Antibodies binding to LRP5 proteins and methods of use

Abstract

The invention relates to antibodies that bind to the LRP5 protein and methods of use. Provided herein are antibodies that specifically bind to LRP5 and methods of use thereof.

Description

[0001] This application is a divisional application of an application filed on August 14, 2020, with application number 202080072086.2, and invention name “Antibodies binding to LRP5 protein and methods of use”. Citation of Related Applications

[0002] This application claims the benefit of the priority date of U.S. Provisional Application No. 62 / 886,913, filed on August 14, 2019, the contents of which are incorporated herein by reference in their entirety. background

[0003] Wnt signaling plays a key role in the regulation of multiple cellular processes such as cell fate determination, proliferation, survival, polarity, and migration. 1 Perturbations due to altered expression or mutations in the Wnt signaling pathway have been associated with defects in embryonic development and a variety of pathologies such as cancer and osteoporosis. 2-4 Wnt signaling leads to activation of both canonical and non-canonical signaling pathways 1,5 . Activation of the atypical pathway does not involve signaling molecules in the nucleus or transcription, but rather activates cytoplasmic signals that regulate the cytoskeleton and calcium stores. This pathway plays a major role in regulating cell polarity or migration. The typical pathway controls transcriptional activity primarily by regulating the cytoplasmic levels of β-catenin. Under non-stimulatory conditions, β-catenin associates with a destruction complex composed of Axin, APC, CK1, and GSK3b, which leads to phosphorylation, ubiquitination, and proteasomal degradation of β-catenin. Wnt signaling destabilizes this complex, causing "free" β-catenin to accumulate in the cytosol, translocate to the nucleus, and act as a co-activator of TCF / LEF-mediated transcription. Wnt binds to the frizzled family of seven-transmembrane domain receptors, as well as LRP5 or LRP6, leading to the initiation of the typical signaling pathway. 6-8 .

[0004] LRP5 and LRP6 are functionally redundant single-pass transmembrane receptors that share approximately 70% homology. Binding of Wnt ligands to Fzd and LRP5 / 6 leads to the recruitment of the destruction complex and Dishvelled (Dsh / Dvl) and phosphorylation of the PPPSPxS motif located in the intracellular domain of LRP5 / 6 9This phosphorylation is mediated by GSK3b and CK1, which in turn leads to attenuation of GSK3b activity, inhibition of β-catenin phosphorylation and subsequent proteasomal degradation, and enhancement of TCF / LEF-mediated transcriptional activity. LRP5 is widely expressed during embryonic development and in adult tissues. Mutations in LRP5 are associated with bone mass disorders, and several mouse models with LRP5 knockout or mutations exhibit alterations in bone development. 10 .

[0005] The expression of LRP5 has 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 attenuated following overexpression of dominant-negative LRP5. 11-13 In addition, LRP5 also appears to play an important role in regulating cell invasion and cell motility. 14-16 Although studies have shown that LRP6 is more effective than LRP5 in transducing Wnt signals, recent genetic experiments have shown that some Wnt ligands require the presence of both receptors to produce a canonical signal (17). Due to the importance of LRP5 in regulating Wnt signaling and its established role in several human diseases, LRP5 is becoming an increasingly important target for therapeutic drug development. Important biology regarding LRP5 and its role in Wnt signaling and the pathogenesis of various diseases remains to be discovered, and a deep toolbox of synthetic antibodies will help to systematically unravel these roles and will also provide additional targeted therapies.

[0006] Wnt signaling leads to the activation of canonical and atypical signaling pathways. Atypical pathway activation does not involve the nucleus or transcription but rather activates signaling molecules in the cytoplasm that regulate the cytoskeleton and calcium levels. This pathway plays a major role in regulating cell polarity or migration.

[0007] The canonical pathway controls transcriptional activity primarily by regulating the cytoplasmic levels of β-catenin. Under unstimulated conditions, β-catenin associates with the destruction complex (comprising Axin, APC, CD1, and GSKβ), leading to phosphorylation, ubiquitination, and proteasomal degradation of β-catenin. When Wnt binds to the 7-transmembrane receptor Frizzled receptor (FZD) and to the co-receptor low-density lipoprotein receptor-related protein (LRP5 or LRP6), Wnt signaling is activated. This signaling destabilizes the destruction complex, in part by attracting dishevelled (Dsh / Dvl) to the plasma membrane, leading to the accumulation of β-catenin, which then travels to the nucleus and activates TCF / LEF-mediated transcription.

[0008] The present invention described below identifies a novel set of synthetic antibodies targeting extracellular epitopes of LRP5 by utilizing state-of-the-art antibody phage display libraries and techniques. Overview

[0009] In one embodiment, provided herein is an antibody that specifically binds to LRP5, which antibody comprises 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, 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 a sequence selected from the following: the CDR sequence set of anti-LRP5 antibodies 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 CDR comprises or consists of a sequence selected from the group consisting of: CDR-H1 is selected from the group consisting of: LSYYYM, ISYSYI, LSYSSM, ISSYSI, ISYSYI, IYSYSI, LSYYYM, FSSSSI, LYYYYI, LSYSSI, IYSYYI, LLYYSSM and FSSSSI; CDR-H2 is selected from the group consisting of: SIYPYYGYTY, SSSYYGYTY, SISSSYGYTY, SIYSSYGSTS, SIYSSYGYTY, SIYPYSSYTS, SIYSSYGYTY, SIYPSYGYTY, SISPYYGYTS, SISSSYGSTS, SIYSYYGYTY, SISSSSYGYTY , SISSSYGYTYSISSYYGYTS and YISPYYGYTS; CDR-H3 is selected from the group consisting of: HGAM, TVRGSKKPYFSGWAM, SSYYSSVSSSVYAL, TVRGSKKPYFSGWAM, HYSYFFYAM, YAVYFPGYYWGM, WSHVSGHYSGM, WGAYHSSGYGM, GGSGVS and CDR-L3 In another embodiment, the antibody comprises a heavy chain variable region comprising: i) a heavy chain amino acid sequence as listed 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 a heavy chain amino acid sequence as listed in Table 2, wherein the CDR sequence is a set of CDR sequences as listed in Table 1, or iii) a conservatively substituted amino acid sequence of i), wherein the CDR sequence is a set of CDR sequences as listed in Table 1.In another embodiment, the antibody comprises a light chain variable region comprising: i) a light chain amino acid sequence as listed 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 a light chain amino acid sequence as listed in Table 2, wherein the CDR sequence is a set of CDR sequences as listed in Table 1, or iii) a conservatively substituted amino acid sequence of i), wherein the CDR sequence is a set of CDR sequences as listed in Table 1. In another embodiment, the CDR sequence is a full 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 light chain or heavy chain CDR sequence set 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 an antibody selected from antibodies 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 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 the group consisting of Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimer, nanobody, minibody. In another embodiment, the antibody is a bispecific antibody that also binds to a FZD receptor. In another embodiment, the antibody comprises a non-native glycosylation pattern. In another embodiment, the antibody comprises a cysteine ​​substitution or addition, for example in a constant region or a framework region.

[0010] In another aspect, provided herein are immunoconjugates comprising antibodies provided herein and detectable markers or cytotoxic agents. In another embodiment, the immunoconjugate comprises a cytotoxic agent selected from the group consisting of maytansine alkaloids, auristatins, dolastatins, tubulysin, scin, pyrrolobenzodiazepine (PBD) dimers, indolebenzodiazepine dimers, α-amanitin, trichothene, SN-38, duocarmycin, CC1065, calicheamicin, enediyne antibiotics, taxanes, doxorubicin derivatives, anthracyclines and stereoisomers thereof, azanofide, isosteres, analogs or derivatives.

[0011] In another aspect, nucleic acid molecules encoding antibodies provided herein are provided herein. In one embodiment, one or more 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 comprising: i) a heavy chain nucleic acid sequence as 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 a heavy chain nucleic acid sequence as listed in Table 2, wherein the CDR sequence is a CDR sequence set as listed in Table 1, or iii) a codon degenerate nucleic acid sequence of i), wherein the CDR sequence is a CDR sequence set as listed in Table 1. In another embodiment, the antibody comprises a light chain variable region encoded by a nucleic acid comprising: i) a light chain nucleic acid sequence as 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 to a light chain nucleic acid sequence as listed in Table 2, wherein the CDR sequence is a set of CDR sequences as listed in Table 1, or iii) a codon degenerate nucleic acid sequence of i), wherein the CDR sequence is a set of CDR sequences as listed in Table 1.

[0012] In another aspect, provided herein are vectors comprising expression control sequences operably linked to a nucleic acid as provided herein.

[0013] In another aspect, provided herein is a host cell comprising a recombinant nucleic acid molecule comprising an expression control sequence operably linked to a nucleic acid provided herein. In another embodiment, the host cell is a Chinese Hamster Ovary (CHO) cell.

[0014] In another aspect, provided herein is a host cell comprising a vector provided herein.

[0015] In another aspect, provided herein is a method for preparing an anti-LRP5 antibody, the method comprising culturing a host cell as provided herein.

[0016] In another aspect, provided herein is a composition comprising an antibody, immunoconjugate, nucleic acid molecule, vector or host cell as provided herein, and optionally a suitable diluent. In one embodiment, the composition comprises one or more antibodies or immunoconjugates, optionally wherein the composition is a pharmaceutical composition.

[0017] In another aspect, provided herein is a kit comprising an antibody, immunoconjugate, nucleic acid molecule, vector or host cell as provided herein.

[0018] On the other hand, provided herein is a method for detecting LRP5 expression, the method comprising contacting a sample comprising one or more cells with one or more antibodies or immunoconjugates as provided herein, and detecting the presence of any antibody complexes, under conditions that allow the formation of antibody: cell complexes. In one embodiment, the detection is by immunofluorescence. In another embodiment, the detection is by flow cytometry. In another embodiment, the method is used to detect LRP5 expression, and the antibody or immunoconjugate comprises a CDR sequence set 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, LRP5-R3O_G6.

[0019] On the other hand, provided herein is a method for inhibiting Wnt ligand binding to LRP5 receptor, destroying Wnt signaling pathway, inhibiting Wnt-induced transcriptional activity, inhibiting the activation of dishevelled proteins, promoting the retention of β-catenin destruction complex, promoting the accumulation of β-catenin or inhibiting cell growth, the method comprising contacting a cell expressing LRP5 receptor with an antibody or immunoconjugate as provided herein. On another hand, provided herein is an antibody or immunoconjugate for inhibiting Wnt ligand binding to LRP5 receptor, destroying Wnt signaling pathway, inhibiting Wnt-induced transcriptional activity, inhibiting the activation of dishevelled proteins, promoting the retention of β-catenin destruction complex, promoting the accumulation of β-catenin or inhibiting cell growth. On the other hand, provided herein is an antibody or immunoconjugate for inhibiting Wnt ligand binding to LRP5 receptor, destroying Wnt signaling pathway, inhibiting Wnt-induced transcriptional activity, inhibiting the activation of dishevelled proteins, promoting the retention of β-catenin destruction complex, promoting the accumulation of β-catenin or inhibiting cell growth. In yet another aspect, an antibody or immunoconjugate as provided herein is provided for the preparation of a drug for inhibiting Wnt ligand binding to LRP5 receptor, disrupting Wnt signaling pathways, inhibiting Wnt-induced transcriptional activity, inhibiting the activation of dishevelled proteins, promoting the retention of β-catenin destruction complexes, promoting β-catenin accumulation, or inhibiting cell growth. In one embodiment, the antibody or immunoconjugate blocks the binding of Wnt ligands to the Wnt3a binding site of LRP5. In another embodiment, the antibody or immunoconjugate blocks the binding of Wnt ligands to non-Wnt3a binding sites of LRP5. In another embodiment, 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, LRP5-R3O_G6.

[0020] In another embodiment, provided herein is a method for treating a cancer of a subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising an antibody or immunoconjugate as provided herein. On the other hand, it is a pharmaceutical composition for use in treating a cancer of a subject in need thereof, the pharmaceutical composition comprising an antibody or immunoconjugate as provided herein. In another embodiment, it is the use of a pharmaceutical composition for treating a cancer of a subject in need thereof, the pharmaceutical composition comprising an antibody or immunoconjugate as provided herein. In yet another embodiment, it is the use of a pharmaceutical composition in the preparation of a drug for treating a cancer of a subject in need thereof, the pharmaceutical composition comprising an antibody or immunoconjugate as provided herein. In one embodiment, cancer is selected from colon cancer, lung cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, gastric cancer, liver cancer, adrenocortical carcinoma, and osteoblastoma cancer. In another embodiment, cancer is selected from acute myeloid leukemia, prostate cancer, glioblastoma, bladder cancer, and cervical cancer. In another embodiment, the method comprises administering to the subject a first antibody or antibody conjugate as provided herein and a second antibody or antibody conjugate, wherein the first antibody or antibody conjugate blocks the binding of the Wnt ligand to the Wnt3a binding site of LRP5, and the second antibody or antibody conjugate blocks the binding of the Wnt ligand to the non-Wnt3a binding site of LRP5. In another embodiment, the first antibody or immunoconjugate comprises a set of CDR sequences of an antibody selected from 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, optionally wherein 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 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, LRP5-R3O_G6.

[0021] In another aspect, provided herein is a method for enhancing the signaling activity of a Wnt ligand that binds to a Wnt3a binding site of LRP5, the method comprising contacting a cell expressing LRP5 with an antibody that blocks 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.

[0022] In another aspect, provided herein is a method for enhancing the signaling activity of a Wnt ligand that binds to a non-Wnt3a binding site of LRP5, the method being contacting a cell 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings, which are incorporated herein and form a part of this specification, illustrate exemplary embodiments and, together with this description, further serve to enable those skilled in the relevant art to make and use these embodiments and other embodiments that will be apparent to those skilled in the art. The present invention will be described in more detail in conjunction with the following drawings, in which:

[0024] Figure 1A and Figure 1B Sixteen (16) anti-LRP antibodies are shown. (A) The amino acid sequences of the heavy chain (H) and light chain (L) complementary determining regions (CDRs) are presented. The antibodies were grouped according to unique epitopes as determined by competitive ELISA. (B) Single-point ELISA was performed on 96-well Maxisorp immunoplates coated with the ECD of mouse LRP5, mouse LRP6, and human LRP6 chimeras. The plates were incubated with the indicated concentrations of purified Fab or IgG1 and then incubated with horseradish peroxidase (HRP)-conjugated anti-κ antibodies. The wells were washed 8 times and then incubated with 3,3',5,5'-tetramethylbenzidine / H2O2 peroxidase (TMB) substrate for 5-10 min. The reaction was stopped by adding 1 M H3PO4 and the absorbance was measured spectroscopically at 450 nm in a microtiter plate reader.

[0025] Figure 2 Analysis of IgG1 binding to cell surface LRP5 by flow cytometry is shown. Binding of LRP5 IgG (5 μg / ml) to the NSCLC cancer cell line H23 was tested. Binding of anti-LRP5 IgG1 protein was detected using an Alexa488-conjugated anti-F(ab')2 secondary antibody. The stained anti-LRP5 population is shown in green, and the population stained only with the secondary antibody is shown as filled blue.

[0026] Figure 3 A- Figure 3D shows the effect of LRP5 IgG on transcriptional activity in cancer cells. In (A) MDAMB231, (B) T407D, (C) U2OS and (D) H23 cells, TOPflash (firefly luciferase gene) is used to carry out TCF / LEF reporter assay. Cells are seeded in 96 well plates with white walls and white bottoms, and treated with IgG of the concentration shown for 1 hour, and then stimulated with conditioned medium. Cells are lysed after 16-20 hours of conditioned medium stimulation, and reporter activity is assessed by measuring the luminescent signal produced by adding firefly luminescent reagent. Values ​​are normalized for the signal observed in cells treated with negative control antibodies (anti-MBP) and stimulated with ConCM. The data presented represent three independent experiments, and each condition is the mean value of three repetitions.

[0027] Figure 4 A and Figure 4 B shows the effect of LRP5-G2 and LRP5-G10 and Fab on transcriptional activity in cancer cells. TCF / LEF reporter assay was performed using TOPflash (firefly luciferase gene) in H23 cells pretreated with increasing concentrations of (A) anti-LRP IgG1 or (B) anti-LRP5 Fab. Figure 3 Basal reporter activity was assessed as described previously.

[0028] Figure 5 A and Figure 5 B shows the effect of LRP5 IgG1 on β-catenin signaling. H23 cell lines were pretreated with LRP5 IgG (100 nM) for the indicated time and then stimulated with conditioned medium. (A) Overall whole cell lysates or (B) membrane fractions and cytoplasmic fractions were generated and separated by SDS / PAGE. PVDF membranes were cut and immunoblotted for phosphorylated LRP6, LRP5, LRP6, Axin1, Dvl3 and β-catenin. Actin was used as a loading control for whole cell lysates, while Na / K ATPase and GAPDH were used as loading controls for membrane fractions and cytoplasmic fractions, respectively.

[0029] Figure 6 Multi-point competitive ELISA is shown. Dose response curves and non-linear regression plots are for LRP5 antibodies.

[0030] Figure 7 Analysis of IgG1 binding to cell surface LRP5 by flow cytometry is shown. Binding of LRP5 IgG (5 μg / ml) to the breast cancer cell line MDAMB231 was tested. Binding of anti-LRP5 IgG1 protein was detected using an Alexa488-conjugated anti-F(ab')2 secondary antibody. The stained anti-LRP5 population is shown in green, and the population stained only with the secondary antibody is shown as filled blue.

[0031] Figure 8 Analysis of IgG1 binding to cell surface LRP5 by flow cytometry is shown. Binding of LRP5 IgG (5 μg / ml) to the breast cancer cell line T47D was tested. Binding of anti-LRP5 IgG1 protein was detected using an Alexa488-conjugated anti-F(ab')2 secondary antibody. The stained anti-LRP5 population is shown in green, and the population stained only with the secondary antibody is shown as filled blue.

[0032] Fig. 9 A schematic diagram showing the typical Wnt signaling pathway. Detailed Description

[0033] I. Definitions Unless otherwise defined, scientific and technical terms used in conjunction with the present disclosure should have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms should include the plural, and plural terms should include the singular. For example, the term "cell" includes a single cell and more than one cell or cell colony. In general, the nomenclature and techniques used in conjunction with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art (see, e.g., Green and Sambrook, 2012).

[0034] As used herein, the term "polypeptide" refers to a molecule having a sequence of natural and / or non-natural amino acids connected by peptide bonds. The term "peptide" refers to a short polypeptide, usually no more than 30 amino acids in length. The amino acid sequence of a polypeptide is referred to as its "primary structure". The term "protein" refers to a polypeptide having a secondary, tertiary and / or quaternary structure (e.g., a structure stabilized by hydrogen bonds, relationships between secondary structures, and a structure formed by more than one protein). Proteins can be further modified by other attached parts such as carbohydrates (glycoproteins), lipids (lipoproteins), phosphate groups (phosphoproteins), etc.

[0035] As used herein, an amino acid sequence "consists of" only the amino acids in that sequence.

[0036] As used herein, a first amino acid sequence "consists essentially of" a second amino acid sequence if the first amino acid sequence (1) comprises the second amino acid sequence and (2) is no longer than the second amino acid sequence by no more than 1, no more than 2, or no more than 3 amino acids.

[0037] As used herein, if the second amino acid sequence comprises 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 that is a fragment of the second amino acid sequence may be less than any of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids than the second amino acid sequence.

[0038] 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, for example, insertions, deletions or substitutions of one or a few amino acids. Functionally equivalent sequences retain the function (e.g., immunogenicity) of the reference sequence to which they are equivalent. If a functionally equivalent amino acid sequence contains substitutions of one or more amino acids relative to the reference sequence, these will generally be conservative amino acid substitutions.

[0039] As used herein, a "conservative amino acid substitution" is an amino acid substitution in which one amino acid residue is replaced by another amino acid residue without eliminating the desired properties of the protein. Suitable conservative amino acid substitutions can be made by replacing each other with amino acids similar in hydrophobicity, polarity, and R chain length. See, e.g., Watson et al., "Molecular Biology of the Gene," Fourth Edition, 1987, The Benjamin / Cummings Pub. Co., Menlo Park, CA, p. 224. Examples of conservative amino acid substitutions include the following (note that some categories are not mutually exclusive):

[0040]

[0041] As used herein, the term "substantially identical" refers to the identity between a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence contains a sufficient or minimum number of i) amino acid residues identical to the amino acid residues in the second amino acid sequence being compared, or ii) amino acid residues that are conservatively substituted for the amino acid residues in the second amino acid sequence being compared, such that the first amino acid sequence and the second amino acid sequence have a common domain and / or a common functional activity and / or a common immunogenicity. For example, amino acid sequences containing a common structure or antigenic domain with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are referred to as being 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 identical to the nucleotides in the second nucleic acid sequence being compared, such that the first nucleotide sequence and the second nucleotide sequence encode a polypeptide having a common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity, or encode a polypeptide having the same immunogenicity.

[0042] 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., can be bound by an antibody). The term can refer to any molecule that can be recognized by an antibody, for example, a polypeptide, a polynucleotide, a carbohydrate, a lipid, a chemical moiety or a combination thereof (e.g., a phosphorylated polypeptide or a glycosylated polypeptide, etc.). Those skilled in the art will understand that the term does not indicate that the molecule is immunogenic in every case, but only indicates that it can be targeted by an antibody.

[0043] As used herein, the term "epitope" refers to a localization site on an antigen that is recognized and combined by an antibody. An epitope can include a small amount of amino acids or a small amount of amino acid parts, for example, 5 or 6 or more, for example, 20 or more amino acids, or parts of these amino acids. In some cases, the epitope includes non-protein components, for example, components from carbohydrates, nucleic acids or lipids. In some cases, the epitope is a three-dimensional part. Therefore, for example, when the target is a protein, the epitope can include continuous amino acids, or amino acids from different parts of the protein approached by protein folding (for example, discontinuous epitopes).

[0044] 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 through at least one antigen recognition site in the immunoglobulin variable region. The variable region is most critical in terms of binding specificity and affinity. As used herein, the term "antibody" includes complete polyclonal antibodies, complete monoclonal antibodies, antibody fragments, single-chain Fv (scFv) mutants, multispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, hybrid antibodies, fusion proteins and any other immunoglobulin molecules comprising antigen recognition sites, as long as the antibody exhibits the desired biological activity. Antibodies can (i) belong to 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) its subclass (isotype) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). The light chain can be λ or κ. The antibodies can be naked or conjugated to other molecules such as toxins, drugs, radioisotopes, chemotherapeutic agents, and the like.

[0045] In one embodiment, a "complete antibody" comprises a tetramer consisting of two pairs of identical polypeptide chains, each pair having one "light chain" (about 25 kDa) and one "heavy chain" (about 50-70 kDa). The heavy and light chains are linked by covalent and non-covalent bonds (e.g., disulfide bonds), the number and amount of these bonds varying between different immunoglobulin classes. In one aspect, each chain comprises a variable region and a constant region. The antigen recognition site of the variable region consists of a hypervariable region or complementarity determining region (CDR) and a framework region. The framework region does not usually contact the antigen, but provides structural support for the CDR. The constant region interacts with other immune cells of the body. Between the constant and variable regions (only IgG, IgD, IgA, not IgM or IgE) is a hinge region located in the center between the two heavy chains, which provides flexibility for articulating antigen binding.

[0046] The following is a non-exhaustive list of different antibody formats, all of which retain antigen binding activity: (1) Whole immunoglobulin (also called "intact" antibody) (two light chains and two heavy chains, e.g., a tetramer).

[0047] (2) Immunoglobulin polypeptide (light chain or heavy chain).

[0048] (3) Antibody fragments, such as Fv (monovalent or bivalent variable region fragments, and may contain only the variable region (e.g., V L and / or V H )、Fab (VL C L V H C H )、F(ab')2、Fv (V L V H ), scFv (single-chain Fv) (comprising V L and V H peptide), (scFv)2, sc(Fv)2, bispecific sc(Fv)2, bispecific (scFv)2, minibody (sc(FV)2 fused to CH3 domain), triabody is trivalent sc(Fv)3 or trispecific sc(Fv)3.

[0049] (4) Multivalent antibodies (antibodies that contain binding regions that bind to two different epitopes or proteins, e.g., “scorpion” antibodies).

[0050] (5) Fusion proteins, which comprise the binding portion of an immunoglobulin fused to another amino acid sequence (such as a fluorescent protein).

[0051] As used herein, the term "antibody fragment" refers to some or a portion of an antibody or antibody chain, which comprises fewer amino acid residues than a complete or complete antibody or antibody chain, and which binds to an antigen or competes with a complete antibody. The fragment can be obtained via chemical or enzyme treatment of a complete or complete antibody or antibody chain. The fragment can also be obtained by recombinant means. For example, F (ab') 2 fragments can be produced by treating antibodies with pepsin. The resulting F (ab') 2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can result in the formation of Fab fragments. Fab, Fab' and F (ab') 2, scFv, dsFv, ds-scFv, dimers, mini antibodies, double antibodies, bispecific antibody fragments and other fragments can also be constructed by recombinant expression technology.

[0052] While various antibody fragments are defined in terms of the digestion products of an intact antibody, one of skill will appreciate that such fragments may also be chemically synthesized de novo or constructed and expressed using recombinant DNA methodologies.

[0053] A single-chain antibody (scFv) is a fragment comprising a V chain linked by a linker (e.g., a peptide linker). L and V HScFv can also be used to form tandem (or bivalent) ScFv or diabodies. The production and properties of tandem scFv and diabodies are described in, for example, Asano et al. J Biol. Chem. 286:1812 (2011); Kenanova et al. (2010) Prot Eng Design Sel 23:789; Asano et al. (2008) Prot Eng Design Sel 21:597.

[0054] Antibody fragments also include Fd (the portion of the heavy chain contained in the Fab fragment) and single domain antibodies. Single domain antibodies (sdAb) are variable domains of heavy or light chains produced by recombinant methods.

[0055] As used herein, the phrase "CDR sequence set" refers to the 3 heavy chain CDRs and / or 3 light chain CDRs of a particular antibody described herein. A "light chain" CDR sequence set refers to a light chain CDR sequence. A "heavy chain" CDR sequence set refers to a heavy chain CDR sequence. A "full" CDR sequence set refers to both a heavy chain CDR sequence and a light chain CDR sequence. For example, for the antibody LRP5-A7 as shown in Table 1, the full CDR sequence set comprises or consists of the following: SVSSA (CDR L1), SASSLYS (CDR L2), AWGWGLF (CDRL3), LYSSSM (CDR H1), SIYPYYGYTY (CDR H2), and HGAM (CDR H3). The CDR sequence of each CDR can, for example, comprise, consist essentially of, or consist of the CDRs in Table 1. CDRs are predicted based on IMGT sequence alignments.

[0056] As used herein, the term "monoclonal antibody" refers to an antibody clone preparation or composition with a single binding specificity and affinity for a particular epitope on an antigen ("monoclonal antibody composition"). A "polyclonal antibody" refers to an antibody preparation or composition raised against a single antigen, but with different binding specificities and affinities ("polyclonal antibody composition").

[0057] As used herein, the term "chimeric antibody" refers to an antibody having amino acid sequences derived from two or more species. In one embodiment, the variable regions of both the light and heavy chains correspond to the variable regions of an antibody with desired specificity, affinity and capability derived from a mammal of one species (e.g., mouse, rat, rabbit, etc.), while the constant regions are homologous to sequences derived from another species (usually a subject to be treated, e.g., human) to avoid eliciting an immune response.

[0058] As used herein, the term "humanized antibody" refers to a chimeric antibody in which CDRs obtained from the VH and VL regions of non-human antibodies with desired specificity, affinity and capacity are grafted onto human framework sequences. In one embodiment, the framework residues of a humanized antibody are modified to improve and optimize the specificity, affinity and capacity of the antibody. Humanization, i.e., replacement of non-human CDR sequences with the corresponding sequences of a human antibody, can be performed as described in, e.g., U.S. Pat. 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).

[0059] As used herein, the term "human antibody" refers to an antibody produced by the human body or an antibody having an amino acid sequence corresponding to an antibody produced by the human body, prepared by any technique known in the art.

[0060] As used herein, the term "hybrid antibody" refers to an antibody in which heavy and light chain pairs from antibodies with different antigenic determining regions are assembled so that the resulting tetramer can recognize and bind to two different epitopes or two different antigens. Hybrid antibodies can be bispecific (binding to 2 different antigens or epitopes) or multispecific (>1 different antigen or epitope).

[0061] As used herein, an antibody is "monospecific" if all of its antigen binding sites bind to the same epitope.

[0062] As used herein, an antibody is "bispecific" if it has at least two different antigen binding sites that each binds to a different epitope or antigen.

[0063] As used herein, an antibody is "polyvalent" if it has more than one antigen binding site. For example, a tetravalent antibody has four antigen binding sites.

[0064] Specificity of binding can be defined based on the comparative dissociation constant (Kd) of the antibody (or other targeting moiety) for the target compared to the dissociation constant between the antibody and other substances in the environment or generally unrelated molecules. A larger (higher) K d is the K describing the lower affinity interaction d In contrast, a smaller (lower) K d is the K describing higher affinity interactions or tighter binding d By way of example only, the K of an antibody specifically binding to a target is d It can be femtomolar, picomolar, nanomolar, or micromolar, while the K of antibodies binding to unrelated substances is d The concentration can be millimolar or higher. The binding affinity can be in the micromolar range (kD = 10 -4 Up to 10 -6 ), nanomolar concentration range (kD = 10 -7 M to 10 -9 M), picomolar concentration range (kD = 10 -10 M to 10 -12 M) or femtomolar concentration range (kD = 10 -13 M to 10 -15 M) inside.

[0065] As used herein, if the antibody is expressed in less than 10 -4 M (i.e., in the micromolar concentration range) Kd binding antigen or epitope, then the antibody "binds" or "recognizes" the antigen or epitope. For cell types (e.g., antibodies that bind to cancer cells), the term "binds" generally means that the agent binds to the majority of cells in a pure population of these cells. For example, antibodies that bind to a specific cell type generally bind to at least 2 / 3 of the cells in the specified cell population (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 determined by comparing the binding of an antibody to cells presenting the polypeptide and the binding (or lack of binding) of an antibody to cells that do not express the polypeptide. The skilled person will recognize that, depending on the method and / or threshold for determining binding, some variability will occur. The affinity of an antibody to a target can be determined according to methods known in the art, for example, as described in Ernst et al. Determination of EquilibriumDissociation Constants, Therapeutic Monoclonal Antibodies (Wiley & Sons ed.2009).

[0066] As used herein, the term "greater affinity" refers to where antibody X binds to target Y more strongly than to target Z (K on ) and / or have a smaller dissociation constant (K off ), and in this case antibody X has a greater affinity for target Y than for target Z. Likewise, the term "lesser affinity" herein refers to the degree of antibody binding in which antibody X binds to target Y less strongly and / or with a greater dissociation constant than to target Z, and in this case antibody X has a lower affinity for target Y than for target Z. The binding affinity between an antibody and its target antigen can be expressed as K A Equal to 1 / K D , where K D Equal to k on / k off . k on and k off Values ​​can be measured using surface plasmon resonance technology, for example, using a Molecular Affinity Screening System (MASS-1) (Sierra Sensors GmbH, Hamburg, Germany). Antagonists or blocking antibodies are antibodies that partially or completely block, inhibit, or neutralize biological activity associated with a target antigen relative to activity under similar physiological conditions when the antibody is not present. Antagonists can be competitive, non-competitive, or irreversible. Competitive antagonists are substances that bind to a natural ligand or receptor at the same site as the natural ligand-receptor interaction, or substances that allosterically bind in a manner that induces changes to prevent normal binding. Non-competitive antagonists bind at a site different from the natural ligand-receptor interaction, but reduce the KD or signal produced by the interaction. Irreversible inhibitors cause covalent modification of the receptor, preventing any subsequent binding.

[0067] As used herein, the term "avidity" refers to the overall stability of the binding complex between an antibody and a target antigen. It is controlled by three factors: (i) the intrinsic affinity of the antibody for the antigen, (2) the valence of the antibody, and (3) the geometric arrangement of the interacting components. Avidity is the strength of the interaction between an antibody and a single target, while avidity is the cumulative strength of more than one affinities. In one embodiment, the antibodies disclosed herein are bivalent.

[0068] As used herein, an antibody "preferentially binds" a first antigen relative to a second antigen if the antibody binds the first antigen with a greater affinity than it binds the second antigen. Preferential binding can be any of at least 2-fold, 5-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1000-fold greater affinity. Thus, for example, if an antibody binds LRP5 with a greater affinity than it binds LRP6, the antibody preferentially binds LRP5 relative to LRP6.

[0069] As used herein, if the antibody is present at 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 "specifically binds" or is "specific for" a target antigen or group of target antigens if the antibody binds to the target antigen or each member of the group of target antigens with at least one of the following affinity M, and binds to the target antigen or each member of the group of target antigens with an affinity at least 2-fold greater than the affinity for the non-target antigen as compared to the target antigen. Typically, specific binding is characterized by binding to the antigen with sufficient affinity to render the antibody useful as a diagnostic agent for detecting the antigen or epitope and / or as a therapeutic agent targeting the antigen or epitope.

[0070] As used herein, an antibody "blocks" or "antagonizes" the binding of a ligand to a receptor when the antibody competitively reduces or prevents all ligand-receptor interactions. In embodiments, the measured reduction level can be any of at least 5%, 10%, 25%, 50%, 80%, 90%, 95%, 97.5%, 99%, 99.5%, 99.9% of a control (e.g., untreated) cell. 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 a Wnt protein with the LRP5 receptor. This results in a reduction or blocking of downstream signaling events associated with Wnt signaling. This includes, for example, activation of dishevelled proteins, dissolution of the β-catenin destruction complex, lower cytoplasmic levels of β-catenin, and / or lower activity of TCF / LEF-mediated transcription.

[0071] For antibody targets (e.g., antigens, analytes, immune complexes), the term "capture" generally indicates that the antibody binds to the majority of the antibody target in a pure population (assuming an appropriate molar ratio). For example, an antibody that binds to a particular antibody target generally binds to at least 2 / 3 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%). The skilled artisan will recognize that some variability will occur depending on the method and / or threshold for determining binding.

[0072] The term "conjugate" refers to a first molecule, e.g., an antibody, chemically coupled to a moiety (e.g., a detectable label or a biologically active moiety, such as a drug, toxin, or chemotherapeutic or cytotoxic agent) ("immunoconjugate"). Thus, the present disclosure contemplates antibodies conjugated to one or more moieties. Furthermore, an antibody may be a "conjugated antibody" or a "non-conjugated antibody" (i.e., not conjugated to a moiety).

[0073] As used herein, the term "antibody-drug conjugate" or ("ADC") refers to an antibody conjugated to a drug. Typically, the conjugation involves covalent bonding through a linker.

[0074] As used herein, the term "labeled" molecule (e.g., nucleic acid, protein or antibody) refers to a molecule to which a detectable marker is covalently (through a linker or chemical bond) or non-covalently (through ionic bonds, van der Waals bonds, electrostatic bonds or hydrogen bonds) bound, such that the presence of the molecule can be detected by detecting the presence of the detectable marker bound to the molecule.

[0075] As used herein, the term "detectable marker" refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means. Examples of detectable markers are described herein and include, but are not limited to, colorimetric markers, fluorescent markers, chemiluminescent markers, enzyme markers, and radioactive markers. For purposes of this disclosure, a detectable marker may also be a moiety (e.g., biotin) that does not itself generate a signal but binds to a second moiety (e.g., labeled avidin) that can generate a signal.

[0076] The term "crosslinking" about antibodies refers to the attachment of antibodies to solid or semisolid matrices (e.g., agarose gel, beads, microtiter plates) or another protein or antibody. For example, antibodies can be polymerized to produce antibody complexes with multiple (more than 2) antigen binding sites. Antibodies can be polymerized by expressing antibodies as high-price isotypes (e.g., IgA or IgM, which usually form a complex of 2 or 5 antibodies, respectively). Antibody polymerization can also be carried out by using a crosslinking agent (e.g., carbodiimide, NHS ester, etc.) containing a reactive group capable of connecting proteins. Methods and compositions for making antibodies crosslinked to matrices are described in, for example, Abcam and New England Biolab catalogs and websites (available at abcam.com and neb.com). Crosslinker compounds with various reactive groups are described in, for example, Thermo Fisher Scientific catalogs and websites (available at piercenet.com).

[0077] 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.

[0078] As used herein, the term "expression construct" refers to a polynucleotide comprising an expression control sequence operably connected to a heterologous nucleotide sequence to be the object of expression (i.e., a sequence to which the expression control sequence is not usually connected in nature). As used herein, the term "expression vector" refers to a polynucleotide comprising an expression construct and a sequence sufficient to replicate in a host cell or insert into a host chromosome. Plasmids and viruses are examples of expression vectors. As used herein, the term "expression control sequence" refers to a nucleotide sequence that regulates the transcription and / or translation of a nucleotide sequence to which it is operably connected. Expression control sequences include promoters, enhancers, repressors (transcriptional regulatory sequences) and ribosome binding sites (translation regulatory sequences).

[0079] The term "vector" as used herein includes any intermediate vehicle of a nucleic acid molecule that enables, for example, the nucleic acid molecule to be introduced into a prokaryotic cell and / or eukaryotic cell and / or integrated into the genome, and includes plasmids, phagemids, phages or viral vectors, such as retroviral-based vectors, adeno-associated viral vectors, etc. The term "plasmid" as used herein generally refers to a construct of extrachromosomal genetic material, usually a circular DNA duplex, which can replicate independently of chromosomal DNA.

[0080] As used herein, a nucleotide sequence is "operably linked" to an expression control sequence when the expression control sequence functions in a cell to regulate transcription of the nucleotide sequence. This includes promoting transcription of the nucleotide sequence through interaction between a polymerase and a promoter.

[0081] As used herein, "host cell" refers to a recombinant cell that contains an expression construct.

[0082] As used herein, the term "biological sample" refers to a sample containing cells (e.g., tumor cells) or biomolecules derived from cells. Biological samples can be obtained from subjects (e.g., patients), from animals (such as, animal models) or from cultured cells (e.g., cell lines or cells taken out from patients and grown in culture medium for observation). Biological samples can include tissues and / or liquids. It can be obtained from any biological source, including but not limited to blood, blood fractions (e.g., serum or plasma), cerebrospinal fluid (CSF), lymph, tears, saliva, sputum, cheek swabs, milk, urine or feces. The biological sample can be a biopsy sample, such as a tissue biopsy sample, such as a needle puncture biopsy sample, a fine needle aspiration biopsy sample, a surgical biopsy sample, etc. The sample can include a tissue sample with a lesion or a suspected lesion, although the biological sample can also be derived from another site, such as a suspected metastatic site, a lymph node or from blood. The biological sample can be a part of a sample taken from a subject. Examples of tissue samples include brain tissue samples or neural tissue samples. Methods for obtaining such biological samples are known in the art, including but not limited to standard blood recovery procedures.

[0083] As used herein, the term "diagnosis" refers to the relative probability that a subject suffers from a disorder such as cancer. Similarly, the term "prognosis" refers to the relative probability that a subject may experience a certain future outcome. For example, in the context of the present disclosure, prognosis can refer to the likelihood that an individual will develop cancer, relapse, that a cancer will metastasize, that a cancer will be cured, or the possible severity of a disease (e.g., severity of symptoms, rate of functional decline, survival, etc.). These terms are not intended to be absolute, as will be understood by any technician in the field of medical diagnosis.

[0084] 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 refer to, for example, a reduction in tumor size, cancer cell number, growth rate, metastatic activity, a reduction in cell death of non-cancerous cells, a reduction in nausea and other chemotherapy or radiotherapy side effects, etc. The terms "treatment" and "prevention" are not intended to be absolute terms. Treatment and prevention may refer to a delay in any onset, an improvement in symptoms, an increase in patient survival, a survival time or a survival rate, etc. Treatment and prevention may be complete (undetectable levels of neoplastic cells) or partial, so that the neoplastic cells found in the patient are less than those that occur when there is no intervention of the present invention. The effect of treatment may be compared with an individual or a collection of individuals who have not received treatment, or compared with different times before or during treatment of the same patient. In some aspects, the severity of the disease is reduced by at least 10% compared with, for example, an individual before administration or a control individual who has not undergone treatment. In some aspects, the severity of the disease is reduced by at least 25%, 50%, 75%, 80% or 90%, or in some cases, is no longer detectable using standard diagnostic techniques.

[0085] As used herein, the terms "effective amount," "effective dose," and "therapeutically effective amount" refer to an amount of an agent (such as an antibody or immunoconjugate) sufficient to produce a desired response (such as reducing or eliminating the signs or symptoms of a condition or ameliorating a disorder). In some instances, an "effective amount" is an amount that treats (including preventing) one or more symptoms and / or potential causes of any disorder or disease and / or prevents disease progression. For example, for a particular parameter, a therapeutically effective amount will show an increase or decrease of any of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100% of the therapeutic effect. The therapeutic efficacy may also be expressed as an increase or decrease "... times." For example, a therapeutically effective amount may have any of at least 1.2 times, 1.5 times, 2 times, 5 times, or more times the effect compared to a control.

[0086] As used herein, the term "pharmaceutical composition" refers to a composition comprising a pharmaceutical compound (eg, drug) and a pharmaceutically acceptable carrier.

[0087] As used herein, the term "pharmaceutically acceptable" refers to a carrier that is compatible with the other ingredients 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 their preparation and use techniques are known to those skilled in the art in light of the present disclosure. For a detailed list of suitable pharmacological compositions and their administration techniques, one can refer to textbooks such as: Remington's Pharmaceutical Sciences, 17th Edition 1985; Brunton et al., "Goodman and Gilman's The Pharmacological Basis of Therapeutics," McGraw-Hill, 2005; University of the Sciences in Philadelphia (ed.), "Remington: The Science and Practice of Pharmacy," Lippincott Williams & Wilkins, 2005; and University of the Sciences in Philadelphia (ed.), "Remington: The Principles of Pharmacy Practice," Lippincott Williams & Wilkins, 2008.

[0088] Pharmaceutically acceptable carriers will generally be sterile, at least for human use. Depending on the route of administration, pharmaceutical compositions will generally contain agents for buffering and preservation during storage, and may contain buffers and carriers for appropriate delivery. Examples of pharmaceutically acceptable carriers include, but are not limited to, physiological (0.9%) saline, phosphate buffered saline (PBS), Hank's balanced salt solution (HBSS), and various electrolyte solutions, such as PlasmaLyte ATM (Baxter).

[0089] Acceptable carriers, excipients and / or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride or a combination thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, d-glutamic acid, d-aspartic acid, d- ... amino acid, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof; monosaccharides, disaccharides, and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents, such as EDTA; sugars, such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbitol, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or nonionic surfactants, such as Tween, Pluronics, Triton-X, or polyethylene glycol (PEG).

[0090] The terms "dose" and "dosage" are used interchangeably herein. Dose refers to the amount of active ingredient given to an individual per administration. For the present invention, dose can refer to the concentration of an antibody or related component, such as the amount of a therapeutic agent or the dose of a radiolabel. The dose will vary according to many 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 mode of the detectable marker (if any). Those skilled in the art will recognize that the dose can be modified based on the above factors or based on the progress of the treatment. The term "dosage form" refers to the specific form of the drug and depends on the route of administration. For example, the dosage form can be a liquid, for example, a saline solution for injection.

[0091] 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 health care provider such as a doctor or nurse. Subjects include mammals, such as humans and non-human primates, such as monkeys, as well as dogs, cats, horses, cows, rabbits, rats, mice, goats, pigs and other mammalian species. Subjects may also include birds. A patient may be an individual seeking treatment, monitoring, adjusting or modifying an existing treatment regimen, etc. The term "cancer subject" refers to an individual who has been diagnosed with cancer. Cancer patients may include individuals who have not yet received treatment, individuals who are currently receiving treatment, individuals who have undergone surgery, and individuals who have stopped treatment.

[0092] In the context of treating cancer, a subject in need of treatment may refer to an individual who has cancer or a precancerous condition, an individual who already has cancer and is at risk of recurrence, an individual suspected of having cancer, an individual who is undergoing standard cancer treatment (such as radiation therapy or chemotherapy, etc.).

[0093] "Cancer", "tumor", "transformed" and the like terms include precancerous cells, neoplastic cells, transformed cells and cancerous cells, and may refer to solid tumors or non-solid cancers (see, e.g., Edge et al. AJCC Cancer Staging Manual (7th edition 2009); Cibas and Ducatman Cytology: Diagnostic principles and clinical correlates (3rd edition 2009)). Cancer includes both benign and malignant neoplasms (abnormal growths). "Transformation" refers to spontaneous or induced phenotypic changes, such as cell immortalization, morphological changes, abnormal cell growth, reduction of contact inhibition and anchorage and / or malignant tumors (see, Freshney, Culture of Animal Cells Manual of Basic Technique (3rd edition 1994)). Although transformation can be caused by infection with transforming viruses and incorporation of new genomic DNA or uptake of exogenous DNA, transformation can also occur spontaneously or after exposure to carcinogens.

[0094] The term "cancer" may refer to any cancer, including but not limited to leukemia, carcinoma, sarcoma, adenocarcinoma, lymphoma, solid cancer and lymphoid cancer, etc. Examples of different types of cancer include but are not limited to lung cancer (e.g., non-small cell lung cancer or NSCLC), breast cancer, prostate cancer, colorectal cancer, bladder cancer, ovarian cancer, leukemia, liver cancer (i.e., hepatocarcinoma), kidney cancer (i.e., renal cell carcinoma), thyroid cancer, pancreatic cancer, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, stomach (stomach, gastric) cancer, kidney cancer, central nervous system cancer, skin cancer, glioblastoma and melanoma.

[0095] As used herein, a chemical entity, such as a polypeptide, is "substantially pure" if it is the predominant chemical entity of that species (e.g., polypeptide) in a composition. This includes chemical entities that represent more than 50%, more than 80%, more than 90%, more than 95%, more than 98%, more than 99%, more than 99.5%, more than 99.9%, or more than 99.99% of the chemical entities of that species in the composition.

[0096] The language "isolated antibody" refers to an antibody produced in vivo or in vitro that has been removed from a source from which the antibody was produced, eg, an animal, hybridoma, or other cell line such as recombinant insect cells, yeast cells, or bacterial cells that produce the antibody.

[0097] "Substantially pure" or "isolated" means that the target species is the predominant species present (i.e., more abundant on a molar basis than any other individual macromolecular species in the composition), and a substantially purified fraction is a composition in which the target species constitutes at least about 50% (on a molar basis) of all macromolecular species present. Generally speaking, a substantially pure composition means that about 80% to 90% or more of the macromolecular species present in the composition are the purified species of interest. If the composition consists primarily of a single macromolecular species, the target species is purified to substantial homogeneity (contaminant species cannot be detected in the composition by conventional detection methods). For purposes of this definition, solvent species, small molecules (<500 Daltons), stabilizers (e.g., BSA), and elemental ionic species are not considered macromolecular species.

[0098] As used herein, the term "sequence identity" refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. In order to determine the percentage of identity of two amino acid sequences or two nucleic acid sequences, the sequences are compared to achieve the best comparison purpose (for example, a room can be introduced in the sequence of the first amino acid or nucleic acid sequence to compare best with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at this position. The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences (that is, % identity = identical overlapping position number / total number of positions multiplied by 100%). In one embodiment, the two sequences are of the same length. The determination of the percentage of identity between the two sequences can also be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameter set, e.g., score=100, wordlength=12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present application. BLAST protein searches can be performed with the XBLAST program parameter set, e.g., score=50, wordlength=3, to obtain amino acid sequences homologous to the protein molecules described herein. In order to obtain the gap comparison for the purpose of comparison, the Gapped BLAST described in, for example, Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402 can be utilized. Alternatively, PSI-BLAST can be used for iterative retrieval (the same) of the distance relationship between the detection molecules. When utilizing BLAST, Gapped BLAST and PSI-Blast programs, the default parameters of the corresponding programs (e.g., the default parameters of XBLAST and NBLAST) can be used (see, e.g., NCBI website). Another preferred non-limiting example of the mathematical algorithm for sequence comparison is Myers and Miller, 1988, CABIOS 4:11-17 algorithm. Such an algorithm is incorporated into the ALIGN program (version 2.0) as a part of the GCG sequence alignment software package.When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between the two sequences can be determined using techniques similar to those described above with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0099] For antibodies, when the antibody sequences are maximally aligned by IMGT, the percent sequence identity can be determined. After alignment, if a test antibody region (e.g., the entire mature variable region of a heavy or light chain) is compared to the same region of a reference antibody, the percent sequence identity between the test antibody region and the reference antibody region is the number of positions occupied by the same amino acid in both the test antibody region and the reference antibody region divided by the total number of aligned positions in the two regions, multiplied by 100 to convert to a percentage.

[0100] Amino acid sequence identity percentage 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 can be obtained from the National Institute of Health, Bethesda, Md. NCBI-BLAST2 uses several search parameters, wherein all of these search parameters are set to default values, including, for example, unmasked = yes, chain = all, expected occurrence = 10, minimum low complexity length = 15 / 5, multi-pass e-value = 0.01, multi-pass constant = 25, final gap comparison drop = 25 and scoring matrix = BLOSUM62.

[0101] When NCBI-BLAST2 is used to compare amino acid sequences, the % amino acid sequence identity of a specific amino acid sequence A to or relative to a specific amino acid sequence B (which can also be alternatively expressed as a specific amino acid sequence A to or relative to a specific amino acid sequence B having or containing a certain amino acid sequence identity %) is calculated as follows: Multiply 100 by the fraction X / Y Wherein X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in the alignment of A and B in the program, and wherein Y is the total number of amino acid residues in B. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % identity of the amino acid sequence of A to B will not be equal to the % identity of the amino acid sequence of B to A. The term "nucleic acid sequence" as used herein refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars, and inter-sugar (main chain) connections, and includes cDNA. The term also includes sequences comprising modifications or substitutions of non-naturally occurring monomers or portions thereof. The nucleic acid sequence of the present application may be a deoxyribonucleic acid sequence (DNA) or a ribonucleic acid sequence (RNA), and may include naturally occurring bases, including adenine, guanine, cytosine, thymine, and uracil. The sequence may also contain modified bases. Examples of such modified bases include aza and deazaadenine, guanine, cytosine, thymine, and uracil; and xanthine and hypoxanthine. It should be understood that polynucleotides comprising non-transcribeable nucleotide bases can be used as probes in, for example, hybridization assays. Nucleic acids can be double-stranded or single-stranded and represent sense or antisense strands. In addition, the term "nucleic acid" includes complementary nucleic acid sequences and codon-optimized or synonymous codon equivalents.

[0102] As used herein, the term "isolated nucleic acid" refers to a nucleic acid that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. An isolated nucleic acid is also substantially free of sequences that naturally flank the nucleic acid from which the nucleic acid is derived (i.e., sequences located at the 5' and 3' ends of the nucleic acid).

[0103] "At least moderately stringent hybridization conditions" means selecting conditions that promote selective hybridization between two complementary nucleic acid molecules in a solution. Hybridization can occur in all or part of a nucleic acid sequence molecule. The length of the hybridized portion is usually at least 15 (e.g., 20, 25, 30, 40 or 50) nucleotides. Those skilled in the art will recognize that the stability of a nucleic acid duplex or hybrid is determined by Tm, which is a function of sodium ion concentration and temperature in a sodium-containing buffer (Tm = 81.5°C - 16.6 (Log10[Na+]) + 0.41(%(G+C) - 600 / l), or similar equations). Therefore, the parameters that determine hybrid stability in the washing conditions are sodium ion concentration and temperature. In order to identify molecules that are similar but not identical to known nucleic acid molecules, it can be assumed that a 1% mismatch results in a Tm reduction of about 1°C, for example, if a nucleic acid molecule with> 95% identity is sought, the final washing temperature will be reduced by about 5°C. Based on these considerations, those skilled in the art will be able to easily select appropriate hybridization conditions. In a preferred embodiment, stringent hybridization conditions are selected. By way of example, stringent hybridization can be achieved using the following conditions: based on the above equation, hybridization at 5× sodium chloride / sodium citrate (SSC) / 5× Denhardt's solution / 1.0% SDS at Tm-5°C, followed by washing at 60°C 0.2× SSC / 0.1% SDS. Moderately stringent hybridization conditions include a washing step at 42°C with 3× SSC. However, it should be understood that alternative buffers, salts, and temperatures can be used to achieve equivalent stringency. Additional guidance on hybridization conditions can be found in: Current Protocols in Molecular Biology, John Wiley & Sons, NY, 2002, and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001.

[0104] The terms "treating" or "treatment" as used herein and well known in the art mean methods for obtaining beneficial or desired results (including clinical results). Beneficial or desired clinical results may include, but are not limited to, alleviating or improving one or more symptoms or conditions, reducing the extent of the disease, stabilizing the disease state (i.e., not worsening), preventing disease spread, delaying or slowing disease progression, improving or alleviating the disease state, reducing disease recurrence, and alleviating (whether partially or completely), whether detectable or undetectable. "Treatment" and "treatment" may also refer to prolonged survival compared to survival expected if not receiving treatment. "Treatment" and "treatment" as used herein also include preventive treatment. For example, a subject with cancer can be treated with an antibody, immunoconjugate, nucleic acid, or composition described herein to prevent progression.

[0105] As used herein, the term "administering" means providing or administering an agent, such as a composition comprising an effective amount of an antibody, to a subject by an effective route, such as an intratumoral or intravenous administration route.

[0106] As used herein, the term "diluent" refers to a pharmaceutically acceptable carrier that does not inhibit the physiological activity or characteristics of the active compound to be administered (such as an antibody or immunoconjugate), does not stimulate the subject, and does not eliminate the biological activity and characteristics of the administered compound. Diluents include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservative salts, preservatives, adhesives, excipients, disintegrants, lubricants, similar materials and combinations thereof, as known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Science, 18th edition. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Any conventional carrier is intended for use in a pharmaceutical composition unless it is incompatible with the active ingredient.

[0107] A composition or method that "comprising" or "includes" one or more recited elements may include other elements not specifically recited. For example, a composition that "comprising" or "includes" an antibody may contain the antibody alone or in combination with other ingredients.

[0108] In understanding the scope of the present disclosure, the term "consisting of" and its derivatives as used herein are intended to be closed terms that specify the presence of stated features, elements, components, groups, integers and / or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0109] Numerical ranges set forth herein by endpoints include all numbers and fractions that fall within the range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It should also be understood that all numbers and fractions thereof are assumed to be modified by the term "about". In addition, it should be understood that the singular forms of the articles "a", "an", and "the" include plural indicators unless the context clearly dictates otherwise. For example, the term "antibody" or "at least one antibody" may include more than one antibody, including mixtures thereof.

[0110] Depending on the context, the terms "Frizzled" and "FZD" refer to any gene or protein member of the Frizzled receptor family. 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.

[0111] "Lipoprotein receptor-related proteins", "low-density lipoprotein receptor-related proteins" (HGNC) or "pre-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 receptor co-receptor group involved in the canonical 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 the protein has NCBI reference sequence: NP_002326. The LRP6 gene has ENTREZ Gene ID: 4040, and the protein has NCBI Reference Sequence: NP_002327. LRP6 is also known as ADCAD2, STHAG7.

[0112] II. Disorders associated with dysregulated LRP signaling The binding of Wnt to LRP5 or LRP6 destabilizes the β-catenin binding complex that leads to the degradation of β-catenin. The result is an increase in intracellular β-catenin levels. Therefore, provided herein is a method for blocking the binding of Wnt to LRP family proteins (such as LRP5 or LRP6).

[0113] "LRP-related disorder" (e.g., "LRP5-related disorder" or "LRP6-related disorder") refers to a condition or disease associated with dysregulation of the specific LRP receptors mentioned. Deregulation refers to an abnormal decrease or increase in signaling that affects normal β-catenin-mediated transcriptional changes or any other intracellular signaling pathway controlled by these receptors. Thus, for example, increased aberrant LRP-related signaling (e.g., through the canonical Wnt signaling pathway (Wnt3 / Wnt3A)) is associated with certain cancers and is associated with increased bone density, while decreased aberrant LRP-related signaling is associated with decreased bone density.

[0114] Antibodies that block the binding of Wnt to LRP5 or LRP6 can be used to treat cancer. In particular, methods of blocking the binding of Wnt to LRP5 can be used to treat brain cancer, breast cancer, colon cancer, endometrial cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, skin cancer, stomach cancer and testicular cancer.

[0115] III. Anti-LRP5 Antibodies A. Antibodies LRP5 and LRP6 each have two Wnt binding sites. Most Wnt activators of β-catenin signaling, including Wnt 1, 2, 2b, 6, 8a, 9a, 9b, 10b, etc., associate with the β propellers 1 and 2 regions of LRP5 and LRP6. This site is referred to as "non-Wnt3a binding site" in this article. Known Wnt proteins Wnt3 and Wnt3a associate with unique sites on the β propeller regions 3 and 4 of LRP5 and LRP6. This site is referred to as "Wnt3a binding site" in this article. The combination of antibodies that block the binding of Wnt ligands to specific binding sites with LRP5 or LRP6 reduces the activity of the signal transduction pathways associated with the Wnt ligands that bind to the site. In some cases, such blocking activity can also enhance the activity of the signal transduction pathways associated with the Wnt ligands that bind to other binding sites. Thus, for example, an antibody that blocks the binding of a Wnt ligand to a Wnt3a binding site inhibits the activity of a Wnt3a signaling pathway and may enhance the activity of a non-Wnt3a signaling pathway. Similarly, an antibody that blocks the binding of a Wnt ligand to a non-Wnt3a binding site inhibits the activity of a non-Wnt3a signaling pathway and may enhance the activity of a Wnt3a signaling pathway.

[0116] Antibodies directed against the LRP5 receptor are described herein. Certain of these antibodies block the binding of Wnt ligands to the Wnt3a binding site of LRP5. Other 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.

[0117] Thus, one aspect of the present disclosure includes an isolated antibody that specifically binds to an LRP5 receptor. The antibody comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region comprising complementary determining regions CDR-H1, CDR-H2, and CDR-H3, the light chain variable region comprising complementary determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of the CDRs comprises, consists essentially of, or consists of a sequence selected from the sequences in Table 1 or Table 2.

[0118] In an embodiment, the antibody comprises a set of CDR sequences selected from the set of CDR sequences in Table 1 (i.e., 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, LRP5-R3O_G6).

[0119] Heavy and light chain variable regions are also described herein. Table 2 provides exemplary variable domain sequences of Fab heavy and light chains 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, LRP5-R3O_G6. Antibodies comprising sequences in Table 2 or sequences substantially identical thereto, wherein the CDRs are the CDR sequence sets identified in Table 1 are also contemplated. In another embodiment, the antibody comprises a heavy chain variable region comprising: i) a heavy chain amino acid sequence as listed 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 as listed in Table 2, wherein the CDR sequence is a set of CDR sequences as listed in Table 1, or iii) a conservatively substituted amino acid sequence of i), wherein the CDR sequence is a set of CDR sequences as listed in Table 1.

[0120] In another embodiment, the antibody comprises a light chain variable region comprising i) a light chain amino acid sequence as listed 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 as listed in Table 2, wherein the CDR sequence is a set of CDR sequences as listed in Table 1, or iii) a conservatively substituted amino acid sequence of i), wherein the CDR sequence is a set of CDR sequences as listed in Table 1.

[0121] In Table 1, the following antibodies are assigned to epitope groups according to their binding properties. The antibodies in epitope group 1 enhance the activity of Wnt ligands binding to non-Wnt3A binding sites. However, they do not inhibit the activity of Wnt3A ligands. The antibodies in epitope group 2 inhibit the activity of Wnt ligands binding to non-Wnt3a binding sites. In addition, antibody LRP5-G 10 enhances the activity of wnt ligands binding to Wnt3a binding sites. Antibodies belonging to epitope groups 3 and 4 bind to LRP5, but do not inhibit or enhance the activity of Wnt ligands. The epitope groups of other antibodies were not determined.

[0122] The amino acid sequences of the CDRs of these antibodies are provided in Table 1.

[0123] Table 1: CDR sequences of anti-LRP5 antibodies

[0124]

[0125] The amino acid sequences and nucleotide sequences of the variable heavy chain domain and variable light chain domain are provided in Table 2: Table 2 – Heavy and light chain DNA sequences and amino acid sequences 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, LRP5 - R3O_G6.

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139] LRP5-A9 VH-DNA GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACATCTCTTCTTATTCTATCCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCATCTTCTTCTTATTATGGCTATACTTATTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCACTGTTCGTGGATCCAAAAAACCGTACTTCTCTGGTTGGGCTATGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG VH-AA EVQLVESGGGLVQPGGSLRLSCAASGFNISSYSIHWVRQAPGKGLEWVASSSYYGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARTVRGSKKPYFSGWAMDYWGQGTLVTVSS VL-DNA GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGTCCGTGTCCAGCGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACTCGGCATCCAGCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAAGTGCACTACAGCCCCTACAGCCTGATCACGTTCGGACAGGGTACCAAGGTGGAGATCAAACGA VL-AA DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ VHYSPYSLITFGQGTKVEIKR LRP5-C5 VH-DNA GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACTTTTCTTCTTCTTCTATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCATCTATTTCTTCTTCTTATGGCTATACTTATTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCTCTTCTTACTACTCTTCTGTTTCTTCTTCTGTTTACGCTTTGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG VH-AA EVQLVESGGGLVQPGGSLRLSCAASGFNFSSSSIHWVRQAPGKGLEWVASISSSYGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSSYYSSVSSSVYALDYWGQGTLVTVSS VL-DNA GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGTCCGTGTCCAGCGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACTCGGCATCCAGCCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAATACCAGTACTCTGGTCTGATCACGTTCGGACAGGGTACCAAGGTGGAGATCAAACGA VL-AA DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ YQYSGLITFGQGTKVEIKR LRP5-C12 VH-DNA GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACTTTTCTTCTTCTTCTATACACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCATCTATTTATTCTTCTTATGGCTCTACTTCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCACTGTTCGTGGATCCAAAAAACCGTACTTCTCTGGTTGGGCTATGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG VH-AA EVQLVESGGGLVQPGGSLRLSCAASGFNFSSSSIHWVRQAPGKGLEWVASIYSSYGSTSYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARTVRGSKKPYFSGWAMDYWGQGTLVTVSS VL-DNA GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGTCCGTGTCCAGCGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGTCCCGAAGCTTCTGATTTACTCGGCATCCAGCCTCT ACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAATTCAGCCACGTGAGCCTGATCACGTTCGGACAGGGTACCAAGGTGGAGATCAAACGA VL-AA DIQMTQSPSSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ FSHVSLITFGQGTKVEIKR In some embodiments, the variable domain sequences are at least 95%, 96%, 97%, 98% or 99% similar outside of the CDR regions, and the set of CDR sequences are 100% identical to the amino acid sequences provided in Table 1.

[0140] In another embodiment, a competing antibody that competes for binding with an antibody comprising a CDR sequence set described herein is also provided. For example, in one embodiment, the competing antibody reduces the binding of an antibody comprising the CDR sequence set to LRP5 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%.

[0141] As demonstrated herein, the antibodies described herein have high affinity for LRP5. For example, in one embodiment, the antibody has a binding affinity of between about 1 nM and about 50 nM as measured by surface plasmon resonance.

[0142] The antibody may be a humanized antibody or a chimeric antibody as described herein.

[0143] In some embodiments, the antibody is a single chain antibody, which can be obtained, for example, by fusing together a heavy chain and a light chain or portions thereof.

[0144] In some embodiments, the antibody is an antibody binding fragment selected from the group consisting of Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, nanobodies, minibodies, diabodies, and multimers thereof.

[0145] In some other embodiments, the antibody is a binding fragment Fab. For some embodiments, binding fragments are preferred, for example, for use in vitro.

[0146] In other embodiments, multivalent antibodies or antibodies comprising an Ig portion may be preferred.

[0147] As demonstrated in the Examples, the Fab fragment may be combined with an Ig such as an IgG. In an embodiment, the IgG is an IgG1, IgG2, IgG3 or IgG4.

[0148] B. Detectably labeled antibodies Detectable labels can include peptide sequences (such as myc tags, HA-tags, V5-tags, or NE-tags), fluorescent proteins or luminescent proteins (e.g., green fluorescent protein or luciferase), which can be attached to or introduced into the antibodies described herein, and which are capable of directly or indirectly producing a detectable signal. For example, the label can be a radio-opaque, positron-emitting radionuclide (e.g., for PET imaging), or a radioisotope, such as 3 H. 13 N. 14 C. 18 F. 32 P. 35 S. 123 I. 125 I. 131 I; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; an enzyme, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase; an imaging agent; or a metal ion.

[0149] C. Antibody-drug conjugates Another aspect includes immunoconjugates comprising an antibody described herein and a detectable label or a cytotoxic agent.

[0150] Chemotherapeutic (anticancer) agents can be any agent that can reduce cancer growth, interfere with cancer cell replication, kill cancer cells directly or indirectly, reduce metastasis, reduce tumor blood supply, etc. Chemotherapeutic agents therefore include cytotoxic agents. Cytotoxic agents include, but are not limited to, saponins, taxanes, vinca alkaloids, anthracyclines, and platinum-based agents. The categories of chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites (e.g., methotrexate), plant alkaloids (e.g., vincristine), and antitumor antibiotics such as anthracyclines (e.g., doxorubicin), and various drugs that do not belong to a specific category, such as hydroxyurea. Platinum-based drugs exemplified by cisplatin and oxaliplatin represent an important class of chemotherapeutic agents. These drugs bind to DNA and interfere with replication. Taxanes exemplified by paclitaxel represent another important class of chemotherapeutic agents. These compounds work by interfering with the formation of the cytoskeleton and spindle to inhibit cell division, and thereby prevent the growth of rapidly dividing cancer cells. Other chemotherapeutic drugs include hormone therapy. Chemotherapeutic agents also include agents that inhibit tubulin assembly or polymerization, such as maytansine, mertansine, and auristatin. Chemotherapeutic agents also include DNA damaging agents, such as calicheamicin.

[0151] Chemotherapeutic agents may include maytansine alkaloids, auristatins, dolastatins, tubulysin, scintilide, pyrrolobenzodiazepine (PBD) dimers, indolebenzodiazepine dimers, α-amanitin, trichothene, SN-38, duocarmycin, CC1065, calicheamicin, enediyne antibiotics, taxanes, doxorubicin derivatives, anthracyclines and their stereoisomers, azanofide, isosteres, analogs or derivatives.

[0152] IV. Nucleic Acids Other aspects include nucleic acid molecules or polynucleotides, recombinant nucleic acid molecules, expression constructs and vectors as described herein.

[0153] A. Nucleic acid molecules Another aspect includes nucleic acid molecules as listed in Table 2 and polynucleotides that hybridize to one of the sequences, for example under stringent hybridization conditions. The CDR and variable domain nucleic acid sequences can be used, for example, to prepare expression constructs.

[0154] B. Expression constructs and vectors Nucleic acid molecules can be incorporated into appropriate expression constructs or expression vectors that ensure protein expression in a known manner. Expression constructs can include expression control sequences, such as promoters, which are operably connected to polynucleotides comprising nucleotide sequences encoding antibodies of the present disclosure. Possible expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses). The vector should be compatible with the host cell used. An expression vector is "suitable for transforming a host cell," which means that the expression vector contains a nucleic acid molecule encoding a peptide corresponding to an epitope or antibody described herein.

[0155] In an embodiment, the vector is suitable for expressing, for example, a single-chain antibody by gene therapy. In an embodiment, the vector comprises an IRES and allows expression of a light chain variable region and a heavy chain variable region. Such a vector can be used to deliver antibodies in vivo.

[0156] Suitable regulatory sequences may be derived from a variety of sources including bacterial, fungal, viral, mammalian, or insect genes.

[0157] Examples of such regulatory sequences include transcriptional promoters and enhancers or RNA polymerase binding sequences, ribosome binding sequences, including translation initiation signals. In addition, depending on the selected host cell and the expression vector used, other sequences (such as replication origin, additional DNA restriction sites, enhancers, and sequences that confer transcriptional inducibility) may be incorporated into the expression vector.

[0158] In embodiments, the regulatory sequence directs or increases expression in neural tissues and / or cells.

[0159] The vector can be any vector, including vectors suitable for producing the antibodies described herein.

[0160] In an embodiment, the vector is a viral vector.

[0161] The recombinant expression vector may also contain a marker gene that facilitates the selection of host cells transformed, infected or transfected with a vector expressing an antibody or epitope peptide described herein.

[0162] The recombinant expression vector can also contain an expression cassette encoding a fusion moiety (i.e., "fusion protein") that provides increased expression or stability of the recombinant peptide; increased solubility of the recombinant peptide; and assisting the purification of the target recombinant peptide by acting as a ligand (including, for example, tags and markers described herein) in affinity purification. In addition, a proteolytic cleavage site can be added to the target recombinant protein to allow the recombinant protein to be separated from the fusion moiety 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.

[0163] Systems for transferring genes in vitro and in vivo include viral-based vectors, most notably herpes simplex virus, adenovirus, adeno-associated virus (AAV), and retroviruses including lentiviruses. Alternative methods of gene delivery include the use of naked plasmid DNA and liposome-DNA complexes.

[0164] In one aspect, the disclosure includes a method for making an antibody described herein, the method comprising synthesizing a nucleic acid molecule comprising the antibody framework and CDR sequence sets described herein.

[0165] V. Recombinant Cells Another aspect is a recombinant host cell expressing an antibody described herein.

[0166] Antibodies as described herein can be prepared by recombinant expression of nucleic acid encoding the antibody sequences.

[0167] Antibodies as disclosed herein can be prepared by culturing cells engineered to express a nucleic acid construct encoding an immunoglobulin polypeptide.

[0168] Recombinant host cells can be produced using any cell suitable for producing polypeptides (e.g., suitable for producing antibodies). For example, in order to introduce nucleic acids (e.g., vectors) into cells, cells can be transfected, transformed or infected, depending on the vector used.

[0169] Suitable host cells include a variety of prokaryotic and eukaryotic host cells. For example, the proteins described herein can be expressed in bacterial cells (such as E. coli), insect cells (using baculovirus), yeast cells or mammalian cells.

[0170] In embodiments, the cell is a eukaryotic cell selected from the group consisting of yeast, plant, worm, insect, avian, fish, reptile, and mammalian cells.

[0171] In another embodiment, the mammalian cell is a CHO cell, a myeloma cell, a spleen cell, or a hybridoma cell.

[0172] Yeast and fungal host cells suitable for expressing antibodies include, but are not limited to, species of S. cerivisiae, Schizosaccharomyces pombe, Pichia or Kluyveromyces and Aspergillus. Examples of vectors for expression in yeast Saccharomyces cerevisiae include pYepSec1, pMFa, pJRY88 and pYES2 (Invitrogen Corporation, San Diego, CA). Protocols for transforming yeast and fungi are well known to those of ordinary skill in the art.

[0173] Potentially suitable mammalian cells include, among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL2), 293 (ATCC No. 1573), and NS-1 cells. Suitable expression vectors for directing expression in mammalian cells generally contain a promoter (e.g., derived from viral material such as polyoma virus, adenovirus 2, cytomegalovirus, and simian virus 40) and other transcriptional and translational control sequences. Examples of mammalian expression vectors include pCDM8 and pMT2PC.

[0174] VI. Pharmaceutical Compositions Another aspect is a composition comprising an antibody, immunoconjugate, nucleic acid molecule, vector or recombinant cell described herein, and optionally a suitable diluent, such as a pharmaceutically acceptable carrier.

[0175] The composition may, for example, comprise one or more antibodies or immunoconjugates.

[0176] Suitable diluents for polypeptides (including antibodies and / or cells) include, but are not limited to, saline solutions, pH buffered solutions, and glycerol solutions or other solutions suitable for freezing polypeptides and / or cells.

[0177] Suitable diluents for nucleic acids include, but are not limited to, water, saline solutions, and ethanol.

[0178] In an embodiment, the composition is a pharmaceutical composition comprising any of the antibodies, nucleic acids or vectors disclosed herein, and optionally a pharmaceutically acceptable vehicle, such as a diluent or carrier.

[0179] The compositions described herein can be prepared by methods known per se for preparing pharmaceutically acceptable compositions that can be administered to a subject, such that an effective amount of the active substance is combined with a pharmaceutically acceptable vehicle in the form of a mixture.

[0180] Pharmaceutical compositions include but are not limited to lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which may also contain antioxidants, buffers, antibacterial agents and solutes that make compositions compatible with tissues or blood of the intended recipient. Other components that may be present in such compositions include water, surfactants (such as Tween), alcohol, polyols, glycerol and vegetable oils. Instant injection solutions and suspensions may be prepared from sterile powders, granules and tablets or concentrated solutions or suspensions. Compositions may, for example but not in a limiting manner, be provided with lyophilized powders that are reconstituted with sterile water or saline before being applied to the patient.

[0181] The pharmaceutical composition may include a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include substantially chemically inert and non-toxic compositions that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition. Examples of suitable pharmaceutical carriers include, but are not limited to, water, saline solutions, glycerol solutions, ethanol, N-(1(2,3-dioleyl)propyl)N,N,N-trimethylammonium chloride (DOTMA), dioleoylphosphatidylethanolamine (DOPE) and liposomes. Such compositions should contain a therapeutically effective amount of the compound and a suitable amount of carrier to provide a form for direct administration to a patient.

[0182] The composition may be in the form of a pharmaceutically acceptable salt, including but not limited to those formed with free amino groups, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with free carboxyl groups, such as those derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, isopropylamine, triethylamine, 2-ethylethanolamine.

[0183] In an embodiment, the composition comprises an antibody described herein. In another embodiment, the composition comprises an antibody described herein and a diluent. In an embodiment, the composition is a sterile composition.

[0184] Another aspect includes an antibody complex comprising an antibody described herein that binds to an LRP protein (eg, LRP5 or LRP6). The complex can be in solution or contained in a tissue, optionally in vitro.

[0185] Also provided are methods for making and using the agents described herein.

[0186] VII. Kit On the other hand is a kit or package, which includes any antibody, immunoconjugate, nucleic acid molecule, vector, recombinant cell and / or composition included herein. Antibodies, immunoconjugates, nucleic acid molecules, vectors, recombinant cells and / or compositions can 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. The kit may optionally include a reference agent and / or its instructions for use. The kit may also include a transport container suitable for accommodating a container (such as a vial) containing a composition as disclosed herein.

[0187] VIII. Methods of Using Antibodies The antibodies described herein can be used in a number of in vitro and in vivo methods.

[0188] A. Methods for detecting LRP5 expression As demonstrated herein, antibodies can be used to detect expression of LRP5.

[0189] Accordingly, the present disclosure provides, in one aspect, a method for detecting LRP5 expression, the method comprising contacting a sample comprising one or more cells with one or more antibodies or immunoconjugates described herein under conditions that allow the formation of an antibody:LRP5 complex, and detecting the presence of any antibody complex. Typically, the antibody is part of an immunoconjugate that comprises an antibody coupled to a detectable label.

[0190] The sample may comprise viable cells or cell extracts. The antibody: LRP5 complex may be detected by immunoassays such as immunofluorescence, flow cytometry, Western blot, ELISA, SPR, and immunoprecipitation followed by SDS-PAGE, immunocytochemistry. In some embodiments, detection is by immunofluorescence. In some embodiments, detection is by flow cytometry.

[0191] As demonstrated herein, some of the identified antibodies preferentially recognize LRP5. Thus, in embodiments where the method is used to detect 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, LRP5-R3O_G6.

[0192] B. Methods for inhibiting the binding of WNT to LRP5 The antibodies disclosed herein inhibit the binding of Wnt to LRP proteins, particularly to LRP5. Without wishing to be limited by theory, inhibition of Wnt binding to LRP5 affects signal transduction induced by the binding of specific Wnt ligands. For example, binding of the antibody to the LRP5 receptor inhibits LRP5's promotion of β-catenin phosphorylation. Because phosphorylated β-catenin is marked for destruction in cells, the non-phosphorylated form gradually accumulates. Accumulation of β-catenin is associated with malignant tumors.

[0193] It may be desirable to reduce or inhibit Wnt ligand signaling through LRP5. Thus, another aspect is a method of inhibiting Wnt ligand binding to LRP5 or Wnt-induced transcriptional activity, the method comprising contacting one or more cells expressing one or more LRP5 polypeptides with an effective amount of an antibody or immunoconjugate described herein.

[0194] In an embodiment, the antibody or immunoconjugate comprises a set of CDR sequences corresponding to an antibody as described herein (all, light chain or heavy chain). As shown herein, some identified antibodies preferentially recognize LRP5. Thus, in embodiments where the method is used to detect LRP5 expression, 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, LRP5-R3O_G6.

[0195] Contacting can be accomplished in vivo, for example, by administering the antibody or immunoconjugate to a subject.Such inhibition may be desirable, particularly when Wnt signaling is dysregulated, such as in cancer cells.

[0196] C. Methods of enhancing the WNT signaling pathway Some antibodies that block the binding of Wnt ligands to the Wnt3a binding site of LRP5 or LRP6 enhance the signal transduction activity of Wnt ligands bound to non-Wnt3a binding sites. Similarly, some antibodies that block the binding of Wnt ligands to the non-Wnt3a binding sites of LRP5 or LRP6 enhance the signal transduction activity of Wnt ligands bound to the Wnt3a binding site. Blocking activity can be competitive or allosteric. Therefore, provided herein is a method for enhancing the signal transduction activity of Wnt ligands bound to the Wnt3a binding site of LRP5 or LRP6 by contacting LRP5 or LRP6 with antibodies that block the binding of Wnt ligands to the non-Wnt3a binding sites of LRP5 or LRP6. Also provided herein is a method for enhancing the signal transduction activity of Wnt ligands bound to the non-Wnt3a binding sites of LRP5 or LRP6 by contacting LRP5 or LRP6 with antibodies that block the binding of Wnt ligands to the Wnt3a binding sites of LRP5 or LRP6. Contact can be performed in vitro or in vivo. In vivo contacting can include administering an appropriate anti-LRP5 or anti-LRP6 antibody to a subject.

[0197] For example, an antibody comprising a set of CDR sequences from an antibody of epitope group 1 enhances the activity of Wnt ligand binding to a non-Wnt3A binding site.

[0198] D. Methods and uses for treating cancer The method for treating cancer comprises using or administering the pharmaceutical composition of the present disclosure comprising an antibody that binds to LRP5 to a subject in need thereof, wherein the subject may be a subject suffering from cancer or at risk of cancer (such as cancer recurrence), for example, a human.

[0199] In one embodiment, the cancer is selected from acute myeloid leukemia, prostate cancer, glioblastoma, bladder cancer, and cervical cancer.

[0200] In another embodiment, the cancer cell is selected from the group consisting of brain cancer, breast cancer, colon cancer, endometrial cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, skin cancer, stomach cancer, and testicular cancer.

[0201] Without wishing to be bound by theory, such treatments may act by inhibiting activation of the canonical Wnt pathway, for example, by inhibiting Wnt binding to LRP5, by inhibiting Wnt-induced transcriptional activity, by inhibiting Dishevelled activation, by inhibiting inhibition of the β-catenin destruction complex, and by promoting β-catenin accumulation.

[0202] Since LRP5 and / or LRP6 are often upregulated in cancer, the disclosure includes, on the other hand, a method for treating cancer, the method comprising administering to a subject in need thereof 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. The disclosure also includes the use of an effective amount of an antibody or immunoconjugate for treating cancer or in the preparation of a medicament for treating cancer, the antibody or immunoconjugate specifically binds to LRP5 or LRP6 in at least one assay and inhibits Wnt3a-induced signaling in at least one assay for treating cancer. The disclosure also includes an effective amount of an antibody or immunoconjugate for use in treating cancer, the antibody or immunoconjugate specifically binds to LRP5 or LRP6 in at least one assay and inhibits Wnt3a-induced signaling in at least one assay.

[0203] In an embodiment, the antibody or immunoconjugate, such as an antibody-drug conjugate, is contained in a pharmaceutical composition.

[0204] In embodiments, 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, stomach cancer and testicular cancer. In embodiments, the antibody or immunoconjugate comprises a CDR sequence set corresponding to an antibody as described herein. Some identified antibodies preferentially recognize LRP5. Therefore, in embodiments in which the method is used to detect LRP5 expression, the antibody or immunoconjugate comprises a CDR sequence set 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.

[0205] As demonstrated herein, antibodies are also capable of inhibiting cancer cell proliferation. Thus, methods for inhibiting cancer cell proliferation are also provided, the method comprising 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.

[0206] In one embodiment, a method for treating cancer comprises administering an antibody or an immunoconjugate comprising the antibody to a subject in need thereof, 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 a non-Wnt3a binding site of LRP5 or LRP6. The present disclosure also provides antibodies or immunoconjugates comprising antibodies for use in treating 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 a non-Wnt3a binding site of LRP5 or LRP6. The present disclosure also provides the use of an antibody or an immunoconjugate comprising an antibody for treating 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 a non-Wnt3a binding site of LRP5 or LRP6. The present disclosure also provides the use of an antibody or an immunoconjugate comprising the antibody in the preparation of a medicament for treating cancer, wherein the first antibody blocks the binding of the Wnt ligand to the Wnt3a binding site of LRP5 or LRP6, and the second antibody blocks the binding of the Wnt ligand to the non-Wnt3a binding site of LRP5 or LRP6.

[0207] In an embodiment, the antibody or immunoconjugate is an antibody or immunoconjugate described herein, e.g., an antibody or immunoconjugate comprising a set of CDR sequences (whole, light chain or heavy chain) 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.

[0208] IX. Method of application The anti-LRP antibodies of the present invention can effectively deliver therapeutic compositions in vivo to cells undergoing Wnt signaling. In some embodiments, the treatment method includes administering to the individual an effective amount of a therapeutic anti-LRP conjugate, such as an anti-LRP antibody attached to a therapeutic agent. In some embodiments, the individual has been diagnosed with cancer. In some embodiments, the individual is receiving or has received cancer treatment, such as surgery, radiotherapy, or chemotherapy. In some embodiments, the individual has been diagnosed but the cancer is in remission.

[0209] In some embodiments, the anti-LRP conjugate comprises a liposome. In some embodiments, the method further comprises monitoring the cancer progression of the individual. In some embodiments, the dosage of the anti-LRP conjugate administered each time is determined based on the individual's treatment progress, for example, if the individual does not respond adequately to treatment, a higher dose of the chemotherapeutic agent is administered.

[0210] In some embodiments, the present invention can include antibodies or antibody targeting compositions and physiologically (i.e., pharmaceutically) acceptable carriers. The term "carrier" refers to a generally inert substance used as a diluent or vehicle for diagnostic or therapeutic agents. The term also includes generally inert substances that impart cohesive quality to the composition. Physiologically acceptable carriers can be liquids, such as normal saline, phosphate buffer, physiologically buffered saline (135-150 mM NaCl), water, buffered water, 0.4% saline, 0.3% glycine, glycoproteins (e.g., albumin, lipoprotein, globulin, etc.) providing enhanced stability, etc. Because physiologically acceptable carriers are determined in part by the particular composition used and the particular method for applying the composition, there are various suitable preparations of the pharmaceutical composition of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17th edition, 1989).

[0211] The compositions of the present invention can be sterilized by conventional, well-known sterilization techniques, or can be produced under aseptic conditions. The aqueous solution can be packaged for use or filtered and lyophilized under aseptic conditions, and the lyophilized preparation is combined with a sterile aqueous solution before administration. The composition can contain pharmaceutically acceptable auxiliary substances as required to approach physiological conditions, such as pH adjusting agents and buffers, tension regulators, wetting agents, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate and triethanolamine oleate. Sugar can also be included for stabilizing the composition, such as a stabilizer for a freeze-dried antibody composition.

[0212] The dosage form can be prepared for mucosal administration (e.g., nasal, sublingual, vaginal, buccal or rectal), parenteral administration (e.g., subcutaneous, intravenous, intramuscular or intraarterial injection, bolus or infusion), oral or transdermal administration to a patient. Examples of dosage forms include, but are not limited to, dispersions, suppositories, ointments, cataplasms, poultices, pastes, powders, dressings, creams, plasters, solutions, patches, aerosols (e.g., nasal sprays or inhalers), gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions or water-in-oil liquid emulsions), solutions and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.

[0213] Injectable (e.g., intravenous) compositions may include a solution of an antibody or antibody targeting composition suspended in an acceptable carrier, such as an aqueous carrier. Any of a variety of aqueous carriers may be used, such as water, buffered water, 0.4% saline, 0.9% isotonic saline, 0.3% glycine, 5% dextrose, etc., and may include glycoproteins for enhancing stability, such as albumin, lipoproteins, globulins, etc. Typically, physiologically buffered saline (135-150 mM NaCl) will be used. The composition may contain pharmaceutically acceptable auxiliary substances to approach physiological conditions, such as pH regulators and buffers, tension regulators, wetting agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. In some embodiments, the antibody targeting composition may be formulated in a kit for intravenous administration.

[0214] Formulations suitable for parenteral administration (such as, for example, by intraarticular (in a joint), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal and subcutaneous routes) include aqueous and nonaqueous, isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats and solutes that make the formulation isotonic with the blood of the intended recipient, and aqueous and nonaqueous sterile suspensions, which 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 practice of the present invention, the composition can be administered, for example, by intravenous infusion, topically, intraperitoneally, intravesically or intrathecally. Parenteral administration and intravenous administration are preferred methods of administration. The formulation of the targeted composition can be present in a sealed container (such as an ampoule and a vial) in a unit dose or multiple doses.

[0215] The selected targeted delivery composition alone or in combination with other suitable components can be formulated into an aerosol formulation for administration via inhalation ("nebulization"). Aerosol formulations can be placed in pressurized acceptable propellants, such as dichlorodifluoromethane, propane and nitrogen.

[0216] Pharmaceutical preparations can be packaged or prepared in unit dosage form. In such a form, the preparation is subdivided into unit doses containing appropriate amounts of active ingredients, for example, according to the dosage of the therapeutic agent or the concentration of the antibody. The unit dosage form can be a packaged preparation containing discrete amounts of the preparation. If desired, the composition can also contain other compatible therapeutic agents.

[0217] The antibody (or antibody targeting composition) can be administered by injection or infusion by any suitable route (including but not limited to intravenous, subcutaneous, intramuscular or intraperitoneal routes). The example of pharmaceutical composition administration includes storing the antibody at 10 mg / ml in a sterile isotonic aqueous saline solution for injection at 4 ° C, and diluting it in 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 the course of 1 hour at a dose between 0.2 mg / kg and 10 mg / kg. In other embodiments, the antibody is administered by intravenous infusion over a period of 15 minutes and 2 hours. In still other embodiments, the administration procedure is via subcutaneous bolus injection.

[0218] The dosage of the antibody is selected so that effective treatment is provided to the patient, and the dosage of the antibody is less than 0.1 mg / kg body weight to about 25 mg / kg body weight, or in the range of 1 mg-2 g in each patient. In some cases, the dosage is in the range of 1-100 mg / kg or about 50 mg-8000 mg / patient. According to the pharmacokinetics (for example, the half-life of the antibody in circulation) and the pharmacodynamic response (for example, the duration of the antibody treatment effect) of the antibody, the dosage can be repeated with appropriate frequency, and the frequency can be in the range of once a day to once every three months. In some embodiments, the half-life in vivo is between about 7 days and about 25 days, and the antibody administration is repeated between once a week and once every three months.

[0219] Administration can be periodic. Depending on the route of administration, the dosage can be, for example, administered once every 1 day, 3 days, 5 days, 7 days, 10 days, 14 days, 21 days or 28 days or longer (e.g., once every 2, 3, 4 or 6 months). In some cases, administration is more frequent, for example, 2 or 3 times a day. As will be appreciated by those skilled in the art, the dosage and frequency of administration can be adjusted based on treatment progress and any adverse side effects.

[0220] Thus, in some embodiments, additional administrations are dependent on the patient's progress, e.g., monitoring the patient between administrations. For example, after the first administration or round of administration, the patient's tumor growth rate, recurrence (e.g., in the case of a postoperative patient), or general disease-related symptoms, such as weakness, pain, nausea, etc., can be monitored.

[0221] In therapeutic uses for treating cancer, antibody targeting compositions (e.g., including therapeutic agents and / or diagnostic agents) can be administered at an initial dose of about 0.001 mg / kg to about 1000 mg / kg per day, and adjusted over time. A daily dose range of about 0.01 mg / kg to about 500 mg / kg, or about 0.1 mg / kg to about 200 mg / kg, or about 1 mg / kg to about 100 mg / kg, or about 10 mg / kg to about 50 mg / kg can be used. The dosage varies according to the needs of the patient, the severity of the condition being treated, and the targeted composition used. For example, the dosage can be determined empirically considering the type and stage of cancer diagnosed in a particular patient. In the case of the present invention, the dosage administered to the patient should be sufficient to achieve a beneficial therapeutic response of the patient over time. As will be appreciated by skilled practitioners, the size of the dosage will also be determined by the presence, nature, and extent of any adverse side effects associated with the administration of a particular targeted composition in a particular patient.

[0222] The above disclosure generally describes the present disclosure. A more complete understanding can be obtained by reference to the following specific examples. These embodiments are described only for the purpose of illustration and are not intended to limit the scope of the present application. When circumstances may suggest or provide convenience, changes in form and replacement of equivalents are also contemplated. Although specific terms have been adopted in this article, such terms are intended to be descriptive, rather than for the purpose of limitation.

[0223] Exemplary embodiments 1. An antibody that specifically binds to LRP5, the antibody comprising a light chain variable region and / or a heavy chain variable region, the heavy chain variable region comprising complementary determining regions CDR-H1, CDR-H2 and CDR-H3, the light chain variable region comprising complementary determining regions CDR-L1, CDR-L2 and CDR-L3, wherein the amino acid sequence of the CDR comprises or consists of a CDR sequence selected from the following: the CDR sequence set of anti-LRP5 antibodies 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.

[0224] 2. The antibody according to embodiment 1, wherein the amino acid sequence of the CDR comprises or consists of a sequence selected from the group consisting of the sequences listed below: CDR-H1 is selected from the group consisting of: LSYYYM, ISYSYI, LSYSSM, ISSYSI, ISYSYI, IYSYSI, LSYYYM, FSSSSI, LYYYYI, LSYSSI, IYSYYI, LLYYSSM, and FSSSSI; CDR-H2 is selected from the group consisting of: SIYPYYGYTY, SSSYYGYTY, SISSSYGYTY, SIYSSYGSTS, SIYSSYGYTY, SIYPYSSYTS, SIYSSYGYTY, SIYPSYGYTY, SISPYYGYTS, SISSSYGSTS, SIYSYYGYTY, SISSSYGYTY, SISSSYGYTY SISSYYGYTS and YISPYYGYTS; CDR-H3 is selected from the group consisting of: HGAM, TVRGSKKPYFSGWAM, SSYYSSVSSSVYAL, TVRGSKKPYFSGWAM, HYSYFFYAM, YAVYFPGYYWGM, WSHVSGHYSGM, WGAYHSSGYGM, GGSGV SHYGSVYYSWWAL, AAPYYGYYYSYAM, SGYGWYAM, GYWAI, SYPAM, SWAM, YWAL, GWGSPASAGYYGL, SSYYSSVSSSVYAL, TVRGSKKPYFSGWAM and TVRGSKKPYFSGWAM; CDR-L1 is SVSSA; CDR-L2 is SASSLYS; and CDR-L3 is selected from the group consisting of: AWGWGLF, VHYSPYSLI, YQYSGLI, FSHVSLI, ASYSPI, YHYYYLF, ASYAPI, SSSSPI, SSYSLI, GVSLI, YWFLI, PVGHYGYPI, SSYSPI, YWAYYSPI, VSYYPLI, SSYSLI, and VHYSPYSLI.

[0225] 3. The antibody according to embodiment 2, wherein the antibody comprises a heavy chain variable region comprising: i) the heavy chain amino acid sequence as listed 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 a heavy chain amino acid sequence as listed in Table 2, wherein the CDR sequence is a set of CDR sequences as listed in Table 1, or iii) a conservatively substituted amino acid sequence of i), wherein the CDR sequence is the CDR sequence set listed in Table 1.

[0226] 4. The antibody according to any one of embodiments 2 to 3, wherein the antibody comprises a light chain variable region comprising: i) the light chain amino acid sequence as listed 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 a light chain amino acid sequence as listed in Table 2, wherein the CDR sequence is a set of CDR sequences as listed in Table 1, or iii) a conservatively substituted amino acid sequence of i), wherein the CDR sequence is the CDR sequence set listed in Table 1.

[0227] 5. An antibody according to any one of embodiments 1 to 4, wherein the CDR sequence is selected from the full set of CDR sequences of the antibodies identified in Table 1.

[0228] 6. An antibody according to any one of embodiments 1 to 5, wherein the antibody cross-reacts with LRP6.

[0229] 7. An antibody according to embodiment 1, wherein the CDR sequence comprises a light chain CDR sequence set or a heavy chain CDR sequence set selected from the antibodies identified in Table 1.

[0230] 8. The antibody of any one of embodiments 1 to 7, wherein the antibody specifically binds to LRP5.

[0231] 9. An antibody according to embodiment 8, wherein the CDR sequence is a set of CDR sequences of antibodies selected from antibodies 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.

[0232] 10. The antibody of embodiments 1 to 9, which blocks the binding of a Wnt ligand to the Wnt3a binding site of LRP5.

[0233] 11. The antibody of embodiments 1 to 9, which blocks the binding of a Wnt ligand to a non-Wnt3a binding site of LRP5.

[0234] 12. The antibody of any one of embodiments 1 to 11, wherein the antibody is a monoclonal antibody.

[0235] 13. The antibody of any one of embodiments 1 to 12, wherein the antibody is a humanized antibody.

[0236] 14. An antibody according to any one of embodiments 1 to 13, wherein the antibody is a single-chain antibody.

[0237] 15. An antibody according to any one of embodiments 1 to 14, wherein the antibody is an antibody binding fragment selected from the group consisting of: Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, nanobodies, minibodies, diabodies and multimers thereof.

[0238] 16. The antibody of any one of embodiments 1 to 14, wherein the antibody is a bispecific antibody.

[0239] 17. The antibody according to any one of embodiments 1 to 14, wherein the antibody is a bispecific antibody that also binds to a FZD receptor.

[0240] 18. An antibody according to any one of embodiments 1 to 17, wherein the antibody comprises a non-native glycosylation pattern.

[0241] 19. The antibody according to any one of embodiments 1 to 17, comprising a cysteine ​​substitution or addition, for example in the constant region or framework region.

[0242] 20. An immunoconjugate comprising the antibody of any one of embodiments 1 to 17 and a detectable label or a cytotoxic agent.

[0243] 21. The immunoconjugate of embodiment 20, comprising a cytotoxic agent selected from the group consisting of maytansine alkaloids, auristatins, dolastatins, tubulysin, moniliformin, pyrrolobenzodiazepine (PBD) dimers, indolebenzodiazepine dimers, α-amanitin, trichothene, SN-38, duocarmycin, CC1065, calicheamicin, enediyne antibiotics, taxanes, doxorubicin derivatives, anthracyclines and their stereoisomers, azanofide, isosteres, analogs or derivatives.

[0244] 22. A nucleic acid molecule encoding the antibody of any one of embodiments 1 to 17.

[0245] 23. A nucleic acid molecule according to embodiment 22, wherein one or more of the CDR sequences are encoded by a nucleic acid in Table 2.

[0246] 24. The nucleic acid molecule of embodiment 22, wherein the antibody comprises a heavy chain variable region encoded by a nucleic acid comprising: i) the heavy chain nucleic acid sequence as 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 to a heavy chain nucleic acid sequence as listed in Table 2, wherein the CDR sequence is a set of CDR sequences as listed in Table 1, or iii) the codon-degenerate nucleic acid sequence of i), wherein the CDR sequence is the CDR sequence set listed in Table 1.

[0247] 25. The nucleic acid molecule of embodiment 22, wherein the antibody comprises a light chain variable region encoded by a nucleic acid comprising: i) the light chain nucleic acid sequence as listed in Table 2, ii) a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, at least 95%, at least 98% or at least 99% sequence identity to a light chain nucleic acid sequence as listed in Table 2, wherein the CDR sequence is a set of CDR sequences as listed in Table 1, or iii) the codon-degenerate nucleic acid sequence of i), wherein the CDR sequence is the CDR sequence set listed in Table 1.

[0248] 26. A vector comprising an expression control sequence operably linked to the nucleic acid of any one of embodiments 22 to 25.

[0249] 27. A host cell comprising a recombinant nucleic acid molecule comprising an expression control sequence operably linked to the nucleic acid of any one of embodiments 22-26.

[0250] 28. The host cell of embodiment 27, wherein the host cell is a Chinese Hamster Ovary (CHO) cell.

[0251] 29. A host cell comprising the vector described in embodiment 26.

[0252] 30. A method for preparing an anti-LRP5 antibody, the method comprising culturing the host cell of any one of embodiments 27 to 29.

[0253] 31. A composition comprising the antibody of any one or more of embodiments 1 to 17, the immunoconjugate of embodiments 20-21, the nucleic acid molecule of embodiments 22-25, the vector of embodiment 26, or the host cell of embodiment 29, and optionally a suitable diluent.

[0254] 32. A composition according to embodiment 31, wherein the composition comprises one or more antibodies or immunoconjugates, optionally wherein the composition is a pharmaceutical composition.

[0255] 33. A kit comprising the antibody of any one or more of embodiments 1 to 17, the immunoconjugate of embodiments 20-21, the nucleic acid molecule of embodiments 22-25, the vector of embodiment 26, or the host cell of embodiments 29-29.

[0256] 34. A method for detecting LRP5 expression, the method comprising contacting a sample comprising one or more cells with one or more of the antibodies or immunoconjugates of any one of embodiments 1 to 21 under conditions that allow formation of antibody:cell complexes, and detecting the presence of any antibody complexes.

[0257] 35. A method according to embodiment 34, wherein the detection is by immunofluorescence.

[0258] 36. A method according to embodiment 34, wherein the detection is by flow cytometry.

[0259] 37. A method according to any one of embodiments 34 to 36, wherein the method is used to detect LRP4 expression and 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, LRP5-R3O_G6.

[0260] 38. A method of inhibiting Wnt ligand binding to an LRP5 receptor, disrupting a Wnt signaling pathway, inhibiting Wnt-induced transcriptional activity, inhibiting Dishevelled protein activation, promoting retention of a β-catenin destruction complex, promoting β-catenin accumulation, or inhibiting cell growth, the method comprising contacting a cell expressing an LRP5 receptor with the antibody or immunoconjugate of any one of embodiments 1 to 21.

[0261] 39. The method of embodiment 38, wherein the antibody or immunoconjugate blocks the binding of the Wnt ligand to the Wnt3a binding site of LRP5.

[0262] 40. The method of embodiment 38, wherein the antibody or immunoconjugate blocks the binding of the Wnt ligand to the non-Wnt3a binding site of LRP5.

[0263] 41. A method according to 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, LRP5-R3O_G6.

[0264] 42. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising the antibody or immunoconjugate of any one of embodiments 1 to 21.

[0265] 43. A method according to embodiment 42, wherein the cancer is selected from colon cancer, lung cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, gastric cancer, liver cancer, adrenocortical carcinoma and osteoblastoma cancer.

[0266] 44. A method according to embodiment 42, wherein the cancer is selected from acute myeloid leukemia, prostate cancer, glioblastoma, bladder cancer and cervical cancer.

[0267] 45. A method according to embodiment 42, comprising administering to the subject a first antibody or antibody conjugate and a second antibody or antibody conjugate of any one of embodiments 1 to 21, wherein the first antibody or antibody conjugate blocks binding of Wnt ligand to the Wnt3a binding site of LRP5, and the second antibody or antibody conjugate blocks binding of Wnt ligand to a non-Wnt3a binding site of LRP5.

[0268] 46. ​​A method according to embodiment 45, wherein the first antibody or immunoconjugate comprises a set of CDR sequences of an antibody selected from epitope group 2.

[0269] 47. The method of embodiment 42, wherein the antibody or immunoconjugate specifically binds LRP5 in at least one assay and inhibits Wnt3a-induced signaling in at least one assay, optionally wherein the antibody or immunoconjugate is the antibody or immunoconjugate of any one of embodiments 1 to 21.

[0270] 48. A method according to 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, LRP5-R3O_G6.

[0271] 49. A method of enhancing the signaling activity of a Wnt ligand that binds to a Wnt3a binding site of LRP5, the method comprising contacting a cell expressing LRP5 with an antibody that blocks binding of the Wnt ligand to a non-Wnt3a binding site of LRP5.

[0272] 50. A method of enhancing the signaling activity of a Wnt ligand that binds to a non-Wnt3a binding site of LRP5 by contacting a cell expressing LRP5 with an antibody that blocks binding of the Wnt ligand to the Wnt3a binding site of LRP5.

[0273] 51. The method of any one of embodiments 49 or 50, wherein the method is performed in vitro.

[0274] 52. The method of any one of embodiments 49 or 50, wherein the method is performed in vivo.

[0275] Example The following non-limiting examples are illustrative of the present disclosure: Example 1 Phage display antibody library Phage display antibody libraries are a powerful technology for generating therapeutic antibodies 17,18 . Will>10 10 The highly complex library of independent antibody fragments is displayed on phage particles as coat protein fusion molecules and screened to separate antibodies that recognize the antigen of interest. The inventors have established a synthetic antibody library in which the antigen binding site is constructed entirely by engineered sequences. The resulting antibodies use optimized human frameworks and therefore have minimal immunogenicity when used as potential therapeutic agents. Synthetic antibodies are highly stable, and their human frameworks and antigen binding sites can be customized to optimize affinity, specificity and effectiveness.

[0276] Characterization and optimization of anti-LRP5 antibodies Example 2 New synthetic antibodies targeting LRP5. A highly optimized Fab-phage library constructed in the laboratory was used to generate specific anti-LRP5 antibodies by direct selection on immobilized recombinant LRP5 ECD. Fab-phage clones from rounds 3 and 4 were screened for binding by ELISA, and the amino acid sequences of the CDRs of positive binders were analyzed in Figure 1A The complete DNA and amino acid sequences of the variable regions (heavy and light chains) of IgG are presented above. Epitope mapping by competitive ELISA revealed that the antibodies bind to four unique epitopes on the ECD of LRP5.

[0277] Full-length IgG1 and antibody fragments (Fab) were purified and subjected to single-point ELISA to determine the specificity of the antibodies. Figure 1BAs shown in , all LRP5 antibodies bound to the recombinant mouse LRP5-His chimera. Interestingly, the LRP5-H5 and LRP5-R3OD3 antibodies showed partial binding to the recombinant mouse LRP6-His chimera. The LRP5-R3OD3 antibody also showed partial binding to the recombinant human LRP6-Fc chimera. The LRP5-R3OD3 antibody also showed partial binding to the recombinant human LRP6-Fc chimera, indicating that the antibody can cross-react with both LRP5 and LRP6. Multipoint competitive ELISA was used to determine the relative affinity of the antibodies to the recombinant antigens. The IC50 was determined by nonlinear regression analysis and is summarized in Table 3. The complete dose response curves and nonlinear regression plots are in Figure 6 Presented in.

[0278] Table 3 reports the IC50 of LRP5 antibodies determined by competitive ELISA. The logarithm of the recombinant human LRP6-Fc chimera concentration (x-axis) is plotted against the OD450 reading of the antibody (y-axis). The IC50 was determined by nonlinear regression analysis.

[0279] Table 3 IgG1 Antibodies <![CDATA[IC 50 (nM)]]> LRP5-A7 4.68 LRP5-D9 25.11 LRP5-G2 24.21 LRP5-G9 26.44 LRP5-G10 3.02 LRP5-G11 25.11 LRP5-H5 4.44 LRP5-H9 37.46 Western blot analysis of whole cell lysates showed that LRP5 was highly expressed in NSCLC cell line H23, triple negative breast cancer cell line MDAMB231, and breast cancer cell line T470 (data not shown). As assessed by FACS analysis, LRP5 IgG1 labeled H23 ( Figure 2 ), MDAMB231 (Appendix 3), and T470 (Appendix 4) cells, and demonstrated that the LRP5 antibody can bind to the full-length receptor expressed on the surface of these cell lines. As mentioned above, Wnt signaling initiates a canonical pathway that primarily regulates the stability and function of β-catenin.

[0280] Upon Wnt stimulation, β-catenin levels increase in the cytosol and nucleus, leading to increased TCF / LEF (transcription factor)-mediated transcription. The TOPflash reporter assay is a widely used tool to assess the in vitro activity of β-catenin. The vector consists of several TCF / LEF binding sites that drive the expression of the firefly luciferase reporter gene. To assess the functional activity of the LRP5 antibody in regulating the Wnt canonical pathway, several cancer cell lines expressing this reporter were generated.

[0281] like Figure 3As shown in A, in MDAMB231 cells pretreated with negative control IgG1 (anti-MBP), conditioned medium expressing Wnt3a (Wnt3aCM) induced a 120-fold increase in luciferase reporter activity compared to treatment with control conditioned medium (ConCM). LRP5-G10 and LRP5-H5 antibodies significantly enhanced Wnt3aCM-induced reporter activity. In breast cancer cell line T47D ( Figure 3 B) and osteosarcoma cell line U20S ( Figure 3 Similar results were observed in C). In the U20S cell line, reporter activity induced by both ConCM and Wnt3aCM was enhanced by LRP5-G10 and LRP5-H5 antibodies (compared to M8P).

[0282] Structural and mutagenesis studies revealed that Wnt3a binds to a unique domain that is distinct from the binding site of the remaining Wnt ligands. In addition, recent studies have shown that certain Wnt ligands require both LRP5 and LRP6 to initiate the canonical pathway. To address the hypothesis that epitope-specific LRP5 antibodies would modulate β-catenin-mediated transcriptional activity in a Wnt-dependent manner, we assessed reporter activity in the NSCLC cell line H23. Previous studies (20) have shown that Wnt2 primarily drives basal TCF / LEF-mediated transcriptional activity and that these cells do not express Wnt3a. As expected, LRP5-G10 and LRP5-H5 antibodies enhanced Wnt3aCM-induced reporter activity (compared to M8P; Figure 3 D). Interestingly, these antibodies (as well as LRP5-A7, another member of this epitope group) inhibited basal (ConCM-induced) reporter activity (compared to M8P). In addition, LRP5-D9 and LRP5-G2 antibodies significantly enhanced ConCM-induced reporter activity (compared to MBP). Our results showed that LRP5-G2 and LRP5-G10 antibodies had the strongest effect on the induction of reporter activity. Consistent with this observation, LRP5-G2 antibody enhanced ConCM-induced reporter activity in a dose-dependent manner ( Figure 4 A). Interestingly, LRP5-G10 antibody inhibited ConCM-induced reporter activity at all concentrations shown. LRP5-G10 Fab also inhibited ConCM-induced reporter activity at the concentrations shown, while LRP5-G2 Fab failed to recapitulate the effect of IgG1 on reporter activity ( Figure 4 B).

[0283] The effects of LRP5-G2 and LRP5-G10 antibodies on proximal events of Wnt signaling were evaluated by Western blot analysis. Figure 5A). In H23 cells, LRP5-G10, but not LRP5-G2, antibody significantly inhibited LRP6 phosphorylation induced by both ConCM and Wnt3aCM. LRP5-G10 antibody also significantly reduced total 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 and then stimulated with conditioned medium ( Figure 5 B). LRP5-G10 antibody also significantly reduced Axin1 protein levels in cells stimulated with Wnt3aCM. Since Axin1 is a component of the destruction complex that regulates the "free" pool level of β-catenin, the reduced expression of Axin1 after treatment with LRP5-G10 antibody followed by stimulation with Wnt3aCM may explain why this antibody effectively enhances Wnt3aCM-induced reporter activity ( Figure 3 ).

[0284] To better understand the effect of the antibodies on β-catenin levels, the cytosolic fraction of H23 cells treated with LRP5-G2 and LRP5-G10 antibodies and then stimulated with conditioned medium was isolated. Figure 5 LRP5-G2 antibody significantly upregulated β-catenin levels in ConCM-treated cells, consistent with the effect of LRP5-G2 antibody on reporter activity, as shown in B. Similarly, LRP5-G10 antibody slightly decreased and increased β-catenin levels in ConCM-treated cells.

[0285] The NSCLC cell line H23 was used as an excellent system to explore the effect of LRP5 antibody co-treatment on TOPflash reporter activity. Our previous results showed that the LRP5-G2 antibody enhanced ConCM-induced reporter activity. In contrast, the LRP5-G10 antibody inhibited and enhanced ConCM-induced reporter activity and Wnt3aCM-induced reporter activity, respectively. Figure 6 As shown in , the enhancement of ConCM-induced reporter activity by LRP5-G2 was significantly inhibited by the presence of LRP5-G10 antibody. Our observations suggest that co-treatment of LRP5-G2 and LRP5-G10 antibodies can have a potent inhibitory effect on Wnt-stimulated TCF / LEF-mediated transcription. Therefore, by exploiting advances in library design strategies, we were able to discover new LRP5 antibodies that can exert both antagonistic and enhancing activities on proximal and distal events associated with β-catenin signaling. These activities also depend on the different interactions between Wnt ligands and LRP5. We are actively studying the therapeutic potential of these antibodies in vitro and in vivo.

[0286] abbreviation: abbreviation Full term APC Adenomatous colon polyps CDR Complementarity determining region CK1 Casein kinase 1 Dsh / Dvl Dishevelled ECD Extracellular domain ELISA ELISA Fab Fab FACS Fluorescence activated cell sorting Fc Crystallizable region fragment GSK3b Glycogen synthase kinase 3β <![CDATA[IC 50 ]]> Half maximum inhibitory concentration IgG1 IgG 1 LRP Low-density lipoprotein receptor-related protein OD Optical density NSCLC Non-small cell lung cancer 1.Saito-Diaz, K. et al. The way Wnt works: components and mechanism.Growth Factors Chur Switz. 31, 1-31 (2013). 2.Clevers, H. & Nusse, R. Wnt / β-catenin signaling and disease. Cell149, 1192-1205 (2012). 3.Anastas, J. N. & Moon, R. T. WNT signalling pathways as therapeutictargets in cancer. Nat. · Rev. Cancer 13, 11-26 (2012). 4.Baron, R. & Kneissel, M. WNT signaling in bone homeostasis anddisease: from human mutations to treatments. Nat. Med. 19, 179-192 (2013). 5.Kim, W., Kim, M. & Jho, E. Wnt / β-catenin signalling: from plasmamembrane to nucleus., Biochem. J. 450, 9-21 (2013). 6.MacDonald, B. T. & He, X. Frizzled and LRP5 / 6 Receptors for Wnt / β-catenin Signaling. Cold Spring Harb. Perspect. Biol. 4, a007880-a007880(2012). 7.Chen, S. et al. Structural and Functional Studies of LRP6Ectodomain Reveal a Platform for Wnt Signaling. Dev. Cell 21, 848-861 (2011). 8.Johnson, M. L. & Summerfield, D. T. Parameters of LRP5 from astructural and molecular perspective. Crit. Rev. Eukaryot. Gene Expr. 15,229-242 (2005). 9.Niehrs, C. & Shen, J. Regulation of Lrp6 phosphorylation. Cell.Mol. Life Sci. CMLS 67, 2551-2562 (2010). 10.Williams, B. 0. & Insogna, K. L. Where Wnts Went: The ExplodingField of Lrp5 and Lrp6 Signaling in Bone. J. Bone Miner. Res. 24, 171-178(2009). 11.Hoang, B. H. et al. Expression of LDL receptor-related protein 5(LRP5) as a novel marker for disease progression in high-grade osteosarcoma.Int. J. Cancer J. Int. Cancer 109, 106-111 (2004). 12.Li, Y. & Bu, G. LRP5 / 6 in Wnt signaling and tumorigenesis. FutureOncol. Lond. Engl. 1, 6781 (2005). 13.Bjorklund, P., Svedlund, J., Olsson, A.-K., Akerstrom, G. &Westin, G. The Internally Truncated LRP5 Receptor Presents a TherapeuticTarget in Breast Cancer. PLoS ONE 4, e4243 (2009). 14.Guo, Y. et al. Blocking Wnt / LRP5 signaling by a soluble receptormodulates the epithelial to mesenchymal transition and suppresses met andmetalloproteinases in osteosarcoma Saos-2 cells. J. Orthop. Res. Soc. 25,964-971 (2007). 15.Guo, Y., Rubin, E. M., Xie, J., Zi, X. & Hoang, B. H. DominantNegative LRP5 Decreases Tumorigenicity and Metastasis of Osteosarcoma in anAnimal Model. Clin. Orthop. 466, 2039-2045 (2008). 16.Rabbani, S. A., Arakelian, A. & Farookhi, R. LRP5 knockdown:effect on prostate cancer invasion growth and skeletal metastasis in vitroand in vivo. Cancer Med. 2 (5), 625-635 (2013). 17.Goel, S. et al. Both LRP5 and LRP6 Receptors Are Required toRespond to Physiological Wnt Ligands in Mammary Epithelial Cells andFibroblasts. J. Biol. Chem. 287, 16454-16466 (2012). 18.Lee, C. V. et al. High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold. J. Mol.Biol. 340, 1073-1093 (2004). 19.Fellouse, F. A. & Sidhu, S. S. in Phage Disp. Drug Discov.Biotechnol. 709-740 (CRC Press / Taylor & Francis, 2006). 20.Akiri, G. et al. Wnt pathway aberrations including autocrine Wntactivation occur at high frequency in human non-small-cell lung carcinoma.Oncogene 28, 2163-2172 (2009). As used herein, unless otherwise specified, the following meanings apply. The word "may" is used in a permissive sense (i.e., meaning possible), rather than a mandatory sense (i.e., meaning must). The words "include", "including", and "includes" etc. mean including but not limited to. The singular forms "a", "a", and "the" include plural indicators. Thus, for example, reference to "an element" includes a combination of two or more elements, although other terms and expressions such as "one or more" are used for one or more elements. The expression "at least one" includes "one or more", "one or a plurality", and "more than one aplurality". Unless otherwise specified, the term "or" is non-exclusive, i.e., covers both "and" and "or". The term "any of" between a modifier and a sequence means that the modifier modifies each member of the sequence. Thus, for example, the phrase "any of at least 1, 2, or 3" means "at least 1, at least 2, or at least 3." The term "consisting essentially of" means comprising the recited elements and other elements that do not materially affect the basic and novel characteristics of the claimed combination.

[0287] It should be understood that this specification and the accompanying drawings are not intended to limit the present invention to the specific form disclosed, but on the contrary, it is intended to cover all modifications, equivalents and alternative forms falling within the spirit and scope of the present invention defined by the appended claims. According to this specification, other modifications and alternative embodiments of various aspects of the present invention will be obvious to those skilled in the art. Therefore, this specification and the accompanying drawings should be interpreted as being only illustrative, and are for the purpose of teaching those skilled in the art to carry out the general mode of the present invention. It should be understood that the form of the present invention shown and described herein should be considered as an example of an embodiment. The elements and materials illustrated and described herein can be replaced, components (part) and processes can be reversed or omitted, and certain features of the present invention can be used independently, all of which will be obvious to those skilled in the art who understand the benefits of the present invention specification. The elements described herein can be changed without departing from the spirit and scope of the present invention as described in the appended claims. The titles used herein are only for the purpose of organization and are not meant to limit the scope of this specification.

[0288] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. An antibody that specifically binds to LRP5, the antibody comprising a light chain variable region and / or a heavy chain variable region, the heavy chain variable region comprising complementary determining regions CDR-H1, CDR-H2 and CDR-H3, the light chain variable region comprising complementary determining regions CDR-L1, CDR-L2 and CDR-L3, wherein the amino acid sequence of the CDR comprises or consists of a CDR sequence selected from the following: the CDR sequence set of anti-LRP5 antibodies 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.

2. The antibody according to claim 1, wherein the amino acid sequence of the CDR comprises or consists of a sequence selected from the group consisting of the sequences listed below: CDR-H1 is selected from the group consisting of: LSYYYM, ISYSYI, LSYSSM, ISSYSI, ISYSYI, IYSYSI, LSYYYM, FSSSSI, LYYYYI, LSYSSI, IYSYYI, LLYYSSM, and FSSSSI; CDR-H2 is selected from the group consisting of: SIYPYYGYTY, SSSYYGYTY, SISSSYGYTY, SIYSSYGSTS, SIYSSYGYTY, SIYPYSSYTS, SIYSSYGYTY, SIYPSYGYTY, SISPYYGYTS, SISSSYGSTS, SIYSYYGYTY, SISSSYGYTY, SISSSYGYTY SISSYYGYTS and YISPYYGYTS; CDR-H3 is selected from the group consisting of: HGAM, TVRGSKKPYFSGWAM, SSYYSSVSSSVYAL, TVRGSKKPYFSGWAM, HYSYFFYAM, YAVYFPGYYWGM, WSHVSGHYSGM, WGAYHSSGYGM, GGSGV SHYGSVYYSWWAL, AAPYYGYYYSYAM, SGYGWYAM, GYWAI, SYPAM, SWAM, YWAL, GWGSPASAGYYGL, SSYYSSVSSSVYAL, TVRGSKKPYFSGWAM and TVRGSKKPYFSGWAM; CDR-L1 is SVSSA; CDR-L2 is SASSLYS; and CDR-L3 is selected from the group consisting of: AWGWGLF, VHYSPYSLI, YQYSGLI, FSHVSLI, ASYSPI, YHYYYLF, ASYAPI, SSSSPI, SSYSLI, GVSLI, YWFLI, PVGHYGYPI, SSYSPI, YWAYYSPI, VSYYPLI, SSYSLI, and VHYSPYSLI.

3. The antibody of claim 2, wherein the antibody comprises a heavy chain variable region comprising: i) the heavy chain amino acid sequence as listed 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 a heavy chain amino acid sequence as listed in Table 2, wherein the CDR sequence is a set of CDR sequences as listed in Table 1, or iii) a conservatively substituted amino acid sequence of i), wherein the CDR sequence is a set of CDR sequences listed in the table.

4. The antibody according to any one of claims 2 to 3, wherein the antibody comprises a light chain variable region comprising: i) the light chain amino acid sequence as listed 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 a light chain amino acid sequence as listed in Table 2, wherein the CDR sequence is a set of CDR sequences as listed in Table 1, or iii) a conservatively substituted amino acid sequence of i), wherein the CDR sequence is the CDR sequence set listed in Table 1.

5. The antibody according to any one of claims 1 to 4, wherein the CDR sequence is selected from the full set of CDR sequences of the antibodies identified in Table 1.

6. The antibody of any one of claims 1 to 5, wherein the antibody cross-reacts with LRP6.

7. The antibody of claim 1, wherein 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.

8. The antibody of any one of claims 1 to 7, wherein the antibody specifically binds to LRP5.

9. The antibody according to claim 8, wherein the CDR sequence is a set of CDR sequences of antibodies selected from the group consisting of antibodies 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.

10. The antibody of any one of claims 1 to 9, which blocks the binding of a Wnt ligand to the Wnt3a binding site of LRP5.

Images