determinant regions are assembled together such that the resulting tetramer can recognize and bind to two different epitopes or two different antigens. A hybrid antibody may be bispecific (binding to two different antigens or epitopes) or multispecific (binding to more than one different antigen or epitope).

[0050] As used herein, an antibody is "single-specific" if all of its antigen-binding sites bind to the same epitope.

[0051] As used herein, an antibody is "bispecific" if it has at least two different antigen-binding sites that bind to different epitopes or antigens, respectively.

[0052] As used herein, an antibody is "polyvalent" if it has multiple antigen-binding sites. For example, a tetravalent antibody has four antigen-binding sites.

[0053] Binding specificity can be defined with respect to the relative dissociation constant (Kd) of the antibody (or other targeting moiety) against the target, compared to the dissociation constants of the antibody and other materials in the environment, or generally unrelated molecules. Relatively large (relatively high) K d K represents a relatively low affinity interaction. d Conversely, a relatively small (relatively low) K d K represents a relatively high affinity interaction or a relatively tight bond. d As just one example, consider the K of an antibody that specifically binds to a target. dThis can be femtomole, picomole, nanomolar or micromolar, and is the K of an antibody that binds to an unrelated material. d This can be millimoles or more. Binding affinity is in the micromolar range (kD=10 -4 ~10 -6 ), nanomolar range (kD=10 -7 M~10 -9 M), picomolar range (kD=10 -10 M~10 -12 M), or femtomole range (kD=10 -13 M~10 -15 M) is a possibility.

[0054] When used in this specification, the antibody is 10 -4 When an antibody binds to an antigen or epitope with a Kd of less than M (i.e., in the micromolar range), it is said to "bind" to or "recognize" the antigen or epitope. The term "bind" in relation to a cell type (e.g., an antibody that binds to cancer cells) typically indicates that the active agent binds to the majority of cells within a pure population of those cells. For example, an antibody that binds to a given cell type will typically bind to at least two-thirds of the cells within the population of cells indicated (e.g., 67, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%). In some cases, binding to a polypeptide can be assayed by comparing the binding of an antibody to cells that present the polypeptide with the binding (or lack thereof) of an antibody to cells that do not express the polypeptide. Those skilled in the art will recognize that some variability will occur depending on the method used to determine binding and / or the threshold. The affinity of an antibody for a target can be determined according to methods known in the art, for example, as outlined in Ernst et al. Determination of Equilibrium Dissociation Constants, Therapeutic Monoclonal Antibodies (Wiley & Sons ed. 2009).

[0055] As used herein, the term "relatively high affinity" means that antibody X binds more strongly to target Y than to target Z (K on ) and / or dissociation constant (K off ) refers to the relative degree of antibody binding when the dissociation constant is small, in this context, antibody X has a greater affinity for target Y than Z. Similarly, the term “relatively low affinity” as used herein refers to the degree of antibody binding when antibody X binds to target Y with a weaker and / or larger dissociation constant than target Z, in this context, antibody X has a smaller affinity for target Y than Z. The affinity of binding between an antibody and its target antigen is 1 / K D K equal to A It can be expressed as, where K D is k on / k off It is equal to k. on and k off The value can be measured using surface plasmon resonance technology, for example, using the Molecular Affinity Screening System (MASS-1) (Sierra Sensors GmbH, Hamburg, Germany). An antagonist or blocking antibody is an antibody that partially or completely blocks, inhibits, or neutralizes the biological activity associated with a target antigen compared to the activity under similar physiological conditions in the absence of the antibody. Antagonists can be competitive, non-competitive, or irreversible. A competitive antagonist is a substance that binds to the native ligand or receptor at the same site as the native ligand-receptor interaction, or binds allosterically in a way that induces a change that prevents normal binding. A non-competitive antagonist binds at a different site than the native ligand-receptor interaction, but reduces the KD or signal resulting from the interaction. An irreversible inhibitor causes covalent modification of the receptor, preventing any subsequent binding.

[0056] As used herein, the term “avidity” refers to the overall stability of the binding complex between an antibody and a target antigen. It is defined by three factors: (i) the intrinsic affinity of the antibody to the antigen, (2) the titer of the antibody, and (3) the geometric arrangement of the interacting components. Affinity is the strength of the interaction between the antibody and a single target, whereas avidity is the cumulative strength of multiple affinities. In one embodiment, the antibody disclosed herein is divalent.

[0057] As used herein, an antibody "preferentially binds" to the first antigen compared to the second antigen if it binds to the first antigen with a greater affinity than the second antigen. Preferential binding can be at least one of two, five, nine, ten, twenty, thirty, forty, fifty, one hundred, five hundred, or one thousand times greater affinity. Therefore, for example, if an antibody binds to LRP5 with a greater affinity than it binds to LRP6, it preferentially binds to LRP5 compared to LRP6.

[0058] When used in this specification, the antibody is 1 × 10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 M, 1×10 -12 An antibody is said to "specifically bind" to or be "specific" to a target antigen or its target group if it binds to the target antigen or each member of its target group with at least one affinity of M, for example, with at least twice the affinity it has for the non-target antigen being compared. Typically, specific binding is characterized by binding to the antigen with sufficient affinity for the antibody to be useful as a diagnostic agent for detecting the antigen or epitope and / or as a therapeutic agent when targeting the antigen or epitope.

[0059] As used herein, an antibody “blocks” or “antagonizes” ligand-receptor binding if it competitively reduces or prevents any interaction between the ligand and the receptor. In one embodiment, the measured reduction level may be at least one of 5%, 10%, 25%, 50%, 80%, 90%, 95%, 97.5%, 99%, 99.5%, or 99.9% of control (e.g., untreated) cells. For example, an antibody that antagonizes or blocks the binding of a Wnt ligand to the LRP5 receptor competitively reduces or prevents the interaction of the Wnt protein with the LRP5 receptor. This results in attenuation or blockage of downstream signaling events associated with Wnt signaling. These include, for example, activation of disheveled, lysis of the β-catenin degradation complex, a decrease in cytosolic levels of β-catenin, and / or a decrease in the activity of TCF / LEF-mediated transcription.

[0060] The term "capture" with respect to antibody targets (e.g., antigens, analytes, immune complexes) typically indicates that the antibody binds to the majority of the antibody target in a pure population (assuming appropriate molar ratios). For example, an antibody that binds to a given antibody target typically binds to at least two-thirds of the antibody target in solution (e.g., at least 67, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%). Those skilled in the art will recognize that some variation will occur depending on the method used to determine binding and / or the threshold.

[0061] The term “conjugate” refers to a first molecule, such as an antibody (“immunoconjugate”), that is chemically bound to a portion, such as a detectable label, or to a biologically active portion, such as a drug, toxin, chemotherapeutic agent, or cytotoxic agent. Therefore, this disclosure intends to describe antibodies conjugated with one or more portions. Furthermore, the antibody may be a “conjugated antibody” or a “non-conjugated antibody” (i.e., not conjugated with a portion).

[0062] As used herein, the term “antibody-drug conjugate,” i.e., “ADC,” refers to an antibody conjugated with a drug. Typically, the conjugation involves a covalent bond via a linker.

[0063] As used herein, the term “labeled” molecule (e.g., nucleic acid, protein, or antibody) means a molecule to which a detectable label is attached, either covalently via a linker or chemical bond, or noncovalently via an ionic bond, van der Waals bond, electrostatic bond, or hydrogen bond, so that the presence of the molecule can be detected by detecting the presence of the detectable label attached to the molecule.

[0064] As used herein, the term “detectable label” refers to a composition that is detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. Examples of detectable labels are described herein and include, but are not limited to, colorimetric labels, fluorescent labels, chemiluminescent labels, enzymatic labels, and radioactive labels. For the purposes of this disclosure, a detectable label may also be a moiety that does not generate a signal itself (e.g., biotin) but binds to a second moiety that can generate a signal (e.g., labeled avidin).

[0065] The term "crosslinked" in relation to antibodies refers to the attachment of an antibody to a solid or semi-solid matrix (e.g., Sepharose, beads, microtiter plates), or to another protein or antibody. For example, antibodies can be polymerized to create antibody complexes with multiple (more than two) antigen-binding sites. Antibodies can be polymerized by expressing them as high-titer isotypes (e.g., IgA or IgM, which typically form complexes of two or five antibodies each). Antibody polymerization can also be carried out by using crosslinking agents containing reactive groups (e.g., carbodiimides, NHS esters, etc.) that can link proteins. Methods and compositions for crosslinking antibodies to a matrix are described, for example, in the catalogs and websites of Abcam and New England Biolab (available at abcam.com and neb.com). Crosslinking agent compounds with various reactive groups are described, for example, in the catalogs and websites of Thermo Fisher Scientific (available at piercenet.com).

[0066] As used herein, the term “immunoassay” refers to a method of detecting an analyte by detecting the binding between an antibody that recognizes the analyte and the analyte itself.

[0067] As used herein, the term “expression construct” refers to a polynucleotide containing a heterogeneous nucleotide sequence to be expressed (i.e., a sequence to which the expression regulatory sequence is not normally ligated) and an expression regulatory sequence functionally linked to it. As used herein, the term “expression vector” refers to a polynucleotide containing an expression construct and a sequence sufficient for replication in a host cell or insertion into a host chromosome. Plasmids and viruses are examples of expression vectors. As used herein, the term “expression regulatory sequence” refers to a nucleotide sequence that regulates the transcription and / or translation of a nucleotide sequence functionally linked to it. Expression regulatory sequences include promoters, enhancers, repressors (transcriptional regulatory sequences), and ribosome-binding sites (translational regulatory sequences).

[0068] As used herein, the term "vector" includes any intermediate vehicle for a nucleic acid molecule that enables the nucleic acid molecule to be introduced into, for example, prokaryotic and / or eukaryotic cells and / or incorporated into the genome, and includes plasmids, phagemids, bacteriophages, or viral vectors, such as retrovirus-based vectors and adeno-associated virus vectors. As used herein, the term "plasmid" generally refers to extrachromosomal genetic material, usually a circular double-stranded DNA construct, that can replicate independently of chromosomal DNA.

[0069] As used herein, when an expression regulatory element functions within a cell to control the transcription of a nucleotide sequence, the nucleotide sequence is "functionally linked" to the expression regulatory element. This includes promoting the transcription of the nucleotide sequence through an interaction between a polymerase and a promoter.

[0070] As used herein, “host cell” refers to a recombinant cell containing an expression construct.

[0071] As used herein, the term “biological sample” means a sample containing cells (e.g., tumor cells) or biological molecules derived from cells. A biological sample may be obtained from a subject, e.g., a patient, from an animal such as an animal model, or from cultured cells, e.g., from a cell line or cells taken from a patient and grown in culture for observation. A biological sample may include tissue and / or fluid. A biological sample may be obtained from any biological source, without limitation, including blood, blood fractions (e.g., serum or plasma), cerebrospinal fluid (CSF), lymph, tears, saliva, sputum, buccal swab, breast milk, urine, or feces. A biological sample may be biopsy material, e.g., tissue biopsy material, e.g., needle biopsy material, fine-needle biopsy material, surgical biopsy material, etc. A sample may include a tissue sample with a lesion or suspected lesion, but a biological sample may also originate from another site, e.g., a suspected site of metastasis, a lymph node, or blood. A biological sample may be part of a sample taken from a subject. Examples of tissue samples include brain tissue samples or nerve tissue samples. Methods for obtaining such biological samples, including, but not limited to, standard blood collection procedures, are known in the art.

[0072] As used herein, the term “diagnosis” refers to the relative probability that a subject has a disorder such as cancer. Similarly, the term “prognosis” refers to the relative probability that a particular future outcome may occur in a subject. For example, in the context of this disclosure, prognosis may refer to the likelihood that an individual will develop cancer, the likelihood that an individual will have recurrences, the likelihood that cancer will metastasize, the likelihood that cancer will be cured, or the possible severity of the disease (e.g., the severity of symptoms, the rate of functional decline, the survival rate, etc.). The terms are not intended to be absolute, as would be understood by those skilled in the art of medical diagnosis.

[0073] As used herein, the terms “therapy,” “treatment,” “therapeutic intervention,” and “improvement” refer to any activity that results in a reduction in the severity of symptoms. In the case of cancer, treatment may include, for example, a reduction in tumor size, the number of cancer cells, growth rate, metastatic activity, a reduction in non-cancerous cell death, a reduction in nausea and other side effects of chemotherapy or radiotherapy. The terms “treat” and “prevent” are not intended to be absolute terms. Treatment and prevention may include delaying the onset of any of the symptoms, improvement in symptoms, improvement in patient survival, or an increase in survival time or survival rate. Treatment and prevention may be complete (undetectable levels of neoplasms) or partial, such that fewer neoplasms are found in the patient than would have occurred without the intervention. The effect of treatment can be compared to an untreated individual, or a pool of individuals, or to the same patient at different points in time before or during treatment. In some aspects, the severity of the disease may be reduced by at least 10% compared to, for example, an individual before administration or an untreated control individual. In some cases, the severity of the disease decreases by at least 25%, at least 50%, at least 75%, at least 80%, or at least 90%, or in some cases, it may no longer be detectable using standard diagnostic techniques.

[0074] As used herein, the terms “effective dose,” “effective dosage,” and “therapeutic effective dose” refer to the amount of an active substance, such as an antibody or immunoconjugate, sufficient to produce a desired response, such as reducing or eliminating the signs or symptoms of a disease or improving a disorder. In some examples, “effective dose” is the effective dose that treats (including prevention) one or more symptoms and / or underlying causes of any disorder or disease and / or prevents the progression of the disease. For example, for a given parameter, a therapeutic effective dose shows an increase or decrease in therapeutic effect by at least one of 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic effectiveness can also be expressed as an increase or decrease of “~ times.” For example, a therapeutic effective dose may have at least one of 1.2 times, 1.5 times, 2 times, 5 times, or more of the effect compared to a control.

[0075] As used herein, the term “pharmaceutical composition” means a composition comprising a pharmaceutical compound (e.g., a drug) and a pharmaceutically acceptable carrier.

[0076] As used herein, the term “pharmaceutically acceptable” refers to a carrier that is compatible with other components of a pharmaceutical composition and can be safely administered to a subject. The term is used synonymously with “physiologically acceptable” and “pharmacologically acceptable.” Pharmaceutical compositions and techniques for their preparation and use are known to those skilled in the art in light of this disclosure. For a detailed list of suitable pharmacological compositions and techniques for their administration, refer to the following documents: Remington's Pharmaceutical Sciences, 17th ed. 1985; Brunton et al., “Goodman and Gilman's The Pharmacological Basis of Therapeutics,” McGraw-Hill, 2005; University of the Sciences in Philadelphia (eds.), “Remington: The Science and Practice of Pharmacy,” Lippincott Williams & Wilkins, 2005; and University of the Sciences in Philadelphia (eds.), “Remington: The Principles of Pharmacy Practice,” Lippincott Williams & Wilkins, 2008.

[0077] Pharmacoherent carriers are generally sterilized, at least for use in humans. Pharmaceutical compositions generally contain an active ingredient for buffering and preservation during storage, and may include buffers and carriers for appropriate delivery depending on the route of administration. Examples of pharmaceutically acceptable carriers include, but are not limited to, ordinary (0.9%) saline, phosphate-buffered saline (PBS) Hanks equilibrium salt solution (HBSS), and multiple electrolyte solutions, such as PlasmaLyte ATM (Baxter).

[0078] Acceptable carriers, excipients, and / or stabilizers are non-toxic to the recipient at the dosage and concentration used, and include buffers, e.g., phosphates, citrates, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine, and citric acid; preservatives (ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methylparaben or propylparaben, benzalkonium chloride, or combinations thereof, etc.); amino acids, e.g., arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, etc. Lanine, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof; monosaccharides, disaccharides, and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, e.g., gelatin or serum albumin; chelating agents, e.g., EDTA; sugars, e.g., trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or nonionic surfactants, e.g., Tween, Pluronics, Triton-X, or polyethylene glycol (PEG).

[0079] The terms “dose” and “administered amount” are used interchangeably herein. Dose refers to the amount of active ingredient given to an individual at each administration. In this invention, dose may refer to the concentration of an antibody or related component, e.g., the amount of a therapeutic agent, or the amount of a radiolabel. The dose varies depending on several factors, including the frequency of administration, the size and tolerance of the individual, the severity of the condition, the risk of side effects, the route of administration, and the imaging modality of the detectable label (if present). Those skilled in the art will recognize that the dose may be modified depending on the factors mentioned above or based on the progress of treatment. The term “dosage form” refers to a specific form of a pharmaceutical product, depending on the route of administration. For example, the dosage form may be a liquid, e.g., saline solution for injection.

[0080] As used herein, the term “subject” refers to an individual animal. As used herein, the term “patient” refers to a subject under the care or supervision of a healthcare provider, such as a physician or nurse. Subjects include mammals, e.g., humans and non-human primates, e.g., monkeys, as well as dogs, cats, horses, cattle, rabbits, rats, mice, goats, pigs, and other mammalian species. Subjects may also include birds. A patient may be an individual seeking treatment, monitoring, adjustment or modification of an existing treatment regimen, etc. The term “cancer subject” refers to an individual diagnosed with cancer. Cancer patients may include individuals not receiving treatment, individuals currently receiving treatment, individuals who have undergone surgery, and individuals who have discontinued treatment.

[0081] In the context of cancer treatment, individuals requiring treatment may include those with cancer or a precancerous condition, those who have previously had cancer and are at risk of recurrence, those suspected of having cancer, and those receiving standard cancer treatments such as radiation therapy or chemotherapy.

[0082] The terms “cancer,” “tumor,” and “transformed” include precancerous cells, neoplastic cells, transformed cells, and cancerous cells, and can refer to solid tumors or non-solid cancers (see, for example, Edge et al. AJCC Cancer Staging Manual (7th ed. 2009); Cibas and Ducatman Cytology: Diagnostic principles and clinical correlates (3rd ed. 2009)). Cancer includes both benign neoplasms and malignant neoplasms (abnormal growth). “Transformation” refers to spontaneous or induced phenotypic changes, such as cell immortalization, morphological changes, abnormal cell growth, contact inhibition, and reduced fixation, and / or malignancy (see Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd ed. 1994)). Transformation can result from infection with transforming viruses and the integration of new genomic DNA, or the uptake of exogenous DNA, but can also occur spontaneously or after exposure to carcinogens.

[0083] The term "cancer" can refer to any cancer, without limitation, including leukemia, carcinoma, sarcoma, adenocarcinoma, lymphoma, solid tumors, and lymphoma. Examples of various types of cancer include, but are not limited to, lung cancer (e.g., non-small cell lung cancer, i.e., NSCLC), breast cancer, prostate cancer, colorectal cancer, bladder cancer, ovarian cancer, leukemia, liver cancer (i.e., hepatocellular carcinoma), renal cancer (i.e., renal cell carcinoma), thyroid cancer, pancreatic cancer, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, stomach cancer, kidney cancer, central nervous system cancer, skin cancer, glioblastoma, and melanoma.

[0084] As used herein, a chemical entity such as a polypeptide is "substantially pure" if it is the primary chemical entity of its kind (e.g., a polypeptide) in the composition. This includes chemical entities that constitute more than 50%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 99.99% of the chemical entities of its kind in the composition.

[0085] The term "isolated antibody" refers to an antibody produced in vivo or in vitro, isolated from the source that produced the antibody, such as an animal, hybridoma, or other cell line (recombinant insect cells, yeast cells, or bacterial cells that produce antibodies).

[0086] "Substantially pure" or "isolated" means that the species of interest is the dominant species present (i.e., more abundant on a molar basis than any other individual polymer species in the composition), and that the substantially purified fraction is a composition in which the species of interest constitutes at least about 50% (on a molar basis) of all polymer species present. Generally, a substantially pure composition means that about 80% to 90% or more of the polymer species present in the composition are the purified species of interest. If the composition essentially consists of a single polymer species, the species of interest is purified to an essential homogeneity (contaminating species cannot be detected in the composition by conventional detection methods). Solvent species, small molecules (<500 Daltons), stabilizers (e.g., BSA), and elemental ion species are not considered polymer species for the purposes of this definition.

[0087] As used herein, the term “sequence identity” refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, gaps can be introduced in the sequence of the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid sequence or nucleic acid sequence). Then, amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical overlapping positions / total number of positions × 100%). In one embodiment, the two sequences are the same length. Determining the percentage identity between two sequences can also be achieved using mathematical algorithms. A preferred non-restrictive example of a mathematical algorithm used to compare two sequences is the algorithm described in Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877. Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215: 403. For example, a BLAST nucleotide search can be performed using NBLAST nucleotide program parameters set to score=100 and word length=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of this application. For example, a BLAST protein search can be performed using XBLAST program parameters set to score-50 and word length=3 to obtain amino acid sequences homologous to the protein molecules described herein.To obtain gapped alignments for comparative purposes, Gapped BLAST, as described in Altschul et al., 1997, Nucleic Acids Res. 25: 3389-3402, can be used. Alternatively, PSI-BLAST can be used to perform iterative searches to detect intermolecular distance relationships (ibid.). When using the BLAST program, Gapped BLAST program, and PSI-Blast program, the default parameters of each program (e.g., XBLAST and NBLAST) can be used (see, for example, the NCBI website). Another preferred non-restrictive example of a mathematical algorithm used for sequence comparison is the algorithm in Myers and Miller, 1988, CABIOS 4: 11-17. Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 weighted residue table, gap length penalty 12, and gap penalty 4 can be used. The percentage identity between two sequences can be determined using the same techniques as described above, with or without allowing gaps. In calculating percentage identity, typically only exact matches are counted.

[0088] In antibodies, percentage sequence identity can be determined when the antibody sequence is maximally aligned by IMGT. After alignment, if the target antibody region (e.g., the entire maturation variable region of the heavy or light chain) is compared to the same region of the reference antibody, the percentage sequence identity between the target antibody region and the reference antibody region is calculated by multiplying the number of positions occupied by the same amino acids in both the target antibody region and the reference antibody region by the total number of aligned positions in these two regions by 100 to convert it into a percentage.

[0089] Percent amino acid sequence identity can also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program is available from the National Institute of Health, Bethesda, Md. NCBI-BLAST2 uses several search parameters, all of which are set to default values, including, for example, unmask=yes, strand=all, expected occurrences=10, minimum low complexity length=15 / 5, multipath e value=0.01, multipath constant=25, final gapped alignment dropoff=25, and scoring matrix=BLOSUM62.

[0090] In situations where NCBI-BLAST2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (or, as can be expressed, a given amino acid sequence A that has or contains a specific % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y In the formula, X is the number of amino acid residues scored as identical in the alignment of A and B by the sequence alignment program NCBI-BLAST2, and Y is the total number of amino acid residues in B. It is understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. As used herein, the term “nucleic acid sequence” refers to a sequence of nucleoside monomers or nucleotide monomers consisting of naturally occurring bases, sugars, and sugar-coated (skeletal) bonds, and includes cDNA. The term also includes modified or substituted sequences containing monomers or parts thereof that do not exist in nature. The nucleic acid sequences of this application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine, and uracil. Sequences may also contain modified bases. Examples of such modified bases include adenine, guanine, cytosine, thymidine, and uracil in aza and deaza, as well as xanthine and hypoxanthine. Polynucleotides containing non-transcribeable nucleotide bases are understood to be useful as probes, for example, in hybridization assays. Nucleic acids can be either double-stranded or single-stranded, representing a sense strand or an antisense strand. Furthermore, the term “nucleic acid” includes complementary nucleic acid sequences and codon-optimized codon equivalents or synonymous codon equivalents.

[0091] As used herein, the term “isolated nucleic acid” refers to a nucleic acid substantially free of cell material or culture medium when produced by recombinant DNA technology, or, if chemically synthesized, to a chemical precursor or other chemical substance. Isolated nucleic acids also substantially free of sequences naturally adjacent to the nucleic acid from which they originate (i.e., sequences located at the 5' and 3' ends of the nucleic acid).

[0092] "At least moderately stringent hybridization conditions" means that conditions are selected that promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization can occur for all or part of a nucleic acid sequence molecule. The hybridized portion is typically at least 15 (e.g., 20, 25, 30, 40, or 50) nucleotides long. Those skilled in the art will recognize that the stability of nucleic acid double-stranded or hybrid nucleic acids is determined by Tm (Tm = 81.5°C - 16.6(Log10[Na+]) + 0.41(%(G+C) - 600 / l), or a similar formula), which is a function of sodium ion concentration and temperature in a sodium-containing buffer. Therefore, the parameters of the washing conditions that determine hybrid stability are sodium ion concentration and temperature. To identify molecules that are similar to but not identical to known nucleic acid molecules, it can be assumed that a 1% mismatch results in a decrease of approximately 1°C in Tm. For example, if nucleic acid molecules with >95% identity are required, the final washing temperature will decrease by approximately 5°C. Based on these considerations, those skilled in the art will be able to easily select appropriate hybridization conditions. In preferred embodiments, stringent hybridization conditions are selected. As an example, to achieve stringent hybridization, the following conditions may be used: hybridization with 5× sodium chloride / sodium citrate (SSC) / 5× Denhardt's solution / 1.0% SDS at Tm-5°C based on the above formula, followed by washing with 0.2× SSC / 0.1% SDS at 60°C. Moderately stringent hybridization conditions include a washing step with 3× SSC at 42°C. However, it is understood that equivalent stringency can be achieved using alternative buffers, salts, and temperatures.Additional guidelines regarding hybridization conditions can be found in *Current Protocols in Molecular Biology*, John Wiley & Sons, NY, 2002, and in *Molecular Cloning: A Laboratory Manual*, Sambrook et al., *Cold Spring Harbor Laboratory Press*, 2001.

[0093] As used herein and as well understood in the art, the terms “to treat” or “to cure” mean a method for obtaining beneficial or desired outcomes, including clinical outcomes. Beneficial or desired clinical outcomes may include, without limitation, reduction or improvement of one or more symptoms or conditions, whether detectable or undetectable; attenuation of the severity of the disease; a state of stabilization (i.e., no worsening) of the disease; prevention of disease progression; prolongation or delay of disease progression; improvement or mitigation of the disease state; reduction of disease recurrence; and remission (whether partial or total), whether detectable or undetectable. “To treat” or “to cure” may also mean extending survival compared to the predicted survival time without treatment. As used herein, “to treat” or “to cure” also includes prophylactic treatment. For example, a subject with cancer may be treated to prevent progression, and to prevent progression, may be treated with antibodies, immunoconjugates, nucleic acids, or compositions described herein.

[0094] As used herein, the term “administration” means providing or delivering to a target an active substance, such as a composition containing an effective amount of antibody, by an effective route, such as an intratumoral or intravenous route.

[0095] As used herein, the term “diluent” refers to a pharmaceutically acceptable carrier that does not inhibit the physiological activity or properties of an active compound, such as an antibody or immunoconjugate, nor does it irritate the subject or invalidate the biological activity and properties of the administered compound. Diluents include, as is well known to those skilled in the art, all kinds of solvents, dispersions, coatings, surfactants, antioxidants, preservatives, antiseptics, binders, excipients, disintegrants, lubricants, such similar materials, and combinations thereof (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289–1329, incorporated herein by reference). Its use in a pharmaceutical composition is intended unless any conventional carrier is incompatible with the active ingredient.

[0096] A composition or method that "comprising" or "including" one or more of the listed elements may also include other elements not specifically listed. For example, a composition that "comprises" or "includes" an antibody may contain the antibody alone or in combination with other components.

[0097] For understanding the scope of this disclosure, the terms “consisting” and their derivatives as used herein are intended to be closed terms that specify the presence of described features, elements, components, groups, integers, and / or processes, and exclude the presence of other features, elements, components, groups, integers, and / or processes not described.

[0098] In this specification, enumerations of numerical ranges by endpoints include all numbers and fractions contained within that range (for example, 1–5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It should also be understood that all such numbers and fractions are presumed to be modified by the term “approximately”. Furthermore, it should be understood that the singular articles “a,” “an,” and “the” include multiple references unless otherwise explicitly indicated in the context. For example, the term “antibody” or “at least one antibody” may include multiple antibodies, including mixtures thereof.

[0099] The terms "Frizzled" and "FZD" refer to any gene or protein member of the Frizzled family, depending on the context. Frizzled proteins are involved in the activation of Dishevelled proteins in the cytosol. Frizzled refers to any of Frizzled-1, Frizzled-2, Frizzled-3, Frizzled-4, Frizzled-5, Frizzled-6, Frizzled-7, Frizzled-8, Frizzled-9, and Frizzled-10.

[0100] "Lipoprotein receptor-related proteins," "low-density lipoprotein receptor-related proteins" (HGNC), or "pro-low-density lipoprotein receptor-related proteins" (UniProt), abbreviated as "LRP," are a group of genes and proteins. They include LRP1, LRP1B, LRP2 (megalin), LRP3, LRP4, LRP5, LRP6, LRP8 (apolipoprotein e receptor), LRP10, LRP11, and LRP12. LRP5 and LRP6 are part of the LRP5 / LRP6 / Frizzled coreceptor group involved in the classical Wnt pathway. LRP5 is also known as LRP5, BMND1, EVR1, EVR4, HBM, LR3, LRP-5, LRP7, OPPG, OPS, OPTA1, VBCH2, and LDL receptor-related protein 5. The LRP5 gene has ENTREZ gene ID: 4041, and its protein has the NCBI reference sequence: NP_002326. The LRP6 gene has ENTREZ gene ID: 4040, and its protein has the NCBI reference sequence: NP_002327. LRP6 is also known as ADCAD2 and STHAG7.

[0101] II. Disorders related to LRP signaling dysregulation Wnt binding to LRP5 or LRP6 destabilizes the β-catenin binding complex and causes β-catenin degradation. As a result, intracellular β-catenin levels increase. Therefore, a method for blocking Wnt binding to LRP family proteins such as LRP5 or LRP6 is provided herein.

[0102] "LRP-related disorders" (e.g., "LRP5-related disorders" or "LRP6-related disorders") refer to medical conditions or diseases associated with dysregulation of the specific LRP receptors mentioned. Dysregulation refers to an abnormal decrease or increase in signaling that affects normal β-catenin-mediated transcriptional changes or any other intracellular signaling pathways regulated by these receptors. For example, an abnormal LRP-related increase in signaling via the classical Wnt signaling pathway (Wnt3 / Wnt3A) is associated with certain cancers and increased bone density, while an abnormal LRP-related decrease in signaling is associated with decreased bone density.

[0103] Antibodies that block Wnt binding to LRP5 or LRP6 are useful in the treatment of cancer. In particular, methods that block Wnt binding to LRP5 are useful in the treatment of brain cancer, breast cancer, colon cancer, endometrial cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, skin cancer, gastric cancer, and testicular cancer.

[0104] III. Anti-LRP5 antibody A. Antibodies LRP5 and LRP6 each possess two Wnt binding sites. The majority of Wnt activators of β-catenin signaling, including Wnt1, 2, 2b, 6, 8a, 9a, 9b, and 10b, associate with the β-propeller 1 and β-propeller 2 regions of LRP5 and LRP6. These sites are referred to herein as “non-Wnt3a binding sites.” Wnt proteins Wnt3 and Wnt3a are known to associate with distinct sites on β-propeller regions 3 and 4 of LRP5 and LRP6. These sites are referred to herein as “Wnt3a binding sites.” Antibody binding to LRP5 or LRP6 that blocks the binding of Wnt ligands to a particular binding site reduces the activity of signaling pathways associated with Wnt ligand binding to that site. Such blocking activity may also, under certain circumstances, enhance the activity of signaling pathways associated with Wnt ligand binding to other binding sites. Therefore, for example, an antibody that blocks the binding of a Wnt ligand to a Wnt3a binding site may inhibit the activity of the Wnt3a signaling pathway and enhance the activity of the non-Wnt3a signaling pathway. Similarly, an antibody that blocks the binding of a Wnt ligand to a non-Wnt3a binding site may inhibit the activity of the non-Wnt3a signaling pathway and enhance the activity of the Wnt3a signaling pathway.

[0105] Antibodies against the LRP5 receptor are described herein. Some of these antibodies block the binding of Wnt ligands to the Wnt3a binding site of LRP5. Others of these antibodies block the binding of Wnt ligands to non-Wnt3a binding sites. These antibodies block ligand-Wnt binding and modulate the activity of the Wnt signaling pathway. These antibodies also have antiproliferative effects and have therapeutic potential for treating cancer and other diseases in which the LRP receptor is dysregulated.

[0106] Accordingly, one aspect of the present disclosure includes an isolated antibody that specifically binds to the LRP5 receptor. The antibody comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region comprising complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprising complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the amino acid sequence of the CDR comprises, essentially consists of, or comprises sequences selected from the sequences in Table 1 or Table 2.

[0107] In one embodiment, the antibody includes a set of CDR sequences selected from the CDR sequence sets in Table 1 for clones LRP5-A7, LRP5-A9, LRP5-C5, LRP5-C12, LRP5-D9, LRP5-E5, LRP5-G2, LRP5-G9, LRP5-G10, LRP5-G11, LRP5-H3, LRP5-H5, LRP5-H9, LRP5-R3O_D3, LRP5-R3_E8, and LRP5-R3O_G6.

[0108] Heavy chain variable regions and light chain variable regions are also described herein. Table 2 provides exemplary variable domain sequences of Fab heavy chains and Fab light chains derived from clones LRP5-A7, LRP5-A9, LRP5-C5, LRP5-C12, LRP5-D9, LRP5-E5, LRP5-G2, LRP5-G9, LRP5-G10, LRP5-G11, LRP5-H3, LRP5-H5, LRP5-H9, LRP5-R3O_D3, LRP5-R3_E8, and LRP5-R3O_G6. Antibodies containing sequences from Table 2 or substantially identical sequences, wherein the CDR is the set of CDR sequences identified in Table 1, are also intended. In another embodiment, the antibody comprises a heavy chain variable region including: (i) a heavy chain amino acid sequence as described in Table 2; (ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the heavy chain amino acid sequence as described in Table 2, wherein the CDR sequence is one of the CDR sequence sets described in Table 1; or (iii) a conservatively substituted amino acid sequence of (i), wherein the CDR sequence is one of the CDR sequence sets described in Table 1.

[0109] In another embodiment, the antibody comprises a light chain variable region including: (i) a light chain amino acid sequence as described in Table 2; (ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to the light chain amino acid sequence as described in Table 2, wherein the CDR sequence is one of the CDR sequence sets described in Table 1; or (iii) a conservatively substituted amino acid sequence of (i), wherein the CDR sequence is one of the CDR sequence sets described in Table 1.

[0110] In Table 1 below, antibodies are assigned to epitope groups based on their binding characteristics. Antibodies in epitope group 1 enhance the activity of Wnt ligands that bind to non-Wnt3A binding sites; however, they do not inhibit Wnt3A ligand activity. Antibodies in epitope group 2 inhibit the activity of Wnt ligands that bind to non-Wnt3a binding sites. Furthermore, antibody LRP5-G 10 enhances the activity of went ligands that bind to Wnt3a binding sites. Antibodies belonging to epitopes 3 and 4 bind to LRP5 but do not inhibit or enhance Wnt ligand activity. The epitope groups of the other antibodies were not determined.

[0111] The amino acid sequences of the CDRs of these antibodies are shown in Table 1.

[0112] (Table 1) CDR sequences of anti-LRP5 antibodies TIFF0007840842000002.tif215170TIFF0007840842000003.tif90170

[0113] Table 2 shows the amino acid and nucleotide sequences of the variable heavy chain domain and the variable light chain domain.

[0114] (Table 2) Heavy chain DNA sequences and light chain DNA sequences, as well as heavy chain amino acid sequences and light chain amino acid sequences for LRP5-A7, LRP5-A9, LRP5-C5, LRP5-C12, LRP5-D9, LRP5-E5, LRP5-G2, LRP5-G9, LRP5-G10, LRP5-G11, LRP5-H3, LRP5-H5, LRP5-H9, LRP5-R3O_D3, LRP5-R3_E8, and LRP5-R3O_G6. Table 2 discloses SEQ ID NO: 58 to 109 in order of appearance. TIFF0007840842000004.tif132129TIFF0007840842000005.tif137131TIFF0007840842000006.tif138132TIFF0007840842000007.t if132130TIFF0007840842000008.tif126128TIFF0007840842000009.tif133128TIFF0007840842000010.tif142135TIFF00078408420 00011.tif135133TIFF0007840842000012.tif149146TIFF0007840842000013.tif129128TIFF0007840842000014.tif131128TIFF000 7840842000015.tif134131TIFF0007840842000016.tif228150TIFF0007840842000017.tif225150TIFF0007840842000018.tif104150

[0115] In some embodiments, the variable domain sequences are at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similar outside the CDR region, and the CDR sequence set is 100% identical to the amino acid sequences shown in Table 1.

[0116] In another embodiment, a competing antibody is also provided that competes for binding with the antibody containing the CDR sequence set described herein. For example, in one embodiment, the competing antibody reduces the binding of the antibody containing the CDR sequence set to LRP5 CDR by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%.

[0117] As shown herein, the antibodies described herein have high affinity for LRP5. For example, in one embodiment, the antibodies have a binding affinity of about 1 nM to about 50 nM, as measured by surface plasmon resonance.

[0118] The antibody may be a humanized antibody or a chimeric antibody as described herein.

[0119] In some embodiments, the antibody is a single-chain antibody, which can be obtained, for example, by fusing a heavy chain and a light chain or a portion thereof together.

[0120] In some embodiments, the antibody is an antibody-conjugated fragment selected from Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, nanobodies, minibodies, diabodies, and their polymers.

[0121] In some other embodiments, the antibody is a conjugated fragment Fab. In some embodiments, the conjugated fragment is preferred, for example, for use in vitro.

[0122] In other embodiments, it may be preferable to have a polyvalent antibody or an antibody containing an Ig moiety.

[0123] As shown in the examples, the Fab fragment can be combined with Ig such as IgG. In one embodiment, IgG is IgG1, IgG2, IgG3, or IgG4.

[0124] B. Detectable labeled antibodies Detectable labels may include peptide sequences (such as myc tags, HA tags, V5 tags, or NE tags) that can be added to or introduced into antibodies described herein and generate a detectable signal directly or indirectly, fluorescent proteins or luminescent proteins (e.g., green fluorescent protein or luciferase). For example, labels may include radiopaque positron-emitting radionuclides (e.g., for use in PET imaging) or radioisotopes, e.g., 3 H, 13 N, 14 C, 18 F, 32 P, 35 S, 123 I, 125 I, 131I; fluorescent (fluorophore) compounds or chemiluminescent (chromophore) compounds, e.g., fluorescein isothiocyanate, rhodamine, or luciferin; enzymes, e.g., alkaline phosphatase, β-galactosidase, or horseradish peroxidase; imaging agents; or metal ions.

[0125] C. Antibody-drug conjugates A further aspect involves an immunoconjugate comprising the antibody described herein and a detectable label or cytotoxic agent.

[0126] Chemotherapy (anti-cancer) agents can be any active substances that can reduce cancer growth, interfere with cancer cell replication, directly or indirectly kill cancer cells, reduce metastasis, and decrease tumor blood supply. Therefore, chemotherapeutic agents include cytotoxic agents. Cytotoxic agents include, but are not limited to, saporins, taxanes, vinca alkaloids, anthracyclines, and platinum-based drugs. Classes of chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, e.g., methotrexate, plant alkaloids, e.g., vincristine, and antitumor antibiotics, e.g., anthracyclines, e.g., doxorubicin, as well as a variety of drugs not classified into a specific class, e.g., hydroxyureas. Platinum-based drugs, exemplified by cisplatin and oxaliplatin, are a major class of chemotherapeutic agents. These drugs bind to DNA and interfere with replication. Taxanes, exemplified by taxol, are another major class of chemotherapeutic agents. These compounds work by interfering with the formation of the cytoskeleton and spindle, thereby inhibiting cell division and preventing the proliferation of rapidly dividing cancer cells. Other chemotherapy drugs include hormone therapy. Chemotherapy drugs also include agents that inhibit the aggregation or polymerization of tubulin, such as meitansine, meltansine, and auristatin. Chemotherapy agents also include DNA damaging agents such as calicheamicin.

[0127] Chemotherapy agents may include maytansinoids, auristatin, drastatin, tubulicin, cryptophycin, pyrrolobenzodiazepine (PBD) dimers, indolinobenzodiazepine dimers, α-amanitin, trichoten, SN-38, duocalmycin, CC1065, calicheamicin, engine antibiotics, taxanes, doxorubicin derivatives, anthracyclines, and their stereoisomers, azanofide, isosteres, analogs, or derivatives.

[0128] IV. Nucleic acids Further aspects include nucleic acid molecules or polynucleotides, recombinant nucleic acid molecules, expression constructs, and vectors as described herein.

[0129] A. Nucleic acid molecules Further aspects include the nucleic acid molecules listed in Table 2, and polynucleotides that hybridize to one of the above sequences under stringent hybridization conditions, for example. CDRs and variable domain nucleic acid sequences can be used, for example, to prepare expression constructs.

[0130] B. Expression constructs and vectors Nucleic acid molecules can be incorporated in known ways into appropriate expression constructs or expression vectors that ensure protein expression. Expression constructs may include a polynucleotide containing the nucleotide sequence encoding the antibody of this disclosure, and a functionally linked expression regulatory sequence, such as a promoter. Possible expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses). The vector should be compatible with the host cell in which it is used. An expression vector is "suitable for transformation of host cells," meaning that the expression vector contains a nucleic acid molecule encoding a peptide corresponding to an epitope or antibody described herein.

[0131] In one embodiment, the vector is suitable for expressing single-chain antibodies, for example, by gene therapy. In one embodiment, the vector comprises IRESs and enables the expression of light chain variable regions and heavy chain variable regions. Such a vector can be used to deliver antibodies in vivo.

[0132] Suitable regulatory sequences can be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes.

[0133] Examples of such regulatory sequences include transcription promoters and enhancers or RNA polymerase binding sequences, and ribosome binding sequences containing translation initiation signals. Furthermore, depending on the selected host cell and the vector used, other sequences such as origins of replication, additional DNA restriction sites, enhancers, and sequences that confer transcriptional induction ability may be incorporated into the expression vector.

[0134] In one embodiment, the regulatory sequence induces or increases expression in nerve tissue and / or within nerve cells.

[0135] The vector may be any vector suitable for producing the antibodies described herein.

[0136] In one embodiment, the vector is a viral vector.

[0137] Recombinant expression vectors may also contain marker genes to facilitate the selection of host cells transformed, infected, or transfected with a vector for expressing an antibody or epitope peptide as described herein.

[0138] Recombinant expression vectors also include an expression cassette encoding a fusion region (i.e., a "fusion protein") that results in increased expression or stability of the recombinant peptide, or increased solubility of the recombinant peptide, and may assist in the purification of the target recombinant peptide by acting as a ligand in affinity purification, including, for example, tags and labels described herein. Furthermore, proteolytic cleavage sites can be added to the target recombinant protein to separate the recombinant protein from the fusion region after purification of the fusion protein. Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA), and pRIT5 (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E-binding protein, or protein A to the recombinant protein, respectively.

[0139] Systems for gene transfer both in vitro and in vivo include retrovirus-based vectors, most notably those based on viruses, including herpes simplex virus, adenovirus, adeno-associated virus (AAV), and lentivirus. Alternative methods for gene delivery include the use of naked plasmid DNA and liposome-DNA complexes.

[0140] In one aspect, this disclosure includes a method for producing an antibody as described herein, comprising the step of synthesizing a nucleic acid molecule comprising an antibody framework and a set of CDR sequences as described herein.

[0141] V. Recombinant Cells A further aspect is recombinant host cells expressing the antibodies described herein.

[0142] The antibodies described herein can be produced by recombinant expression of nucleic acids encoding antibody sequences.

[0143] The antibodies disclosed herein can be produced by culturing cells that have been engineered to express nucleic acid constructs encoding immunoglobulin polypeptides.

[0144] Recombinant host cells can be generated using any cell suitable for polypeptide production, for example, suitable for antibody production. For example, cells may be transfected, transformed, or infected to introduce nucleic acids (e.g., vectors) into them, depending on the vector used.

[0145] Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the proteins described herein can be expressed in bacterial cells, such as Escherichia coli (E. coli), insect cells (using baculoviruses), yeast cells, or mammalian cells.

[0146] In one embodiment, the cells are eukaryotic cells selected from yeast, plant, insect, bird, fish, reptile, and mammalian cells.

[0147] In another embodiment, the mammalian cells are CHO cells, myeloma cells, spleen cells, or hybridoma cells.

[0148] Suitable yeast and fungal host cells for antibody expression include, but are not limited to, various species of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia, Kluyveromyces, and Aspergillus. Examples of vectors for expression in the yeast S. cerevisiae include pYepSec1, pMFa, pJRY88, and pYES2 (Invitrogen Corporation, San Diego, CA). Protocols for yeast and fungal transformation are well known to those skilled in the art.

[0149] Suitable mammalian cells include, among others, COS cells (e.g., ATCC numbers CRL 1650 or 1651), BHK cells (e.g., ATCC number CRL 6281), CHO cells (ATCC number CCL 61), HeLa cells (e.g., ATCC number CCL 2), 293 cells (ATCC number 1573), and NS-1 cells. Suitable expression vectors for guiding expression in mammalian cells generally include promoters (e.g., derived from viral material such as polyoma, adenovirus 2, cytomegalovirus, and Simianvirus 40), as well as other transcriptional and translational regulatory sequences. Examples of mammalian expression vectors include pCDM8 and pMT2PC.

[0150] VI. Pharmaceutical Compositions A further aspect is a composition comprising an antibody, immunoconjugate, nucleic acid molecule, vector, or recombinant cell as described herein, along with an optional and suitable diluent, such as a pharmaceutically acceptable carrier.

[0151] The composition may, for example, include one or more antibodies or immunoconjugates.

[0152] Suitable diluents for polypeptides containing antibodies and / or cells include, but are not limited to, physiological saline, pH buffer, and glycerol solution, or other solutions suitable for freezing polypeptides and / or cells.

[0153] Suitable diluents for nucleic acids include, without limitation, water, saline solution, and ethanol.

[0154] In one embodiment, the composition is a pharmaceutical composition comprising any of the antibodies, nucleic acids, or vectors disclosed herein, and optionally comprising a pharmaceutically acceptable vehicle such as a diluent or carrier.

[0155] The compositions described herein can be prepared by methods known to themselves for preparing pharmaceutically acceptable compositions that can be administered to a subject, such as by mixing an effective amount of an active substance with a pharmaceutically acceptable vehicle.

[0156] Pharmaceutical compositions include, but are not limited to, lyophilized powders, or aqueous or non-aqueous sterile injection solutions or suspensions, which may further contain antioxidants, buffers, bacteriostatic agents, and solutes to make the composition substantially compatible with the tissue or blood of the intended recipient. Other components that may be present in such compositions include, for example, water, surfactants (such as Tween), alcohols, polyols, glycerin, and vegetable oils. Immediate injection solutions and suspensions may be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions. Compositions may be supplied, for example, as lyophilized powders that are reconstituted with sterile water or saline before administration to the patient, but are not limited to these.

[0157] Pharmaceutical compositions may contain pharmaceutically acceptable carriers. Suitable pharmaceutically acceptable carriers include compositions that are essentially chemically inert and non-toxic and do not interfere with the biological activity of the pharmaceutical composition. Examples of suitable pharmaceutical carriers, without limitation, include water, physiological saline, glycerol solution, ethanol, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA), diolesylphosphotidylethanolamine (DOPE), and liposomes. Such compositions should contain a therapeutically effective amount of the compound together with a suitable amount of carrier to provide a form for direct administration to a patient.

[0158] The composition may be in the form of pharmaceutically acceptable salts, without limitation, that are formed by free amino groups such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed by free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, etc.

[0159] In one embodiment, the composition comprises the antibody described herein. In another embodiment, the composition comprises the antibody described herein and a diluent. In one embodiment, the composition is a sterile composition.

[0160] A further aspect involves an antibody conjugate comprising an antibody described herein, conjugated to an LRP protein, such as LRP5 or LRP6. The conjugate may be in solution or optionally contained in tissue in vitro.

[0161] Methods for preparing and using the reagents described herein are also provided.

[0162] VII. Kit Another aspect is a kit or package comprising any of the antibodies, immunoconjugates, nucleic acid molecules, vectors, recombinant cells, and / or compositions disclosed herein. The antibodies, immunoconjugates, nucleic acid molecules, vectors, recombinant cells, and / or compositions may be contained in vials, such as sterile vials or other housings. As used herein, the term “kit” refers to a collection of articles intended to be used together. A kit may optionally include a reference agent and / or instructions for its use. A kit may further include a transport container adapted to hold a container, such as a vial, containing the compositions disclosed herein.

[0163] VIII. Methods using antibodies The antibodies described herein can be used in several in vitro and in vivo procedures.

[0164] A. Method for detecting LRP5 expression As shown herein, antibodies can be used to detect LRP5 expression.

[0165] Accordingly, in one aspect, this disclosure provides a method for detecting LRP5 expression, comprising the steps of contacting a sample containing one or more cells with one or more antibodies or immunoconjugates described herein under conditions that allow the formation of antibody:LRP5 complexes, and detecting the presence of any antibody complex. Typically, the antibody is part of an immunoconjugate containing an antibody conjugated to a detectable label.

[0166] The sample may include live cells or cell extracts. The antibody:LRP5 complex can be detected by immunoassays, such as immunofluorescence, flow cytometry, Western blotting, ELISA, SPR, and immunoprecipitation, followed by SDS-PAGE immunocytochemistry. In some embodiments, detection is by immunofluorescence. In some embodiments, detection is by flow cytometry.

[0167] As shown herein, several identified antibodies preferentially recognize LRP5. Therefore, in embodiments where the method is for detecting LRP5 expression, the antibody or immunoconjugate comprises a set of CDR sequences corresponding to an antibody selected from LRP5-A7, LRP5-A9, LRP5-C5, LRP5-C12, LRP5-D9, LRP5-E5, LRP5-G2, LRP5-G9, LRP5-G10, LRP5-G11, LRP5-H3, LRP5-H5, LRP5-H9, LRP5-R3O_D3, LRP5-R3_E8, and LRP5-R3O_G6.

[0168] B. Method to inhibit WNT binding to LRP5 The antibodies disclosed herein inhibit the binding of Wnt to LRP proteins, particularly LRP5. While we do not wish to be limited by theory, inhibition of Wnt binding to LRP5 affects signaling induced by the binding of specific Wnt ligands. For example, antibodies that bind to the LRP5 receptor inhibit LRP5-promoting β-catenin phosphorylation. Since phosphorylated β-catenin is marked for degradation within cells, the unphosphorylated form accumulates. The accumulation of β-catenin is associated with malignancies.

[0169] It may be desirable to reduce or inhibit Wnt ligand signaling mediated by LRP5. Therefore, another aspect is a method for inhibiting Wnt ligand binding to LRP5 or Wnt-inducible transcriptional activity, comprising contacting one or more cells expressing one or more LRP5 polypeptides with an effective amount of an antibody or immunoconjugate described herein.

[0170] In one embodiment, the antibody or immunoconjugate comprises a set of CDR sequences (complete light chain or heavy chain) corresponding to the antibody described herein. As shown herein, several identified antibodies preferentially recognize LRP5. Therefore, in embodiments where the method is for detecting LRP5 expression, the antibody or immunoconjugate comprises a set of CDR sequences corresponding to an antibody selected from LRP5-A7, LRP5-A9, LRP5-C5, LRP5-C12, LRP5-D9, LRP5-E5, LRP5-G2, LRP5-G9, LRP5-G10, LRP5-G11, LRP5-H3, LRP5-H5, LRP5-H9, LRP5-R3O_D3, LRP5-R3_E8, and LRP5-R3O_G6.

[0171] Contact can be performed in vivo, for example, by administering an antibody or immunoconjugate to the target. Such inhibition may be desirable, especially when Wnt signaling is dysregulated, such as in cancer cells.

[0172] C. Methods to enhance the WNT signaling pathway Certain antibodies that block the binding of Wnt ligands to the Wnt3a binding site of LRP5 or LRP6 enhance the signaling activity of Wnt ligands that bind to non-Wnt3a binding sites. Similarly, certain antibodies that block the binding of Wnt ligands to non-Wnt3a binding sites of LRP5 or LRP6 enhance the signaling activity of Wnt ligands that bind to Wnt3a binding sites. The blocking activity may be competitive or allosteric. Therefore, a method for enhancing the signaling activity of Wnt ligands that bind to the Wnt3a binding site of LRP5 or LRP6 is provided herein by contacting LRP5 or LRP6 with an antibody that blocks the binding of Wnt ligands to the non-Wnt3a binding site of LRP5 or LRP6. A method for enhancing the signaling activity of a Wnt ligand that binds to a non-Wnt3a binding site of LRP5 or LRP6 is also provided herein, by contacting LRP5 or LRP6 with an antibody that blocks the binding of a Wnt ligand to the Wnt3a binding site of LRP5 or LRP6. The contact can be performed in vitro or in vivo. In vivo contact may involve administering a suitable anti-LRP5 antibody or anti-LRP6 antibody to the target.

[0173] For example, an antibody containing a set of CDR sequences from an antibody of epitope group 1 enhances the activity of Wnt ligands that bind to non-Wnt3A binding sites.

[0174] D. Methods and uses for treating cancer Methods of treating cancer include the use of a pharmaceutical composition comprising the antibody of this disclosure that binds to LRP5, or administration thereof to a subject that needs it. A subject that needs it may be a person who has cancer or is at risk of cancer, such as a recurrence of cancer.

[0175] In one embodiment, the cancer is selected from acute myeloid leukemia, prostate cancer, glioblastoma, bladder cancer, and cervical cancer.

[0176] In another embodiment, the cancer cells are selected from brain cancer, breast cancer, colon cancer, endometrial cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, skin cancer, gastric cancer, and testicular cancer.

[0177] While we do not wish to be limited by theory, such therapies may function by inhibiting the activation of the classical Wnt pathway, for example by inhibiting Wnt binding to LRP5, by inhibiting Wnt-induced transcriptional activity, by inhibiting disheveled activation, by inhibiting the inhibition of the β-catenin degradation complex, and by promoting the accumulation of β-catenin.

[0178] Because LRP5 and / or LRP6 are often upregulated in cancer, the Disclosure, in another aspect, includes a method for treating cancer, comprising the step of administering an effective amount of an antibody or immunoconjugate that specifically binds to LRP5 or LRP6 in at least one assay and inhibits Wnt3a-induced signaling in at least one assay to a subject in need. The Disclosure also includes the use of an effective amount of an antibody or immunoconjugate that specifically binds to LRP5 or LRP6 in at least one assay and inhibits Wnt3a-induced signaling in at least one assay for treating cancer or in the manufacture of a drug for treating cancer. The Disclosure further includes an effective amount of an antibody or immunoconjugate that specifically binds to LRP5 or LRP6 in at least one assay and inhibits Wnt3a-induced signaling in at least one assay for use in treating cancer.

[0179] In one embodiment, an antibody or immunoconjugate, such as an antibody-drug conjugate, is included in the pharmaceutical composition.

[0180] In one embodiment, the cancer is selected from brain cancer, breast cancer, colon cancer, endometrial cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, skin cancer, gastric cancer, and testicular cancer. In one embodiment, the antibody or immunoconjugate comprises a set of CDR sequences corresponding to the antibody described herein. Some of the identified antibodies preferentially recognize LRP5. Therefore, in an embodiment in which the method is for detecting LRP5 expression, the antibody or immunoconjugate includes a set of CDR sequences corresponding to an antibody selected from LRP5-A7, LRP5-A9, LRP5-C5, LRP5-C12, LRP5-D9, LRP5-E5, LRP5-G2, LRP5-G9, LRP5-G10, LRP5-G11, LRP5-H3, LRP5-H5, LRP5-H9, LRP5-R3O_D3, LRP5-R3_E8, and LRP5-R3O_G6.

[0181] As shown herein, antibodies can also inhibit cancer cell proliferation. Therefore, a method for inhibiting cancer cell proliferation is also provided, comprising the step of contacting one or more cancer cells expressing LRP5 with an effective amount of an antibody or immunoconjugate that specifically binds to LRP5 in at least one assay and inhibits Wnt3a-induced signaling in at least one assay.

[0182] In one embodiment, a method for treating cancer comprises the step of administering an antibody, or an antibody-containing immunoconjugate, to a subject in need thereof, wherein the first antibody blocks the binding of a Wnt ligand to a Wnt3a binding site of LRP5 or LRP6, and the second antibody blocks the binding of a Wnt ligand to a non-Wnt3a binding site of LRP5 or LRP6. The disclosure also provides an antibody, or an antibody-containing immunoconjugate, for use in the treatment of cancer, wherein the first antibody blocks the binding of a Wnt ligand to a Wnt3a binding site of LRP5 or LRP6, and the second antibody blocks the binding of a Wnt ligand to a non-Wnt3a binding site of LRP5 or LRP6. This disclosure further provides the use of an antibody, or an antibody-containing immunoconjugate, for the treatment of cancer, wherein the first antibody blocks the binding of a Wnt ligand to the Wnt3a binding site of LRP5 or LRP6, and the second antibody blocks the binding of a Wnt ligand to the non-Wnt3a binding site of LRP5 or LRP6. This disclosure also provides the use of an antibody, or an antibody-containing immunoconjugate, for the treatment of cancer, wherein the first antibody blocks the binding of a Wnt ligand to the Wnt3a binding site of LRP5 or LRP6, and the second antibody blocks the binding of a Wnt ligand to the non-Wnt3a binding site of LRP5 or LRP6.

[0183] In one embodiment, the antibody or immunoconjugate is an antibody or immunoconjugate comprising a CDR sequence set (complete light chain or heavy chain) corresponding to an antibody selected from the antibodies or immunoconjugates described herein, such as LRP5-A7, LRP5-A9, LRP5-C5, LRP5-C12, LRP5-D9, LRP5-E5, LRP5-G2, LRP5-G9, LRP5-G10, LRP5-G11, LRP5-H3, LRP5-H5, LRP5-H9, LRP5-R3O_D3, LRP5-R3_E8, and LRP5-R3O_G6.

[0184] IX. Method of Administration The anti-LRP antibody of the present invention can efficiently deliver a therapeutic composition in vivo to cells that receive Wnt signaling. In some embodiments, the therapeutic method includes administering an effective amount of a therapeutic anti-LRP conjugate, e.g., an anti-LRP antibody attached to a therapeutic agent, to an individual. In some embodiments, the individual has been diagnosed with cancer. In some embodiments, the individual is currently undergoing or has undergone cancer therapy, e.g., surgery, radiation therapy, or chemotherapy. In some embodiments, the individual has been diagnosed, but the cancer is in remission.

[0185] In some embodiments, the anti-LRP conjugate comprises liposomes. In some embodiments, the method further includes a step of monitoring the individual for cancer progression. In some embodiments, the dose of the anti-LRP conjugate for each administration is determined based on the individual's treatment progress, for example, if the individual is not responding well to treatment and a higher dose of chemotherapy is administered.

[0186] In some embodiments, the present invention may include an antibody or antibody-targeted composition and a physiologically (i.e., pharmaceutically) acceptable carrier. The term “carrier” refers to a typically inert substance used as a diluent or vehicle for a diagnostic or therapeutic agent. The term also includes typically inert substances that impart cohesiveness to the composition. Physiologically acceptable carriers may be liquids, e.g., saline, phosphate buffer, ordinary buffered saline (135–150 mM NaCl), water, buffer water, 0.4% saline, 0.3% glycine, glycoproteins to enhance stability (e.g., albumin, lipoprotein, globulin, etc.). Since the physiologically acceptable carrier is partially determined by the specific composition administered and the specific method used to administer the composition, a wide variety of suitable formulations of the pharmaceutically acceptable compositions of the present invention exist (see, for example, Remington's Pharmaceutical Sciences, 17th ed., 1989).

[0187] The compositions of the present invention may be sterilized by conventional, well-known sterilization techniques or may be produced under sterile conditions. The aqueous solutions may be packaged for use or filtered and lyophilized under sterile conditions, and the lyophilized preparations may be combined with the sterile aqueous solution before administration. The compositions may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, isotonic adjusters, and wetting agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. Sugars for stabilizing the composition may also be included, such as stabilizers for lyophilized antibody compositions.

[0188] Dosage forms can be prepared for mucosal administration to patients (e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral administration (e.g., subcutaneous, intravenous, intramuscular, or intra-arterial injection, bolus, or infusion), oral administration, or transdermal administration. Examples of dosage forms, without limitation, include dispersions; suppositories; ointments; poultices; pastes; powders; bandages; creams; adhesive bandages; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water or water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for oral or mucosal administration to patients; liquid dosage forms suitable for parenteral administration to patients; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to patients.

[0189] Injectable (e.g., intravenous) compositions may include solutions of antibodies or antibody-targeted compositions suspended in an acceptable carrier, such as an aqueous carrier. For example, any of the various aqueous carriers may be used, such as water, buffered water, 0.4% saline, 0.9% isotonic saline, 0.3% glycine, or 5% dextrose, and may contain glycoproteins to enhance stability, such as albumin, lipoprotein, or globulin. Often, standard buffered saline (135-150 mM NaCl) is used. The composition may contain pharmaceutically acceptable adjuncts to approximate physiological conditions, such as pH adjusters and buffers, isotonic adjusters, and wetting agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, or triethanolamine oleate. In some embodiments, the antibody-targeted composition may be formulated into a kit for intravenous administration.

[0190] For example, formulations suitable for parenteral administration via intra-articular, intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Injection solutions and suspensions can also be prepared from sterile powders, granules, and tablets. In the embodiment of the present invention, the composition can be administered locally, intraperitoneally, intravesically, intrathecally, or intrathecally, for example, by intravenous infusion. Parenteral and intravenous administration are preferred methods of administration. Formulations of the target-directed composition can be provided in sealed containers of unit doses or multiple doses, such as ampoules and vials.

[0191] The selected target-directed delivery composition can be used alone or in combination with other suitable components to form an aerosol formulation ("spray") administered by inhalation. The aerosol formulation can be encapsulated in a pressurized, acceptable propellant such as dichlorodifluoromethane, propane, and nitrogen.

[0192] Pharmaceutical preparations can be packaged or prepared in unit dosage forms. In such forms, the preparation is subdivided into unit doses containing an appropriate amount of the active ingredient, for example, according to the dose of the therapeutic agent or the concentration of the antibody. The unit dosage form may be a packaged preparation, and the package contains individual amounts of the preparation. The composition may also contain other suitable therapeutic agents, if desired.

[0193] Antibodies (or antibody-targeted compositions) can be administered by injection or infusion via any preferred route, including, but not limited to, intravenous, subcutaneous, intramuscular, or intraperitoneal routes. An example of administration of a pharmaceutical composition is storing the antibody at 10 mg / ml in sterile isotonic saline for injection at 4°C, and diluting it in either 100 ml or 200 ml of 0.9% sodium chloride for injection before administration to the patient. The antibody is administered by intravenous infusion over 1 hour at doses of 0.2–10 mg / kg. In other embodiments, the antibody is administered by intravenous infusion over a period of 15 minutes to 2 hours. In yet another embodiment, the administration procedure is by subcutaneous bolus injection.

[0194] The antibody dose is selected to provide effective treatment to the patient and ranges from less than 0.1 mg / kg body weight to approximately 25 mg / kg body weight or from 1 mg to 2 g per patient. In some cases, the dose ranges from 1 to 100 mg / kg or from approximately 50 mg to approximately 8000 mg per patient. Depending on the pharmacokinetics of the antibody (e.g., half-life of the antibody in circulation) and the pharmacodynamic response (e.g., duration of therapeutic effect of the antibody), the dose may be repeated at an appropriate frequency, which may range from once daily to once every three months. In some embodiments, antibody administration with an in vivo half-life of approximately 7 to 25 days is repeated once weekly to once every three months.

[0195] Administration may be cyclical. Depending on the route of administration, the dose may be administered once every 1, 3, 5, 7, 10, 14, 21, or 28 days, or at longer intervals (e.g., once every 2, 3, 4, or 6 months). In some cases, administration may be more frequent, such as twice or three times a day. As recognized by those skilled in the art, the patient may be monitored and the dose and frequency of administration adjusted in response to the progression of treatment and any adverse side effects.

[0196] Therefore, in some embodiments, additional doses depend on the patient's progress, and for example, the patient is monitored between doses. For example, after the first dose or a series of doses, the patient may be monitored for tumor growth rate, recurrence (e.g., in postoperative patients), or common disease-related symptoms such as weakness, pain, and nausea.

[0197] For therapeutic use in treating cancer, antibody-targeted compositions (e.g., including therapeutic and / or diagnostic agents) are administered at an initial dose of approximately 0.001 mg / kg to approximately 1000 mg / kg per day and may be adjusted over time. Daily dose ranges of approximately 0.01 mg / kg to approximately 500 mg / kg, or approximately 0.1 mg / kg to approximately 200 mg / kg, or approximately 1 mg / kg to approximately 100 mg / kg, or approximately 10 mg / kg to approximately 50 mg / kg may be used. The dose varies depending on the patient's requirements, the severity of the disease being treated, and the targeted composition used. For example, the dose may be determined empirically in a particular patient, taking into account the type and stage of cancer diagnosed. In the context of the present invention, the dose administered to a patient should be sufficient to influence the patient's beneficial therapeutic response over time. The size of the dose is also determined by the presence, nature, and extent of any adverse side effects associated with the administration of a particular targeted composition to a particular patient, as will be recognized by those skilled in the art.

[0198] The above disclosure provides a general overview of this disclosure. A more complete understanding can be obtained by referring to the following specific examples. These examples are provided for illustrative purposes only and are not intended to limit the scope of this application. Modifications of form and substitutions of equivalents are considered as suggested or convenient in the context. Certain terms are used herein, but such terms are intended to be descriptive and not limiting.

[0199] Exemplary examples 1. An antibody that specifically binds to LRP5, comprising a light chain variable region and / or a heavy chain variable region, The heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of the CDR comprises or consists of CDR sequences selected from the anti-LRP5 antibody CDR sequence set: LRP5-A7, LRP5-A9, LRP5-C5, LRP5-C12, LRP5-D9, LRP5-E5, LRP5-G2, LRP5-G9, LRP5-G10, LRP5-G11, LRP5-H3, LRP5-H5, LRP5-H9, LRP5-R3O_D3, LRP5-R3_E8, and LRP5-R3O_G6. antibody. 2. The amino acid sequence of the CDR includes or consists of sequences selected from the sequences described below. The CDR-H1 is LSYYYM (SEQ ID NO: 7) , ISYSYI (SEQ ID NO: 5) LSYSSM (SEQ ID NO: 2) ISSYSI (SEQ ID NO: 3) , ISYSYI (SEQ ID NO: 5) IYSYSI (SEQ ID NO: 6) , LSYYYM (SEQ ID NO: 7) FSSSSI (SEQ ID NO: 4) ,LYYYYI (SEQ ID NO: 8) LSYSSI (SEQ ID NO: 9) IYSYYI (SEQ ID NO: 10) LLYYSSM (SEQ ID NO: 122) , and FSSSSI (SEQ ID NO: 4) Selected from the group consisting of, The CDR-H2 is SIYPYYGYTY (SEQ ID NO: 11) SSSYYGYTY (SEQ ID NO: 12) SISSSYGYTY (SEQ ID NO: 13) , SIYSSYGSTS (SEQ ID NO: 14) , SIYSSYGYTY (SEQ ID NO: 15) , SIYPYSSYTS (SEQ ID NO: 16) , SIYSSYGYTY (SEQ ID NO: 15) , SIYPSYGYTY (SEQ ID NO: 17) SISPYYGYTS (SEQ ID NO: 18) SISSSYGSTS (SEQ ID NO: 19) , SIYSYYGYTY (SEQ ID NO: 20)SISSSYGYTY (SEQ ID NO: 13) SISSSYGYTY (SEQ ID NO: 13) SISSYYGYTS (SEQ ID NO: 53) , and YISPYYGYTS (SEQ ID NO: 56) Selected from the group consisting of, The CDR-H3 is HGAM (SEQ ID NO: 21) TVRGSKKPYFSGWAM (SEQ ID NO: 22) SSYYSSVSSSVYAL (SEQ ID NO: 23) TVRGSKKPYFSGWAM (SEQ ID NO: 22) HYSYOFFYAM (SEQ ID NO: 24) ,YAVYFPGYYWGM (SEQ ID NO: 25) WSHVSGHYSGM (SEQ ID NO: 26) WGAYHSSGYGM (SEQ ID NO: 27) ,GGSGVSHYGSVYYSWWAL (SEQ ID NO: 28) ,AAPYYGYYYGam (SEQ ID NO: 29) SGYGWYAM (SEQ ID NO: 30) GYWAI (SEQ ID NO: 31) SYPAM (SEQ ID NO: 32) SWAM (SEQ ID NO: 33) ; YWAL (SEQ ID NO: 54) GWGSPASAGYYGL (SEQ ID NO: 57) SSYYSSVSSSVYAL (SEQ ID NO: 23) TVRGSKKPYFSGWAM (SEQ ID NO: 22) , and TVRGSKKPYFSGWAM (SEQ ID NO: 22) Selected from the group consisting of, CDR-L1 is SVSSA (SEQ ID NO: 34) And, CDR-L2 is SASSLYS (SEQ ID NO: 35) and CDR-L3 is AWGWGLF (SEQ ID NO: 36) , VHYSPYSLI (SEQ ID NO: 37) , YQYSGLI (SEQ ID NO: 38) , FSHVSLI (SEQ ID NO: 39) ASYSPI (SEQ ID NO: 40) , YHYYYLF (SEQ ID NO: 41) ASYAPI (SEQ ID NO: 42) , SSSSPI (SEQ ID NO: 43) SSYSLI (SEQ ID NO: 44) GVSLI (SEQ ID NO: 45) YWFLI (SEQ ID NO: 46) , PVGHYGYPI (SEQ ID NO: 47) , SSYSPI (SEQ ID NO: 48) , YWAYYSPI (SEQ ID NO: 49) , VSYYPLI (SEQ ID NO: 51) , SSYSLI (SEQ ID NO: 44) , and VHYSPYSLI (SEQ ID NO: 37) Selected from the group consisting of The antibody of Aspect 1. 3. (i) The heavy chain amino acid sequence described in Table 2, (ii) An amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the heavy chain amino acid sequence described in Table 2, wherein the CDR sequences are the CDR sequence sets described in Table 1, or (iii) A conservatively substituted amino acid sequence of (i), wherein the CDR sequences are the CDR sequence sets described in the table, the conservatively substituted amino acid sequence An antibody of Aspect 2 comprising a heavy chain variable region containing 4. (i) The light chain amino acid sequence described in Table 2, (ii) An amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the light chain amino acid sequence described in Table 2, wherein the CDR sequences are the CDR sequence sets described in Table 1, or (iii) A conservatively substituted amino acid sequence of (i), wherein the CDR sequences are the CDR sequence sets described in Table 1, the conservatively substituted amino acid sequence An antibody of any one of Aspects 2 to 4 comprising a light chain variable region containing 5. An antibody of any one of Aspects 1 to 4, wherein the CDR sequences are a complete set of CDR sequences selected from the antibodies identified in Table 1. 6. An antibody of any one of Aspects 1 to 5 that cross-reacts with LRP6. [[ID=I44]] 7. An antibody of embodiment 1, wherein the CDR sequences comprise a set of light-chain CDR sequences or a set of heavy-chain CDR sequences selected from the antibodies identified in Table 1. 8. An antibody of any one of embodiments 1-7 that specifically binds to LRP5. 9. An antibody of embodiment 8, wherein the CDR sequences are a set of CDR sequences of an antibody selected from LRP5-A7, LRP5-A9, LRP5-C5, LRP5-C12, LRP5-D9, LRP5-E5, LRP5-G2, LRP5-G9, LRP5-G10, LRP5-G11, LRP5-H3, LRP5-H5, LRP5-H9, LRP5-R3O_D3, LRP5-R3_E8, LRP5-R3O_G6. 10. An antibody of any one of embodiments 1-9 that blocks the binding of a Wnt ligand to the Wnt3a binding site of LRP5. 11. An antibody of any one of embodiments 1-9 that blocks the binding of a Wnt ligand to a non-Wnt3a binding site of LRP5. 12. An antibody of any one of embodiments 1-11 that is a monoclonal antibody. 13. An antibody of any one of embodiments 1-12 that is a humanized antibody. 14. An antibody of any one of embodiments 1-13 that is a single-chain antibody. 15. An antibody of any one of embodiments 1-14 that is an antibody binding fragment selected from Fab, Fab’, F(ab’)2, scFv, dsFv, ds-scFv, dimer, nanobody, minibody, diabody, and multimers thereof. 16. An antibody of any one of embodiments 1-14 that is a bispecific antibody. 17. An antibody of any one of embodiments 1-14 that is a bispecific antibody that further binds to a FZD receptor. 18. An antibody of any one of embodiments 1-17 that comprises a non-natural glycosylation pattern. 19. For example, one antibody from any one of embodiments 1 to 17, comprising substitution or addition of cysteine ​​to a constant region or framework region. 20. An immunoconjugate comprising one antibody from any of embodiments 1 to 17 and a detectable label or cytotoxic agent. twenty one. An immunoconjugate according to embodiment 20, comprising a cytotoxic agent selected from meitansinoids, auristatin, dorastatin, tubulicin, cryptophycin, pyrrolobenzodiazepine (PBD) dimers, indolinobenzodiazepine dimers, α-amanitin, trichoten, SN-38, duocalmycin, CC1065, calicheamicin, engine antibiotics, taxanes, doxorubicin derivatives, anthracyclines, and their stereoisomers, azanofide, isosteres, analogs, or derivatives. twenty two. A nucleic acid molecule encoding one of the antibodies described in embodiments 1 to 17. twenty three. A nucleic acid molecule according to embodiment 22, wherein one or more of the CDR sequences are encoded by the nucleic acids in Table 2. twenty four. The aforementioned antibody (i) Heavy chain nucleic acid sequences listed in Table 2, (ii) A nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to the heavy chain nucleic acid sequences listed in Table 2, wherein the CDR sequence is one of the CDR sequence sets listed in Table 1, or (iii)(i) Codon-degenerate nucleic acid sequences, wherein the CDR sequence is one of the CDR sequence sets listed in Table 1. A nucleic acid molecule according to embodiment 22, comprising a heavy chain variable region encoded by a nucleic acid containing the following: twenty five. The aforementioned antibody (i) Light chain nucleic acid sequences listed in Table 2, (ii) A nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to the light chain nucleic acid sequences listed in Table 2, wherein the CDR sequence is one of the CDR sequence sets listed in Table 1, or (iii)(i) Codon-degenerate nucleic acid sequences, wherein the CDR sequence is one of the CDR sequence sets listed in Table 1. A nucleic acid molecule according to embodiment 22, comprising a light chain variable region encoded by a nucleic acid containing the following: 26. A vector comprising an expression control sequence functionally linked to one nucleic acid according to any one of embodiments 22 to 25. 27. A host cell comprising a recombinant nucleic acid molecule containing an expression regulatory sequence functionally linked to one of the nucleic acids described in any one of embodiments 22 to 26. 28. Host cells of aspect 27, which are Chinese hamster ovary (CHO) cells. 29. A host cell containing the vector according to embodiment 26. 30. A method for producing an anti-LRP5 antibody, comprising the step of culturing one of the host cells described in any one of embodiments 27 to 29. 31. A composition comprising, optionally, a suitable diluent, one or more antibodies from embodiments 1 to 17, an immunoconjugate from embodiments 20 to 21, a nucleic acid molecule from embodiments 22 to 25, a vector from embodiment 26, or a host cell from embodiment 29. 32. A composition according to embodiment 31, comprising one or more antibodies or immunoconjugates, and optionally being a pharmaceutical composition. 33. A kit comprising one or more antibodies from embodiments 1 to 17, an immunoconjugate from embodiments 20 to 21, a nucleic acid molecule from embodiments 22 to 25, a vector from embodiment 26, or host cells from embodiments 29 to 29. 34. A method for detecting LRP5 expression, comprising the steps of contacting a sample containing one or more cells with one or more antibodies or immunoconjugates of any one of Aspects 1 to 21 under conditions that permit the formation of antibody: cell complexes, and detecting the presence of any antibody complexes. 35. The method of Aspect 34, wherein the detection is by immunofluorescence. 36. The method of Aspect 34, wherein the detection is by flow cytometry. 37. The method of any one of Aspects 34 to 36, wherein the method is for detecting LRP4 expression and the antibody or immunoconjugate comprises a set of CDR sequences corresponding to an antibody selected from LRP5 - A7, LRP5 - A9, LRP5 - C5, LRP5 - C12, LRP5 - D9, LRP5 - E5, LRP5 - G2, LRP5 - G9, LRP5 - G10, LRP5 - G11, LRP5 - H3, LRP5 - H5, LRP5 - H9, LRP5 - R3O_D3, LRP5 - R3_E8, LRP5 - R3O_G6. 38. A method of inhibiting Wnt ligand binding to the LRP5 receptor, interfering with the Wnt signaling pathway, inhibiting Wnt - induced transcriptional activity, inhibiting the activation of disheveled, promoting the preservation of the β - catenin degradation complex, promoting the accumulation of β - catenin, or inhibiting cell proliferation, comprising the step of contacting a cell expressing the LRP5 receptor with an antibody or immunoconjugate of any one of Aspects 1 to 21. 39. The method of Aspect 38, wherein the antibody or immunoconjugate blocks the binding of the Wnt ligand to the Wnt3a - binding site of LRP5. 40. The method of Aspect 38, wherein the antibody or immunoconjugate blocks the binding of the Wnt ligand to the non - Wnt3a - binding site of LRP5. 41. The method of embodiment 38, wherein the antibody or immunoconjugate comprises a set of CDR sequences corresponding to an antibody selected from the group consisting of LRP5-A7, LRP5-A9, LRP5-C5, LRP5-C12, LRP5-D9, LRP5-E5, LRP5-G2, LRP5-G9, LRP5-G10, LRP5-G11, LRP5-H3, LRP5-H5, LRP5-H9, LRP5-R3O_D3, LRP5-R3_E8, and LRP5-R3O_G6. 42. A method for treating a cancer of a subject that requires such treatment, comprising the step of administering to the subject an effective amount of a pharmaceutical composition comprising one antibody or immunoconjugate from any one of embodiments 1 to 21. 43. A method according to aspect 42, wherein the cancer is selected from cancer cells of the colon, lung, breast, ovary, endometrium, pancreas, stomach, liver, adrenal cortex, and osteoblastoma. 44. The method according to aspect 42, wherein the cancer is selected from acute myeloid leukemia, prostate cancer, glioblastoma, bladder cancer, and cervical cancer. 45. The process includes administering one of the first and second antibodies or antibody conjugates from embodiments 1 to 21 to the subject, The first method blocks the binding of Wnt ligand to the Wnt3a binding site of LRP5, and the second method blocks the binding of Wnt ligand to the non-Wnt3a binding site of LRP5. The method according to embodiment 42. 46. The method according to embodiment 45, wherein the first antibody or immunoconjugate comprises a set of CDR sequences selected from antibodies of epitope group 2. 47. The method of embodiment 42, wherein the antibody or immunoconjugate specifically binds to LRP5 in at least one assay and inhibits Wnt3a-induced signaling in at least one assay, and optionally the antibody or immunoconjugate is one of the antibodies or immunoconjugates of embodiments 1 to 21. 48. The method of embodiment 42, wherein the antibody or immunoconjugate comprises a set of CDR sequences corresponding to an antibody selected from the group consisting of LRP5-A7, LRP5-A9, LRP5-C5, LRP5-C12, LRP5-D9, LRP5-E5, LRP5-G2, LRP5-G9, LRP5-G10, LRP5-G11, LRP5-H3, LRP5-H5, LRP5-H9, LRP5-R3O_D3, LRP5-R3_E8, and LRP5-R3O_G6. 49. A method for enhancing the signaling activity of a Wnt ligand that binds to the Wnt3a binding site of LRP5, comprising the step of contacting cells expressing LRP5 with an antibody that blocks the binding of a Wnt ligand to the non-Wnt3a binding site of LRP5. 50. A method for enhancing the signaling activity of a Wnt ligand that binds to the non-Wnt3a binding site of LRP5 by contacting cells expressing LRP5 with an antibody that blocks the binding of a Wnt ligand to the Wnt3a binding site of LRP5. 51. A method performed in vitro, as described in either embodiment 49 or 50. 52. The method described in either embodiment 49 or 50, performed in vivo. [Examples]

[0200] The following non-limiting examples illustrate the present disclosure.

[0201] Example 1 Phage display antibody library Phage display antibody libraries are a powerful technology for generating therapeutic antibodies. 17、18 10 10A highly complex library of over 100 independent antibody fragments is presented on phage particles as coat protein fusion molecules and screened to isolate antibodies that recognize the antigen of interest. We have established a synthetic antibody library with antigen-binding sites entirely constructed from manipulated sequences. Because the resulting antibodies utilize an optimized human framework, they exhibit minimal immunogenicity when used as potential therapeutic agents. The synthetic antibodies are highly stable, and their human frameworks and antigen-binding sites can be tuned to optimize affinity, specificity, and efficacy.

[0202] Characterization and optimization of anti-LRP5 antibodies Example 2 Specific anti-LRP5 antibodies were generated by direct selection using a novel synthetic antibody targeting LRP5, from a highly optimized laboratory-constructed Fab phage library, with immobilized recombinant LRP5 ECDs. Fab phage clones obtained from rounds 3 and 4 were screened for binding by ELISA. The amino acid sequence of the positive binder CDR is shown in Figure 1A. The complete DNA and amino acid sequences of the variable regions (heavy and light chains) of IgG are presented in the above specification. Epitope mapping by competitive ELISA revealed that the antibody binds to four unique epitopes on the LRP5 ECO.

[0203] Full-length IgG1 and antibody fragments (Fab) were purified, and antibody specificity was determined by single-point ELISA. As shown in Figure 1B, all LRP5 antibodies bind to recombinant mouse LRP5-His chimeras. Interestingly, the LRP5-H5 antibody and LRP5-R30D3 antibody show partial binding to recombinant mouse LRP6-His chimeras. The LRP5-R30D3 antibody also shows partial binding to recombinant human LRP6-Fc chimeras. The LRP5-R3OD3 antibody also shows partial binding to recombinant human LRP6-Fc chimeras, suggesting that this antibody may cross-react with both LRP5 and LRP6. Relative affinity of antibodies to recombinant antigens was determined using multi-point competitive ELISA. IC50 was determined by nonlinear regression analysis. This is summarized in Table 3. The complete dose-response curve and nonlinear regression plot are shown in Figure 6.

[0204] Table 3 reports the IC50 values ​​of LRP5 antibodies determined by competitive ELISA. The log (x-axis) of the recombinant human LRP6-Fc chimeric concentration was plotted against the OD450 reading of the antibody (y-axis). The IC50 was determined by nonlinear regression analysis.

[0205] (Table 3) TIFF0007840842000019.tif80128

[0206] Western blot analysis of whole cell lysates showed that LRP5 is highly expressed in NSCLC cell line H23, triple-negative breast cancer cell line MOAMB231, and breast cancer cell line T470 (data not shown). LRP5 IgG1 labeled the surface of H23 cells (Figure 2), MOAMB231 cells (Appendix 3), and T470 cells (Appendix 4), as assessed by FACS analysis, demonstrating that the LRP5 antibody can bind to the full-length receptor expressed on the surface of these cell lines. As described above, Wnt signaling initiates a classical pathway that primarily regulates the stability and function of β-catenin.

[0207] Wnt stimulation elevates cytosolic and nuclear β-catenin levels, leading to increased TCF / LEF (transcription factor)-mediated transcription. The TOPflash reporter assay is a widely used method for evaluating β-catenin activity in vitro. The vector consists of several TCF / LEF binding sites that promote the expression of a firefly luciferase reporter gene. To evaluate the functional activity of the LRP5 antibody in regulating the classical Wnt pathway, several cancer cell lines expressing this reporter were created.

[0208] As shown in Figure 3A, Wnt3a-expressing conditioned medium (Wnt3aCM) induced a 120-fold increase in luciferase reporter activity compared to treatment with control conditioned medium (ConCM) of MOAMB231 cells pretreated with negative control IgG1 and anti-MBP. LRP5-G10 and LRP5-H5 antibodies significantly enhanced Wnt3aCM-inducible reporter activity. Similar results were observed in breast cancer cell line T47D (Figure 38) and osteosarcoma cell line U20S (Figure 3C). In the U20S cell line, both ConCM-inducible and Wnt3aCM-inducible reporter activity were enhanced by LRP5-G10 and LRP5-H5 antibodies (compared to M8P).

[0209] Structural and mutagenesis studies revealed that Wnt3a binds to a unique domain derived from the binding sites of the remaining Wnt ligands. Furthermore, recent studies have shown that certain Wnt ligands require both LRP5 and LRP6 to initiate the classical pathway. To address the hypothesis that epitope-specific LRP5 antibodies modulate β-catenin-mediated transcriptional activity in a Wnt-dependent manner, we evaluated reporter activity in the NSCLC cell line H23. Previous studies (20) have shown that Wnt2 primarily promotes basal TCF / LEF-mediated transcriptional activity, and these cells do not express Wnt3a. As predicted, LRP5-G10 and LRP5-H5 antibodies enhanced Wnt3aCM-inducible reporter activity (compared to M8P; Figure 3D). Interestingly, these antibodies (another member of this epitope group, as well as LRP5-A7) inhibited basal (ConCM-inducible) reporter activity (compared to M8P). Furthermore, the LRP5-D9 antibody and the LRP5-G2 antibody significantly enhance ConCM-inducible reporter activity (compared to MBP). Our results show that the LRP5-G2 antibody and the LRP5-G10 antibody induce the most potent effect on reporter activity. Consistent with this observation, the LRP5-G2 antibody enhances ConCM-inducible reporter activity in a dose-dependent manner (Figure 4A). Interestingly, the LRP5-G10 antibody inhibits ConCM-inducible reporter activity at all indicated concentrations. Also, while the LRP5-G10 Fab inhibits ConCM-inducible reporter activity at the indicated concentrations, the LRP5-G2 Fab fails to replicate the effect of IgG1 on reporter activity (Figure 4B).

[0210] Western blot analysis evaluated the effects of LRP5-G2 and LRP5-G10 antibodies on proximal events of Wnt signaling (Figure 5A). LRP5-G10 antibody, rather than LRP5-G2 antibody, significantly inhibited both ConCM-induced and Wnt3aCM-induced LRP6 phosphorylation in H23 cells. LRP5-G10 antibody also significantly reduced overall LRP6 phosphorylation in H23 cells. Similar effects on LRP6 phosphorylation were observed in membrane fractions isolated from H23 cells treated with LRP5-G2 and LRP5-G10 antibodies before stimulation with conditioned medium (Figure 5B). LRP5-G10 antibody also significantly reduced Axin1 protein levels in Wnt3aCM-stimulated cells. Since Axin1 is a component of the degradation complex that regulates the "free" pool of β-catenin levels, the decrease in its expression upon treatment with LRP5-G10 antibody before Wnt3aCM stimulation may explain why this antibody effectively enhances Wnt3aCM-inducible reporter activity (Figure 3).

[0211] To further understand the effects of antibodies on β-catenin levels, cytosolic fractions of H23 cells treated with LRP5-G2 and LRP5-G10 antibodies were isolated before stimulation with conditioned medium. As shown in Figure 5B, the LRP5-G2 antibody significantly upregulates β-catenin levels in ConCM-treated cells, consistent with its effect on reporter activity. Similarly, the LRP5-G10 antibody slightly decreases and increases β-catenin levels in ConCM-treated cells.

[0212] The NSCLC cell line H23 serves as an excellent system for investigating the effects of co-treatment with LRP5 antibodies on TOPflash reporter activity. Our previous results have shown that the LRP5-G2 antibody enhances ConCM-inducible reporter activity. In contrast, the LRP5-G10 antibody inhibits and enhances ConCM-inducible reporter activity and Wnt3aCM-inducible reporter activity, respectively. As shown in Figure 6, the enhancement of ConCM-inducible reporter activity by LRP5-G2 is significantly inhibited in the presence of the LRP5-G10 antibody. Our observations suggest that co-treatment with LRP5-G2 and LRP5-G10 antibodies may have a potent inhibitory effect on Wnt-stimulated TCF / LEF-mediated transcription. Therefore, by utilizing advances in library design strategies, we were able to discover a novel LRP5 antibody capable of exhibiting both antagonistic and enhancing activity against proximal and distal events related to β-catenin signaling. These activities also depend on various interactions between Wnt ligand and LRP5. The inventors are actively studying the therapeutic potential of these antibodies in vitro and in vivo.

[0213] Abbreviation TIFF0007840842000020.tif149150

[0214] TIFF0007840842000021.tif142150TIFF0007840842000022.tif179150

[0215] Where used herein, unless otherwise specified, the following meanings apply: The word “may” is used in a permissive sense (i.e., meaning it is possible) rather than a compulsory sense (i.e., meaning it must). The words “include,” “including,” and “includes,” etc., mean to include without limitation. The singular forms “a,” “an,” and “the” include multiple referents. Thus, for example, a reference to “one element” includes a combination of two or more elements, despite the use of other terms and phrases for one or more elements, such as “one or more.” The phrase “at least one” includes “one or more,” “one or a plurality,” and “a plurality.” The term “or,” unless otherwise indicated, is non-exclusive, i.e., encompasses both “and” and “or.” The term “any of” between a modifier and a set of things means that the modifier modifies each member of the set of things. Therefore, for example, the phrase "at least one of 1, 2, or 3" means "at least 1, at least 2, or at least 3." The term "essentially consisting of" refers to the inclusion of enumerated and other elements that do not substantially affect the basic and novel features of the claimed combination.

[0216] The description and drawings are not intended to limit the invention to any particular form disclosed, but rather to encompass all modifications, equivalents, and substitutions that fall within the spirit and scope of the invention as defined by the appended claims. Further modifications and substitutions of various aspects of the invention will be apparent to those skilled in the art in consideration of this description. Accordingly, this description and drawings should be interpreted as illustrative only and are intended to teach those skilled in the art a general way of carrying out the invention. The forms of the invention shown and described herein should be interpreted as examples of embodiments. As will be apparent to any person skilled in the art after taking advantage of this description of the invention, elements and materials may be replaced with elements and materials illustrated and described herein, parts and processes may be reversed or omitted, and certain features of the invention may be used independently. Modifications to the elements described herein may be made without departing from the spirit and scope of the invention as set forth in the following claims. The headings used herein are for structural purposes only and are not intended to limit the scope of the description.

[0217] All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference.

Claims

1. An antibody that binds to LRP5, comprising a light chain variable region and a heavy chain variable region, The heavy chain variable region includes complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region includes complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of the CDR includes or consists of the CDR sequence described below. CDR-H1 is FSSSSI (SEQ ID NO: 4), CDR-H2 is SISSSYGYTY (SEQ ID NO: 13), CDR-H3 is SWAM (SEQ ID NO: 33), CDR-L1 is SVSSA (SEQ ID NO: 34), CDR-L2 is SASSLYS (SEQ ID NO: 35), and CDR-L3 is YWAYYSPI (SEQ ID NO: 49). antibody.

2. The aforementioned antibody (i) The heavy chain variable region amino acid sequence of SEQ ID NO: 99, or (ii) Amino acid sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the heavy chain variable region amino acid sequence of SEQ ID NO: 99 It includes a heavy chain variable region, and The aforementioned antibody (i) The amino acid sequence of the light chain variable region of SEQ ID NO: 101, or (ii) Amino acid sequences having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the light chain variable region amino acid sequence of SEQ ID NO: 101 Including a light chain variable region, The antibody according to claim 1.

3. The antibody according to claim 1 or 2, comprising CDR-H1 consisting of SEQ ID NO: 4, CDR-H2 consisting of SEQ ID NO: 13, CDR-H3 consisting of SEQ ID NO: 33, CDR-L1 consisting of SVSSA (SEQ ID NO: 34), CDR-L2 consisting of SASSLYS (SEQ ID NO: 35), and CDR-L3 consisting of YWAYYSPI (SEQ ID NO: 49).

4. An antibody according to any one of claims 1 to 3, which is a monoclonal antibody, a humanized antibody, and / or a single-chain antibody.

5. The antibody according to any one of claims 1 to 4, which is an antibody-conjugated fragment selected from Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimer, minibody, diabody, and their polymers.

6. An antibody according to any one of claims 1 to 5, which is a bispecific antibody.

7. The antibody according to any one of claims 1 to 6, which is a bispecific antibody that further binds to the FZD receptor.

8. An antibody according to any one of claims 1 to 7, comprising a non-natural glycosylation pattern.

9. An immunoconjugate comprising an antibody according to any one of claims 1 to 7 and a detectable label or cytotoxic agent.

10. The immunoconjugate according to claim 9, wherein the cytotoxic agent is selected from meitansinoids, auristatin, drastatin, tubulicin, cryptophycin, pyrrolobenzodiazepine (PBD) dimers, indolinobenzodiazepine dimers, α-amanitin, trichoten, SN-38, duocalmycin, CC1065, calicheamicin, engine antibiotics, taxanes, doxorubicin derivatives, anthracyclines, and stereoisomers thereof.

11. A nucleic acid molecule encoding the antibody according to any one of claims 1 to 7.

12. The aforementioned antibody (i) SEQ ID NO: 98 heavy chain nucleic acid sequence, (ii) A nucleotide sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to the heavy chain nucleic acid sequence of SEQ ID NO: 98, or (iii) Codon degenerate nucleic acid sequence of (i) It includes a heavy chain variable region encoded by a nucleic acid, and The aforementioned antibody (i) SEQ ID NO: 100 light chain nucleic acid sequences, (ii) SEQ ID NO: A nucleic acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to the light chain nucleic acid sequence of 100, (iii) Codon degenerate nucleic acid sequence of (i) Including a light chain variable region encoded by nucleic acids, The nucleic acid molecule according to claim 11.

13. A vector comprising an expression control sequence functionally linked to the nucleic acid according to claim 11 or 12.

14. A host cell comprising a recombinant nucleic acid molecule containing an expression regulatory sequence functionally linked to the nucleic acid according to claim 11 or 12, or the vector according to claim 13.

15. The host cell according to claim 14, wherein the host cell is a Chinese hamster ovary (CHO) cell.

16. A method for producing an anti-LRP5 antibody, comprising the step of culturing host cells according to claim 14 or 15.

17. A composition comprising an antibody according to any one of claims 1 to 7, an immunoconjugate according to claim 9 or 10, a nucleic acid molecule according to claim 11 or 12, a vector according to claim 13, or a host cell according to claim 14 or 15.

18. The composition according to claim 17, wherein the composition comprises a diluent.

19. The composition according to claim 17 or 18, wherein the composition comprises one or more antibodies or immunoconjugates.

20. The composition according to any one of claims 17 to 19, wherein the composition is a pharmaceutical composition.

21. A kit comprising an antibody according to any one of claims 1 to 7, an immunoconjugate according to claim 9 or 10, a nucleic acid molecule according to claim 11 or 12, a vector according to claim 13, or a host cell according to claim 14 or 15.

22. A pharmaceutical composition for detecting LRP5 expression, comprising one or more antibodies according to any one of claims 1 to 7 or an immunoconjugate according to claim 9 or 10.

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