Curling receptor antibodies and uses thereof

Abstract

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

Description

[0001] field

[0002] This disclosure relates to antibodies that bind to frizzled receptors, and particularly antibodies that bind to the cysteine-rich domain of frizzled receptor 4, and their uses.

[0003] Citation of relevant applications

[0004] This application claims the priority date of U.S. Provisional Application 62 / 885,781, filed August 12, 2019, and U.S. Provisional Application 62 / 886,292, filed August 13, 2019, the contents of which are incorporated herein by reference in their entirety.

[0005] background

[0006] Coiled receptors (FZDs) are an important class of seven-transmembrane receptors involved in many vital biological processes, such as development, cell proliferation, survival, migration, and stem cell maintenance. When Wnt ligands interact with the coiled receptor family of seven-transmembrane receptors, signaling pathways are activated, controlling the renewal and differentiation of stem cells and progenitor cells during embryonic development and tissue homeostasis in adult animals. Aberrant expression and signaling of these receptors and their ligands (Wnts) are associated with many cancers, including colon, lung, breast, and ovarian cancers. Typically, more than one Wnt ligand and / or coiled receptor is upregulated, leading to aberrant signaling that drives tumorigenesis. Therefore, inhibiting more than one coiled receptor may be necessary to achieve better anticancer effects. Furthermore, coiled receptors are also associated with cancer stem cells (a small group of cancer cells believed to contribute to drug resistance, tumor recurrence, and metastasis). Therefore, inhibiting FZD receptors, including FZD4 (one or more receptors), may be an effective approach to targeting cancer stem cells and treating various cancer types.

[0007] Wnt signaling leads to the activation of both typical and atypical signaling pathways. Atypical pathway activation does not involve the nucleus or transcription but rather activates cytoplasmic signaling molecules that regulate the cytoskeleton and calcium levels. This pathway primarily functions in regulating cell polarity and migration.

[0008] The typical pathway primarily controls transcriptional activity by regulating cytoplasmic levels of β-agonist. Under unstimulated conditions, β-agonist associates with the disruption complex (containing Axin, APC, CD1, and GSKβ), leading to phosphorylation, ubiquitination, and proteasome degradation of β-agonist. Wnt signaling is activated when Wnt binds to the 7-transmembrane receptor coiled-up receptor (FZD) and its co-receptor low-density lipoprotein receptor-associated proteins (LRP5 or LRP6). This signaling destabilizes the disruption complex, partly by attracting disheveled proteins (Dsh / Dvl) to the plasma membrane, leading to β-agonist accumulation. β-agonist 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, leading to the activation of ligand-independent Wnt target genes. For example, inactive APC mutations and activating β-linkin mutations are major potential causes of colorectal cancer in humans. Because this pathway is activated downstream of cell surface receptors, developing targeted therapies against 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 these mutations are associated with ligand-dependent tumor growth. Indeed, RNF43 and ZNRF3 are Wnt target genes encoding transmembrane E3 ubiquitin ligases targeting coiled receptors; loss-of-function mutations in RNF43 and ZNRF3 lead to high expression of FZD and may sensitize tumor cells to inhibition of Wnt-dependent signaling.

[0010] Overview

[0011] The following overview is intended to introduce the various aspects of this disclosure to the reader and is not intended to define or limit any invention.

[0012] In one aspect, this disclosure provides antibodies that specifically bind to a cysteine-rich domain (CRD) of one or more of the human coiled receptors selected from FZD1, FZD2, FZD4, FZD5, FZD7, FZD8, and FZD9. The antibody comprises 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, and the light chain variable region comprising complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3. The amino acid sequence of said CDR comprises or consists of sequences selected from or composed ...

[0013] In another implementation, the CDR comprises or consists of the following sequences, 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 implementation, the CDR comprises or consists of the following sequences, 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, SISSYSSTY, 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, this disclosure provides an antibody as previously described, the antibody comprising a heavy chain variable region, the heavy chain variable region comprising:

[0028] i) Heavy chain amino acid sequences 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 with the heavy chain amino acid sequences listed in Table 2, wherein the CDR sequence is a set of CDR sequences listed in Table 1a or Table 3a, or

[0030] iii) The conserved substituted amino acid sequences of i), wherein the CDR sequences are the set of CDR sequences listed in Table 1a or Table 3a.

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

[0032] i) Light chain amino acid sequences as 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 with the light chain amino acid sequences listed in Table 2, wherein the CDR sequence is a set of CDR sequences listed in Table 1a or Table 3a; or

[0034] iii) The conserved substituted amino acid sequences of i), wherein the CDR sequences are the set of CDR sequences listed in Table 1a or Table 3a.

[0035] In another embodiment, this disclosure provides an antibody that specifically binds to FZD4. In yet another embodiment, the CDR sequence of the antibody that specifically binds to FZD4 is a set of CDR sequences 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, this 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 antibodies selected from the following: 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 coiled receptor 4 (FZD4) relative to another FZD receptor. In yet another embodiment, the CDR sequence is a set of CDR sequences of antibodies selected from the following: 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 between approximately 0.2 nM and approximately 15.3 nM, as measured by surface plasmon resonance.

[0037] In yet another embodiment, the CDR sequence is a set of CDR sequences of antibodies 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.

[0038] In yet another embodiment, the antibody is a monoclonal, humanized, single-chain, antibody fragment, multivalent, bispecific, containing a non-natural glycosylation pattern, containing cysteine ​​substitution or addition, or blocking the binding of Wnt to FZD. In yet another embodiment, the antibody fragment is selected from the group consisting of fragments such as Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, nanobodies, microbodies, biantibodies, and multimers. In yet another embodiment, the multivalent antibody is bivalent, trivalent, or tetravalent. In yet another embodiment, the cysteine ​​substitution is present in a constant region or frame region. In yet another embodiment, the bispecific antibody also binds LRP 5 and / or LRP 6. In yet another embodiment, the antibody contains a non-natural glycosylation pattern. In yet another embodiment, the cysteine ​​substitution is in a constant region or frame region. In yet another embodiment, the antibody as described herein blocks the binding of Wnt to FZD.

[0039] On the other hand, this disclosure provides immunoconjugates comprising antibodies and detectable markers or cytotoxic agents as described herein. In one embodiment, the cytotoxic agent is selected from maytansinoid, auristatin, dolastatin, tubulysin, novozygain, pyrrolobenzodiazepine (PBD) dimers, indolebenzodiazepine dimers, α-amanitin, trichothene, SN-38, pyroximide, CC1065, calicheamicin, enediyne antibiotics, taxanes, doxorubicin derivatives, anthracyclines and their stereoisomers, azanofide, isosteres, analogs, or derivatives.

[0040] On the other hand, this 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 Tables 1b, 1c, 3b, and 3c.

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

[0042] i) Heavy chain nucleic acid sequences as listed in Table 2;

[0043] ii) Nucleic acid sequences that have at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with the heavy chain nucleic acid sequences listed in Table 2, wherein the CDR sequence is a set of CDR sequences listed in Table 1a or Table 3a, or

[0044] iii)i) codon degenerate nucleic acid sequences, wherein the CDR sequences are the sets of CDR sequences 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 the nucleic acid, which includes:

[0046] i) Light chain nucleic acid sequences as listed in Table 2,

[0047] ii) Nucleic acid sequences having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with the light chain nucleic acid sequences listed in Table 2, wherein the CDR sequence is a set of CDR sequences listed in Table 1a or Table 3a, or

[0048] iii)i) codon degenerate nucleic acid sequences, wherein the CDR sequences are the sets of CDR sequences listed in Table 1a or Table 3a.

[0049] On another front, this disclosure provides a carrier containing an expression control sequence operatively linked to a nucleic acid encoding an antibody described herein.

[0050] In another aspect, this disclosure provides a host cell containing a recombinant nucleic acid molecule that includes an expression control sequence operatively 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 another aspect, this disclosure provides a method for preparing anti-FZD antibodies, the method comprising culturing host cells as described herein.

[0052] In another aspect, this disclosure provides compositions comprising one or more antibodies, immunoconjugates, nucleic acids, vectors, or host cells described herein, and optionally suitable diluents. In one embodiment, the composition comprises one or more antibodies or immunoconjugates, optionally wherein the composition is a pharmaceutical composition.

[0053] On the other hand, this disclosure provides kits containing one or more antibodies, immunoconjugates, nucleic acids, vectors or host cells described herein.

[0054] In another aspect, this disclosure provides a method for detecting FZD expression, the method comprising contacting a sample containing one or more cells with one or more antibodies or immunoconjugates described herein under conditions allowing for the formation of antibody:cell complexes, and detecting the presence of any antibody complex. 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 set of CDR sequences corresponding to antibodies selected from the following: 5017, 5027, 5030, 6499, 5038, 5040-5044, 5046, 5047, 5049-5053, 5055, 5058-5064, 5066, 5068-5072, and 5077-5081.

[0055] In another aspect, this disclosure provides methods for inhibiting the binding of Wnt ligands to FZD receptors, disrupting Wnt signaling pathways, inhibiting Wnt-induced transcriptional activity, inhibiting disheveled protein activation, promoting the maintenance of the β-linkin disruption complex, promoting β-linkin accumulation, or inhibiting cell growth, the method comprising contacting cells expressing the FZD receptor with an antibody or immunoconjugate described herein. In another aspect, this disclosure provides antibodies or immunoconjugates described herein for inhibiting the binding of Wnt ligands to FZD receptors, disrupting Wnt signaling pathways, inhibiting Wnt-induced transcriptional activity, inhibiting disheveled protein activation, promoting the maintenance of the β-linkin disruption complex, promoting β-linkin accumulation, or inhibiting cell growth. In yet another aspect, this disclosure provides the use of antibodies or immunoconjugates described herein for inhibiting the binding of Wnt ligands to FZD receptors, disrupting Wnt signaling pathways, inhibiting Wnt-induced transcriptional activity, inhibiting disheveled protein activation, promoting the maintenance of the β-linkin disruption complex, promoting β-linkin accumulation, or inhibiting cell growth. In another aspect, this disclosure provides the use of the antibodies or immunoconjugates described herein in the preparation of medicaments for inhibiting the binding of Wnt ligands to FZD receptors, disrupting Wnt signaling pathways, inhibiting Wnt-induced transcriptional activity, inhibiting scattered protein activation, promoting the retention of β-linkin disruption complexes, promoting β-linkin 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 antibodies selected from: 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 another aspect, this disclosure provides a method of treating a subject with cancer in need, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising an antibody or immunoconjugate as described herein. In another aspect, this disclosure provides the use of the antibody or immunoconjugate as described herein for treating cancer. In yet another aspect, this disclosure provides the use of the antibody or immunoconjugate as described herein for treating cancer. In yet another aspect, this disclosure provides the use of the 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 includes 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 the 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 antibodies selected from: 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 antibodies selected from 5019 and 5020. In yet another embodiment, the cancer treated by the method or use comprises one or more cancer cells containing a mutation in the RNF43 gene, and the antibody or immunoconjugate comprises a set of CDR sequences corresponding to antibody 5020.

[0057] Other features and advantages of this 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 embodiments, and the scope of the claims should not be limited to 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 attached diagram

[0059] The embodiments of this disclosure will now be described with reference to the accompanying drawings, in which:

[0060] Figure 1 This diagram illustrates the binding of phage clones to FZD4-CRD-Fc and Fc. Single colonies were inoculated into 96-well plates, and overnight phage supernatant was diluted 1:2 in 0.05% Tween 20 / 0.5% BSA / PBS (dilution buffer) for ELISA testing of binding. Phages were detected with anti-M13-HRP secondary antibody (1:5000 in dilution buffer), and the plate was stopped with TMB substrate and acid. The absorbance of the FZD4-Fc-coated wells and the control Fc-coated wells was read at 450 nm, as shown.

[0061] Figure 2 This is a graph showing the competitive ELISA binding of anti-FZD4 Fab. ELISA was performed to assess the affinity of the FZD4 Fab group. A 384-well ELISA plate was coated overnight at 4°C with 2 μg / ml FZD4 CRD-Fc (R&D systems) in PBS. The plate was 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 0.5 μg / 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. This Fab antigen mixture was transferred to a 384-well plate and incubated at room temperature for 20 minutes. The plate was washed six times, and Fab binding was detected using a 1:5000 dilution of anti-FLAG-HRP secondary antibody (Sigma) in 0.05% Tween 20 / 0.5% BSA / PBS. The secondary antibody was incubated at room temperature for 45 minutes, the plate was washed, and the color development was terminated with TMB substrate and acid. The absorbance was read at 450 nm. The binding percentage was calculated as the absorbance of the Fab well containing the competitively soluble FZD4 CRD-Fc divided by the absorbance of the Fab well without the competitively soluble FZD4 CRD-Fc, multiplied by 100.

[0062] Figure 3 These are a series of immunofluorescence staining images demonstrating the binding of anti-FZD4 Fab to FZD expressed on CHO cells. Immunofluorescence (IF) staining was used to test the responsiveness of Fab from an FZD4 selection to CHO overexpression lines stably expressing the FZD4 CRD region (as a GPI-linked domain with a myc tag). Fab was detected with anti-F(ab')2-FITC secondary antibody (Jackson Immuno), and white areas indicate FITC staining.

[0063] Figure 4 These are a series of immunofluorescence staining images showing the binding of anti-FZD4 Fab to CHO cells. The responsiveness of Fab from the FZD4 selection to control CHO cell lines stably transfected with GPI adapters and myc tags was tested by immunofluorescence (IF). Fab was detected with anti-F(ab')2-FITC secondary antibody (Jackson Immuno), and white areas indicate FITC staining.

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

[0065] Figure 6 These are a series of immunofluorescence staining images showing the binding of Fab 5019 and Fab 5020 with FZD in various human pancreatic cancer cell lines.

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

[0067] Figure 8 AD is a series of diagrams illustrating the combination of anti-FZD4 Fab with FZD.

[0068] Figure 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 binding; ++ = weak binding; +++ = good binding; ++++ = very good binding.

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

[0070] Figure 11 This is a diagram showing the binding of anti-FZD4 Fab to pancreatic cancer cells, as determined by flow cytometry.

[0071] Figure 12Figures AB show a series of diagrams illustrating the 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 BSA-blocked 96-well TC-treated plates at room temperature for 1 hour. Control wells were also included where a separate buffer was added instead of biotinylated Wnt5a. Biotinylated Wnt5a was added at a final concentration of 150 ng / µl. Samples were transferred to pre-blocked streptavidin-coated plates (R&D systems) and allowed to capture at room temperature for 1 hour. Wash the wells four times with 0.05% Tween 20 / PBS, and then add anti-Fc-HRP (1:5000 in dilution buffer, Jackson Immuno) to the wells and incubate at room temperature for 45 minutes. Wash the wells four times and terminate the color development with TMB reagent and acid. Read the absorbance at 450 nm. (B) Dilute FZD4-CRD-Fc to the concentration previously determined in (A) to provide an ELISA signal within the linear range and incubate 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 are also included. Add biotinylated Wnt5a to the wells and incubate the plate for another 1 hour. Control wells in which a separate buffer is added instead of biotinylated Wnt5a are also included. Biotinylated Wnt5a is added at a final concentration of 150 ng / µl. The final concentration of 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 Fab proteins specific to different protein antigens, as well as controls against the effects of the neutralization elution buffer in which the Fab proteins are stored (Fab buffer controls), were included. The binding percentage was calculated.

[0072] Figure 13 This is a table showing the effect of Fab on β-linkin-driven transcription (TOPFLASH assay).

[0073] Figure 14 AH is a series of graphs illustrating the effect of anti-FZD4 Fab on cancer cell proliferation.

[0074] Figure 15This is a table showing the effect of anti-FZD4 Fab on the proliferation of pancreatic cancer cells, as determined by SRB (sulfonylrhodamine B) assay, where "na" indicates untested.

[0075] Figure 16 This is an overview table of anti-FZD4 Fab activity, where na indicates untested. Antiproliferative activity was demonstrated in association 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] Figure 17 This diagram illustrates the inhibition of Axin2, a gene regulating the Wnt pathway, by anti-FZD4 antibody in the human pancreatic cancer cell line (HPAF II). Gene expression after treatment with 200 nM Fab / IgG (RT-qPCR) is normalized to β-actin.

[0077] Figure 18 AB are a series of graphs illustrating the inhibition of proliferation by IgG 5019 and IgG 5020. A) shows that IgG 5019 and IgG 5020 inhibit the proliferation of pancreatic cancer cells using an Almar Blue proliferation assay (200 nM Fab / IgG), and B) shows that IgG 5020 inhibits cell proliferation in a dose-dependent manner.

[0078] Figure 19 These are a series of colony photographs showing how IgG 5020 inhibits colony formation.

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

[0080] Figure 21 This figure shows the effects of Fab 5019, Fab 5020, and IgG 5020 on the proliferation of PDAC PDX cell lines in the Almar Blue proliferation assay (200 nM Fab / IgG).

[0081] Figure 22 A schematic diagram of a typical signal transmission path in Wnt is shown.

[0082] Detailed description of the publicly disclosed information

[0083] I. Definition

[0084] Unless otherwise defined, scientific and technical terms used in conjunction with this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. For example, the term “cell” includes a single cell as well as more than one cell or a population of cells. Generally, the nomenclature and techniques described herein for cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization are those well known and commonly used in the art (see, for example, 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 linked by peptide bonds. The term "peptide" refers to a short polypeptide, typically 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 secondary, tertiary, and / or quaternary structures (e.g., structures stabilized by hydrogen bonds, relationships between secondary structures, and structures formed by more than one protein). Proteins can be further modified by other attachment parts such as carbohydrates (glycoproteins), lipids (lipoproteins), phosphate groups (phosphoproteins), etc.

[0086] As used in this article, an amino acid sequence is "composed only of the amino acids in that sequence".

[0087] As used herein, if the first amino acid sequence (1) contains the second amino acid sequence and (2) is no more than 1, 2, or 3 amino acids longer than the second amino acid sequence, then the first amino acid sequence is "mainly composed of the second amino acid sequence".

[0088] As used herein, if a second amino acid sequence contains a first amino acid sequence, then the first amino acid sequence is a “fragment” of the second amino acid sequence. In some embodiments, the first amino acid sequence that is a fragment of the second amino acid sequence may be no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fewer 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 different from the reference sequence but contains minor changes, such as, for example, the insertion, deletion, or substitution of one or a few amino acids. The functionally equivalent sequence retains the function (e.g., immunogenicity) of its equivalent reference sequence. If the functionally equivalent amino acid sequence contains substitutions of one or more amino acids relative to the reference sequence, these are generally conserved amino acid substitutions.

[0090] As used herein, a “conservative amino acid substitution” is an amino acid substitution in which an amino acid residue is replaced by another amino acid residue without eliminating the desired property of the protein. Suitable conservative amino acid substitutions can be made by substituting each other with amino acids of similar hydrophobicity, polarity, and R-chain length. See, for example, Watson et al., “Molecular Biology of the Gene,” 4th ed., 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):

[0092] As used herein, the term "substantially identical" means the identity between a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence contains a sufficient or minimum number of i) amino acid residues identical to those in the aligned second amino acid sequence, or ii) conserved substituted amino acid residues that are amino acid residues in the aligned second amino acid sequence, such that the first and second amino acid sequences have common structural domains and / or common functional activities and / or common immunogenicity. For example, amino acid sequences containing common structural or antigenic domains with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity are described as sufficiently or substantially identical. In the context of nucleotide sequences, the term "substantially identical" herein is used to mean that the first nucleic acid sequence contains a sufficient or minimum number of nucleotides identical to those in the aligned second nucleic acid sequence, such that the first and second nucleotide sequences encode polypeptides with common functional activities, or encode common structural polypeptide domains or common functional polypeptide activities, or encode polypeptides with 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 capable of being recognized by an antibody, such as peptides, polynucleotides, carbohydrates, lipids, chemical moieties, or combinations thereof (e.g., phosphorylated peptides or glycosylated peptides, etc.). Those skilled in the art will understand that the term does not indicate that the molecule is immunogenic in every case, but only 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 bound by an antibody. An epitope may contain a small number of amino acids or portions thereof, such as five or six or more, for example, 20 or more amino acids, or portions thereof. In some cases, an epitope contains non-protein components, such as components derived from carbohydrates, nucleic acids, or lipids. In some cases, an epitope is a three-dimensional portion. Thus, for example, when the target is a protein, an epitope may contain continuous amino acids or amino acids from different parts of a protein that are accessible through protein folding (e.g., a discontinuous epitope).

[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 via at least one antigen recognition site within the variable region of the immunoglobulin. 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 molecule containing an antigen recognition site, provided that the antibody exhibits the desired biological activity. Antibodies may (i) belong to any of the five major classes of immunoglobulins—α (IgA), δ (IgD), ε (IgE), γ (IGg), and μ (IGm)—based on their heavy chain constant domain identity, or (ii) their subclass (isotype) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The light chain can be λ or κ. Antibodies can be naked or conjugated with other molecules such as toxins, drugs, radioisotopes, chemotherapeutic agents, etc.

[0096] In one embodiment, the "intact antibody" comprises a tetramer consisting of two pairs of identical polypeptide chains, each pair having a "light chain" (approximately 25 kDa) and a "heavy chain" (approximately 50-70 kDa). The heavy and light chains are linked by covalent and non-covalent bonds (e.g., disulfide bonds), the number and amount of which vary among different immunoglobulin classes. In one embodiment, each chain contains a variable region and a constant region. The antigen recognition site of the variable region consists of a hypervariable region or complementarity-determining region (CDR) and a framework region. The framework region typically does not contact the antigen but provides structural support for the CDR. The constant region interacts with other immune cells in the body. Between the constant and variable regions (only IgG, IgD, and IgA, not IgM or IgE) is a hinge region located centrally between the two heavy chains, which provides flexibility for articulating antigen binding.

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

[0098] (1) Whole immunoglobulin (also known as “intact” antibody) (two light chains and two heavy chains, e.g., tetramer).

[0099] (2) Immunoglobulin polypeptides (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) (containing V linked by a linker (e.g., a peptide linker) L and V H The peptides, (scFv)2, sc(Fv)2, bispecific sc(Fv)2, bispecific (scFv)2, minibody (sc(FV)2 fused with the CH3 domain), and triabody are trivalent sc(Fv)3 or trispecific sc(Fv)3.

[0101] (4) Multivalent antibodies (antibodies that bind to the binding regions of two different epitopes or proteins, such as “scorpion” antibodies).

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

[0103] As used herein, the term "antibody fragment" refers to some or a portion of an antibody or antibody chain that contains fewer amino acid residues than a complete or whole antibody or antibody chain and that binds to an antigen or competes with a complete antibody. Fragments can be obtained via chemical or enzymatic treatment of a complete or whole antibody or antibody chain. Fragments can also be obtained through recombinant methods. For example, an F(ab')2 fragment can be generated by treating an antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to generate a Fab' fragment. Papain digestion can lead to the formation of Fab fragments. Fab, Fab', and F(ab')2, scFv, dsFv, ds-scFv, dimers, microantibodies, biantibodies, bispecific antibody fragments, and other fragments can also be constructed using recombinant expression techniques.

[0104] Although various antibody fragments are defined in terms of digestion products of intact antibodies, those skilled in the art will understand that such fragments can also be de novo chemically synthesized or constructed and expressed using recombinant DNA methods.

[0105] Single-chain antibodies (scFv) refer to antibodies containing V chains linked by linkers (e.g., peptide linkers). L and V H The polypeptide. ScFv can also be used to form tandem (or bivalent) scFv or biantibodies. The generation and properties of tandem scFv and biantibodies are described in the following: e.g., 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 heavy chain portion contained in the Fab fragment) and single-domain antibodies. Single-domain antibodies (sdAbs) are variable domains of either the heavy or light chain generated through recombination methods.

[0107] As used herein, “CDR sequence set” refers to the three heavy chain CDRs and / or three light chain CDRs of a specific antibody described herein. The “light chain” CDR sequence set refers to the light chain CDR sequence. The “heavy chain” CDR sequence set refers to the heavy chain CDR sequence. The “complete” CDR sequence set refers to both heavy chain and light chain CDR sequences. For example, for antibody 5017 as shown in Table 1a, the complete 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 may, for example, include, the CDRs in Table 1a or Table 3a, consist primarily of the CDRs in Table 1a or Table 3a, or consist 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 or composition that has a single binding specificity and affinity for a specific epitope on an antigen ("monoclonal antibody composition"). "Polyclonal antibody" refers to an antibody clone or composition that produces antibodies 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 derived from a mammal of one species (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capability, while the constant regions are homologous to sequences derived from another species (typically the treated subject, e.g., human) to avoid triggering an immune response.

[0110] As used herein, the term "humanized antibody" refers to a chimeric antibody in which a CDR obtained from the VH and VL regions of a non-human antibody, possessing the desired specificity, affinity, and potency, is grafted onto a human framework sequence. In one embodiment, the framework residues of the humanized antibody are modified to improve and optimize the antibody's specificity, affinity, and potency. Humanization, i.e., replacing a non-human CDR sequence with the corresponding sequence of a human antibody, can be performed according to the methods described below: for example, U.S. Patents 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 prepared by any technique known in the art that has an amino acid sequence corresponding to that of an antibody produced by the human body.

[0112] As used herein, the term "hybrid antibody" refers to an antibody in which heavy and light chain pairs from antibodies with different antigenic determinant regions are assembled together such that the resulting tetramer can recognize and bind to two different epitopes or two different antigens. Hybrid antibodies can be bispecific (binding to two different antigens or epitopes) or multispecific (binding to more than one different antigen or epitope).

[0113] As used in this article, an antibody is "single-specific" if all its antigen-binding sites bind to the same epitope.

[0114] As used herein, an antibody is "bispecific" if it has at least two distinct antigen-binding sites, each binding 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 comparing the dissociation constant of the antibody (or other targeting moiety) to the target with the dissociation constant (Ki) of the antibody and other substances or generally unrelated molecules in the environment. d Defined by ) . Larger (higher) K d K describes lower affinity interactions. d Conversely, smaller (lower) K d K describes higher affinity interactions or tighter binding. d To illustrate, just by example, the antibody binds specifically to the K... d It can be a femtomolar concentration, picomolar concentration, nanomolar concentration, or micromolar concentration, while the Kc of the antibody binding to irrelevant substances... d It can be a millimolecular concentration or higher. Binding affinity can be in the micromolar concentration range (kD = 10). -4 Up to 10 -6 Nanomolar concentration range (kD = 10) -7 M to 10 -9 M), Pimolar 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 in this article, if the antibody is less than 10 -4 If an antibody "binds" or "recognizes" an antigen or epitope whose Kd-binding concentration M (i.e., within the micromolar concentration range) is M, then the antibody "binds" or "recognizes" that antigen or epitope. For cell types (e.g., antibodies binding to cancer cells), the term "binding" typically indicates binding to the majority of cells in a pure population of those cells. For example, antibodies binding to a specific cell type typically bind to at least two-thirds of the cells in that 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 peptide can be determined by comparing the binding of the antibody to cells presenting the peptide with the binding (or lack thereof) of the antibody to cells not expressing the peptide. Those skilled in the art will recognize that some variability arises depending on the method used to determine binding and / or the threshold. The affinity of an antibody for a target can be determined according to methods known in the art, such as Ernst et al., Determination of Equilibrium Dissociation Constants. Therapeutic Monoclonal AntibodiesAs described in (Wiley & Sons ed. 2009).

[0118] As used in this article, the term "greater affinity" refers to antibody X binding more strongly to target Y than to target Z (K). on ) and / or have a smaller dissociation constant (K) off The relative degree of antibody binding to target Y, where antibody X has a greater affinity for target Y than for target Z. Similarly, the term "less affinity" in this context refers to the degree of antibody binding where antibody X binds less strongly to target Y than to target Z and / or has a larger dissociation constant, where 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 equals 1 / K D K D equals k on / k off k on and k off Values ​​can be measured using surface plasmon resonance techniques, for example, using the 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 the biological activity associated with a target antigen, relative to their 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 the natural ligand or receptor at the same site as the natural ligand-receptor interaction, or allosterically bind by inducing a change to prevent normal binding. Non-competitive antagonists bind at a site different from the natural ligand-receptor interaction, but reduce the KD or signaling generated 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 inherent affinity of the antibody for the antigen, (2) the valence of the antibody, and (3) the geometric arrangement of the interacting components. Affinity is the strength of the interaction between an antibody and a single target, while affinity is the cumulative strength of more than one affinity. In one embodiment, the antibody disclosed herein is bivalent.

[0120] As used herein, if an antibody binds to a first antigen with a greater affinity than it binds to a second antigen, then the antibody “preferentially binds” to the first antigen relative to the second antigen. Preferential binding can be any of an affinity that is at least 2, 5, 9, 10, 20, 30, 40, 50, 100, 500, or 1000 times greater. Therefore, for example, if an antibody binds to a first FZD protein with a greater affinity than it binds to a second FZD protein, then the antibody preferentially binds to the first FZD protein relative to the second FZD protein.

[0121] As used in this article, if the antibody is in the form of 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 M, 1×10 -12 An antibody is considered "specifically bound" to a target antigen or a group of target antigens if it binds to the target antigen or each member of the target antigen group with an affinity at least twice that of a non-target antigen compared to the target antigen, for example, such that the antibody binds to the target antigen or each member of the target antigen group with an affinity at least twice that of a non-target antigen compared to the target antigen. Typically, specific binding is characterized by binding to the antigen with sufficient affinity to allow the antibody to be used as a diagnostic agent for detecting the antigen or epitope and / or as a therapeutic agent targeting the antigen or epitope.

[0122] If an antibody specifically binds to a group of target proteins (e.g., some or all members of the coiled receptor protein family), the antibody has a greater affinity for the member of the target group with which it binds weakest than for the non-target antigen. In one embodiment, an antibody that specifically binds to the cysteine-rich domain (CRD) of each of one or more human coiled (FZD) receptors selected from FZD1, FZD2, FZD4, FZD5, FZD7, FZD8, and FZD9 means that the antibody specifically binds to the selected member of that group compared to unselected members or, more generally, other antigens. Thus, for example, an antibody that specifically binds to the cysteine-rich domain of a target group consisting of FZD1, FZD2, FZD4, FZD5, and FZD7 specifically binds to these proteins instead of FZD3, FZD8, FZD9, and FZD10.

[0123] As used herein, an antibody “blocks” or “antagonizes” ligand-receptor binding when it 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%, or 99.9% of control (e.g., untreated) cells. For example, an antibody that antagonizes or blocks the binding of Wnt ligands to the FZD receptor competitively reduces or prevents the interaction of the Wnt protein with the FZD receptor. This results in attenuation or blockage of downstream signaling events associated with Wnt signaling. This includes, for example, activation of the scattered protein, dissolution of the β-linkin disruption complex, lower cytoplasmic levels of β-linkin, and / or lower activity of TCF / LEF-mediated transcription.

[0124] For antibody targets (e.g., antigens, analytes, immune complexes), the term "capture" typically 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 binding to a specific antibody target typically binds to at least two-thirds of the antibody target in solution (e.g., at least 67%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%). Those skilled in the art will recognize that some variability arises depending on the method used to determine the binding and / or the threshold.

[0125] The term "conjugate" refers to a first molecule chemically conjugated to a portion (such as a detectable marker or a biologically active portion, such as a drug, toxin, or chemotherapeutic agent or cytotoxic agent), for example, an antibody ("immunoconjugate"). Therefore, this disclosure contemplates antibodies conjugated to one or more portions. Furthermore, antibodies can be "conjugated antibodies" or "unconjugated antibodies" (i.e., not conjugated to any portion).

[0126] As used herein, the term "antibody-drug conjugate" or ("ADC") refers to an antibody conjugated to a drug. Typically, conjugation involves covalent binding via a linker.

[0127] As used herein, the term “tagged” molecule (e.g., nucleic acid, protein, or antibody) refers to a molecule that is covalently (through a linker or chemical bond) or non-covalently (through an ionic bond, van der Waals bond, electrostatic bond, or hydrogen bond) bound to a detectable marker, such that the presence of the molecule can be detected by detecting the presence of the detectable marker bound to that molecule.

[0128] As used herein, the term "detectable label" refers to a composition that can be detected by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. Examples of detectable labels are described herein, and they include, but are not limited to, colorimetric labels, fluorescent labels, chemiluminescent labels, enzyme labels, and radiolabels. For the purposes of this disclosure, a detectable label may also be a portion (e.g., biotin) that does not generate a signal itself but is combined with a second portion (e.g., labeled avidin) capable of generating a signal.

[0129] The term "crosslinking" in antibody contexts refers to the attachment of an antibody to a solid or semi-solid matrix (e.g., agarose gel, beads, microtiter plates) or to another protein or antibody. For example, antibodies can be polymerized to produce antibody complexes with multiple (more than two) antigen-binding sites. Antibodies can be polymerized by expressing the antibody as a high-valent isoform (e.g., IgA or IgM, which typically form complexes of two or five antibodies, respectively). Antibody polymerization can also be performed using crosslinking agents (e.g., carbodiimides, NHS esters, etc.) containing reactive groups capable of linking proteins. Methods and compositions for crosslinking antibodies to a matrix are described, for example, in the Abcam and New England Biolab catalogues and websites (available at abcam.com and neb.com). Crosslinking agent compounds with various reactive groups are described, for example, in the Thermo Fisher Scientific catalogue and website (available at piercenet.com).

[0130] As used herein, the term "immunoassay" refers to a method for detecting an analyte by detecting the binding between an antibody that identifies the analyte and the analyte.

[0131] As used herein, the term "expression construct" refers to a polynucleotide containing an expression control sequence operatively linked to a heterologous nucleotide sequence to which the expression will be performed (i.e., a sequence to which the expression control sequence is not normally linked in nature). As used herein, the term "expression vector" refers to a polynucleotide containing 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 transcription and / or translation of a nucleotide sequence operatively linked to it. Expression control sequences include promoters, enhancers, repressors (transcriptional regulatory sequences), and ribosome binding sites (translational regulatory sequences).

[0132] As used herein, the term "vector" includes any intermediate medium for nucleic acid molecules that enables the nucleic acid molecules to be introduced, for example, into prokaryotic and / or eukaryotic cells and / or integrated into the genome, and includes plasmids, phage particles, bacteriophages, or viral vectors, such as retrovirus-based vectors, adeno-associated virus vectors, etc. As used herein, the term "plasmid" generally refers to a construct of extrachromosomal genetic material, typically a circular double-stranded DNA that can replicate independently of chromosomal DNA.

[0133] As used herein, when the expression control sequence plays a role in regulating the transcription of nucleotide sequences in the cell, the nucleotide sequence is "operably linked" to the expression control sequence. This includes promoting the transcription of the nucleotide sequence through interactions between polymerase and promoter.

[0134] As used in this article, “host cell” refers to a recombinant cell containing the 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 a subject (e.g., a patient), from an animal (such as an animal model), or from cultured cells (e.g., a cell line or cells taken from a patient and grown in a culture medium for observation). Biological samples can include tissues and / or fluids. They 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, buccal swabs, milk, urine, or feces. Biological samples can be biopsy samples, such as tissue biopsy samples, such as needle biopsy samples, fine-needle aspiration biopsy samples, surgical biopsy samples, etc. Samples can include tissue samples with lesions or suspected lesions, although biological samples can also originate from another site, such as suspected metastatic sites, lymph nodes, or from blood. Biological samples can be 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 has 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 this disclosure, prognosis may refer to the likelihood that an individual will develop cancer, relapse, metastasize, or be cured, or the likely severity of the disease (e.g., the severity of symptoms, the rate of functional decline, survival, etc.). These terms are not intended to be absolute, as will be understood by anyone skilled in the art of medical diagnostics.

[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, number of cancer cells, growth rate, metastatic activity, reduction in cell death of non-cancer cells, reduction in nausea, and other side effects of chemotherapy or radiation therapy. The terms “treatment” and “prevention” are not intended to be absolute terms. Treatment and prevention may refer to any delay in onset, improvement in symptoms, improvement in patient survival, increase in survival time, or increase in survival rate. Treatment and prevention may be complete (undetectable levels of neoplastic cells) or partial, resulting in fewer neoplastic cells found in the patient than would have been present without the intervention of the present invention. The effects of treatment may be compared to untreated individuals or a collection of individuals, or to the same patient before treatment or at different times during treatment. In some aspects, the severity of the disease is reduced by at least 10% compared to, for example, an individual before administration or a control individual who has not experienced treatment. In some aspects, the severity of the disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, it is no longer detectable using standard diagnostic techniques.

[0138] As used herein, the terms “effective amount,” “effective dose,” and “therapeutic effective amount” refer to the amount of an agent (such as an antibody or immune conjugate) sufficient to produce a desired response (such as a reduction or elimination of the signs or symptoms of the condition or improvement of the disorder). In some instances, an “effective amount” is the amount that treats (including prevents) one or more symptoms and / or underlying causes of any disorder or disease and / or prevents disease progression. For example, for a specific parameter, a therapeutic effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100% of the therapeutic effect. Therapeutic efficacy may also be expressed as an increase or decrease of “... times.” For example, a therapeutic effective amount may have an effect of at least 1.2 times, 1.5 times, 2 times, 5 times, or more times compared to a control.

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

[0140] As used herein, the term “pharmaceuticalally acceptable” means a carrier that is compatible with the other components of a pharmaceutical composition and can be safely administered to a subject. This term is used synonymously with “physiologically acceptable” and “pharmacologically acceptable.” Pharmaceutical compositions and techniques for their preparation and use are known to those skilled in the art based on this disclosure. For a detailed list of suitable pharmacological compositions and their administration techniques, one may 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 non-toxic to recipients at the doses and concentrations used and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine, and citrate; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methylparaben or propylparaben, benzalkonium chloride, or combinations thereof); and amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, etc. Amino acids, 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. A dose refers to the amount of active ingredient administered to an individual at each administration. For the purposes of this invention, a dose may refer to the concentration of an antibody or related component, such as the amount of a therapeutic agent or the dose of a radiolabeled substance. A dose will vary depending on many factors, including the frequency of administration, individual size and tolerability, severity of the condition, risk of side effects, route of administration, and imaging modality of the detectable marker (if present). Those skilled in the art will recognize that the dose can be modified based on the above factors or on treatment progress. The term “dosage form” refers to a specific form of the drug and depends on the route of administration. For example, a dosage form may be a liquid, such as a saline solution for injection.

[0144] As used herein, the term "subject" refers to an individual animal. The term "patient" as used herein refers to a subject under the care or supervision of a healthcare provider such as a physician or nurse. Subjects include mammals such as humans and non-human primates such as monkeys, as well as dogs, cats, horses, cattle, rabbits, rats, mice, goats, pigs, and other mammal species. Subjects may also include birds. A patient can be an individual seeking treatment, monitoring, adjustment, or modification of an existing treatment regimen. The term "cancer subject" refers to an individual who has been diagnosed with cancer. Cancer patients can include individuals who have not yet received treatment, individuals currently receiving treatment, individuals who have undergone surgery, and individuals who have discontinued treatment.

[0145] In the context of cancer treatment, subjects requiring treatment may refer to individuals with cancer or precancerous conditions, individuals who already have cancer and are at risk of recurrence, individuals suspected of having cancer, or individuals undergoing standard cancer treatment (such as radiation therapy or chemotherapy).

[0146] The terms “cancer,” “tumor,” and “transformed” encompass precancerous cells, neoplastic cells, transformed cells, and cancerous cells, and can refer to solid tumors or non-solid cancers (see, for example, Edge et al., AJCC Cancer Staging Manual (7th edition, 2009); Cibas and Ducatman Cytology: Diagnostic principles and clinical correlates (3rd edition, 2009)). Cancer includes both benign and malignant neoplastics (abnormal growth). “Transformation” refers to spontaneous or induced phenotypic changes, such as immortalization of cells, morphological changes, abnormal cell growth, reduced contact inhibition and anchoring, and / or malignancy (see Freshney, Culture of Animal Cells: a Manual of Basic Technique (3rd edition, 1994)). While transformation can be caused by infection with transforming viruses and the incorporation of new genomic DNA or the uptake of exogenous DNA, it can also occur spontaneously or after exposure to carcinogens.

[0147] The term "cancer" can refer to any type of cancer, including but not limited to leukemia, carcinoma, sarcoma, adenocarcinoma, lymphoma, solid carcinoma, and lymphoid carcinoma. 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 (gastric) cancer, kidney cancer, central nervous system cancer, skin cancer, glioblastoma, and melanoma.

[0148] As used herein, if a chemical entity, such as a polypeptide, is the dominant chemical entity of that kind (e.g., polypeptide) in a composition, then it is “substantially pure.” This includes chemical entities representing 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 that kind of chemical entity in the composition.

[0149] The term "isolated antibody" refers to antibodies produced in vivo or in vitro that have been removed from the source of the antibody (e.g., animal, hybridoma, or other cell line such as recombinant insect cells, yeast cells, or bacterial cells that produce antibodies).

[0150] "Substantially pure" or "isolated" means that the target class is the dominant class present (i.e., more abundant, on a molar basis, than any other individual macromolecular class in the composition), and a substantially purified fraction is a composition in which the target class constitutes at least about 50% (on a molar basis) of all present macromolecular classes. Generally, a substantially pure composition means that about 80% to 90% or more of the macromolecular classes present in the composition are purified to the class of interest. If the composition consists primarily of a single macromolecular class, the target class is purified to substantially homogeneity (contaminant classes are undetectable in the composition by conventional detection methods). For the purposes of this definition, solvents, small molecules (<500 Daltons), stabilizers (e.g., BSA), and elemental ions are not considered macromolecular classes.

[0151] As used herein, the term "sequence identity" refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced into the first amino acid or nucleic acid sequence to achieve optimal alignment with the second amino acid or nucleic acid sequence). The corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between two sequences is a function of the number of shared positions (i.e., identity % = number of 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 two sequences can also be accomplished using mathematical algorithms. Preferred, non-limiting examples of mathematical algorithms for comparing two sequences are those described in Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, and modified as described in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such algorithms are incorporated into the NBLAST and XBLAST procedures in Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed using the NBLAST nucleotide procedure parameter set, e.g., score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of this application. BLAST protein searches can be performed using the XBLAST procedure parameter set, e.g., score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain vacancy alignments for comparative purposes, Gapped BLAST, as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402, can be used. Alternatively, PSI-BLAST can be used for iterative retrieval to detect distance relationships between molecules (ibid.). When using BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs can be used (e.g., the default parameters of XBLAST and NBLAST) (see, for example, the NCBI website). Another preferred, non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.When comparing amino acid sequences using the ALIGN program, a PAM120 weighted residue table, a 12-fold vacancy length penalty, and a 4-fold vacancy penalty can be used. The percentage of identity between two sequences can be determined using techniques similar to those described above, allowing or disallowing vacancy. When calculating the percentage of identity, typically only exact matches are counted.

[0152] For antibodies, the percentage sequence identity can be determined when the antibody sequences are maximally aligned via IMGT. After alignment, if a region of the test antibody (e.g., the entire mature variable region of the heavy or light chain) is compared with the same region of the reference antibody, the percentage of sequence identity between the test antibody region and the reference antibody region is the number of positions in both regions occupied by the same amino acid divided by the total number of aligned positions in both regions, multiplied by 100 to convert to a percentage.

[0153] The percentage of amino acid sequence identity can also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program is available from the National Institutes 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 dropoff = 25, and score matrix = BLOSUM62.

[0154] When comparing amino acid sequences using NCBI-BLAST2, the percentage of amino acid sequence identity of a specific amino acid sequence pair A with or relative to a specific amino acid sequence B (which can also be expressed as a percentage of amino acid sequence identity of a specific amino acid sequence pair A with or relative to a specific amino acid sequence B) is calculated as follows:

[0155] 100 multiplied by the fraction X / Y

[0156] Where X is the number of amino acid residues that the sequence alignment program NCBI-BLAST2 scores as identical matches in the alignment of A and B, and 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 amino acid sequence identity % of A to B will not be equal to the amino acid sequence identity % of B to A. As used herein, the term "nucleic acid sequence" refers to a sequence of nucleosides or nucleotide monomers composed of naturally occurring bases, sugars, and inter-sugar (backbone) linkages, and includes cDNA. This term also includes sequences containing modifications or substitutions of non-naturally occurring monomers or portions thereof. The nucleic acid sequence of this application may be a deoxyribonucleic acid (DNA) sequence or a ribonucleic acid (RNA) sequence 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 deazoadenine, guanine, cytosine, thymine, and uracil; and xanthine and hypoxanthine. It should be understood that polynucleotides containing non-transcribed nucleotide bases can be used as probes, for example, in hybridization assays. Nucleic acids can be double-stranded or single-stranded and can represent sense or antisense strands. Furthermore, the term "nucleic acid" includes complementary nucleic acid sequences as well as codon-optimized or synonymous codon equivalents.

[0157] As used herein, “isolated nucleic acid” refers to nucleic acid that is substantially free of cellular material or culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemical substances when chemically synthesized. Isolated nucleic acid also substantially lacks the sequences naturally located on the flanks of the nucleic acid from which it originates (i.e., the 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 solution. Hybridization can occur in all or part of the nucleic acid sequence molecule. The length of the hybridization portion is typically at least 15 (e.g., 20, 25, 30, 40, or 50) nucleotides. Those skilled in the art will recognize that the stability of the nucleic acid duplex or hybrid is determined by Tm, which, in a sodium-containing buffer, is a function of sodium ion concentration and temperature (Tm = 81.5°C - 16.6(Log10[Na+]) + 0.41(%(G+C) - 600 / l), or a similar equation). Therefore, the parameters determining the stability of the hybrid in the washing conditions are sodium ion concentration and temperature. To identify molecules similar to but not identical to known nucleic acid molecules, it can be assumed that a 1% mismatch results in a decrease in Tm of about 1°C; for example, if searching for nucleic acid molecules with >95% identity, the final washing temperature will be reduced by about 5°C. Based on these considerations, those skilled in the art will be able to readily select appropriate hybridization conditions. In a preferred embodiment, stringent hybridization conditions are selected. By way of example, stringent hybridization can be achieved under the following conditions: based on the equation above, hybridize at Tm -5°C with 5× sodium chloride / sodium citrate (SSC) / 5× Denhardt's solution / 1.0% SDS, followed by washing at 60°C with 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 equivalent stringency can be achieved using alternative buffers, salts, and temperatures. Further 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] As used herein and as is well known in the art, the terms “treating” or “treatment” mean a method used to obtain a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes may include, but are not limited to, the reduction or improvement of one or more symptoms or conditions, a reduction in the severity of disease, stabilization of the disease state (i.e., no worsening), prevention of disease spread, delay or slowing of disease progression, improvement or mitigation of the disease state, reduction of disease recurrence, and remission (whether partial or complete), whether detectable or undetectable. “Treating” and “treatment” may also mean prolonged survival compared to expected survival without treatment. As used herein, “treating” and “treatment” also include preventative treatment. For example, a subject with cancer may be treated with antibodies, immune conjugates, nucleic acids, or compositions described herein to prevent progression.

[0160] As used herein, the term “administration” means the delivery or administration of an agent, such as a composition containing an effective amount of antibody, to a subject via an effective route (such as intratumoral or intravenous administration).

[0161] As used herein, the term "diluent" means 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 irritate 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, binders, excipients, disintegrants, lubricants, similar materials, and combinations thereof, as known to those skilled 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 contemplated for use in pharmaceutical compositions unless incompatible with the active ingredient.

[0162] A composition or method that “contains” or “includes” one or more of the listed elements may include other elements not specifically listed. For example, a composition that “contains” or “includes” an antibody may contain an antibody alone or in combination with other components.

[0163] In understanding the scope of this 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 described in this document by endpoints include all numbers and fractions falling within that 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 their fractions are assumed to be modified by the term “about.” Furthermore, it should be understood that the singular forms of the articles “a,” “an,” and “the” include plural indicators unless the context explicitly specifies otherwise. For example, the terms “antibody” or “at least one antibody” can include more than one antibody, including mixtures thereof.

[0165] In context, the terms "frizzled receptor" and "FZD" refer to any gene or protein member of the frizzled receptor family. Frizzled receptor proteins are involved in the activation of scattered proteins in the cytosol. Frizzled receptors refer to any one of frizzled receptor-1, frizzled receptor-2, frizzled receptor-3, frizzled receptor-4, frizzled receptor-5, frizzled receptor-6, frizzled receptor-7, frizzled receptor-8, frizzled receptor-9, and frizzled receptor-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. This gene has ENTREZ gene ID: 8322. This protein has NCBI reference sequence: NP_036325.2.

[0166] Lipoprotein receptor-associated proteins (LRPs), also known as low-density lipoprotein receptor-associated proteins (HGNCs) or pre-low-density lipoprotein receptor-associated proteins (UniProts), 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 / curly receptor co-receptor group involved in the typical Wnt pathway. LRP5 is also known as LRP5, BMND1, EVR1, EVR4, HBM, LR3, LRP-5, LRP7, OPPG, OPS, OPTA1, VBCH2, and LDL receptor-associated protein 5. The LRP5 gene has ENTREZ gene ID: 4041, and the protein has an NCBI reference sequence: NP_002326. The LRP6 gene has an ENTREZ gene ID of 4040, and the protein has an NCBI reference sequence of NP_002327. LRP6 is also known as ADCAD2 or STHAG7.

[0167] II. Disorders associated with FRZ signal transduction dysregulation

[0168] The binding of Wnt to FZD destabilizes the β-linkin binding complex, leading to β-linkin degradation. The effect is an increase in intracellular β-linkin levels. Therefore, this paper provides a method to block the binding of Wnt to coiled receptor proteins (particularly FZD4, but also other members of the coiled 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. Dysregulation refers to abnormal signaling that increases normal β-linkin-mediated transcriptional changes or any other intracellular signaling pathways controlled by these receptors.

[0170] Different coil 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 gland-like 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] This article describes antibodies targeting the coiled receptor (FZD), including antibodies that bind to more than one FZD and other antibodies that preferentially bind to FZD4. These antibodies bind to the coiled receptor, block the binding of the ligand WNT, and modulate coiled receptor signaling. These antibodies also exhibit antiproliferative effects and have the potential to treat cancers and other diseases in which the coiled receptor is dysregulated.

[0174] Accordingly, one aspect of this disclosure includes an isolated antibody that specifically binds to the cysteine-rich domain (CRD) of a coiled receptor (FZD). The isolated antibody comprises a light chain variable region and a heavy chain variable region. The heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3. The amino acid sequence of the CDR comprises a sequence selected from the sequences in Table 1a or Table 3a, is mainly composed of sequences selected from the sequences in Table 1a or Table 3a, or is composed of sequences selected from the sequences in Table 1a or Table 3a.

[0175] In the implementation scheme, the antibody comprises a set of CDR sequences selected from the CDR sequence set in Table 1a (i.e., clones 5016 to 5037 and 6498 to 6500).

[0176] Table 1a–FZD4 antibody CDR amino acid sequence

[0178] Table 1b–FZD4 antibody CDR nucleic acid sequence

[0180] Table 1. CDR nucleic acid sequence of c–FZD4 antibody

[0182] This document also describes heavy chain variable regions and light chain variable regions. Table 2 provides exemplary variable domain sequences of the Fab heavy and light chains from clone 5017. Antibodies comprising sequences in Table 2 or substantially identical thereto are also envisioned, wherein the CDR is the 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 the heavy chain amino acid sequence as listed in Table 2, wherein the CDR sequence is the set of CDR sequences listed in Table 1a or Table 3a; or iii) a conserved substituted amino acid sequence of i), wherein the CDR sequence is the set of CDR sequences 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 with the light chain amino acid sequence as listed in Table 2, wherein the CDR sequence is a set of CDR sequences as listed in Table 1a or Table 3a, or iii) a conserved 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 – Examples of the full-length sequence of FZD4 antibody 5017

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

[0188] Table 3a–FZD4 antibody CDR amino acid sequence

[0189] Table 3a–FZD4 antibody CDR amino acid sequence

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

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

[0194] In some implementations, the variable domain sequence is at least 95%, 96%, 97%, 98%, or 99% similar outside the CDR region, and the CDR sequence set is 100% identical to the amino acid sequences provided in Table 1a or Table 3a.

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

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

[0199] In another embodiment, a competitive antibody is also provided that competes with an antibody containing the CDR sequence set described herein for binding. For example, in one embodiment, the competitive antibody reduces the binding of an antibody containing the CDR sequence set to the 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 described antibodies 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 may be an antibody in which the CDR sequence is selected from the CDR sequence set of antibodies 5016, 5018-5023, 5025, 6495, 6496, 5039, 5045, 5048, 5054, 5056, 5057, 5067, and 5073-5076.

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

[0202] Compared to FZD4, certain antibodies, such as those with a CDR set from antibodies 5018, 5019, 5022, 6494, and 5025, preferentially bind to other FZD proteins. (See, for example, Figure 5 ).

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

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

[0205] Antibodies can be humanized antibodies or chimeric antibodies as described in this article.

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

[0207] In some implementations, the antibody is selected from the following antibody-binding fragments: Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, their dimers, nanobodies, microbodies, biantibodies, and multimers.

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

[0209] In other embodiments, antibodies having multivalent antibodies or containing an Ig moiety may be preferred.

[0210] As shown in the embodiments, the Fab fragment of this disclosure can be combined with a constant region of immunoglobulin (Ig), such as IgG. In the embodiments, IgG is IgG1, IgG2, IgG3, or IgG4.

[0211] B. Detectable labeled antibodies

[0212] Detectable markers may include peptide sequences (such as myc tags, HA-tags, V5-tags, or NE-tags), fluorescent proteins, or luminescent proteins (such as green fluorescent protein or luciferase), which may be attached to or introduced into the antibodies described herein, and which are capable of generating a detectable signal directly or indirectly. For example, markers may be radio-opaque, positron-emitting radionuclides (e.g., for PET imaging), or radioisotopes, such as... 3 H, 13 N、 14 C 18 F, 32 P, 35 S, 123 I, 125I, 131 I; fluorescent (fluorophore) or chemiluminescent (chromophore) compounds, such as fluorescein isothiocyanate, rhodamine, or luciferin; enzymes, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase; imaging agents; or metal ions.

[0213] C. Antibody-drug conjugates

[0214] On the other hand, this includes immunoconjugates containing antibodies and detectable markers or cytotoxic agents as described herein.

[0215] Chemotherapy (anticancer) agents can be any agent capable of reducing cancer growth, interfering with cancer cell replication, directly or indirectly killing cancer cells, reducing metastasis, reducing tumor blood supply, etc. Chemotherapy agents therefore include cytotoxic agents. Cytotoxic agents include, but are not limited to, saponins, taxanes, vinblastine alkaloids, anthracyclines, and platinum-based agents. Classes of chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites (e.g., methotrexate), phytoalkaloids (e.g., vincristine), and antitumor antibiotics such as anthracyclines (e.g., doxorubicin), as well as various drugs not belonging to a specific class, 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 inhibit cell division by interfering with the formation of the cytoskeleton and spindle, thereby preventing the growth of rapidly dividing cancer cells. Other chemotherapeutic agents include hormone therapy. Chemotherapy agents also include those that inhibit microtubule assembly or polymerization, such as maytansine, mertansine, and auratestatin. Chemotherapy agents also include DNA-damaging agents, such as calicheamicin.

[0216] Chemotherapy agents may include maytansine alkaloids, auristatin, dolalastatin, tubulysin, novozygain, pyrrolobenzodiazepine (PBD) dimers, indolebenzodiazepine dimers, α-amaminine, trichothene, SN-38, pyroximide, CC1065, cazithromycin, 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 in this article.

[0219] A. Nucleic acid molecules

[0220] On the other hand, this includes nucleic acid molecules as listed in Tables 1b, 1c, 3b, and 3c, as well as polynucleotides, for example, that hybridize with one of the sequences described under stringent hybridization conditions. CDR and variable domain nucleic acid sequences can be used, for example, to prepare expression constructs.

[0221] B. Expression Components and Carriers

[0222] Nucleic acid molecules can be incorporated in a known manner into appropriate expression constructs or vectors to ensure protein expression. Expression constructs may contain expression control sequences, such as promoters, operatively linked to a polynucleotide containing a nucleotide sequence encoding an antibody encoded by this disclosure. Possible expression vectors include, but are not limited to, entrapment, plasmid, or modified viruses (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses). The vector should be compatible with the host cells used. An expression vector “suitable for transformation of host cells” means that the expression vector contains a nucleic acid molecule encoding a peptide corresponding to an epitope or antibody described herein.

[0223] In this embodiment, the vector is adapted to express, for example, single-chain antibodies via gene therapy. In this embodiment, the vector contains IRES and allows expression of both light chain variable regions and heavy chain variable regions. Such a vector can be used for in vivo antibody delivery.

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

[0225] Examples of such regulatory sequences include transcription promoters and enhancers or RNA polymerase-binding sequences, ribosome-binding sequences, and translation initiation signals. Furthermore, depending on the chosen host cell and the expression vector used, other sequences (such as origins of replication, additional DNA restriction sites, enhancers, and sequences that confer transcriptional induction) may be incorporated into the expression vector.

[0226] In the implementation scheme, the regulatory sequence guides or increases expression in neural tissue and / or cells.

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

[0228] In the implementation plan, the vector is a viral vector.

[0229] Recombinant expression vectors may also contain biomarker genes, which help select host cells to be transformed, infected, or transfected with vectors expressing the antibodies or epitope peptides described herein.

[0230] Recombinant expression vectors may also contain expression cassettes encoding a fusion moiety (i.e., a "fusion protein") that provide increased expression or stability of the recombinant peptide; increased solubility of the recombinant peptide; and aid in the purification of the target recombinant peptide by acting as a ligand (including, for example, the tags and markers described herein) during affinity purification. Furthermore, proteolytic cleavage sites may be added to the target recombinant protein to allow for the separation of the recombinant protein from the fusion moiety after purification. 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, respectively, to a recombinant protein.

[0231] Systems used for gene transfer in vitro and in vivo include virus-based vectors, most notably herpes simplex virus, adenovirus, adeno-associated virus (AAV), and retroviruses including lentiviruses. Alternative methods for gene delivery include the use of naked plasmid DNA and liposome-DNA complexes.

[0232] In one aspect, this disclosure includes methods for preparing the antibodies described herein, the methods comprising synthesizing nucleic acid molecules comprising the antibody framework and CDR sequence set described herein.

[0233] V. Recombinant cells

[0234] On the other hand, there are recombinant host cells expressing the antibodies described in this article.

[0235] As described in this article, antibodies can be prepared by recombinant expression of nucleic acids encoding antibody sequences.

[0236] The antibodies disclosed herein can be prepared by culturing cells engineered to express nucleic acid constructs encoding immunoglobulin peptides.

[0237] Recombinant host cells can be produced using any cell suitable for producing peptides (e.g., suitable for producing antibodies). For example, 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 wide 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 baculoviruses), yeast cells, or mammalian cells.

[0239] In the implementation plan, the cells are eukaryotic cells selected from yeast, plant, worm, insect, bird, fish, reptile and mammal cells.

[0240] In another implementation, the mammalian cells are CHO cells, myeloma cells, spleen cells, or hybridoma cells.

[0241] Suitable yeast and fungal host cells for antibody expression include, but are not limited to, *Saccharomyces cerivisiae*, *Schizosaccharomyces pombe*, *Pichia*, *Kluyveromyces*, and various species of *Aspergillus*. Examples of vectors used for expression in *Saccharomyces cerivisiae* include pYepSec1, pMFa, pJRY88, and pYES2 (Invitrogen Corporation, San Diego, CA). Protocols for transforming yeasts and fungi are well known to those skilled in the art.

[0242] Suitable mammalian cell types include, in particular: COS (e.g., ATCC No. CRL1650 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 promoters (e.g., viral materials such as polyomavirus, adenovirus 2, cytomegalovirus, and simian virus 40) as well as other transcriptional and translational control sequences. Examples of mammalian expression vectors include pCDM8 and pMT2PC.

[0243] VI. Pharmaceutical Composition

[0244] On the other hand, there are compositions comprising antibodies, immunoconjugates, nucleic acid molecules, vectors or recombinant cells as described herein, and optionally suitable diluents, such as pharmaceutically acceptable vectors.

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

[0246] Suitable diluents for peptides (including antibodies and / or cells) include, but are not limited to, saline solutions, pH buffer solutions, and glycerol solutions, or other solutions suitable for freezing peptides and / or cells.

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

[0248] In the embodiments, the composition is a pharmaceutical composition comprising any of the antibodies, nucleic acids or carriers disclosed herein, and optionally includes a pharmaceutically acceptable medium, such as a diluent or carrier.

[0249] The compositions described herein can be prepared by methods known per se, methods 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 carrier 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 the composition substantially compatible with the tissues or blood of the intended recipient. Other components that may be present in such compositions include water, surfactants (such as Tween), alcohols, polyols, glycerol, and vegetable oils. Immediate-use solutions and suspensions can be prepared from sterile powders, granules, and tablets, or concentrated solutions or suspensions. Compositions may, for example but not in a limited manner, be provided as lyophilized powders, which are reconstituted with sterile water or saline prior to administration to a patient.

[0251] Pharmaceutical compositions may contain a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include compositions that are substantially chemically inert and non-toxic, and do not interfere with the efficacy of the bioactivity 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-dioleoyloxy)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 the carrier to provide a form for direct administration to the patient.

[0252] The composition may be in the form of pharmaceutically acceptable salts, including but not limited to those salts that form with a free amino group, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those salts that form with a free carboxyl group, such as those derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, iron hydroxide, isopropylamine, triethylamine, 2-ethylethanolamine.

[0253] In one embodiment, the composition comprises the antibody described herein. In another embodiment, the composition comprises the antibody described herein and a diluent. In another embodiment, the composition is a sterile composition.

[0254] On the other hand, it includes antibody complexes containing antibodies that bind to FZD proteins (e.g., FZD4) as described herein. The complexes may be in solution or contained in tissues, optionally in vitro.

[0255] Methods for preparing and using the reagents described herein are also provided.

[0256] VII. Methods of application and use

[0257] The anti-FZD antibody 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 includes 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 liposomes. In some embodiments, the method further includes 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; for example, if the individual does not respond adequately to treatment, a higher dose of chemotherapy 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 medium 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 physiological saline, phosphate-buffered saline, physiologically buffered saline (135-150 mM NaCl), water, buffered water, 0.4% saline, 0.3% glycine, glycoproteins that provide enhanced stability (e.g., albumin, lipoprotein, globulin, etc.), and so on. Because a physiologically acceptable carrier is determined in part by the specific composition administered and the specific method of administration, a wide variety of suitable formulations of the pharmaceutical compositions of the present invention exist (see, for example, Remington's Pharmaceutical Sciences, 17th edition, 1989).

[0260] The compositions of this invention can be sterilized using 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; the lyophilized formulation is combined with the sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable excipients to approximate physiological conditions, such as pH adjusters and buffers, tonicants, wetting agents, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. Sugars may also be included for stabilizing the composition, such as stabilizers for lyophilized antibody compositions.

[0261] Dosage forms can be prepared for administration to a patient's mucous membranes (e.g., nasal, sublingual, vaginal, sublingual, or rectal), parenteral (e.g., subcutaneous, intravenous, intramuscular, or intra-arterial injection, bolus, or infusion), oral, or transdermal administration. Examples of dosage forms include, but are not limited to: dispersions, suppositories, ointments, pouches, pastes, powders, dressings, creams, plasters, solutions, patches, aerosols (e.g., nasal sprays or inhalers), gels; liquid dosage forms suitable for oral or mucous membrane administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil 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 reconfigured to provide a liquid dosage form suitable for parenteral administration to a patient.

[0262] Injectable (e.g., intravenous) compositions may comprise 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% dextran, etc., and may contain glycoproteins for enhancing stability, such as albumin, lipoproteins, globulins, etc. Typically, physiologically buffered saline (135-150 mM NaCl) will be used. The composition may contain pharmaceutically acceptable excipients to approximate physiological conditions, such as pH adjusters and buffers, tonicants, 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 (e.g., via intra-articular, intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes) include aqueous and non-aqueous, isotonic sterile injectable solutions that may contain antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Injectable solutions and suspensions can also be prepared from sterile powders, granules, and tablets. In the practice of this invention, the composition can be administered, for example, via intravenous infusion, topically, intraperitoneally, intravesically, or intrathecally. Parenteral and intravenous administration are preferred methods of administration. Formulations of the targeted composition can be presented in single-dose or multi-dose sealed containers (such as ampoules and vials).

[0264] The selected targeted delivery composition, alone or in combination with other suitable components, can be formulated into an aerosol formulation (“nebulization”) administered via inhalation. The aerosol formulation can be placed in a pressurized, acceptable propellant, such as dichlorodifluoromethane, propane, and nitrogen.

[0265] Pharmaceutical formulations can be packaged or prepared in unit dosage forms. In such forms, the formulation is subdivided into unit doses containing appropriate amounts of the active ingredient, for example, based on the dosage of the therapeutic agent or the concentration of the antibody. Unit dosage forms can be packaged formulations containing discrete amounts of the formulation. If desired, the composition may also contain other compatible therapeutic agents.

[0266] Antibodies (or antibody-targeting compositions) may be administered or used via injection or infusion through any suitable route, including but not limited to intravenous, subcutaneous, intramuscular, or intraperitoneal routes. Examples of administration of the pharmaceutical composition include storing the antibody at 10 mg / ml in a sterile isotonic saline solution for injection at 4°C, and diluting it in 100 ml or 200 ml of 0.9% sodium chloride for injection prior to administration to a patient. Antibodies are administered via intravenous infusion over a 1-hour period at doses between 0.2 mg / kg and 10 mg / kg. In other embodiments, antibodies are administered via intravenous infusion over a time period between 15 minutes and 2 hours. In still other embodiments, the administration procedure is via subcutaneous bolus injection.

[0267] The antibody dosage is selected to provide effective treatment to the patient, and the antibody dosage is in the range of less than 0.1 mg / kg body weight to about 25 mg / kg body weight, or in the range of 1 mg to 2 g per patient. In some cases, the dosage is in the range of 1-100 mg / kg or about 50 mg to 8000 mg per patient. Depending on the pharmacokinetics of the antibody (e.g., the half-life of the antibody in circulation) and the pharmacodynamic response (e.g., the duration of the antibody's therapeutic effect), the dosage can be repeated at an appropriate frequency, ranging from once daily to once every three months. In some embodiments, the in vivo half-life is between about 7 days and about 25 days, and antibody administration is repeated between once weekly and once every three months.

[0268] Application or use can be periodic. Depending on the route of administration, the dosage can be given once every 1, 3, 5, 7, 10, 14, 21, or 28 days or longer (e.g., once every 2, 3, 4, or 6 months). In some cases, administration is more frequent, such as 2 or 3 times daily. As those skilled in the art will recognize, the dosage and frequency of administration can be adjusted based on treatment progress and any adverse side effects.

[0269] Therefore, in some implementations, additional administration depends on the patient's progress, such as monitoring the patient between administrations. For example, after the first administration or a round of administration, the patient's tumor growth rate, recurrence (e.g., in the case of postoperative patients), or general disease-related symptoms such as weakness, pain, nausea, etc., may be monitored.

[0270] In therapeutic uses for treating cancer, antibody-targeted compositions (e.g., including therapeutic and / or diagnostic agents) may be administered at an initial dose of about 0.001 mg / kg to about 1000 mg / kg daily, adjusted over time. Daily dose ranges 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 may be used. The dose varies depending on the patient's needs, the severity of the condition being treated, and the targeted composition used. For example, the dose may be determined empirically, taking into account the type and stage of cancer diagnosed in a particular patient. In the case of this invention, the dose administered to the patient should be sufficient to achieve a beneficial therapeutic response over time. As will be appreciated by those skilled in the art, the magnitude of the dose will also be determined by the presence, nature, and extent of any adverse side effects accompanying the administration of the particular targeted composition in a particular patient.

[0271] VIII. Reagent Kit

[0272] On the other hand, there are kits or packages containing any antibodies, immunoconjugates, nucleic acid molecules, vectors, recombinant cells, and / or compositions included herein. Antibodies, immunoconjugates, nucleic acid molecules, vectors, recombinant cells, and / or compositions may be contained in vials such as sterile vials or other housings. As used herein, the term "kit" refers to... Figure 1 A collection of items used in the kit. The kit may optionally include a reference reagent and / or instructions for use. The kit may also include a shipping container suitable for containing containers (such as vials) containing compositions as disclosed herein.

[0273] IX. Methods using antibodies

[0274] The antibodies described in this article can be used in many in vitro and in vivo methods.

[0275] A. Methods for detecting FZD expression

[0276] As shown in this article, antibodies can be used to detect FZD expression.

[0277] Accordingly, this disclosure provides, in one aspect, a method for detecting FZD expression, comprising contacting a sample containing one or more cells with one or more antibodies or immunoconjugates described herein under conditions allowing for the formation of antibody:FZD complexes, and detecting the presence of any antibody complex. Typically, the antibody is part of an immunoconjugate, which contains an antibody conjugated to a detectable marker.

[0278] The sample may contain viable cells or cell extracts. The antibody:FZD complex can be detected by immunoassays such as immunofluorescence, flow cytometry, Western blotting, ELISA, SPR, and immunoprecipitation followed by SDS-PAGE and immunocytochemistry. In some embodiments, detection is by immunofluorescence. In some embodiments, detection is by flow cytometry.

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

[0280] B. Methods to inhibit the binding of WNT and FZD

[0281] This paper discloses antibody inhibition of Wnt binding to the coiled receptor, particularly FZD4. Not wishing to be limited by theory, the inhibition of Wnt-FZD protein binding affects signal transduction in which FZD plays a role in initiation. For example, antibody binding to the FZD receptor inhibits the promotion of FZD-mediated β-linkin phosphorylation. Unphosphorylated β-linkin evades destruction and accumulates in cells. β-linkin accumulation is associated with malignant tumors.

[0282] Reducing or inhibiting Wnt ligand signaling via FZD may be desirable. Therefore, another approach is to inhibit the binding of Wnt ligands to FZD or Wnt-induced transcriptional activity by contacting one or more cells expressing one or more FZD peptides with an effective amount of the antibody or immunoconjugate described herein.

[0283] In an embodiment, the antibody or immunoconjugate comprises a set of CDR sequences (all, light chain, or heavy chain) corresponding to an antibody selected from clones 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 implementation, the antibody or immunoconjugate comprises a set of CDR sequences (all, light chain, 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] Contact can be achieved in vivo, for example, by administering antibodies or immune conjugates to the subject. Such inhibition may be desirable, especially when Wnt signaling is dysregulated, as in cancer cells.

[0286] C. Methods of treating cancer

[0287] Methods of treating cancer include administering a pharmaceutical composition comprising an antibody that binds to FZD to a subject in need. The subject may be a person who has cancer or is at risk of cancer (such as cancer recurrence).

[0288] Without being limited by theory, such treatments can work by inhibiting the activation of the typical Wnt pathway, for example, by inhibiting the binding of Wnt to FZD, by inhibiting Wnt-induced transcriptional activity, by inhibiting the activation of scattered proteins, by inhibiting the inhibition of the β-linkin disruption complex, and by promoting the accumulation of β-linkin.

[0289] This disclosure, in another aspect, includes a method for treating cancer, comprising administering an effective amount of an antibody or immunoconjugate to a subject in need, the antibody or immunoconjugate specifically binding to FZD1, FZD2, FZD4, FZD5, FZD7, FZD8, and FZD9 in at least one assay and inhibiting Wnt-induced signaling in at least one assay. This disclosure also includes the use of an effective amount of an antibody or immunoconjugate for treating cancer, the antibody or immunoconjugate specifically binding to FZD1, FZD2, FZD4, FZD5, FZD7, FZD8, and FZD9 in at least one assay and inhibiting Wnt-induced signaling in at least one assay. This disclosure further provides the use of an effective amount of an antibody or immunoconjugate for treating cancer, the antibody or immunoconjugate specifically binding to FZD1, FZD2, FZD4, FZD5, FZD7, FZD8, and FZD9 in at least one assay and inhibiting Wnt-induced signaling in at least one assay. This disclosure also provides the use of effective amounts of antibodies or immunoconjugates in the preparation of medicaments for treating cancer, wherein the antibodies or immunoconjugates specifically bind to FZD1, FZD2, FZD4, FZD5, FZD7, FZD8 and FZD9 in at least one assay and inhibit Wnt-induced signaling in at least one assay.

[0290] In the implementation scheme, an antibody or immune conjugate, such as an antibody-drug conjugate, is included in the 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 pancreatic cancer. In another embodiment, the antibody or immunoconjugate comprises a set of CDR sequences (all, light chain, or heavy chain) corresponding to antibodies selected from 5014, 5017-5023, 5025, 5035-5037, 6495, and 6500.

[0292] As demonstrated in this paper, antibodies can also inhibit cancer cell proliferation. Therefore, a method for inhibiting cancer cell proliferation is also provided, 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 the implementation scheme, the antibody or immunoconjugate is the antibody or immunoconjugate described herein, for example, an antibody or immunoconjugate comprising a set of CDR sequences (all, light chain, or heavy chain) corresponding to antibodies selected from 5014, 5017-5023, 5025, 5035-5037, 6495, and 6500.

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

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

[0296] In another implementation, the cancer cells are selected from colon cancer cells, lung cancer cells, breast cancer cells, ovarian cancer cells, endometrial cancer cells, pancreatic cancer cells, gastric cancer cells, liver cancer cells, adrenocortical cancer cells, and osteoblastoma cells.

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

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

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

[0300] The foregoing disclosure provides a general description of this disclosure. A more complete understanding can be obtained by referring to the following specific embodiments. These embodiments are described for illustrative purposes only and are not intended to limit the scope of this application. Variations of form and substitutions of equivalents are also contemplated where circumstances may suggest or provide convenience. Although specific terminology has been used herein, such terminology is intended to be descriptive and not for limiting purposes. X. Example

[0301] Example 1

[0302] Antibody selection and functional testing of FZD4 FAB

[0303] Antibody selection: Two selection methods were used to identify FZD4 conjugates.

[0304] 1. Selection was performed using a library designed based on previously developed FZD7-derived conjugates. Antibodies had previously been identified through selection using FZD7 CRD-Fc as the antigen. These antibodies bound to FZD1, FZD2, FZD5, FZD7, FZD8, and FZD9 and exhibited antagonistic activity against the Wnt pathway and inhibition of pancreatic cancer cell proliferation and tumor growth. This library was used to identify antibodies that bind to FZD4 and possess both Wnt antagonistic and antitumor activity.

[0305] a). Design and preparation of Fab phage display libraries.

[0306] The design and preparation of Fab phage display libraries were accomplished using an IPTG-inducible display vector encoding Fab that recognizes MBP. The Fab template was identical to library F and included a FLAG-tagged light chain. The dimerization domains L1, L2, and L3, mutated to the parental Fab sequences H1, H2, and H3, were soft randomized to allow 50% wild-type amino acid bias and 50% bias towards any other amino acid (using a 70:10:10:10 nucleotide mix). All six CDR regions were mutated in a single kunkel mutagenesis reaction. A second-generation library was constructed based on the Fab antagonist group. An IPTG-inducible display vector encoding Fab specific for maltose-binding proteins was used as the library template. Site-specific kunkel mutagenesis was performed using light chain oligonucleotides to mutate CDRs L1, L2, and L3 to the parental Fab sequences and soft randomize CDRs H1, H2, and H3 (50% wild-type and 50% any other amino acid). The purified mutagenic material was electroporated into SR320 cells pre-infected with M13 K07. The library was rescued overnight in 500 ml of culture, double-precipitated with PEG / NaCl, and resuspended in PBS containing 50% glycerol for storage at -20°C.

[0307] b) Selection and production of FZD4 Fab

[0308] Second-generation libraries (e.g., CFU) were collected and screened four times for recombinant FZD4 cysteine-rich domain (CRD) fused with the Fc tag (R&D systems). Input and output phage titers for carbenicillin (CARB)-resistant library phages and kanamycin (KAN)-resistant helper phages were calculated. Maxisorp plates were coated overnight at 4°C with 5 μg / ml FZD4-CRD-Fc or Fc protein in PBS, and wells were numbered and blocked with 0.5% BSA. Coated wells were washed four times with PBS / 0.5% Tween 20 (wash buffer), and library phages (PEG-precipitated and resuspended in 0.5% BSA / 0.05% Tween 20 / PBS) were first incubated in Fc protein wells at room temperature for 1 hour. Unbound phages were transferred to blocked FZD4-CRD-Fc plates and incubated at room temperature for 1 hour. Wash wells as shown, and then elute with 100 mM HCl. The eluted phages were amplified in the laboratory using standard protocols for subsequent rounds of selection. Input and output titers for the library phages (carb-resistant) and helper phages (kan-resistant) were specified. DNA from the site-specific Kunkel mutagenesis of the output phage library from the FZD4 selection (designed to add a His6-succinate terminator between phage gene III and Fab CH1) was transformed into Omnimax cells and plated to obtain single colonies. Single colonies were used to inoculate 96-well cassettes, and overnight phage supernatant was diluted 1:2 in 0.05% Tween 20 / 0.5% BSA / PBS (dilution buffer) to test ELISA binding. Phages were detected with anti-M13-HRP secondary antibody (1:5000 in dilution buffer), and the plates were developed with TMB substrate and acid-stopped staining. The absorbance of FZD4-Fc coated wells and control Fc coated wells at 450 nm was read, as shown. Heavy and light chains were sequenced to determine the CDR sequence of individual Fab proteins. The CH1-gene III adapter was also sequenced to determine the successful incorporation of the His tag and amber stop codon for Fab expression. The phage conjugate was cloned into a bacterial expression vector and purified as the Fab protein for characterization.

[0309] c). Characterization of FZD4 conjugates.

[0310] The CDR sequences of the antibodies described here are shown in Table 1. First, phage conjugates are tested in an ELISA assay to confirm their binding to the antigen. (As...) Figure 1As shown, all phage clones bound to FZD4 CRD-Fc but not to the Fc protein alone, indicating that these phage conjugates bind to FZD4 CRD but not to Fc. Secondly, purified Fab was tested in the presence of an incremental amount of non-immobilized antigen in a competitive ELISA assay to assess their binding affinity. Figure 2 In the presence of 50 nM competitive free antigen (FZD4 CRD), binding of all Fabs decreased by more than 50%. Binding of five Fabs (5022, 5031, 6497, 6498, and 6500) decreased by more than 50% in the presence of 10 nM competitive antigen (FZD4 CRD), suggesting that these five Fabs may have higher binding affinity than the remaining Fabs. It should be noted that binding of Fab 5025 and Fab 6494 was not observed in this assay. The reason for this is unknown, but it may be due to physical interference. Next, the binding of Fabs to FZD4 expressed on the cell surface was tested by immunofluorescence staining. Membrane staining patterns of all FZD4 Fabs were observed on CHO cells stably expressing FZD4. Figure 3 ), but not on CHO cells ( Figure 4 IF staining was also used to determine whether these Fab cells bind to other FZDs. Binding was tested on 10 CHO cell lines that stably express individual FZDs on their surfaces. Figure 5 As shown, Fabs exhibited different binding profiles. Fab 5017, Fab 5027, Fab 5030, and Fab 6499 bound only to CHO cells expressing FZD4, while other Fabs (5014, 5018-5023, 5025, 6495, 6496) bound to more than one FZD, including FZD1, FZD2, FZD4, FZD5, FZD7, FZD8, and FZD9. However, no Fab showed binding to FZD3, FZD6, or FZD10 in this assay. Next, Fab 5019 and Fab 5020 were used as examples, and their binding to cancer cells was tested by both immunofluorescence staining and flow cytometry. Figure 6 As shown, both Fabs exhibited clear, concentrated staining on the membrane in the five pancreatic cell lines tested. Flow cytometry confirmed the binding of these Fabs to these cancer cell lines, such as... Figure 7 As shown in the image.

[0311] 2. Use the initial (naive) Fab library (Library F) for selection.

[0312] a) Recombinant FZD4-CRD-Fc was used to select Fabs that bind to FZD4. Specifically, a Fab phage library (library F) was pre-cleaned to remove non-specific binders of unrelated proteins. Several rounds of selection were performed on the pre-cleaned Fab library to enrich binders that bind to FZD4-CRD-Fc. Selected clonal phages were screened by ELISA for binders that bind to FZD4-CRD-Fc but not specifically to Fc. Forty-four Fabs with unique CDR sequences were identified (sequences are shown in Table 3).

[0313] b) Characterization of resistance to FZD4 Fab

[0314] The anti-FZD4 phage conjugate clone was then cloned into a bacterial expression vector, and 42 Fabs were expressed and purified for further characterization. Multiple methods were used to determine FZD binding selectivity. First, the purified Fabs were tested in an ELISA assay to confirm their binding to the recombinant antigen (FZD4-CRD-Fc) and their binding to other FZDs (FZD1, 2, 5, 6, 7, 8, 9, and 10). Figure 8 As shown, all Fabs were observed to bind to FZD4-CRD-Fc (the antigen used for selection), but rarely to Fc or the unrelated protein BSA. Furthermore, these Fabs also showed varying degrees of binding to other FZDs. Figure 8 For example, Fab was detected to bind in small amounts to FZD2 CRD-Fc and FZD8 CRD-Fc, in addition to binding to FZD4-CRD-Fc. Figure 8 A). Fab 5076, in addition to being combined with FZD4, is also combined with FZD1, FZD2, FZD5, FZD7, and FZD8. Figure 8 D). Next, the binding of anti-FZD4 Fab to various FZDs was determined by immunofluorescence staining of FZD-expressing CHO cells, and the results are summarized in Figure 9. In this assay, most Fabs showed binding only to FZD4 (including Fabs 5038, 5040-5044, 5046, 5047, 5049-5053, 5055, 5058-5064, 5066, 5068-5072, and 5077-5081), while other Fabs bound to two or more FZDs. Furthermore, surface plasmon resonance (SPR) was used to determine the binding affinity of anti-FZD4 Fab to FZD4 CRD-Fc. Figure 10 As shown in the figure. FZD4-derived Fabs exhibit 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. Figure 11As outlined in the report, most Fabs were able to bind HPAFII and PATU8988s. Limited binding (PATU8988s cells) was observed in Fab 5049, Fab 5064, and Fab 5072.

[0315] Functional testing of FZD4 Fab: Several assays were performed to characterize the resistance to FZD4 Fab.

[0316] 1. Effect of anti-FZD4 Fab on the binding of Wnt ligand to FZD4-CRD. To optimize assay conditions, incremental concentrations of FZD4-CRD-Fc or Fc were mixed with biotinylated Wnt5A, and the complex was captured by streptavidin-coated plates. The binding of FZD4-CRD-Fc or Fc was detected by anti-Fc-HRP. Figure 12 A). The blocking activity against FZD4 Fab was tested by selecting an FZD4-CRD-Fc concentration that provided an ELISA signal within a linear range. In the presence of various indicated Fabs, FZD4-CRD-Fc was mixed with biotinylated Wnt5A, and the bound FZD4-CRD-Fc was detected by anti-Fc-HRP, as shown in Figure 1. Figure 12 As shown in A. (As indicated by...) Figure 12 As shown in B, greater than 80% binding inhibition was observed for Fab 5014, 5017-5023, 5027-5031, 5034, 5036, 5037, 6496, 6498, 6499, and 6500; greater than 60% binding inhibition 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.

[0317] 2. Effects of anti-FZD4 Fab on β-linkin-driven transcription. To observe whether anti-FZD4 Fab affects β-linkin-dependent signaling, the effect of Fab on Wnt3a-induced transcriptional activity was tested in a TOPFLASH assay. Figure 13 As shown, effective inhibitory activity (>80%) was observed for Fab 5014, 5018-5023, 5025, 5036, 5037, and 6495. Smaller amounts of inhibition (10%-35%) were observed for Fab 5027-5031 and 6497-6499; no inhibition was observed for Fab 5017, 5034, and 5035.

[0318] 3. Effects 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 Fab, and cell proliferation was measured (see [link to study]). Figure 14 AH). Data in Figure 15 Overview. Fabs exhibiting dose-dependent anti-proliferative effects include: Fab 5018-5021, 5023, 5036, and 6495. Fab 5022 and 6500 were tested only at a single dose level (2 μg / ml) and were inhibitory against both tested cell lines. In the assay, several Fabs (5017, 5025, 5035, and 5037) showed inhibitory effects against the HPAFII cell line. Based on... Figure 16 The data summarized in [the original text] show that the antiproliferative activity of anti-FZD4 Fabs is associated with the following observations: their binding to FZD1, FZD2, FZD4, FZD5, FZD7, FZD8, and FZD9, and their ability to inhibit Wnt3a-induced transcriptional activity. The most potent antiproliferative Fabs include 5014, 5019-5023, and 6495 (…). Figure 16 ).

[0319] 4. Effects on the expression of the Wnt-regulated gene Axin2. To further characterize the anti-FZD4 antibody, several Fab strains were converted into IgG. IgG 5020, Fab 5019, and Fab 5020 were tested in gene expression assays, where the mRNA level of the Axin2 gene was measured by RT-qPCR. Figure 17 As shown in the figure, after antibody treatment, IgG5020, Fab 5019 and Fab 5020 all reduced the level of Axin2 mRNA in HPAFII cells, indicating that these antibodies inhibit the Wnt pathway.

[0320] 5. Effects of anti-FZD4 IgG on cancer cell proliferation. The effects of IgG on the proliferation of four pancreatic cancer cell lines—HPAFII, CAPAN2, AsPC11, and PATU8988s—were also tested. These cell lines are known to contain a disruptive mutation in the RNF43 gene. As determined using Almar Blue assays... Figure 18 As shown in Figure A, the proliferation of these pancreatic cells was inhibited by IgG 5019 and IgG 5020, as well as their corresponding Fab. Furthermore, IgG 5020 showed a dose-dependent inhibition of cancer cell proliferation. Figure 18B). Interestingly, several pancreatic cancer cell lines (BxPC3 and PANC 1) without disruptive mutations in the RNF43 gene were also tested, but they 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 targeting coiled receptors. Loss-of-function mutations in RNF43 and ZNRF3 lead to high expression of FZD and may sensitize tumor cells to inhibition of Wnt-dependent signaling. To observe whether the FZD4 antibody also affects colony formation, IgG5020 was tested on five pancreatic cancer cell lines. Figure 19 ).and Figure 18 Consistent with the results, IgG5020 inhibited colony formation in cell lines carrying the RNF43 mutation (HPAFII, AsPC1, and PATU8988s), but did not inhibit colony formation in cell lines without the RNF43 mutation (BxPC3 and PANC 1).

[0321] 6. The effect of anti-FZD4 Fab binding to tumor-derived cells from pancreatic cancer patients on their proliferation. Next, the binding of Fab 5019 and 5020 to pancreatic cancer patient tumor-derived cells (PDX) was tested by immunofluorescence staining. Figure 20 As shown, membrane staining patterns were clearly observed in both Fab cell lines, PDX cell lines GP2A and GP14A, and in the pancreatic cell line CAPAN 2. Furthermore, these antibodies were tested in cell proliferation assays. Figure 21 ).and Figure 18 and 19 Consistent with previous observations, 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.

[0322] In vivo efficacy studies are underway to demonstrate the antitumor activity of these publicly disclosed anti-FZD antibodies.

[0323] XI. Exemplary Implementation Scheme

[0324] 1. An antibody that specifically binds to a cysteine-rich domain (CRD) of one or more of a human coiled receptor 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 complementation-determining regions CDR-H1, CDR-H2, and CDR-H3, the light chain variable region comprising complementation-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the amino acid sequence of the CDR comprises or consists of sequences selected from or consist ...

[0325] 2. The antibody according to embodiment 1, wherein the amino acid sequence of the CDR comprises or consists of a sequence selected from the sequences listed below:

[0326] 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;

[0327] 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;

[0328] CDR-H3 is selected from the group consisting of: YHPFGYAL, 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;

[0329] CDR-L1 is SVSSA;

[0330] CDR-L2 is SASSLYS; and / or

[0331] CDR-L3 can be selected from the following groups: AAYHWPPLF, GVYLF, SSYSLI, WAYGPF, YYHPI, and YYSLF.

[0332] 3. The antibody according to embodiment 1, wherein the amino acid sequence of the CDR comprises or consists of sequences selected from the following:

[0333] 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;

[0334] 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;

[0335] 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;

[0336] CDR-L1 is SVSSA;

[0337] CDR-L2 is SASSLYS; and / or

[0338] 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.

[0339] 4. The antibody according to embodiment 2 or 3, wherein the antibody comprises a heavy chain variable region, the heavy chain variable region comprising:

[0340] i) Heavy chain amino acid sequences as listed in Table 2;

[0341] 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%, or at least 99% sequence identity with the heavy chain amino acid sequences listed in Table 2, wherein the CDR sequence is a set of CDR sequences listed in Table 1a or Table 3a, or

[0342] iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is a set of CDR sequences listed in Table 1a or Table 3a.

[0343] 5. The antibody according to any one of embodiments 2 to 4, wherein the antibody comprises a light chain variable region, the light chain variable region comprising:

[0344] i) Light chain amino acid sequences as listed in Table 2;

[0345] ii) An amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the light chain amino acid sequences listed in Table 2, wherein the CDR sequence is a set of CDR sequences listed in Table 1a or Table 3a; or

[0346] iii) The conserved substituted amino acid sequence of i), wherein the CDR sequence is a set of CDR sequences listed in Table 1a or Table 3a.

[0347] 6. The antibody according to any one of embodiments 1 to 5, wherein the CDR sequence is a set of complete CDR sequences selected from the antibodies identified in Table 1a or Table 3a.

[0348] 7. The 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.

[0349] 8. The antibody according to any one of embodiments 1 to 7, wherein the antibody specifically binds to FZD4.

[0350] 9. The antibody according to embodiment 8, wherein the CDR sequence is a set of CDR sequences of antibodies 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.

[0351] 10. The antibody according to any one of embodiments 1 to 7, wherein the antibody specifically binds to FZD4 and at least one other FZD receptor selected from FZD1, FZD2, FZD5, FZD7, FZD8 and FZD9.

[0352] 11. The antibody according to embodiment 9, wherein the CDR sequence is a set of CDR sequences of antibodies 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.

[0353] 12. The antibody according to any one of embodiments 1 to 11, wherein the antibody preferentially binds to coiled receptor 4 (FZD4) compared to FZD1, FZD2, FZD5, FZD7, FZD8 or FZD9.

[0354] 13. The antibody according to any one of embodiments 1 to 11, wherein the antibody preferentially binds to FZD4 relative to another FZD receptor.

[0355] 14. The antibody according to embodiment 13, wherein the CDR sequence is a set of CDR sequences of antibodies selected from the following antibodies: 5028, 5029, 5031, 5034, 5035, 6497, 6498, 5039, 5045, 5048, 5054, 5056, 5057, 5067, 5073, 5074, 5075.

[0356] 15. The antibody according to any one of embodiments 1 to 13, wherein the antibody has a binding affinity between about 0.2 nM and about 15.3 nM as measured by surface plasmon resonance.

[0357] 16. The antibody according to any one of embodiments 1 to 15, wherein the antibody is a monoclonal antibody.

[0358] 17. The antibody according to any one of embodiments 1 to 16, wherein the antibody is a humanized antibody.

[0359] 18. The antibody according to any one of embodiments 1 to 17, wherein the antibody is a single-chain antibody.

[0360] 19. The antibody according to any one of embodiments 1 to 18, wherein the antibody is selected from antibody-binding fragments selected from Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, nanobodies, microbodies, biantibodies, and multimers.

[0361] 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 quadrivalent antibody.

[0362] 21. The antibody according to any one of embodiments 1 to 18, wherein the antibody is a bispecific antibody that also binds to LPR 5 / 6.

[0363] 22. The antibody according to any one of embodiments 1 to 18, wherein the antibody comprises a non-natural glycosylation pattern.

[0364] 23. The antibody according to any one of embodiments 1 to 18, wherein the antibody comprises cysteine ​​substitution or addition in a constant region or frame region.

[0365] 24. The antibody according to any one of embodiments 1 to 18, wherein the antibody blocks the binding of Wnt to FZD.

[0366] 25. An immunoconjugate comprising an antibody and a detectable marker or cytotoxic agent as described in any one of embodiments 1 to 21.

[0367] 26. The immunoconjugate according to embodiment 25, wherein the immunoconjugate comprises a cytotoxic agent selected from: maytansine alkaloids, auristatin, dolalastatin, tubulysin, novozygain, pyrrolobenzodiazepine (PBD) dimers, indobenzobenzodiazepine dimers, α-amaminine, trichothene, SN-38, pyroximide, CC1065, cazithromycin, enediyne antibiotics, taxanes, doxorubicin derivatives, anthracyclines and their stereoisomers, azanofide, isosteres, analogs or derivatives.

[0368] 27. A nucleic acid molecule, said nucleic acid molecule encoding an antibody according to any one of embodiments 1 to 21.

[0369] 28. The nucleic acid molecule according to embodiment 27, wherein one or more of the CDR sequences are encoded by nucleic acids in Table 1b, Table 1c, Table 3b or Table 3c.

[0370] 29. The nucleic acid molecule according to embodiment 27, wherein the antibody comprises a heavy chain variable region encoded by a nucleic acid, the nucleic acid comprising:

[0371] i) Heavy chain nucleic acid sequences as listed in Table 2;

[0372] ii) A nucleotide 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 listed in Table 2, wherein the CDR sequence is a set of CDR sequences listed in Table 1a or Table 3a, or

[0373] iii)i) codon degenerate nucleic acid sequences, wherein the CDR sequences are sets of CDR sequences listed in Table 1a or Table 3a.

[0374] 30. The nucleic acid molecule according to embodiment 27, wherein the antibody comprises a light chain variable region encoded by a nucleic acid, the nucleic acid comprising:

[0375] i) Light chain nucleic acid sequences as listed in Table 2,

[0376] ii) Nucleic acid sequences having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the light chain nucleic acid sequences listed in Table 2, wherein the CDR sequence is a set of CDR sequences listed in Table 1a or Table 3a, or

[0377] iii)i) codon degenerate nucleic acid sequences, wherein the CDR sequences are sets of CDR sequences listed in Table 1a or Table 3a.

[0378] 31. A vector comprising an expression control sequence operatively linked to a nucleic acid according to any one of embodiments 27 to 30.

[0379] 32. A host cell comprising a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising an expression control sequence operatively linked to a nucleic acid according to any one of embodiments 27 to 30.

[0380] 33. The host cell according to embodiment 32, wherein the host cell is a Chinese hamster ovary (CHO) cell.

[0381] 34. A host cell comprising the vector described in embodiment 31.

[0382] 35. A method for preparing an anti-FZD antibody, the method comprising culturing a host cell according to any one of embodiments 32 to 34.

[0383] 36. A composition comprising an antibody as described in any one or more of embodiments 1 to 24, an immunoconjugate as described in embodiments 25-26, a nucleic acid molecule as described in embodiments 27-30, a vector as described in embodiment 31 or a host cell as described in embodiments 34-34, and optionally a suitable diluent.

[0384] 37. The composition according to embodiment 3636, wherein the composition comprises one or more antibodies or immunoconjugates, optionally wherein the composition is a pharmaceutical composition.

[0385] 38. A kit comprising an antibody as described in any one or more of embodiments 1 to 24, an immunoconjugate as described in embodiments 25-26, a nucleic acid molecule as described in embodiments 27-30, a vector as described in embodiment 31, or a host cell as described in embodiments 34-34.

[0386] 39. A method for detecting FZD expression, the method comprising contacting a sample containing one or more cells with an antibody or immunoconjugate of any one or more embodiments 1 to 26 under conditions that allow for the formation of antibody:cell complexes, and detecting the presence of any antibody complex.

[0387] 40. The method according to embodiment 39, wherein the detection is performed by immunofluorescence.

[0388] 41. The method according to embodiment 39, wherein the detection is performed by flow cytometry.

[0389] 42. The method according to 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 antibodies selected from the following: 5017, 5027, 5030, 6499, 5038, 5040-5044, 5046, 5047, 5049-5053, 5055, 5058-5064, 5066, 5068-5072, and 5077-5081.

[0390] 43. A method for inhibiting the binding of Wnt ligand to FZD receptor, disrupting the Wnt signaling pathway, inhibiting Wnt-induced transcriptional activity, inhibiting scattered protein activation, promoting the maintenance of the β-linkin destruction complex, promoting β-linkin accumulation, or inhibiting cell growth, said method comprising contacting cells expressing the FZD receptor with an antibody or immunoconjugate as described in any one of embodiments 1 to 26.

[0391] 44. The method according to embodiment 43, wherein the Wnt ligand is Wnt3a.

[0392] 45. The method according to embodiment 43, wherein the antibody or immunoconjugate comprises a set of CDR sequences corresponding to antibodies selected from: 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.

[0393] 46. ​​A method of treating cancer in a subject with a corresponding need, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising an antibody or immune conjugate according to any one of embodiments 1 to 26.

[0394] 47. The method according to 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.

[0395] 48. The method according to embodiment 46, wherein the cancer is selected from acute myeloid leukemia, neuroblastoma, liver cancer, lung cancer, endometrial cancer, salivary gland 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.

[0396] 49. The method according to embodiment 46, wherein the antibody or immunoconjugate specifically binds to FZD1, FZD2, FZD4, FZD5, FZD7, FZD8 and FZD9 in at least one assay and inhibits Wnt3a-induced signal transduction in at least one assay, optionally wherein the antibody or immunoconjugate is the antibody or immunoconjugate of any one of embodiments 1 to 26.

[0397] 50. The method according to embodiment 46, wherein the antibody or immunoconjugate comprises a set of CDR sequences corresponding to antibodies selected from: 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.

[0398] 51. The method according to embodiment 46, wherein the antibody or immunoconjugate comprises a set of CDR sequences corresponding to antibodies selected from 5019 and 5020.

[0399] 52. The method according to embodiment 51, wherein the cancer treated by the method comprises one or more cancer cells containing a mutation of the RNF43 gene, and the antibody or immune conjugate comprises a set of CDR sequences corresponding to antibody 5020.

[0400] As used herein, unless otherwise specified, the following meanings apply. The word “may” is used in a permissive sense (i.e., meaning possible), not a mandatory sense (i.e., meaning required). The words “include,” “including,” and “includes” mean including, but not limited to. The singular forms “a,” “one,” and “the” include plural indicators. Thus, for example, references to “an element” include a combination of two or more elements, although other terms and wording, such as “one or more,” are used for one or more elements. The wording “at least one” includes “one or more,” “one or a plurality,” and “a plurality.” Unless otherwise specified, the term “or” is non-exclusive, i.e., encompasses both “and” and “or.” The term "any one of..." between a modifier and a sequence means that the modifier modifies each member of the sequence. Thus, for example, the phrase "any one of at least 1, 2, or 3" means "at least 1, at least 2, or at least 3". The term "consisting primarily of..." means including the listed elements and other elements that do not substantially affect the essential and novel features of the claimed combination.

[0401] As used in this paper, degree terms such as “about,” “substantially,” and “approximately” indicate a reasonable amount of deviation of the modified term such that the final result is not significantly altered. These degree terms should be interpreted as including at least ±5% deviation of the modified term, provided that such deviation does not invalidate the meaning of the word it modifies.

[0402] Furthermore, as those skilled in the art will understand, the definitions and embodiments described in particular sections are intended to be applied to other embodiments described herein to which they apply. For example, different aspects of the invention are defined in more detail in the following paragraphs. Each aspect so defined may be combined with any other one or more aspects unless the contrary is clearly indicated. In particular, any feature indicated as preferred or advantageous may be combined with any other one or more features indicated as preferred or advantageous.

[0403] It should be understood that this specification and accompanying drawings are not intended to limit the invention to the specific forms disclosed, but rather, on the contrary, to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims. Other modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art based on this specification. Therefore, this specification and accompanying drawings should be construed as illustrative only and for the purpose of teaching those skilled in the art the general manner in which the invention is carried out. It should be understood that the forms of the invention shown and described herein should be considered as examples of embodiments. Elements and substances described herein may be substituted, components and processes may be reversed or omitted, and certain features of the invention may be used independently, all of which will be apparent to those skilled in the art who will understand the benefits of this specification. Changes may be made to the elements described herein without departing from the spirit and scope of the invention as set forth in the appended claims. The headings used herein are for organizational purposes only and are not intended to limit the scope of this specification.

[0404] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference.

Claims

1. An antibody that specifically binds to the cysteine-rich domain of the human coiled receptor FZD4, the antibody comprising a light chain variable region and a heavy chain variable region, the heavy chain variable region comprising complementation-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprising complementation-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the amino acid sequence of the CDRs consists of the sequences 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: 103, CDR-L2, consisting of SEQ ID NO: 104, and CDR-L3 consisting of SEQ ID NO:

111.

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

300.

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

5. According to the claims 1 The antibody, wherein the antibody is a monoclonal antibody.

6. According to the claims 1 The antibody, wherein the antibody is a humanized antibody.

7. According to the claims 1 The antibody, wherein the antibody is a single-chain antibody.

8. According to the claims 1 The antibody, wherein the antibody is selected from the antibody-binding fragments selected from Fab, F(ab')2, scFv, dsFv, ds-scFv, their biantibodies and multimers.

9. According to the claims 1 The antibody, wherein the antibody is a multivalent antibody that is a bivalent, trivalent, or quadrivalent antibody.

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

11. According to the claims 1 The antibody described herein contains a non-natural glycosylation pattern.

12. According to the claims 1 The antibody comprises cysteine ​​substitutions or additions in a constant region or frame region.

13. According to the claims 1 The antibody described herein blocks the binding of Wnt to FZD.

14. An immunoconjugate comprising the claims 1 to 10 The antibody and detectable marker or cytotoxic agent described in any one of the following.

15. According to the claims 14 The immunoconjugate comprises a cytotoxic agent selected from the following: maytansine alkaloids, auristatin, dolalastatin, tubulysin, novozygain, pyrrolobenzodiazepine (PBD) dimer, indobenzobenzodiazepine dimer, α-amaminine, trichothene, SN-38, pyroximin, CC1065, chalcogenin, enediyne antibiotics, taxanes, doxorubicin, and anthracyclines.

16. A nucleic acid molecule, said nucleic acid molecule encoding the claims 1 to 10 The antibody mentioned in any one of the above.

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

303.

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

301.

19. A carrier comprising the claims 16 to 18 The nucleic acid mentioned in any one of the above.

20. According to the claims 19 The vector wherein the nucleic acid is operatively linked to an expression control sequence. twenty one. A host cell comprising a recombinant nucleic acid molecule, the recombinant nucleic acid molecule comprising the claims of the present invention. 16 to 18 The nucleic acid mentioned in any one of the following, wherein the host cell is not a plant cell.

22. According to claim 21 The host cell wherein the nucleic acid is operatively linked to an expression control sequence. twenty three. According to the claims 21 or 22 The host cell is a Chinese hamster ovary (CHO) cell. twenty four. A host cell, said host cell comprising the claims 19 or 20 The carrier wherein the host cell is not a plant cell.

25. A method for preparing anti-FZD antibody, the method comprising culturing as claimed in the claim 21 to 24 The host cell mentioned in any one of the above.

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

27. According to the claims 26 The composition further comprises a suitable diluent.

28. According to the claims 26 The composition, wherein the composition comprises an antibody or an immunoconjugate.

29. According to claims 28 The composition described herein, wherein the composition is a pharmaceutical composition.

30. A kit comprising the claims 1 to 13 The antibody mentioned in any one of the claims, 14-15 The immunoconjugate described in any one of the claims, 16-18 The nucleic acid molecule mentioned in any one of the claims 19 or 20 The carrier or as claimed in the claims 21-24 The host cell mentioned in any one of the above.

31. Claims 1 to 13 The antibody or claim in any one of the following statements 14 to 15 The use of any one of the immunoconjugates in the preparation of a kit for detecting FZD expression, wherein, when using the kit, a sample containing one or more cells is contacted with the antibody or immunoconjugate under conditions that allow for the formation of antibody:cell complexes, and the presence of any antibody complex is detected.

32. According to the claims 31 The described use, wherein the detection is performed by immunofluorescence.

33. According to claims 32 The described use, wherein the detection is performed by flow cytometry.

34. Including claims 1 to 13 The antibody or claim in any one of the following statements 14 to 15 Use of any one of the immunoconjugate pharmaceutical compositions 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, and glioblastoma.

35. Including the claims 1 to 13 The antibody or claim in any one of the following statements 14 to 15 Use of any one of the immunoconjugate pharmaceutical compositions in the preparation of a medicament for treating cancer, wherein the cancer is selected from acute myeloid leukemia, neuroblastoma, salivary gland-like cystic carcinoma, colorectal cancer, prostate cancer, bladder cancer, cervical cancer, esophageal cancer, glioma, astrocytoma, and osteosarcoma.

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