Frizzled receptor antibodies and their uses

Abstract

Isolated antibodies and immunoconjugates that specifically bind to the cysteine-rich domain (CRD) of Frizzled receptor (FZD) 4, comprising 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 comprising or consisting of a sequence selected from the sequence in Table 1a or Table 3a. Methods of using the antibodies and immunoconjugates are also provided.

Description

[0001] field

[0002] The present disclosure relates to antibodies that bind to the frizzled receptor and in particular to antibodies that bind to the cysteine-rich domain of the frizzled receptor 4 and uses thereof.

[0003] Citation of Related Applications

[0004] This application claims the benefit of the priority dates of U.S. Provisional Application No. 62 / 885,781, filed on August 12, 2019, and U.S. Provisional Application No. 62 / 886,292, filed on August 13, 2019, the contents of which are incorporated herein by reference in their entireties.

[0005] background

[0006] Frizzled receptors (FZDs), an important class of seven transmembrane receptors, are involved in many important biological processes, such as development, cell proliferation, survival, migration, and stem cell maintenance. When Wnt ligands interact with the Frizzled receptor family of seven transmembrane receptors, signal transduction pathways are activated and control the renewal and cell differentiation of stem and progenitor cells during embryonic development and tissue homeostasis in adult animals. Abnormal expression and signal transduction of these receptors and their ligands (Wnt) are associated with many cancers, including colon cancer, lung cancer, breast cancer, and ovarian cancer. Typically, more than one Wnt ligand and / or Frizzled receptor is upregulated and leads to abnormal signal transduction that drives tumorigenesis. Therefore, inhibiting more than one Frizzled receptor may be necessary to achieve better anti-cancer effects. In addition, Frizzled receptors are also associated with cancer stem cells (a small group of cancer cells that are considered to cause drug resistance, tumor recurrence, and metastasis). Therefore, inhibiting FZD receptors (one or more than one receptor) including FZD4 may be an effective method for targeting cancer stem cells and treating various cancer types.

[0007] 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.

[0008] The typical pathway controls transcriptional activity mainly by regulating the cytoplasmic level of β-catenin. Under unstimulated conditions, β-catenin associates with the destruction complex (comprising Axin, APC, CD1 and GSKβ), which leads to phosphorylation, ubiquitination and proteasomal degradation of β-catenin. When Wnt binds to the 7-transmembrane receptor frizzled receptor (FZD) and binds 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.

[0009] Several human cancers are caused by mutations within the cytoplasmic components of the WNT pathway, which lead to ligand-independent Wnt target gene activation. For example, inactivating APC mutations and activating β-catenin mutations are the main potential causes of human colorectal cancer. Since this pathway is activated downstream of cell surface receptors, the development of targeted therapies for the Wnt pathway has proven challenging. However, cancer-causing mutations in the negative regulator of Wnt signaling RNF43 (colon cancer, endometrial cancer, pancreatic cancer, gastric cancer, ovarian cancer, liver cancer) and its homolog ZNRF3 (adrenocortical carcinoma and osteosarcoma) have recently been identified, and the mutations are associated with ligand-dependent tumor growth. In fact, RNF43 and ZNRF3 are Wnt target genes encoding transmembrane E3 ubiquitin ligases targeting frizzled receptors, and loss-of-function mutations in RNF43 and ZNRF3 lead to high expression of FZDs and may sensitize tumor cells to inhibition of Wnt-dependent signaling.

[0010] Overview

[0011] The following summary is intended to introduce the reader to various aspects of the disclosure without defining or circumscribing any invention.

[0012] In one aspect, the disclosure provides an antibody that specifically binds to a cysteine-rich domain (CRD) of each of one or more than one human frizzled receptors selected from FZD1, FZD2, FZD4, FZD5, FZD7, FZD8, and FZD9, 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, and the amino acid sequence of the CDR comprises or consists of a sequence selected from a sequence in Table 1a or Table 3a. In one embodiment, the CDR comprises (a) a full sequence set or (b) a light chain sequence set or (c) a heavy chain sequence set of an antibody identified in Table 1a or Table 3a.

[0013] In another embodiment, the CDR comprises or consists of the following sequence, wherein:

[0014] CDR-H1 is selected from the group consisting of: ISYYYM, IYSYYM, LSYYYM, IYYYSI, LYSYYM, LSSYSM, ISYYYI, LSYSSM, IYYYYM, LYYYSI, IS SYYI, FSSSSI, LSYYSI, LYSYYI, LSSYYM, LSYYYI, ISSYYM, LSYYSM, LYSYSI, LYYYYI, IYSYYI, ISYSYI and ISYYSM;

[0015] CDR-H2 is selected from the group consisting of: SIYSYYGYTY, SIYSSSSSTY, SIYPSSSSYTY, SIYSSSSYTS, YISSYSGSTY, SIYSSYGYTY, YISSYYGYTY, SIYPSSSSTY ,SIYSSSGYTY,YISSYSGSTS,SISSYYGSTY,SIYSYYGSTY,SIYPYSGYTY,YISPYYGYTS,SISSSSGYTY,SIYSYSSSTY,SISPSSSYTY,YIS PYYGYTY, SISPYSSSTY, SIYSSYGSTY, SIYSSSSYTY, SIYPSSGYTY, SIYPYSGSTY, SIYPSYGSTY, YISSYSSYTY, SIYSYYSSTY, YISSSYG YTS, SISPYSSYTY, YISPYSGYTS, SIYPYYSYTY, SISPYYGYTS, SISPSYSSTY, SISSSYSSTY, SIYPYSGSTS, SISSYYSSTS and SIYSYSGYTY;

[0016] CDR-H3 is selected from the group consisting of: SSFSWAM, SSFYWAL, SWFGWGI, YWFSYGYASYPAF, HPWYGM, SAFYWAL, PAPGHWGF, SSFFWAM, SAFYWAM, HFFA M, SWWAWAF, SAFGWAL, SSFFFAM, PYYWSGGF, HPSSSWFSFGAL, SAFYWAF, SSYAWAM, SSFYWAI, SPWGSGWAGF, PAVWVGL, SWVFWAL ,SWVYWGM,SWVYWAL,SSYAWAI,SSFYWAM,HGASFGSGAPAF,SCFFWAM,WAFFGL,SSFYFAM,SAFSWAI,SGFYWAL,PSVGYAAF,SWVG WGL, SSVGYVAM, SWVYWAF, YYYSSSVYFWYAAL, SSFFWAI, SWVYWAI, SWVGWGI, SSVYWAL, WGGWGSGGYFYAAL, FWYPGM and SSFAWAF;

[0017] CDR-L1 is SVSSA;

[0018] CDR-L2 is SASSLYS; and

[0019] CDR-L3 is selected from the group consisting of: HPWSGGYLI, PVGYWGVPI, VSGGAHALI, VSSAYPI, FWGVPI, SYYHYAALI, WYYAPI, SHSYSLI, SGYGPF, SWSSPI, HYSVYASLI, PHPPSLI, VAYSHVGLI, GYGAPI, SWYSLI, PGYLF, VWFGLI, VYYGSPLF, HAHSPLI, SSAYYPF, GHASPI , SSGGWSLI, VAWSSFLI, SVAAASLI, SGWWGVSLI, SYAAYLF, HGSLF, YAGVSNLF, GWPYSALF, SGYYPSLF, SYHSGSGLI, HGYS ASLI, APGWALF, GHSSPI, GWPSLF, VPGYPVPI, HYYSHLI, GPASSLI, SVGSSYYLI, YYGPWVLI, AASWGYPF, HWSYPI and GGWGPF.

[0020] In yet another embodiment, the CDR comprises or consists of the following sequence, wherein:

[0021] CDR-H1 is selected from the group consisting of: ISYYYM, IYSYYM, LSYYYM, IYYYYSI, LYSYYM, LSSYSM, ISYYYI, LSYSSM, IYYYYM, LYYYSI, ISSYYI, FSSSSI, LSYYSI, LYSYYY, LSSYYM, LSYYYI, ISYYM, LSYYSM, LYSYSI, LYYYYI, IYSYYI, ISYSYI, and ISYYSM;

[0022] The CDR-H2 selection consists of the following groups: PYYYTY, SISPYSSSTY, SYYSSYGSTY, SYYSSSSYTY, SYPSSYGSTY, SYYPYSGSTY, SYYPSYGSTY, YISSSYGYTS, SISPYSSTY, YISSSYGYTS, SISPYYSTY, SISPYGYTS, SISPSYSSTY, SISSSYSSTY, SIYPYSGSTS, SISSYYSSTS and SIYSYSGYTY;

[0023] CDR-H3 is selected from the group consisting of: SSFSWAM, SSFYWAL, SWFGWGI, YWFSYGYASYPAF, HPWYGM, SAFYWAL, PAPGHWGF, SSFFWAM, SAFYWAM, HFFA M, SWWAWAF, SAFGWAL, SSFFFAM, PYYWSGGF, HPSSSWFSFGAL, SAFYWAF, SSYAWAM, SSFYWAI, SPWGSGWAGF, PAVWVGL, SWVFWAL ,SWVYWGM,SWVYWAL,SSYAWAI,SSFYWAM,HGASFGSGAPAF,SCFFWAM,WAFFGL,SSFYFAM,SAFSWAI,SGFYWAL,PSVGYAAF,SWVG WGL, SSVGYVAM, SWVYWAF, YYYSSSVYFWYAAL, SSFFWAI, SWVYWAI, SWVGWGI, SSVYWAL, WGGWGSGGYFYAAL, FWYPGM and SSFAWAF;

[0024] CDR-L1 is SVSSA;

[0025] CDR-L2 is SASSLYS; and

[0026] CDR-L3 is selected from the group consisting of: HPWSGGYLI, PVGYWGVPI, VSGGAHALI, VSSAYPI, FWGVPI, SYYHYAALI, WYYAPI, SHSYSLI, SGYGPF, SWSSPI, HYSVYASLI, PHPPSLI, VAYSHVGLI, GYGAPI, SWYSLI, PGYLF, VWFGLI, VYYGSPLF, HAHSPLI, SSAYYPF, GHASPI , SSGGWSLI, VAWSSFLI, SVAAASLI, SGWWGVSLI, SYAAYLF, HGSLF, YAGVSNLF, GWPYSALF, SGYYPSLF, SYHSGSGLI, HGYS ASLI, APGWALF, GHSSPI, GWPSLF, VPGYPVPI, HYYSHLI, GPASSLI, SVGSSYYLI, YYGPWVLI, AASWGYPF, HWSYPI and GGWGPF.

[0027] In another embodiment, the disclosure provides an antibody as previously described, comprising a heavy chain variable region comprising:

[0028] i) the heavy chain amino acid sequence as listed in Table 2;

[0029] ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80% or at least 90% 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 1a or Table 3a, or

[0030] iii) a conservatively substituted amino acid sequence of i), wherein the CDR sequence is a CDR sequence set as listed in Table 1a or Table 3a.

[0031] In another embodiment, the disclosure provides an antibody further comprising a light chain variable region comprising:

[0032] i) the light chain amino acid sequence listed in Table 2;

[0033] ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80% or at least 90% 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 1a or Table 3a; or

[0034] iii) a conservatively substituted amino acid sequence of i), wherein the CDR sequence is a CDR sequence set as listed in Table 1a or Table 3a.

[0035] In another embodiment, the disclosure provides an antibody that specifically binds to FZD4. In another embodiment, the antibody CDR sequence that specifically binds to FZD4 is a set of CDR sequences of an antibody selected from the following antibodies: 5017, 5027, 5030, 6499, 5038, 5040-5044, 5046, 5047, 5049-5053, 5055, 5058-5064, 5066, 5068-5072, 5077-5080 or 5081.

[0036] In another embodiment, the disclosure provides antibodies that specifically bind to FZD4 and at least one other receptor selected from FZD1, FZD2, FZD5, FZD7, FZD8, and FZD9. In yet another embodiment, the CDR sequence is a set of CDR sequences of an antibody selected from the following antibodies: 5014, 5016, 5018-5023, 5025, 5028, 5029, 5031, 5034, 5035, 5036, 5037, 6494, 6495, 6496, 6497, 6498, 6500, 5039, 5045, 5048, 5054, 5056, 5057, 5067, and 5073-5076. In yet another embodiment, the antibody preferentially binds to frizzled receptor 4 (FZD4) relative to another FZD receptor. In yet another embodiment, the CDR sequence is the set of CDR sequences of an antibody selected from the group consisting of 5028, 5029, 5031, 5034, 5035, 6497, 6498, 5039, 5045, 5048, 5054, 5056, 5057, 5067, 5073, 5074, 5075. In yet another embodiment, the antibody has a binding affinity of between about 0.2 nM and about 15.3 nM as measured by surface plasmon resonance.

[0037] In yet another embodiment, the CDR sequence is a set of CDR sequences of an antibody selected from the group consisting of 5017, 5027, 5030, 6499, 5038, 5040-5044, 5046, 5047, 5049-5053, 5055, 5058-5064, 5066, 5068-5072, 5077-5080, or 5081.

[0038] In yet another embodiment, the antibody is monoclonal, humanized, single chain, antibody fragment, multivalent, bispecific, comprises a non-native glycosylation pattern, comprises cysteine ​​substitutions or additions, or blocks the binding of Wnt to FZD. In yet another embodiment, the antibody fragment is selected from the group consisting of fragments selected from the group consisting of Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers thereof, nanobodies, minibodies, diabodies, and multimers. In yet another embodiment, the multivalent antibody is bivalent, trivalent, or tetravalent. In yet another embodiment, the cysteine ​​substitutions are present in the constant region or the framework region. In yet another embodiment, the bispecific antibody also binds to LRP 5 and / or LRP 6. In yet another embodiment, the antibody comprises a non-native glycosylation pattern. In yet another embodiment, the cysteine ​​substitutions are in the constant region or the framework region. In yet another embodiment, the antibody as described herein blocks the binding of Wnt to FZD.

[0039] In another aspect, the present disclosure provides an immunoconjugate comprising an antibody as described herein and a detectable marker or a cytotoxic agent. In one embodiment, the cytotoxic agent is selected from maytansinoid, auristatin, dolastatin, tubulysin, scin, pyrrolobenzodiazepine (PBD) dimer, indolebenzodiazepine dimer, α-amanitin, trichothene, SN-38, duocarmycin, CC1065, calicheamicin, enediyne antibiotics, taxanes, doxorubicin derivatives, anthracyclines and their stereoisomers, azanofide, isosteres, analogs or derivatives.

[0040] In another aspect, the disclosure provides nucleic acids encoding antibodies as described herein. In one embodiment, one or more CDR sequences encoded by the nucleic acid are described in Table 1b, Table 1c, Table 3b, and Table 3c.

[0041] In another embodiment, the antibody encoded by the nucleic acid comprises a heavy chain variable region encoded by a nucleic acid comprising:

[0042] i) a heavy chain nucleic acid sequence as listed in Table 2;

[0043] ii) a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity with 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 1a or Table 3a, or

[0044] iii) the codon-degenerate nucleic acid sequence of i), wherein the CDR sequence is a CDR sequence set as listed in Table 1a or Table 3a.

[0045] In another embodiment, the antibody encoded by the nucleic acid comprises a light chain variable region encoded by a nucleic acid comprising:

[0046] i) the light chain nucleic acid sequence listed in Table 2,

[0047] ii) a nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 80% or at least 90% 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 1a or Table 3a, or

[0048] iii) the codon-degenerate nucleic acid sequence of i), wherein the CDR sequence is a CDR sequence set as listed in Table 1a or Table 3a.

[0049] In yet another aspect, the present disclosure provides a vector comprising an expression control sequence operably linked to a nucleic acid encoding an antibody described herein.

[0050] In yet another aspect, the disclosure provides a host cell comprising a recombinant nucleic acid molecule comprising an expression control sequence operably linked to a nucleic acid encoding an antibody described herein. In one embodiment, the host cell is a Chinese Hamster Ovary (CHO) cell.

[0051] In yet another aspect, the disclosure provides a method for preparing an anti-FZD antibody, the method comprising culturing a host cell as described herein.

[0052] In yet another aspect, the disclosure provides compositions comprising one or more antibodies, immunoconjugates, nucleic acids, vectors or host cells described 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.

[0053] In yet another aspect, the disclosure provides a kit comprising one or more antibodies, immunoconjugates, nucleic acids, vectors, or host cells described herein.

[0054] In yet another aspect, the disclosure provides a method for detecting FZD 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 antibody: cell complexes, and detecting the presence of any antibody complexes. In one embodiment, the detection method is by immunofluorescence. In another embodiment, the detection method is by flow cytometry. In yet another embodiment, the method is used to detect FZD4 expression, and the antibody or immunoconjugate comprises a CDR sequence set corresponding to an antibody selected from the group consisting of: 5017, 5027, 5030, 6499, 5038, 5040-5044, 5046, 5047, 5049-5053, 5055, 5058-5064, 5066, 5068-5072, and 5077-5081.

[0055] In yet another aspect, the disclosure provides a method of inhibiting Wnt ligand binding to FZD receptors, disrupting Wnt signaling pathways, inhibiting Wnt-induced transcriptional activity, inhibiting dishevelled activation, promoting the retention of β-catenin destruction complexes, promoting β-catenin accumulation, or inhibiting cell growth, the method comprising contacting a cell expressing a FZD receptor with an antibody or immunoconjugate described herein. In another aspect, the disclosure provides an antibody or immunoconjugate described herein for inhibiting Wnt ligand binding to FZD receptors, disrupting Wnt signaling pathways, inhibiting Wnt-induced transcriptional activity, inhibiting dishevelled activation, promoting the retention of β-catenin destruction complexes, promoting β-catenin accumulation, or inhibiting cell growth. In another aspect, the disclosure provides the use of an antibody or immunoconjugate described herein for inhibiting Wnt ligand binding to FZD receptors, disrupting Wnt signaling pathways, inhibiting Wnt-induced transcriptional activity, inhibiting dishevelled activation, promoting the retention of β-catenin destruction complexes, promoting β-catenin accumulation, or inhibiting cell growth. In yet another aspect, the disclosure provides the use of an antibody or immunoconjugate described herein in the preparation of a medicament for inhibiting Wnt ligand binding to a FZD receptor, disrupting a Wnt signaling pathway, inhibiting Wnt-induced transcriptional activity, inhibiting the activation of dishevelled proteins, promoting the retention of a β-catenin destruction complex, promoting β-catenin accumulation, or inhibiting cell growth. In one embodiment, the Wnt ligand is Wnt3a. In another embodiment, the antibody or immunoconjugate comprises a set of CDR sequences corresponding to an antibody selected from the group consisting of: a) 5017, 5027, 5030, 6499, 5038, 5040-5044, 5046, 5047, 5049-5053, 5055, 5058-5064, 5066, 5068-5072, and 5077-5081, or b) 5014, 5016, 5018-5023, 5025, 5028, 5029, 5031, 5034, 5035, 5036, 5037, 6494, 6495, 6496, 6497, 6498, 6500, 5039, 5045, 5048, 5054, 5056, 5057, 5067 and 5073-5076.

[0056] In yet another aspect, the disclosure provides a method for treating cancer in 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 described herein. On the other hand, the disclosure provides the use of an antibody or immunoconjugate as described herein for treating cancer. On yet another aspect, the disclosure provides an antibody or immunoconjugate as described herein for treating cancer. On yet another aspect, the disclosure provides the use of an antibody or immunoconjugate as described herein in the preparation of a medicament for treating cancer. In one embodiment, the cancer is selected from acute myeloid leukemia, neuroblastoma, liver cancer, lung cancer, endometrial cancer, salivary gland-like cystic carcinoma, colorectal cancer, prostate cancer, glioblastoma, bladder cancer, cervical cancer, pancreatic cancer, colon cancer, breast cancer, esophageal cancer, glioma, gastric cancer, astrocytoma and osteosarcoma. In another embodiment, the method or use comprises an antibody or immunoconjugate that specifically binds to FZD1, FZD2, FZD4, FZD5, FZD7, FZD8 and FZD9 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 described herein. In yet another embodiment, the antibody or immunoconjugate of the method or use comprises a set of CDR sequences corresponding to an antibody selected from the group consisting of: a) 5017, 5027, 5030, 6499, 5038, 5040-5044, 5046, 5047, 5049-5053, 5055, 5058-5064, 5066, 5068-5072 and 5077-508 1, or b) 5014, 5016, 5018-5023, 5025, 5028, 5029, 5031, 5034, 5035, 5036, 5037, 6494, 6495, 6496, 6497, 6498, 6500, 5039, 5045, 5048, 5054, 5056, 5057, 5067 and 5073-5076. In yet another embodiment, the antibody or immunoconjugate comprises a set of CDR sequences corresponding to an antibody selected from 5019 and 5020. In yet another embodiment, the cancer treated by the method or use comprises one or more cancer cells comprising a mutation of the RNF43 gene, and the antibody or immunoconjugate comprises a set of CDR sequences corresponding to antibody 5020.

[0057] Other features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples are provided by way of illustration only while indicating the embodiments, and the scope of the claims should not be limited by the embodiments set forth in the examples, but should be provided with the broadest interpretation consistent with the description as a whole. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Embodiments of the present disclosure will now be described with reference to the accompanying drawings, in which:

[0060] Figure 1 Is a diagram showing the binding of phage clones to FZD4-CRD-Fc and Fc. Single colonies were used to inoculate 96-well culture plates, and overnight phage supernatants were diluted 1:2 in 0.05% Tween20 / 0.5% BSA / PBS (dilution buffer) to test binding in ELISA. Phage was detected with anti-M13-HRP secondary antibody (1:5000 in dilution buffer), and the plate was developed with TMB substrate and acid stop. Read the absorbance at 450nm for FZD4-Fc coated wells and control Fc coated wells as shown.

[0061] Figure 2 : is a figure showing the competitive ELISA binding of anti-FZD4 Fab. ELISA was performed to evaluate the affinity of the FZD4 Fab group. 384-well ELISA plates were coated overnight at 4°C with 2μg / ml FZD4 CRD-Fc (R&D systems) in PBS. The plates were blocked with 0.5% BSA / PBS at room temperature for 1 hour, and then washed three times with 0.05% Tween20 / PBS. In a non-binding 96-well ELISA plate, a final concentration of 0.5μg / ml of Fab was pre-incubated with a specified concentration of FZD4 CRD-Fc in a solution (0.05% Tween20 / 0.5% BSA / PBS) at room temperature for 1 hour. The Fab antigen mixture was transferred to a 384-well plate and incubated at room temperature for 20 minutes. The plates were washed six times and bound Fab was detected with an anti-FLAG-HRP secondary antibody (Sigma) at 1:5000 in 0.05% Tween20 / 0.5% BSA / PBS. The secondary antibody was incubated for 45 minutes at room temperature, the plates were washed, and then the color was stopped with TMB substrate and acid. The absorbance at 450 nm was read. The binding % was calculated as the absorbance of the Fab well containing competitive soluble FZD4 CRD-Fc divided by the absorbance of the Fab well without competitive soluble FZD4 CRD-Fc multiplied by 100.

[0062] Figure 3 is a series of immunofluorescence staining photos showing the binding of anti-FZD4 Fab to FZD expressed on CHO cells. Fabs from the FZD4 panel were tested for reactivity by immunofluorescence staining (IF) against a CHO overexpression line stably expressing the FZD4 CRD region as a GPI-linked domain with a myc tag. 2 -FITC secondary antibody (Jackson Immuno) detected Fab, and white areas indicate FITC staining.

[0063] Figure 4 is a series of immunofluorescence staining photos showing the binding of anti-FZD4 Fab to CHO cells. The reactivity of Fabs from the FZD4 panel to a control CHO cell line stably transfected with a GPI linker and a myc tag was tested by immunofluorescence staining (IF). 2 -FITC secondary antibody (Jackson Immuno) detected Fab, and white areas indicate FITC staining.

[0064] Figure 5 is a table showing the binding of Fab to FZD determined by IF staining.

[0065] Figure 6 is a series of immunofluorescence staining photographs showing the binding of Fab 5019 and Fab 5020 to FZD in various human pancreatic cancer cell lines.

[0066] Figure 7 is a series of graphs showing the binding of Fab 5019 and Fab 5020 to pancreatic cell lines as determined by flow cytometry. Numbers indicate the fold increase in MFI over the secondary antibody control.

[0067] Figure 8 AD is a series of graphs showing binding of anti-FZD4 Fab to FZD.

[0068] Fig. 9A -B is a table showing the binding of anti-FZD4 Fab to FZD as determined by IF. CHO myc GPI cell line. 200 nM Fab. - = no binding; + = very weak binder; ++ = weak binder; +++ = good binder; ++++ = very good binder.

[0069] Fig.10 is a table showing the binding affinity of anti-FZD4 Fab to FZD4 determined by SPR.

[0070] Fig.11 is a graph showing the binding of anti-FZD4 Fab to pancreatic cancer cells determined by flow cytometry.

[0071] Fig.12AB is a series of graphs showing inhibition of WNT5A binding by anti-FZD4 Fab. (A) Wnt5a (R&D systems) was biotinylated using a commercial kit (Thermo 21329EZ-linked NHS-PEG4-biotin), and excess biotin was removed by buffer exchange using a 3000MWCO Amicon filter. FZD4-CRD-Fc or control Fc protein (R&D systems) was diluted to the specified concentration in 1% BSA / 0.05% Tween20 / PBS (dilution buffer) and incubated with a constant amount of biotinylated Wnt5a in a BSA-blocked 96-well TC-treated plate at room temperature for 1 hour. Control wells in which a separate buffer was added instead of biotinylated wnt5a were also included. Biotinylated Wnt5a was added at a final concentration of 150ng / ul. The samples were transferred to a pre-blocked streptavidin-coated plate (R&D systems) and allowed to capture for 1 hour at room temperature. The wells were washed four times with 0.05% Tween20 / PBS, and then anti-Fc-HRP (1:5000 in dilution buffer, Jackson Immuno) was added to the wells for 45 minutes at room temperature. The wells were washed four times and color development was stopped with TMB reagent and acid. The absorbance at 450 nm was read. (B) FZD4-CRD-Fc was diluted to the concentration previously determined in (A) to provide an ELISA signal within the linear range and incubated with the desired Fab or buffer control in a 96-well TC plate pre-blocked with 1% BSA at room temperature for 1 hour. As above, control wells containing Fc protein were also included. Biotinylated wnt5a was added to the wells and the plate was incubated for an additional 1 hour. Control wells in which buffer alone was added instead of biotinylated wnt5a were also included. Biotinylated Wnt5a was added at a final concentration of 150ng / ul. The final concentration of the Fab proteins was 400 nM, with the following exceptions: Fab 6494 at 180 nM, Fab 6406 at 135 nM, and Fab 6500 at 159 nM. Negative control Fabs specific for the different protein antigens were included as well as controls for the effects of the neutralization elution buffer in which the Fab proteins were stored (Fab buffer control). Percent binding was calculated.

[0072] Fig.13 Is a table showing the effects of Fabs on β-catenin driven transcription (TOPFLASH assay).

[0073] Fig.14 AH is a series of graphs showing the effects of anti-FZD4 Fab on cancer cell proliferation.

[0074] Fig.15is a table showing the effects of anti-FZD4 Fabs on pancreatic cancer cell proliferation as determined by SRB (sulforhodamine B) assay, where "na" means not tested.

[0075] Fig.16 is a summary table of anti-FZD4 Fabs, where na means not tested. Antiproliferative activity was shown to be associated with binding to FZD1, FZD2, FZD4, FZD5, FZD7, FZD8 and FZD9 and inhibition of wnt3a activity. As shown in this assay, the most potent inhibitors were 5014, 5019-5023 and 6495.

[0076] Fig.17 is a graph showing that anti-FZD4 antibodies inhibit the expression of Wnt pathway regulatory gene Axin2 in a human pancreatic cancer cell line (HPAF II).Gene expression (RT-qPCR) after treatment with 200 nM Fab / IgG was normalized to β-actin.

[0077] Fig.18 AB is a series of graphs showing inhibition of proliferation by IgG 5019 and IgG 5020. A) shows that IgG 5019 and IgG5020 inhibit pancreatic cancer cell proliferation using an Alamar Blue proliferation assay (200 nM Fab / IgG), and B) shows that IgG 5020 inhibits cell proliferation in a dose-dependent manner.

[0078] Fig.19 is a series of colony photographs showing that IgG 5020 inhibits colony formation.

[0079] Fig. 20 is a series of immunofluorescence staining photographs showing the binding of Fab 5019 and Fab 5020 to FZD in PDAC patient-derived xenograft (PDX) cell lines (GP2A and GP14A).

[0080] Fig.21 is a graph showing the effect of Fab 5019, Fab 5020 and IgG5020 on the proliferation of PDAC PDX cell lines in an Alamar Blue proliferation assay (200 nM Fab / IgG).

[0081] Fig. 22 Schematic diagram showing the typical Wnt signaling pathway.

[0082] Details of public content

[0083] I. Definitions

[0084] 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).

[0085] 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.

[0086] As used herein, an amino acid sequence "consists of" only the amino acids in that sequence.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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 one another with amino acids that are 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):

[0091]

[0092] As used herein, the term "substantially identical" refers to the identity between a first amino acid sequence and a second amino acid sequence, the first amino acid sequence containing a sufficient or minimum number of i) amino acid residues identical to the amino acid residues in the aligned second amino acid sequence, or ii) amino acid residues that are conservative substitutions of the amino acid residues in the aligned second amino acid sequence, so 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 aligned second nucleic acid sequence, so 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.

[0093] 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.

[0094] 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).

[0095] 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.

[0096] 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 embodiment, each chain comprises a variable region and a constant region. The antigen recognition site of the variable region consists of a hypervariable region or complementary 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.

[0097] The following is a non-exhaustive list of different antibody formats, all of which retain antigen binding activity:

[0098] (1) Whole immunoglobulin (also called "intact" antibody) (two light chains and two heavy chains, e.g., a tetramer).

[0099] (2) Immunoglobulin polypeptide (light chain or heavy chain).

[0100] (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(V L 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.

[0101] (4) Multivalent antibodies (antibodies that contain binding regions that bind to two different epitopes or proteins, e.g., “scorpion” antibodies).

[0102] (5) Fusion proteins, which comprise the binding portion of an immunoglobulin fused to another amino acid sequence, such as a fluorescent protein.

[0103] 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.

[0104] 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.

[0105] A single-chain antibody (scFv) is a fragment comprising a V chain linked by a linker (e.g., a peptide linker). L and V H ScFv 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.

[0106] 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 the variable domains of heavy or light chains produced by recombinant methods.

[0107] As used herein, the wording "CDR sequence set" refers to 3 heavy chain CDRs and / or 3 light chain CDRs of a particular antibody described herein. "Light chain" CDR sequence set refers to light chain CDR sequences. "Heavy chain" CDR sequence set refers to heavy chain CDR sequences. "Full" CDR sequence set refers to both heavy chain CDR sequences and light chain CDR sequences. For example, for antibody 5017 as shown in Table 1a, the full CDR sequence set includes or consists of the following: SVSSA (CDR L1), SASSLYS (CDR L2), AAYHWPPLF (CDR L3), LYYTDM (CDR H1), SISLFFGYVS (CDR H2) and YLAM (CDR H3). The CDR sequence of each CDR can, for example, include the CDRs in Table 1a or Table 3a, be mainly composed of the CDRs in Table 1a or Table 3a, or be composed of the CDRs in Table 1a or Table 3a. CDRs are predicted based on IMGT sequence alignment.

[0108] 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").

[0109] 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.

[0110] 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., replacing non-human CDR sequences with the corresponding sequences of a human antibody, can be performed according to the methods described in, for example, U.S. Pat. Nos. 5,545,806, 5,569,825, 5,633,425, and 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).

[0111] 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.

[0112] 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).

[0113] As used herein, an antibody is "monospecific" if all of its antigen binding sites bind to the same epitope.

[0114] 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.

[0115] 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.

[0116] The specificity of binding can be determined by measuring the relative dissociation constant (K) of the antibody (or other targeting moiety) for the target compared to the dissociation constants between the antibody and other substances in the environment or generally unrelated molecules. d ) is defined. The 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, and the K of the antibody binding to an unrelated substance 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.

[0117] As used herein, if the antibody is expressed in less than 10 -4 M (i.e., in the micromolar concentration range), 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 indicates that the agent binds to the majority of cells in a pure population of these cells. For example, an antibody that binds to a particular cell type generally binds 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 the antibody to cells presenting the polypeptide and the binding (or lack of binding) of the antibody to cells that do not express the polypeptide. The skilled person will recognize that some variability will occur depending on the method and / or threshold for determining binding. The affinity of an antibody for a target can be determined according to methods known in the art, for example, as described in Ernst et al. Determination of Equilibrium Dissociation Constants, Therapeutic Monoclonal Antibodies(Wiley&Sons ed.2009).

[0118] 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 the activity in the absence of the antibody under similar physiological conditions. 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.

[0119] 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.

[0120] As used herein, an antibody "preferentially binds" a first antigen relative to a second antigen if the antibody binds to the first antigen with an affinity greater than that of the antibody binding to the second antigen. Preferential binding can be any of an affinity 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. Thus, for example, if the antibody binds to a first FZD protein with an affinity greater than that of the antibody binding to a second FZD protein, the antibody preferentially binds to the first FZD protein relative to the second FZD protein.

[0121] 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.

[0122] If the antibody specifically binds to a target protein group (e.g., some or all members of the Frizzled receptor protein family), the antibody has a greater binding affinity to the target group member to which it binds the weakest than the antibody has a binding affinity to a non-target antigen. In one embodiment, an antibody that specifically binds to a cysteine-rich domain (CRD) of each of one or more than one human Frizzled (FZD) receptors selected from FZD1, FZD2, FZD4, FZD5, FZD7, FZD8, FZD9 means that the antibody specifically binds to the selected member of the group compared to unselected group members or more generally other antigens. Thus, for example, an antibody that specifically binds to a cysteine-rich domain of a target group consisting of FZD1, FZD2, FZD4, FZD5, and FZD7 specifically binds to these proteins but not FZD3, FZD8, FZD9, and FZD10.

[0123] 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 a FZD receptor competitively reduces or prevents the interaction of a Wnt protein with a FZD 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.

[0124] 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 any of 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.

[0125] 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).

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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).

[0130] 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.

[0131] 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).

[0132] 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.

[0133] 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.

[0134] As used herein, "host cell" refers to a recombinant cell that contains an expression construct.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] As used herein, the term "pharmaceutical composition" refers to a composition comprising a pharmaceutical compound (eg, drug) and a pharmaceutically acceptable carrier.

[0140] 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.

[0141] 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).

[0142] 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).

[0143] 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.

[0144] 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.

[0145] 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.).

[0146] "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 a 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] By "substantially pure" or "isolated" is meant 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, 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 predominantly 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.

[0151] 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 vacancy can be introduced in the sequence of the first amino acid or nucleic acid sequence to be optimally compared 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 as the corresponding position in the second sequence, the molecules are identical at that 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 = number of identical overlapping positions / 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 gap comparisons for comparison purposes, Gapped BLAST as described in Altschul et al., 1997, Nucleic Acids Res. 25: 3389-3402 can be utilized. Alternatively, PSI-BLAST can be used for iterative searches (ibid.) for distance relationships between detection molecules. When using 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 a 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.

[0152] 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.

[0153] The 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, all of which 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 alignment drop = 25 and scoring matrix = BLOSUM62.

[0154] When NCBI-BLAST2 is used for amino acid sequence comparison, 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:

[0155] Multiply 100 by the fraction X / Y

[0156] 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.

[0157] 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).

[0158] "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 hybridization 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 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.

[0159] 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.

[0160] 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.

[0161] As used herein, the term "diluent" refers to a pharmaceutically acceptable carrier that does not inhibit the physiological activity or properties 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 properties 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] Depending on the context, the terms "Frizzled" and "FZD" refer to any gene or protein member of the Frizzled receptor family. Frizzled receptor proteins are involved in the activation of dishevelled proteins in the cytosol. Frizzled receptors refer to any of Frizzled receptors-1, Frizzled receptors-2, Frizzled receptors-3, Frizzled receptors-4, Frizzled receptors-5, Frizzled receptors-6, Frizzled receptors-7, Frizzled receptors-8, Frizzled receptors-9 and Frizzled receptors-10. Frizzled receptor 4 ("FZD4") (also known as CD344, EVR1, FEVR, FZD4S, Fz-4, Fz4, FzE4, GPCR, hFz4 and Frizzled receptor 4) is a member of the Frizzled receptor gene family of proteins. The gene has ENTREZ gene ID: 8322. The protein has NCBI reference sequence: NP_036325.2.

[0166] "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.

[0167] II. Disorders associated with dysregulated FRZ signaling

[0168] The binding of Wnt to FZD destabilizes the β-catenin binding complex, leading to β-catenin degradation. The effect is an increase in intracellular β-catenin levels. Therefore, provided herein are methods for blocking the binding of Wnt to frizzled receptor proteins (particularly to FZD4, but also to other members of the frizzled receptor family, such as FZD1, FZD2, FZD4, FZD5, FZD7, FZD8 and FZD9).

[0169] "FZD-related disorders" (e.g., "FZD4-related disorders" or "FZD5-related disorders") refer to conditions or diseases associated with dysregulation of the specific FZD receptors mentioned. Deregulation refers to abnormal signaling that increases normal β-catenin-mediated transcriptional changes or any other intracellular signaling pathway controlled by these receptors.

[0170] Different frizzled receptors are associated with different cancers. More specifically, FZD1 is associated with neuroblastoma. FZD2 is associated with liver cancer, lung cancer, endometrial cancer, and salivary adenoid cystic carcinoma. FZD3 is associated with colorectal cancer. FZD4 is associated with acute myeloid leukemia, prostate cancer, glioblastoma, bladder cancer, and cervical cancer. FZD5 is associated with pancreatic cancer, colon cancer, and prostate cancer. FZD6 is associated with colorectal cancer and breast cancer. FZD7 is associated with esophageal cancer, glioma, breast cancer, gastric cancer, and colorectal cancer. FZD8 is associated with prostate cancer, breast cancer, and lung cancer. FZD9 is associated with astrocytoma and osteosarcoma. FZD10 is associated with colorectal cancer and synovial sarcoma.

[0171] III. Anti-FZD4 Antibody

[0172] A. Antibodies

[0173] Antibodies to the Frizzled receptors (FZDs) are described herein, including antibodies that bind more than one FZD and other antibodies that preferentially bind to FZD4. These antibodies bind to the Frizzled receptors, block ligand WNT binding and modulate Frizzled receptor signaling. These antibodies also show antiproliferative effects and have the potential to treat cancer and other diseases in which the Frizzled receptors are dysregulated.

[0174] Accordingly, one aspect of the present disclosure includes an isolated antibody that specifically binds to a cysteine-rich domain (CRD) of a frizzled receptor (FZD), the isolated antibody comprising a light chain variable region and a heavy chain variable region, the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2 and CDR-H3, the light chain variable region comprising complementarity 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 1a or Table 3a.

[0175] In an embodiment, the antibody comprises a set of CDR sequences selected from the set of CDR sequences in Table 1a (ie, clones 5016 to 5037 and 6498 to 6500).

[0176] Table 1a - CDR amino acid sequences of FZD4 antibody

[0177]

[0178] Table 1b - CDR nucleic acid sequences of FZD4 antibodies

[0179]

[0180] Table 1c - CDR nucleic acid sequences of FZD4 antibody

[0181]

[0182] Heavy chain variable regions and light chain variable regions are also described herein. Table 2 provides exemplary variable domain sequences of the Fab heavy and light chains from clone 5017. Antibodies comprising sequences in Table 2 or sequences substantially identical thereto are also contemplated, wherein the CDR is a set of CDR sequences identified in Table 1a or Table 3a. 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 with 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 1a or Table 3a, or iii) a conservatively substituted amino acid sequence of i), wherein the CDR sequence is a set of CDR sequences as listed in Table 1a or Table 3a.

[0183] 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 1a or Table 3a, or iii) a conservatively substituted amino acid sequence of i), wherein the CDR sequence is a set of CDR sequences as listed in Table 1a or Table 3a.

[0184] Table 2 - Example of full length sequence of FZD4 antibody 5017

[0185]

[0186]

[0187] In another embodiment, the antibody comprises a set of CDR sequences selected from the set of CDR sequences in Table 3a (ie, clones 5038 to 5081).

[0188] Table 3a - CDR amino acid sequences of FZD4 antibody

[0189] Table 3a - CDR amino acid sequences of FZD4 antibody

[0190]

[0191] Table 3b - CDR light chain nucleic acid sequences of FZD4 antibody

[0192] Table 3b - CDR nucleic acid sequences of FZD4 antibody

[0193]

[0194] 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 is 100% identical to the amino acid sequences provided in Table 1a or Table 3a.

[0195] Table 3c - CDR heavy chain nucleic acid sequences of FZD4 antibody

[0196] Table 3c-CDR nucleic acid sequences of FZD4 antibody

[0197]

[0198]

[0199] In another embodiment, a competing antibody is provided that competes for binding with an antibody comprising a CDR sequence set described herein. For example, in one embodiment, the competing antibody reduces the binding of an antibody comprising a CDR sequence set to a FZD4 CDR by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%.

[0200] Some of the antibodies described are capable of binding to more than one FZD. Accordingly, in some embodiments, the antibody is an antibody that specifically binds to FZD4 and additionally specifically binds to one or more of FZD1, FZD2, FZD5, FZD7, FZD8, and FZD9. For example, the antibody can be an antibody in which the CDR sequence is a set of CDR sequences of antibodies selected from antibodies 5016, 5018-5023, 5025, 6495, 6496, 5039, 5045, 5048, 5054, 5056, 5057, 5067, and 5073-5076.

[0201] Some of the antibodies described preferentially bind to FZD4. In embodiments, the antibody is an antibody that preferentially binds to frizzled receptor 4 (FZD4) compared to any of FZD1, FZD2, FZD5, FZD7, FZD8, FZD9, or FZD10. In embodiments, the antibody preferentially binds to FZD 4 compared to FZD1, FZD5, FZD7, and FZD9. In one aspect, the antibody comprises the set of CDR sequences of antibody 6497. In another embodiment, the antibody preferentially binds to FZD4 compared to FZD1 and FZD7. In one aspect, the antibody comprises the set of CDR sequences of an antibody selected from 5028, 5035, 5039, 5073. In yet another embodiment, the antibody preferentially binds to FZD4 compared to FZD9. In one aspect, the antibody comprises the set of CDR sequences of antibody 5029. In yet another embodiment, the antibody preferentially binds to FZD4 compared to FZD1, FZD2, and FZD7. In one aspect, the antibody comprises a set of CDR sequences of an antibody selected from 5031, 6498, 5054 or 5075. In yet another embodiment, the antibody preferentially binds to FZD4 compared to FZD1, FZD2, FZD5 and FZD7. In one aspect, the antibody comprises a set of CDR sequences of antibody 5034. In yet another embodiment, the antibody preferentially binds to FZD4 compared to FZD1. In one aspect, the antibody comprises a set of CDR sequences of antibody 5045 or 5048. In yet another embodiment, the antibody preferentially binds to FZD4 compared to FZD1, FZD7 and FZD9. In one aspect, the antibody comprises a set of CDR sequences of antibody 5056. In yet another embodiment, the antibody preferentially binds to FZD4 compared to FZD9 and FZD10. In one aspect, the antibody comprises a set of CDR sequences of antibody 5057. In yet another embodiment, the antibody preferentially binds to FZD4 compared to FZD1 and FZD2. In one aspect, the antibody comprises the CDR sequence set of antibody 5067.

[0202] Certain antibodies, such as antibodies having CDR sets from antibodies 5018, 5019, 5022, 6494, and 5025, preferentially bind to other FZD proteins compared to FZD4. (See, e.g., Figure 5 ).

[0203] In another embodiment, the antibody comprises a CDR sequence from the set of CDR sequences of antibodies selected from antibodies 5022, 5031, 6497, 6498 and 6500.

[0204] As demonstrated herein, the antibodies described herein have high affinity for FZD4. For example, in one embodiment, the antibody has a binding affinity of between about 0.2 nM and about 15.3 nM as measured by surface plasmon resonance.

[0205] The antibody may be a humanized antibody or a chimeric antibody as described herein.

[0206] In some embodiments, the antibody is a single chain antibody, which can be obtained, for example, by fusing the heavy and light chains or portions thereof together.

[0207] 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.

[0208] In some other embodiments, the antibody is a binding fragment Fab. For some embodiments, binding fragments are preferred.

[0209] In other embodiments, it may be preferred to have multivalent antibodies or antibodies that comprise an Ig portion.

[0210] As demonstrated in the Examples, the Fab fragments of the present disclosure can be combined with an immunoglobulin (Ig) constant region such as IgG. In embodiments, the IgG is IgG1, IgG2, IgG3, or IgG4.

[0211] B. Detectably Labeled Antibodies

[0212] 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.

[0213] C. Antibody-Drug Conjugates

[0214] Another aspect includes immunoconjugates comprising an antibody described herein and a detectable label or a cytotoxic agent.

[0215] 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.

[0216] 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.

[0217] IV. Nucleic Acids

[0218] Other aspects include nucleic acid molecules or polynucleotides, recombinant nucleic acid molecules, expression constructs and vectors as described herein.

[0219] A. Nucleic acid molecules

[0220] Another aspect includes nucleic acid molecules as listed in Table 1b, Table 1c, Table 3b and Table 3c 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.

[0221] B. Expression Constructs and Vectors

[0222] 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.

[0223] 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.

[0224] Suitable regulatory sequences may be derived from a variety of sources including bacterial, fungal, viral, mammalian, or insect genes.

[0225] 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.

[0226] In embodiments, the regulatory sequence directs or increases expression in neural tissues and / or cells.

[0227] The vector can be any vector, including vectors suitable for producing the antibodies described herein.

[0228] In an embodiment, the vector is a viral vector.

[0229] 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.

[0230] The recombinant expression vector can also contain an expression cassette encoding a fusion moiety (i.e., a "fusion protein") that provides increased expression or stability of the recombinant peptide; increased solubility of the recombinant peptide; and assists in 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.

[0231] 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.

[0232] 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.

[0233] V. Recombinant Cells

[0234] Another aspect is a recombinant host cell expressing an antibody described herein.

[0235] Antibodies as described herein can be prepared by recombinant expression of nucleic acid encoding the antibody sequences.

[0236] Antibodies as disclosed herein can be prepared by culturing cells engineered to express a nucleic acid construct encoding an immunoglobulin polypeptide.

[0237] 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.

[0238] 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.

[0239] In embodiments, the cell is a eukaryotic cell selected from the group consisting of yeast, plant, worm, insect, avian, fish, reptile, and mammalian cells.

[0240] In another embodiment, the mammalian cell is a CHO cell, a myeloma cell, a spleen cell, or a hybridoma cell.

[0241] Yeast and fungal host cells suitable for expressing antibodies include, but are not limited to, species of S.cerevisiae, 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.

[0242] 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. CCL 2), 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.

[0243] VI. Pharmaceutical Compositions

[0244] 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.

[0245] The composition may, for example, comprise one or more antibodies or immunoconjugates.

[0246] 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.

[0247] Suitable diluents for nucleic acids include, but are not limited to, water, saline solutions, and ethanol.

[0248] 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.

[0249] 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.

[0250] 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.

[0251] 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.

[0252] 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.

[0253] 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.

[0254] Another aspect includes an antibody complex comprising an antibody described herein that binds to a FZD protein (eg, FZD4). The complex can be in solution or contained in a tissue, optionally in vitro.

[0255] Also provided are methods for making and using the agents described herein.

[0256] VII. Methods of Administration and Use

[0257] The anti-FZD antibodies of the present invention can effectively deliver therapeutic compositions in vivo to cells undergoing Wnt signaling. In some embodiments, the method of treatment or use comprises administering or using an effective amount of a therapeutic anti-FZD conjugate, such as an anti-FZD antibody attached to a therapeutic agent, to an individual. In some embodiments, the individual has been diagnosed with cancer. In some embodiments, the individual is receiving or has received cancer treatment, such as surgery, radiation therapy, or chemotherapy. In some embodiments, the individual has been diagnosed but the cancer is in remission.

[0258] In some embodiments, the anti-FZD conjugate comprises a liposome. In some embodiments, the method further comprises monitoring the individual's cancer progression. In some embodiments, the dose of the anti-FZD conjugate administered each time is determined based on the individual's treatment progress, e.g., if the individual does not respond adequately to treatment, a higher dose of the chemotherapeutic agent is administered.

[0259] In some embodiments, the present invention may include an antibody or antibody targeting composition and a physiologically (i.e., pharmaceutically) acceptable carrier. The term "carrier" refers to a generally inert substance used as a diluent or vehicle for a diagnostic or therapeutic agent. The term also includes generally inert substances that impart cohesive quality to the composition. A physiologically acceptable carrier may be a liquid, such as normal saline, phosphate buffer, physiologically buffered saline (135-150 mM NaCl), water, buffered water, 0.4% saline, 0.3% glycine, a glycoprotein (e.g., albumin, lipoprotein, globulin, etc.) providing enhanced stability, and the like. Because a physiologically acceptable carrier is determined in part by the specific composition administered and the specific method for administering the composition, there is a variety of suitable formulations of the pharmaceutical composition of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17th edition, 1989).

[0260] 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.

[0261] 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.

[0262] 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 such as albumin, lipoproteins, globulins, etc., for enhancing stability. 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 adjusters and buffers, tension adjusters, 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.

[0263] 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.

[0264] 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.

[0265] 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.

[0266] The antibody (or antibody targeting composition) can be administered or used 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.

[0267] 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 0.1mg / kg body weight to about 25mg / kg body weight, or in the range of 1mg-2g in each patient. In some cases, the dosage is in the range of 1-100mg / kg or about 50mg-8000mg / 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.

[0268] Administration or use can be periodic. Depending on the route of administration, 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 according to treatment progress and any adverse side effects.

[0269] 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.

[0270] In therapeutic applications 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 targeting composition used. For example, the dosage can be determined empirically based on the type and stage of the 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 degree of any adverse side effects associated with the administration of a particular targeting composition in a particular patient.

[0271] VIII. Kit

[0272] Another aspect is a kit or package comprising any of the antibodies, immunoconjugates, nucleic acid molecules, vectors, recombinant cells and / or compositions contained herein. The antibodies, immunoconjugates, nucleic acid molecules, vectors, recombinant cells and / or compositions may be contained in a vial such as a sterile vial or other housing. As used herein, the term "kit" means Figure 1 The kit may optionally include a reference agent and / or instructions for use thereof. The kit may also include a transport container suitable for containing a container (such as a vial) containing a composition as disclosed herein.

[0273] IX. Methods using antibodies

[0274] The antibodies described herein can be used in a number of in vitro and in vivo methods.

[0275] A. Methods for detecting FZD expression

[0276] As demonstrated herein, antibodies can be used to detect FZD expression.

[0277] Accordingly, the disclosure provides, in one aspect, a method for detecting FZD 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:FZD 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.

[0278] The sample may comprise viable cells or cell extracts. Antibody: FZD complexes 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.

[0279] As demonstrated herein, some of the identified antibodies preferentially recognize FZD4. Thus, in embodiments where the method is used to detect FZD4 expression, the antibody or immunoconjugate comprises a set of CDR sequences corresponding to an antibody selected from: 5017, 5027, 5030, 6499, 5038, 5040-5044, 5046, 5047, 5049-5053, 5055, 5058-5064, 5066, 5068-5072, and 5077-5081.

[0280] B. Methods for inhibiting the binding of WNT to FZD

[0281] The antibodies disclosed herein inhibit the binding of Wnt to the Frizzled receptor, in particular to FZD4. Without wishing to be bound by theory, inhibition of Wnt binding to the FZD protein affects signal transduction in which FZD plays a role in initiation. For example, binding of the antibody to the FZD receptor inhibits FZD's promotion of β-catenin phosphorylation. Unphosphorylated β-catenin will escape destruction and accumulate in the cell. Accumulation of β-catenin is associated with malignant tumors.

[0282] It may be desirable to reduce or inhibit Wnt ligand signaling by FZDs. Thus, another aspect is a method of inhibiting binding of a Wnt ligand to a FZD or Wnt-induced transcriptional activity, the method comprising contacting one or more cells expressing one or more FZD polypeptides with an effective amount of an antibody or immunoconjugate described herein.

[0283] In an embodiment, the antibody or immunoconjugate comprises a set of CDR sequences (whole, light or heavy) corresponding to an antibody selected from a clone as described herein (e.g., 5014, 5017-5023, 5027-5031, 5034, 5036, 5037, 6496, 6498, 6499 and 6500, 5035, 6495 and 5025).

[0284] In an embodiment, the antibody or immunoconjugate comprises a set of CDR sequences (whole, light or heavy chain) corresponding to an antibody selected from clones as described herein (e.g., 5014, 5018-5023, 5025, 5036, 5037, 6495, 5027-5031, and 6497-6499).

[0285] 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.

[0286] C. Methods of treating cancer

[0287] The method of treating cancer comprises administering a pharmaceutical composition comprising an antibody of the present disclosure that binds to FZD 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.

[0288] 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 FZDs, by inhibiting Wnt-induced transcriptional activity, by inhibiting the activation of Dishevelled protein, by inhibiting inhibition of the β-catenin destruction complex and by promoting accumulation of β-catenin.

[0289] The present 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 FZD1, FZD2, FZD4, FZD5, FZD7, FZD8, and FZD9 in at least one assay, and inhibits Wnt-induced signaling in at least one assay. The present disclosure also includes an effective amount of an antibody or immunoconjugate for treating cancer, the antibody or immunoconjugate specifically binds to FZD1, FZD2, FZD4, FZD5, FZD7, FZD8, and FZD9 in at least one assay, and inhibits Wnt-induced signaling in at least one assay. The present disclosure also provides an effective amount of an antibody or immunoconjugate for treating cancer, the antibody or immunoconjugate specifically binds to FZD1, FZD2, FZD4, FZD5, FZD7, FZD8, and FZD9 in at least one assay, and inhibits Wnt-induced signaling in at least one assay. The present disclosure also provides the use of an effective amount of an antibody or immunoconjugate in the preparation of a medicament for treating cancer, wherein the antibody or immunoconjugate specifically binds to FZD1, FZD2, FZD4, FZD5, FZD7, FZD8 and FZD9 in at least one assay and inhibits Wnt-induced signaling in at least one assay.

[0290] In an embodiment, the antibody or immunoconjugate, such as an antibody-drug conjugate, is contained in a pharmaceutical composition.

[0291] In one embodiment, 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, optionally the cancer is pancreatic cancer. In embodiments, the antibody or immunoconjugate comprises a CDR sequence set (all, light chain or heavy chain) corresponding to an antibody selected from 5014, 5017-5023, 5025, 5035-5037, 6495 and 6500.

[0292] As demonstrated herein, antibodies are also capable of inhibiting cancer cell proliferation. Thus, also provided is a method of inhibiting cancer cell proliferation, the method comprising contacting one or more FZD-expressing cancer cells with an effective amount of an antibody or immunoconjugate that specifically binds to FZD1, FZD2, FZD4, FZD5, FZD7, FZD8, and FZD9 in at least one assay and inhibits Wnt3a-induced signaling in at least one assay.

[0293] 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 or heavy) corresponding to an antibody selected from 5014, 5017-5023, 5025, 5035-5037, 6495 and 6500.

[0294] In an embodiment, the antibody comprises a variable region sequence as described herein corresponding to a set of CDR sequences (all, light or heavy) of antibodies selected from 5014, 5017-5023, 5025, 5035-5037, 6495 and 6500.

[0295] In one embodiment, the cancer is selected from acute myeloid leukemia, prostate cancer, glioblastoma, bladder cancer, and cervical cancer.

[0296] In another embodiment, the cancer cell is selected from the group consisting of a colon cancer cell, a lung cancer cell, a breast cancer cell, an ovarian cancer cell, an endometrial cancer cell, a pancreatic cancer cell, a gastric cancer cell, a liver cancer cell, an adrenocortical carcinoma cancer cell, and an osteoblastoma cancer cell.

[0297] In another embodiment, the cancer cell is a pancreatic cancer cell. In an embodiment, the antibody or immunoconjugate comprises a set of CDR sequences corresponding to an antibody selected from 5019 and 5020 (whole, light chain or heavy chain).

[0298] It was also demonstrated that antibody 5020 is effective for treating cancers with RNF43 mutations. Thus, in one embodiment, the cancer cells are known or determined to contain RNF43 gene mutations, and the antibody or immunoconjugate used contains a set of CDRs corresponding to antibody 5020.

[0299] In embodiments, the method comprises determining that the subject's cancer is associated with dysregulated Wnt signaling; optionally determining the specific Wnt protein that is dysregulated; optionally determining a FZD protein family member to be targeted; and administering to the subject an anti-FZD antibody to block binding of the selected one or more Wnts to the selected one or more FZD receptors.

[0300] 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 for the purpose of illustration only 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.

[0301] X. Examples

[0302] Example 1

[0303] Antibody selection and functional testing of FZD4 Fab

[0304] Antibody Selection: Two selection methods were performed to identify FZD4 binders.

[0305] 1. The selection was done using a library designed based on previous FZD7-derived binders. Antibodies have been previously identified through selections using FZD7 CRD-Fc as an antigen. These antibodies bind to FZD1, FZD2, FZD5, FZD7, FZD8, and FZD9 and show antagonistic activity against the Wnt pathway and inhibit proliferation and tumor growth of pancreatic cancer cells. This library was used to identify antibodies that can bind to FZD4 and have wnt antagonistic activity and anti-tumor activity.

[0306] a). Design and preparation of Fab phage display library.

[0307] Fab phage display library design and preparation are completed with the IPTG inducible display vector of the Fab that encodes recognition MBP.Fab template is identical with library F, and comprises the light chain of FLAG tag, and the dimerization domain L1, L2, L3 that mutates to parental Fab sequence H1, H2 and H3 are soft randomized (soft randomized), to allow 50% to be biased to wild-type amino acid and 50% to be biased to any other amino acid (using 70:10:10:10 nucleotide mixing).All six CDR districts are mutated in a single kunkel mutagenesis reaction.Based on the Fab antagonist group, the second generation library is constructed.The IPTG inducible display vector of the Fab that encodes specificity to maltose binding protein is used as the library template.Site-specific kunkel mutagenesis reaction is carried out using light chain oligonucleotide, to mutate CDR L1, L2 and L3 to parental Fab sequence and to make CDR H1, H2 and H3 soft randomization (50% wild type and 50% any other amino acid). Purified mutagenesis reactions were electroporated into SR320 cells pre-infected with M13 K07. Libraries were rescued overnight in 500 ml cultures, double precipitated with PEG / NaCl, and resuspended in PBS containing 50% glycerol for storage at -20°C.

[0308] b). Selection and generation of anti-FZD4 Fab

[0309] The second generation library (equal cfu) was pooled and four rounds of screening were performed for the recombinant FZD4 cysteine-rich domain (CRD) (R&D systems) fused to the Fc tag. The input and output phage titers of carbenicillin (carb)-resistant library phage and kanamycin (kan)-resistant helper phage were calculated. Maxisorp plates were coated with 5 μg / ml of FZD4-CRD-Fc or Fc protein in PBS overnight at 4 degrees and the well numbers were designated and blocked with 0.5% BSA. The coated wells were washed four times with PBS / 0.5% Tween 20 (wash buffer), and the library phage (PEG precipitated and resuspended in 0.5% BSA / 0.05% Tween20 / PBS) was first incubated at room temperature in the Fc protein wells for 1 hr. Unbound phages were transferred to the blocked FZD4-CRD-Fc plates and incubated at room temperature for 1 hour. The washing wells as shown, and then eluted with 100mM HCl. The eluted phages are amplified in the laboratory using standard protocols for subsequent rounds of selection. The input and output titers of the designated library phage (carb resistance) and the auxiliary phage (kan resistance) are determined. The DNA of the site-specific kunkel mutagenesis reaction (designed to add His6-amber termination between phage gene III and Fab CH1 region) of the output phage library from the FZD4 selection is transformed into Omnimax cells, and plated to obtain single colonies. Single colonies are used to inoculate 96-well culture boxes, and overnight phage supernatants are diluted in 0.05% Tween20 / 0.5% BSA / PBS (dilution buffer) with 1:2 to test ELISA binding. Phages are detected with anti-M13-HRP secondary antibodies (1:5000 in dilution buffer), and the plate is developed with TMB substrate and acid termination. The absorbance at 450 nm of the FZD4-Fc coated wells and control Fc coated wells was read as indicated. The heavy and light chains were sequenced to determine the CDR sequences of the individual Fabs. The CH1-gene III linker was also sequenced to determine the successful incorporation of the His tag and amber stop codon for Fab expression. The phage binders were cloned into a bacterial expression vector and purified as Fab proteins for characterization.

[0310] c). Characterization of anti-FZD4 conjugates.

[0311] The CDR sequences of the antibodies described here are shown in Table 1. First, phage binders were tested in an ELISA assay to confirm their binding to the antigen. Figure 1As shown in Figure 3, all phage clones bound to FZD4 CRD-Fc but not to Fc protein alone, indicating that these phage binders bind to FZD4 CRD but not to Fc. Next, the purified Fabs were tested in the presence of increasing amounts of non-immobilized antigen in a competitive ELISA assay to assess their binding affinity ( Figure 2 ). In the presence of 50 nM competing free antigen (FZD4 CRD), the binding of all Fabs was reduced by more than 50%. The binding of 5 Fabs (5022, 5031, 6497, 6498 and 6500) was reduced by more than 50% in the presence of 10 nM competing antigen (FZD4 CRD), indicating that these 5 Fabs may have higher binding affinity than the remaining Fabs. It should be noted that no binding of Fab 5025 and Fab 6494 was observed in this assay. The reason for this is unknown, but it may be due to physical interference. Next, the binding of the Fabs to FZD4 expressed on the cell surface was tested by immunofluorescence staining. A membrane staining pattern of all FZD4 Fabs was observed on CHO cells stably expressing FZD4 ( Figure 3 ), but not in CHO cells ( Figure 4 IF staining was also used to determine whether these Fabs bind to other FZDs. Binding was tested on 10 CHO cell lines that stably express individual FZDs on their surface. Figure 5 As shown in , Fabs show different binding profiles. Fab 5017, Fab 5027, Fab 5030 and Fab 6499 only bind to CHO cells expressing FZD4, while other Fabs (5014, 5018-5023, 5025, 6495, 6496) bind to more than one FZD including FZD1, FZD2, FZD4, FZD5, FZD7, FZD8 and FZD9. However, no Fab was shown to bind to FZD3, FZD6 or FZD10 in this assay. Next, Fab 5019 and Fab 5020 were used as examples to test their binding to cancer cells by both immunofluorescence staining and flow cytometry. As Figure 6 As shown in Figure 5, both Fabs showed clear membrane-localized staining on the five pancreatic cell lines tested. Binding of these Fabs to these cancer cell lines was confirmed by flow cytometry, as shown in Figure 5. Figure 7 As shown in .

[0312] 2. Selection was performed using the naive Fab library (library F).

[0313] a). Recombinant FZD4-CRD-Fc is used to select Fabs that bind to FZD4. Specifically, the Fab phage library (library F) is pre-cleared of non-specific binders of non-related proteins. The pre-cleared Fab library is subjected to several rounds of selection to enrich for binders that bind to FZD4-CRD-Fc. Phages from the selected clones are screened by ELISA for binders that specifically bind to FZD4-CRD-Fc but not to Fc. 44 Fabs with unique CDR sequences were identified (see Table 3 for sequences).

[0314] b). Characterization of anti-FZD4 Fab

[0315] The anti-FZD4 phage binder clones were then cloned into a bacterial expression vector and 42 Fabs were expressed and purified for further characterization. More than one approach was used to determine the binding selectivity to FZDs. First, the purified Fabs were tested in an ELISA assay to confirm their binding to the recombinant antigen (FZD4-CRD-Fc) as well as their binding to other FZDs (FZD1, 2, 5, 6, 7, 8, 9, and 10). Figure 8 As shown in Figure 1, all Fabs were observed to bind to FZD4-CRD-Fc (the antigen used for selection), but very little to Fc or the non-related protein BSA. In addition, these Fabs also showed varying degrees of binding to other FZDs ( Figure 8 For example, it was detected that Fab bound to FZD2 CRD-Fc and FZD8 CRD-Fc in addition to FZD4-CRD-Fc ( Figure 8 A). Fab 5076 binds to FZD1, FZD2, FZD5, FZD7, and FZD8 in addition to FZD4 ( Figure 8 D). Next, the binding of anti-FZD4 Fab to various FZDs was determined by immunofluorescence staining of CHO cells expressing FZDs, and the results are summarized in FIG9. In this assay, most Fabs showed binding only to FZD4 (including Fab5038, 5040-5044, 5046, 5047, 5049-5053, 5055, 5058-5064, 5066, 5068-5072, 5077-5081), while other Fabs bound to two or more FZDs. In addition, surface plasmon resonance (SPR) was used to determine the binding affinity of anti-FZD4 Fab to FZD4 CRD-Fc. Fig.10 As shown in FIG. . The FZD4-derived Fabs showed high affinity for FZD4, ranging from 0.2 nM to 15.3 nM. To observe whether these Fabs bind to FZD expressed on pancreatic cancer cells, their binding was tested using flow cytometry. Fig.11As outlined in , most Fabs were able to bind to HPAFII and PATU8988s. Small amounts of binding were observed for Fab5049, Fab 5064 and Fab 5072 (PATU8988s cells).

[0316] Functional testing of FZD4 Fab: Several assays were performed to characterize the anti-FZD4 Fab.

[0317] 1. Effect of anti-FZD4 Fab on Wnt ligand binding to FZD4-CRD. To optimize the assay conditions, increasing concentrations of FZD4-CRD-Fc or Fc were mixed with biotinylated Wnt5A and the complex was captured by streptavidin-coated plates. Binding of FZD4-CRD-Fc or Fc was detected by anti-Fc-HRP ( Fig.12 A). The blocking activity of anti-FZD4 Fabs was tested by selecting a FZD4-CRD-Fc concentration that gave an ELISA signal in the linear range. FZD4-CRD-Fc was mixed with biotinylated Wnt5A in the presence of the indicated various Fabs and bound FZD4-CRD-Fc was detected by anti-Fc-HRP, as shown in Fig.12 As shown in A. Fig.12 As shown in B, greater than 80% inhibition of binding was observed for Fab 5014, 5017-5023, 5027-5031, 5034, 5036, 5037, 6496, 6498, 6499 and 6500; greater than 60% inhibition of binding was observed for Fab 5035 and Fab 6495; ~30% inhibition was observed for Fab 5025; and no inhibition was observed for Fab 6494 and Fab 6497.

[0318] 2. Effect of anti-FZD4 Fab on β-catenin-driven transcription. To observe whether anti-FZD4 Fab affects β-catenin-dependent signaling, the effect of Fab on Wnt3a-induced transcriptional activity was tested in a TOPFLASH assay. Fig.13 As shown in , potent inhibitory activity (>80%) was observed for Fabs 5014, 5018-5023, 5025, 5036, 5037, and 6495. Smaller amounts of inhibition (10%-35%) were observed for Fabs 5027-5031, 6497-6499; no inhibition was observed for Fabs 5017, 5034, and 5035.

[0319] 3. Effect on cancer cell proliferation. To test whether anti-FZD4 Fab affects cancer cell proliferation, pancreatic cancer cells (HPAFII and PATU8988s) were treated with 2 μg / ml and 10 μg / ml of Fab, and cell proliferation was measured (see Fig.14 AH). Data in Fig.15 Fabs that were antiproliferative in a dose-dependent manner included: Fabs 5018-5021, 5023, 5036, and 6495. Fabs 5022 and 6500 were tested only at a single dose level (2 μg / ml) and were inhibitory to both cell lines tested. Several Fabs (5017, 5025, 5035, and 5037) were shown to be inhibitory to the HPAFII cell line in the assay. Fig.16 Based on the data summarized in , the anti-proliferative activity of anti-FZD4 Fabs was shown to correlate with the following observations: their binding to FZD 1, FZD 2, FZD 4, FZD 5, FZD 7, FZD 8, and FZD 9, and their ability to inhibit Wnt3a-induced transcriptional activity. The most potent anti-proliferative Fabs included 5014, 5019-5023, and 6495 ( Fig.16 ).

[0320] 4. Effect on the expression of the Wnt-regulated gene Axin2. To further characterize the anti-FZD4 antibodies, several Fabs were converted to IgG. IgG 5020 and Fab 5019 and Fab 5020 were tested in a gene expression assay in which the mRNA levels of the Axin2 gene were measured by RT qPCR. Fig.17 As shown in , IgG5020, Fab 5019, and Fab 5020 all reduced Axin2 mRNA levels in HPAFII cells after antibody treatment, indicating that these antibodies inhibit the Wnt pathway.

[0321] 5. Effect of anti-FZD4 IgG on cancer cell proliferation. The effect of IgG on the proliferation of four pancreatic cancer cell lines, HPAFII, CAPAN2, AsPC11, and PATU8988s, was also tested. These cell lines are all known to contain disruptive mutations in the RNF43 gene. Fig.18 As shown in A, the proliferation of these pancreatic cells was inhibited by IgG 5019 and IgG 5020 and their corresponding Fab. In addition, IgG5020 was shown to inhibit the proliferation of cancer cells in a dose-dependent manner ( Fig.18B). Interestingly, several pancreatic cancer cell lines without disruptive mutations in the RNF43 gene (BxPC3 and PANC 1) were also tested, but were insensitive to IgG5020, suggesting that sensitivity to the FZD4 antibody IgG 5020 may depend on mutations in the RNF43 gene. RNF43 and ZNRF3 are Wnt target genes encoding transmembrane E3 ubiquitin ligases that target the frizzled receptors. Loss-of-function mutations in RNF43 and ZNRF3 result in high expression of FZDs and may sensitize tumor cells to inhibition of Wnt-dependent signaling. To see if the FZD4 antibody also affects colony formation, IgG5020 was tested on five pancreatic cancer cell lines ( Fig.19 ).and Fig.18 Consistent with the results in , IgG5020 inhibited colony formation of cell lines carrying RNF43 mutations (HPAFII, AsPC1, and PATU8988s), but did not inhibit colony formation of cell lines not carrying RNF43 mutations (BxPC3 and PANC 1).

[0322] 6. Effect of Anti-FZD4 Fab Binding to Pancreatic Cancer Patient Tumor-derived Cells on Their Proliferation Next, the binding of Fab 5019 and 5020 to pancreatic cancer patient tumor-derived cells (PDX) was tested by immunofluorescence staining. Fig. 20 As shown in Figure 2, for both Fabs, a clear membrane staining pattern was observed on the PDX cell lines GP2A and GP14A as well as the pancreatic cell line CAPAN 2. In addition, these antibodies were tested in a cell proliferation assay ( Fig.21 ).and Fig.18 and 19 Consistent with the observations in Figure 5, both Fab and IgG 5020 were able to inhibit the proliferation of PDX cell lines GP2A and GP14A, both of which contain mutations in the RNF43 gene.

[0323] In vivo efficacy studies are ongoing to demonstrate the anti-tumor activity of these disclosed anti-FZD antibodies.

[0324] XI. Exemplary Implementations

[0325] 1. An antibody that specifically binds to a cysteine-rich domain (CRD) of each of one or more than one human frizzled receptors selected from FZD1, FZD2, FZD4, FZD5, FZD7, FZD8 and FZD9, the antibody comprising a light chain variable region and / or a heavy chain variable region, the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2 and CDR-H3, the light chain variable region comprising complementarity determining regions CDR-L1, CDR-L2 and CDR-L3, wherein the amino acid sequence of the CDR comprises or consists of a sequence selected from the sequence in Table 1a or Table 3a.

[0326] 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:

[0327] CDR-H1 is selected from the group consisting of: LYYTDM, IYFSSI, IGSSSI, VNSSSI, IHFSSI, IYSASI, IHSSSI, IYFSSI, IYSSSI, LSYSFF , LSFYFL, LSSYYM, SSFYFM, LSYYYM, IASYFT, FSSSSI, LSYYFM, IYYYPM, FSAYNI, IYYFGM, IHSSSI and ISYHYM;

[0328] CDR-H2 is selected from the group consisting of: SISLFFGYVS, SNYPSFGSNS, SIYSAFASTS, AFYSSFGATS, AYYSAFASSS, CSYPSFGSTS, SRYPSFGSTS, AIYSSFSANS, SNYPAFGSTS, SIYSAFLSTT, SIYPSSGYTY, SIYPYSGYTY, SIYPFHAS TY, TVYPYLDYTY, SIYPYSRNTF, SIYPFSGYST, SIYPYYAYTY, SIYLSFGYGY, CCNSAYRYGP, SIYPYAGNTY, SFYSYYSFTY, SLYTSYGYTY, YIYPFNGYSY, YIYPSYDYTY, YISPPYGFTY, ATYSSFGSIT and SIYPNLGYTY;

[0329] CDR-H3 is selected from the group consisting of: YHHPFGYAL, YLAM, YHFPFAYSL, YHFPFGFAL, YHFPFGHAL, YHYPFGHAL, YHYPFGHAL, YHYPFGTAL, YHYPFGYAL, YHYPFGYAM, YHYPHGHAL, PAPFSYHVL, AAPGSYHPM, AAPYFY GVM, AFPGSYHPM, AYPFSYHFM, PSAFSYHPM, PVAGAYHPM, SSLGFYNGM, TVRGSKKPYFSGWAM, TYPGYYYIL, SGVGGDHAL, VWYVVQ, GYFYTWGGM, GYFYTWGGM, GYYYSWGGM, YHPFGYAL and AYPFSYHYM;

[0330] CDR-L1 is SVSSA;

[0331] CDR-L2 is SASSLYS; and / or

[0332] CDR-L3 is selected from the group consisting of AAYHWPPLF, GVYLF, SSYSLI, WAYGPF, YYHPI and YYSLF.

[0333] 3. 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:

[0334] CDR-H1 is selected from the group consisting of: ISYYYM, IYSYYM, LSYYYM, IYYYSI, LYSYYM, LSSYSM, ISYYYI, LSYSSM, IYYYYM, LYYYSI, IS SYYI, FSSSSI, LSYYSI, LYSYYI, LSSYYM, LSYYYI, ISSYYM, LSYYSM, LYSYSI, LYYYYI, IYSYYI, ISYSYI and ISYYSM;

[0335] CDR-H2 is selected from the group consisting of: SIYSYYGYTY, SIYSSSSSTY, SIYPSSSYTY, SIYSSSSYTS, YISSYSGSTY, SIYSSYGYTY, YISSYYGYTY, SIYPSSSSTY, SIYSSSGYTY, YISSYSGSTS, SISSYYGSTY, SIYSYYGSTY, SIYPYSGYTY, YISPYYGYTS, SISSSSGYTY, SIYSYSSSTY, SISPSSSYTY, YISPYYGYTY, SISPYSSSTY, SIYSSYGSTY, SIYSSSSYTY, SIYPSSGYTY, SIYPYSGSTY, SIYPSYGSTY, YISSYSSYTY, SIYSYYSSTY, YISSSYGYTS, SISPYSSYTY, YISPYSGYTS, SIYPYYSYTY, SISPYYGYTS, SISPSYSSTY, SISSSYSSTY, SIYPYSGSTS, SISSYYSSTS and SIYSYSGYTY;

[0336] CDR-H3 is selected from the group consisting of: SSFSWAM, SSFYWAL, SWFGWGI, YWFSYGYASYPAF, HPWYGM, SAFYWAL, PAPGHWGF, SSFFWAM, SAFYWAM, HFFAM, SWWAWAF, SAFGWAL, SSFFFAM, PYYWSGGF, HPSSSWFSFGAL, SAFYWAF, SSYAWAM, SSFYWAI, SPWGSGWAGF, PAVWVGL, SWVFWAL, SWVYWGM, SWVYWAL, SSYAWAI, SSFYWAM, HGASFGSGAPAF, SCFFWAM, WAFFGL, SSFYFAM, SAFSWAI, SGFYWAL, PSVGYAAF, SWVGWGL, SSVGYVAM, SWVYWAF, YYYSSSVYFWYAAL, SSFFWAI, SWVYWAI, SWVGWGI, SSVYWAL, WGGWGSGGYFYAAL, FWYPGM and SSFAWAF;

[0337] CDR-L1 is SVSSA;

[0338] CDR-L2 is SASSLYS; and / or

[0339] CDR-L3 is selected from the group consisting of: HPWSGGYLI, PVGYWGVPI, VSGGAHALI, VSSAYPI, FWGVPI, SYYHYAALI, WYYAPI, SHSYSLI, SGYGPF, SWSSPI, HYSVYASLI, PHPPSLI, VAYSHVGLI, GYGAPI, SWYSLI, PGYLF, VWFGLI, VYYGSPLF, HAHSPLI, SSAYYPF, GHASPI , SSGGWSLI, VAWSSFLI, SVAAASLI, SGWWGVSLI, SYAAYLF, HGSLF, YAGVSNLF, GWPYSALF, SGYYPSLF, SYHSGSGLI, HGYS ASLI, APGWALF, GHSSPI, GWPSLF, VPGYPVPI, HYYSHLI, GPASSLI, SVGSSYYLI, YYGPWVLI, AASWGYPF, HWSYPI and GGWGPF.

[0340] 4. The antibody according to embodiment 2 or 3, wherein the antibody comprises a heavy chain variable region comprising:

[0341] i) the heavy chain amino acid sequence as listed in Table 2;

[0342] ii) an amino 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%, 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 1a or Table 3a, or

[0343] iii) the conservatively substituted amino acid sequence of i), wherein the CDR sequence is a CDR sequence set as listed in Table 1a or Table 3a.

[0344] 5. The antibody of any one of embodiments 2 to 4, wherein the antibody comprises a light chain variable region comprising:

[0345] i) the light chain amino acid sequence listed in Table 2;

[0346] 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%, 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 1a or Table 3a; or

[0347] iii) the conservatively substituted amino acid sequence of i), wherein the CDR sequence is a CDR sequence set as listed in Table 1a or Table 3a.

[0348] 6. The antibody of any one of embodiments 1 to 5, wherein the CDR sequence is selected from the full set of CDR sequences of the antibodies identified in Table 1a or Table 3a.

[0349] 7. An antibody according to any one of embodiments 1 to 5, wherein the CDR sequence comprises a set of light chain or heavy chain CDR sequences selected from the antibodies identified in Table 1a or Table 3a.

[0350] 8. The antibody of any one of embodiments 1 to 7, wherein the antibody specifically binds to FZD4.

[0351] 9. The antibody of embodiment 8, wherein the CDR sequence is a set of CDR sequences of an antibody selected from the group consisting of 5017, 5027, 5030, 6499, 5038, 5040-5044, 5046, 5047, 5049-5053, 5055, 5058-5064, 5066, 5068-5072, 5077-5080, or 5081.

[0352] 10. The antibody of any one of embodiments 1 to 7, wherein the antibody specifically binds FZD4 and at least one other FZD receptor selected from FZD1, FZD2, FZD5, FZD7, FZD8, and FZD9.

[0353] 11. An antibody according to embodiment 9, wherein the CDR sequence is a set of CDR sequences of an antibody selected from the following antibodies: 5014, 5016, 5018-5023, 5025, 5028, 5029, 5031, 5034, 5035, 5036, 5037, 6494, 6495, 6496, 6497, 6498, 6500, 5039, 5045, 5048, 5054, 5056, 5057, 5067 and 5073-5076.

[0354] 12. The antibody of any one of embodiments 1 to 11, wherein the antibody preferentially binds to frizzled 4 (FZD4) compared to FZD1, FZD2, FZD5, FZD7, FZD8 or FZD9.

[0355] 13. The antibody of any one of embodiments 1 to 11, wherein the antibody preferentially binds FZD4 over another FZD receptor.

[0356] 14. An antibody according to embodiment 13, wherein the CDR sequence is a set of CDR sequences of an antibody selected from the following antibodies: 5028, 5029, 5031, 5034, 5035, 6497, 6498, 5039, 5045, 5048, 5054, 5056, 5057, 5067, 5073, 5074, 5075.

[0357] 15. The antibody of any one of embodiments 1 to 13, wherein the antibody has a binding affinity of between about 0.2 nM and about 15.3 nM as measured by surface plasmon resonance.

[0358] 16. The antibody according to any one of embodiments 1 to 15, wherein the antibody is a monoclonal antibody.

[0359] 17. The antibody of any one of embodiments 1 to 16, wherein the antibody is a humanized antibody.

[0360] 18. The antibody of any one of embodiments 1 to 17, wherein the antibody is a single chain antibody.

[0361] 19. An antibody according to any one of embodiments 1 to 18, 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.

[0362] 20. The antibody according to any one of embodiments 1 to 18, wherein the antibody is a multivalent antibody that is a bivalent, trivalent or tetravalent antibody.

[0363] 21. The antibody of any one of embodiments 1 to 18, wherein the antibody is a bispecific antibody that also binds to LPR 5 / 6.

[0364] 22. The antibody of any one of embodiments 1 to 18, comprising a non-native glycosylation pattern.

[0365] 23. The antibody according to any one of embodiments 1 to 18, comprising a cysteine ​​substitution or addition in the constant region or framework region.

[0366] 24. The antibody of any one of embodiments 1 to 18, which blocks binding of Wnt to FZD.

[0367] 25. An immunoconjugate comprising the antibody of any one of embodiments 1 to 21 and a detectable label or a cytotoxic agent.

[0368] 26. The immunoconjugate of embodiment 25, 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.

[0369] 27. A nucleic acid molecule encoding the antibody of any one of embodiments 1 to 21.

[0370] 28. The nucleic acid molecule of embodiment 27, wherein one or more of the CDR sequences are encoded by a nucleic acid in Table 1b, Table 1c, Table 3b or Table 3c.

[0371] 29. The nucleic acid molecule of embodiment 27, wherein the antibody comprises a heavy chain variable region encoded by a nucleic acid comprising:

[0372] i) a heavy chain nucleic acid sequence as listed in Table 2;

[0373] ii) a nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80% or at least 90% 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 1a or Table 3a, or

[0374] iii) the codon-degenerate nucleic acid sequence of i), wherein the CDR sequence is a CDR sequence set as listed in Table 1a or Table 3a.

[0375] 30. The nucleic acid molecule of embodiment 27, wherein the antibody comprises a light chain variable region encoded by a nucleic acid comprising:

[0376] i) the light chain nucleic acid sequence listed in Table 2,

[0377] 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%, 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 1a or Table 3a, or

[0378] iii) the codon-degenerate nucleic acid sequence of i), wherein the CDR sequence is a CDR sequence set as listed in Table 1a or Table 3a.

[0379] 31. A vector comprising an expression control sequence operably linked to the nucleic acid of any one of embodiments 27 to 30.

[0380] 32. 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 27 to 30.

[0381] 33. The host cell of embodiment 32, wherein the host cell is a Chinese Hamster Ovary (CHO) cell.

[0382] 34. A host cell comprising the vector of embodiment 31.

[0383] 35. A method for preparing an anti-FZD antibody, the method comprising culturing the host cell of any one of embodiments 32 to 34.

[0384] 36. A composition comprising the antibody of any one or more of embodiments 1 to 24, the immunoconjugate of embodiments 25-26, the nucleic acid molecule of embodiments 27-30, the vector of embodiment 31, or the host cell of embodiments 34-34, and optionally a suitable diluent.

[0385] 37. A composition according to embodiment 3636, wherein the composition comprises one or more antibodies or immunoconjugates, optionally wherein the composition is a pharmaceutical composition.

[0386] 38. A kit comprising the antibody of any one or more of embodiments 1 to 24, the immunoconjugate of embodiments 25-26, the nucleic acid molecule of embodiments 27-30, the vector of embodiment 31, or the host cell of embodiments 34-34.

[0387] 39. A method of detecting FZD 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 26 under conditions that allow formation of antibody:cell complexes, and detecting the presence of any antibody complexes.

[0388] 40. The method of embodiment 39, wherein the detection is by immunofluorescence.

[0389] 41. The method of embodiment 39, wherein the detection is by flow cytometry.

[0390] 42. The method of any one of embodiments 39 to 41, wherein the method is used to detect FZD4 expression and the antibody or immunoconjugate comprises a set of CDR sequences corresponding to an antibody selected from the group consisting of: 5017, 5027, 5030, 6499, 5038, 5040-5044, 5046, 5047, 5049-5053, 5055, 5058-5064, 5066, 5068-5072, and 5077-5081.

[0391] 43. A method of inhibiting Wnt ligand binding to a FZD 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 a FZD receptor with the antibody or immunoconjugate of any one of embodiments 1 to 26.

[0392] 44. A method according to embodiment 43, wherein the Wnt ligand is Wnt3a.

[0393] 45. The method of embodiment 43, wherein the antibody or immunoconjugate comprises a set of CDR sequences corresponding to an antibody selected from the group consisting of: a) 5017, 5027, 5030, 6499, 5038, 5040-5044, 5046, 5047, 5049-5053, 5055, 5058-5064, 5066, 5068-5072, and 5077-5 081, or b) 5014, 5016, 5018-5023, 5025, 5028, 5029, 5031, 5034, 5035, 5036, 5037, 6494, 6495, 6496, 6497, 6498, 6500, 5039, 5045, 5048, 5054, 5056, 5057, 5067 and 5073-5076.

[0394] 46. ​​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 26.

[0395] 47. The method of embodiment 46, 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.

[0396] 48. The method of embodiment 46, wherein the cancer is selected from acute myeloid leukemia, neuroblastoma, liver cancer, lung cancer, endometrial cancer, salivary adenoid cystic carcinoma, colorectal cancer, prostate cancer, glioblastoma, bladder cancer, cervical cancer, pancreatic cancer, colon cancer, breast cancer, esophageal cancer, glioma, gastric cancer, astrocytoma and osteosarcoma.

[0397] 49. The method of embodiment 46, wherein the antibody or immunoconjugate specifically binds FZD1, FZD2, FZD4, FZD5, FZD7, FZD8, and FZD9 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 26.

[0398] 50. The method of embodiment 46, wherein the antibody or immunoconjugate comprises a set of CDR sequences corresponding to an antibody selected from the group consisting of a) 5017, 5027, 5030, 6499, 5038, 5040-5044, 5046, 5047, 5049-5053, 5055, 5058-5064, 5066, 5068-5072, and 5077-5 081, or b) 5014, 5016, 5018-5023, 5025, 5028, 5029, 5031, 5034, 5035, 5036, 5037, 6494, 6495, 6496, 6497, 6498, 6500, 5039, 5045, 5048, 5054, 5056, 5057, 5067 and 5073-5076.

[0399] 51. A method according to embodiment 46, wherein the antibody or immunoconjugate comprises a set of CDR sequences corresponding to an antibody selected from 5019 and 5020.

[0400] 52. A method according to embodiment 51, wherein the cancer treated by the method comprises one or more cancer cells comprising a mutation in the RNF43 gene, and the antibody or immunoconjugate comprises a set of CDR sequences corresponding to antibody 5020.

[0401] 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.

[0402] As used herein, terms of degree, such as "about", "substantially" and "approximately" mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ± 5% of the modified term, provided that such deviation does not invalidate the meaning of the word it modifies.

[0403] In addition, as will be appreciated by those skilled in the art, the definitions and embodiments described in a particular section are intended to be applicable to other embodiments described herein to which they are applicable. For example, in the following paragraphs, different aspects of the present invention are defined in more detail. Unless clearly indicated to the contrary, each aspect so defined can be combined with any other aspect or aspects. In particular, any feature indicated as being preferred or advantageous can be combined with any other feature or features indicated as being preferred or advantageous.

[0404] 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.

[0405] 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 the cysteine-rich domain of human frizzled receptor FZD4, the antibody comprising 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, wherein the amino acid sequence of the CDR consists of the sequence listed below: CDR-H1 consisting of SEQ ID NO:6, CDR-H2 consisting of SEQ ID NO:30, CDR-H3 consisting of SEQ ID NO:66, CDR-L1 consisting of SEQ ID NO:34, CDR-L2 consisting of SEQ ID NO:35, and CDR-L3 consisting of SEQ ID NO:

111.

2. The antibody of claim 1, wherein the antibody comprises a heavy chain variable region comprising: An amino acid sequence having at least 95% sequence identity to the heavy chain amino acid sequence of SEQ ID NO:

302.

3. The antibody of claim 1, wherein the antibody comprises a light chain variable region comprising: An amino acid sequence having at least 95% sequence identity to the light chain amino acid sequence of SEQ ID NO:

300.

4. The antibody of claim 1, wherein the antibody has a binding affinity between 0.2 nM and 15.3 nM as measured by surface plasmon resonance.

5. The antibody of claim 1, wherein the antibody is a monoclonal antibody. The antibody according to claim 1 , wherein the antibody is a humanized antibody. The antibody according to claim 1 , wherein the antibody is a single chain antibody.

8. The antibody of claim 1, wherein the antibody is an antibody binding fragment selected from the group consisting of Fab, F(ab')2, scFv, dsFv, ds-scFv, diabodies and multimers thereof.

9. The antibody according to claim 1, wherein the antibody is a multivalent antibody that is a bivalent, trivalent or tetravalent antibody.

10. The antibody of claim 1, wherein the antibody is a bispecific antibody that also binds to LPR 5 / 6.

11. The antibody of claim 1, comprising a non-native glycosylation pattern.

12. The antibody of claim 1, comprising a cysteine ​​substitution or addition in the constant region or framework region.

13. The antibody of claim 1, which blocks the binding of Wnt to FZD.

14. An immunoconjugate comprising the antibody of any one of claims 1 to 10 and a detectable label or a cytotoxic agent.

15. The immunoconjugate of claim 14, 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, and anthracyclines.

16. A nucleic acid molecule encoding the antibody of any one of claims 1 to 10.

17. The nucleic acid molecule of claim 16, wherein the antibody comprises a heavy chain variable region encoded by a nucleic acid comprising: A nucleotide sequence having at least 90% sequence identity to the heavy chain nucleic acid sequence of SEQ ID NO:

303.

18. The nucleic acid molecule of claim 16, wherein the antibody comprises a light chain variable region encoded by a nucleic acid comprising: A nucleic acid sequence having at least 90% sequence identity to the light chain nucleic acid sequence of SEQ ID NO:

301.

19. A vector comprising the nucleic acid of any one of claims 16 to 18.

20. The vector of claim 19, wherein the nucleic acid is operably linked to an expression control sequence.

21. A host cell comprising a recombinant nucleic acid molecule comprising the nucleic acid of any one of claims 16 to 18, wherein the host cell is not a plant cell.

22. The host cell of claim 21, wherein the nucleic acid is operably linked to an expression control sequence.

23. The host cell according to claim 21 or 22, which is a Chinese Hamster Ovary (CHO) cell.

24. A host cell comprising the vector of claim 19 or 20, wherein the host cell is not a plant cell.

25. A method for preparing an anti-FZD antibody, the method comprising culturing the host cell of any one of claims 21 to 24.

26. A composition comprising the antibody of any one of claims 1 to 13, the immunoconjugate of any one of claims 14-15, the nucleic acid molecule of any one of claims 16-18, the vector of claim 19 or 20, or the host cell of any one of claims 21-24.

27. The composition of claim 26, further comprising a suitable diluent.

28. The composition of claim 26, wherein the composition comprises an antibody or an immunoconjugate.

29. The composition of claim 28, wherein the composition is a pharmaceutical composition.

30. A kit comprising the antibody of any one of claims 1 to 13, the immunoconjugate of any one of claims 14-15, the nucleic acid molecule of any one of claims 16-18, the vector of claim 19 or 20, or the host cell of any one of claims 21-24.

31. Use of the antibody of any one of claims 1 to 13 or the immunoconjugate of any one of claims 14 to 15 in the preparation of a kit for detecting FZD expression, wherein a sample comprising one or more cells is contacted with the antibody or immunoconjugate under conditions that allow formation of antibody:cell complexes, and the presence of any antibody complexes is detected.

32. Use according to claim 31, wherein the detection is by immunofluorescence.

33. The use according to claim 32, wherein the detection is by flow cytometry.

34. Use of a pharmaceutical composition comprising the antibody of any one of claims 1 to 13 or the immunoconjugate of any one of claims 14 to 15 in the preparation of a medicament for treating cancer, wherein the cancer is selected from colon cancer, lung cancer, breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, gastric cancer, liver cancer, adrenocortical carcinoma, osteoblastoma cancer and glioblastoma.

35. Use of a pharmaceutical composition comprising the antibody of any one of claims 1 to 13 or the immunoconjugate of any one of claims 14 to 15 in the preparation of a medicament for treating cancer, wherein the cancer is selected from acute myeloid leukemia, neuroblastoma, salivary adenoid cystic carcinoma, colorectal cancer, prostate cancer, bladder cancer, cervical cancer, esophageal cancer, glioma, astrocytoma and osteosarcoma.

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