Monoclonal Antibodies Against Human Frizzled Receptors and Their Uses

Abstract

The present invention relates to an antibody or an antigen-binding fragment thereof, which binds to a Frizzled receptor, preferably a human Frizzled receptor, and the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment that binds to a class I site in FZD10, or an antibody or antigen-binding fragment that binds to a class II site in a Frizzled receptor.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to monoclonal antibodies targeting human Frizzled receptors and their uses. Background Art

[0002] The Wnt signaling pathway is an extremely important signal transduction pathway in the human body. Wnt ligands activate downstream signaling pathways by binding to Frizzled receptors (FZD), regulating various biological processes such as embryonic development, organ formation, and maintenance of stem cell pluripotency. Frizzled receptors have a cysteine-rich N-terminal domain (CRD) and a transmembrane domain composed of seven transmembrane helices. As the main receptors of the Wnt signaling pathway, 10 types (FZD1 - FZD10) have been found in the human body. According to the sequence differences in the CRD region, Frizzled receptors can be divided into four subfamilies: FZD1 / 2 / 7, FZD5 / 8, FZD4 / 9 / 10, and FZD3 / 6. There is a complex cross-binding network between Frizzled receptors and Wnt ligands. Each receptor can interact with multiple Wnt ligands, and each Wnt ligand can also bind to multiple Frizzled receptors.

[0003] According to the reported complex crystal structure model, Wnt ligands bind to two epitopes in the CRD domain of Frizzled receptors simultaneously through the "thumb" domain at the amino terminus and the "index finger" domain at the carboxyl terminus, which are named class I site and class II site respectively. Structural and sequence analysis show that the class I site is quite conserved, especially in FZD1 / 2 / 7 and FZD5 / 8, while the class II site is relatively less conserved.

[0004] Studies have shown that the Wnt signaling pathway is involved in the proliferation, invasion, and metastasis of tumor tissues and can regulate tumor angiogenesis. For example, the FZD2 receptor is involved in the epithelial-mesenchymal transition and metastasis of various tumors. The FZD7 receptor is abnormally expressed in various types of cancer tissues such as triple-negative breast cancer, non-small cell lung cancer, and liver cancer, and promotes tumor cell proliferation and metastasis by activating the canonical Wnt signaling pathway. The FZD10 receptor is significantly upregulated in approximately 40% of primary gastric cancers, primary colon cancers, and most synovial sarcoma tissues.

[0005] Given the important role of the dysregulated Wnt signaling pathway in tumorigenesis and development, treating tumors by inhibiting the overactivation of the Wnt signaling pathway has always been a hot topic in life science research. So far, a variety of small molecule Wnt signaling pathway inhibitors have been developed and entered clinical trials, such as the Porcupine enzyme inhibitor LGK-974, the Tankyrase inhibitor E7449, etc. However, due to the lack of specificity of small molecule drugs, while inhibiting the Wnt signaling pathway in cancer tissues, they will also affect the normal signal transduction of the body, resulting in serious toxic and side effects. To improve the treatment specificity and reduce side effects, antibody drugs have gradually become the focus of research and development by scientific researchers. Among them, the monoclonal antibody OMP-18R5 targeting FZD7 developed by OncoMed Company in the United States is in Phase I clinical trial. The Sachdev Sidhu research group in Canada developed the F2.I antibody and related antibodies, and at the same time analyzed the crystal structure of the complex of the F2.I antibody and the FZD5 CRD. It is worth noting that the reported antibodies of this kind all bind to the Class I site in the Frizzled receptor and can inhibit the corresponding Wnt signaling pathway by antagonizing the binding of Wnt ligands to the Class I site of the Frizzled receptor. On the other hand, both of these antibodies can bind to the Frizzled receptor subfamilies FZD1 / 2 / 7 and FZD5 / 8 simultaneously, which is mainly due to the high conservation of the Class I site in FZD1 / 2 / 7 and FZD5 / 8.

[0006] The antibodies reported currently still cannot meet the clinical or research needs. On the one hand, neither OMP-18R5, the F2.I antibody or related antibodies can bind to the FZD10 receptor. Although other anti-FZD10 antibodies have been reported currently, the binding epitopes of these antibodies are unknown, and most of the antibodies play a targeting role in the form of antibody-drug conjugates, and it is impossible to determine whether these antibodies can effectively inhibit the activation of the corresponding Wnt signaling pathway. On the other hand, both OMP-18R5, the F2.I antibody or related antibodies can bind to the two Frizzled receptor subfamilies FZD1 / 2 / 7 and FZD5 / 8 simultaneously, so there is a risk of off-target in the treatment process of this kind of antibody. If antibodies that bind to specific functional epitopes (such as Class II sites) and have recognition specificity can be screened, the off-target effect can be reduced and the anti-tumor activity can be enhanced. At the same time, this specific antibody can also be used to explore the different functions of each Frizzled receptor in a certain biological process, which is helpful for the basic research of the Wnt signaling pathway. Summary of the Invention

[0007] The present invention develops a variety of monoclonal antibodies that bind to specific functional sites in the Frizzled receptor and have different recognition specificities through a directed epitope antibody screening method. After functional experimental verification, these antibodies can regulate the Wnt signaling pathway mediated by specific receptors.

[0008] On the one hand, the present invention screened and obtained an antibody that binds to the class I site in the FZD10 receptor, which can directly regulate the Wnt signaling pathway mediated by the FZD10 receptor and can be used for the treatment of tumors related to the FZD10 receptor. On the other hand, the present invention screened and obtained an antibody that binds to the class II functional epitope and has recognition specificity, providing a tool for the specific regulation of the Wnt signaling pathway and being used for the treatment of related diseases and basic research.

[0009] Specifically, the present invention provides the following technical solutions:

[0010] On the one hand, the present invention provides an antibody or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment thereof binds to a frizzled receptor, preferably a human frizzled receptor, wherein: the antibody comprises:

[0011] HCDR1, which comprises the sequences shown in SEQ ID NO: 8, 18, 28, 38, 48, 58, 68, a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence, or an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitution, insertion or deletion) compared with the sequence, or consisting of the same,

[0012] HCDR2, which comprises the sequences shown in SEQ ID NO: 9, 19, 29, 39, 49, 59, 69, a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence, or an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitution, insertion or deletion) compared with the sequence, or consisting of the same, and

[0013] HCDR3, which comprises the sequences shown in SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence, or an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitution, insertion or deletion) compared with the sequence, or consisting of the same,

[0014] LCDR1, which comprises the amino acids shown in SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, or a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with said sequence, or an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with said sequence, or consisting of the same,

[0015] LCDR2, which comprises the amino acid sequences shown in SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with said sequence, or an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with said sequence, or consisting of the same, and

[0016] LCDR3, which comprises the sequences shown in SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, a sequence having at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with said sequence, or an amino acid sequence having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with said sequence, or consisting of the same.

[0017] In some embodiments, the antibody comprises:

[0018] (i) a heavy chain variable region, which comprises the following sequence or consists of the following sequence:

[0019] the amino acid sequences shown in SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, or

[0020] a sequence having at least 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequences shown in SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, or

[0021] an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared with the amino acid sequences shown in SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, and

[0022] (ii) The light chain variable region, which comprises or consists of the following sequences:

[0023] The amino acid sequences shown in SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, or

[0024] Sequences having at least 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequences shown in SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, or

[0025] Amino acid sequences having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequences shown in SEQ ID NO: 3, 13, 23, 33, 43, 53, 63.

[0026] In some embodiments, the antibody comprises:

[0027] (i) A heavy chain, which comprises or consists of the following sequences:

[0028] The amino acid sequences shown in SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, or

[0029] Sequences having at least 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequences shown in SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, or

[0030] Amino acid sequences having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequences shown in SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, and

[0031] (ii) A light chain, which comprises or consists of the following sequences:

[0032] The amino acid sequences shown in SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, or

[0033] Sequences having at least 80%, 85%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequences shown in SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, or

[0034] An amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequences shown in SEQ ID NO: 1, 11, 21, 31, 41, 51, 61.

[0035] Further, the antibody or antigen-binding fragment thereof further comprises an Fc domain, preferably an IgG1 Fc domain, more preferably a human IgG1 Fc domain, and the sequence of the human IgG1 Fc domain is as shown in SEQ ID NO: 73.

[0036] In some embodiments, the Frizzled receptor includes but is not limited to FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9 and FZD10.

[0037] In some embodiments, the Frizzled receptor is selected from the group consisting of FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9 and FZD10.

[0038] In some embodiments, the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof.

[0039] On the other hand, the present invention provides a polynucleotide encoding the antibody or antigen-binding fragment thereof as described above.

[0040] On the other hand, the present invention provides an expression vector comprising the polynucleotide as described above.

[0041] On the other hand, the present invention provides a host cell comprising the expression vector as described above, and the host cell is a host cell for expressing foreign proteins, such as bacteria, yeast, insect cells, mammalian cells.

[0042] On the other hand, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof as described above, which comprises culturing the host cell as described above and recovering the antibody or antigen-binding fragment thereof from the cell culture.

[0043] On the other hand, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described above and a pharmaceutical carrier.

[0044] Further, the pharmaceutical composition is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection.

[0045] On the other hand, the present invention provides the use of the antibody or its antigen-binding fragment as described above in the preparation of a kit or a medicament for preventing, treating, and / or diagnosing a disorder associated with a Frizzled receptor.

[0046] Further, the disorder associated with a Frizzled receptor is selected from cancer, such as breast cancer, lung cancer, liver cancer, gastric cancer, colon cancer, synovial sarcoma, pancreatic cancer, cervical cancer, oral squamous cell carcinoma, bladder cancer, angiosarcoma, prostate cancer, renal cell carcinoma, ovarian cancer, melanoma, and osteogenesis-related disorders.

[0047] On the other hand, the present invention provides a kit, which comprises the antibody or its antigen-binding fragment as described above. Preferably, the antibody further comprises a second antibody that specifically recognizes the antibody or its antigen-binding fragment. Optionally, the second antibody comprises a detectable label, such as a radioisotope, a luminescent substance, a colored substance, an enzyme, or polyethylene glycol.

[0048] On the other hand, the present invention provides a multispecific antibody, preferably a bispecific antibody, which comprises the antibody or its antigen-binding fragment as described above.

[0049] On the other hand, the present invention provides a fusion protein, which comprises the antibody or its antigen-binding fragment as described above.

[0050] More specifically, the present invention provides the following:

[0051] 1) The present invention provides an antibody or antigen-binding fragment that binds to the class I site in FZD10. Based on the reported crystal structure model of the F2.I-FZD5 CRD complex (refer to Raman, S., et al. (2019). "Structure-guided design fine-tunes pharmacokinetics, tolerability, and antitumor profile of multispecific frizzled antibodies."), the inventors introduced directed mutations into the sequences of the three CDR regions of the heavy chain and the CDR-L3 region of the light chain of the F2.I antibody to construct a phage display library. In vitro screening was performed using in vitro recombinant expression of the FZD10 CRD antigen with a human IgG Fc fusion tag. Three antibodies were isolated in the present invention, named F10_A9, F10_E10, and F10_H7, respectively. Further, the present invention analyzed the crystal structure of the complex of the F10_A9 antibody binding to the FZD10 CRD, and determined that F10_A9 binds to the class I site in the FZD10 CRD, which site includes two regions, namely alanine at position 72 to tyrosine at position 77 (SEQ ID NO: 95) and isoleucine at position 120 to phenylalanine at position 124 (SEQ ID NO: 96). Further, the present invention determined that the binding epitopes of F10_E10 and F10_H7 overlap with the binding epitope of F10_A9 by competitive binding. Specifically, the antibody or its antigen-binding fragment that binds to FZD10 has any one of the following characteristics:

[0052] (1) An antibody or its antigen-binding fragment that competitively binds to an antibody having the amino acid sequences shown in CDR1-3 of the heavy chain variable region (VH) of the antibody (SEQ ID NO: 8-10 or SEQ ID NO: 18-20 or SEQ ID NO: 28-30), or the amino acid sequences shown in CDR1-3 of the light chain variable region (VL) of the antibody (SEQ ID NO: 5-7 or SEQ ID NO: 15-17 or SEQ ID NO: 25-27);

[0053] (2) An antibody or its antigen-binding fragment that binds to the epitope bound by an antibody having the amino acid sequences shown in CDR1-3 of the heavy chain variable region (VH) of the antibody (SEQ ID NO: 8-10 or SEQ ID NO: 18-20 or SEQ ID NO: 28-30), or the amino acid sequences shown in CDR1-3 of the light chain variable region (VL) of the antibody (SEQ ID NO: 5-7 or SEQ ID NO: 15-17 or SEQ ID NO: 25-27);

[0054] (3) An antibody or an antigen-binding fragment thereof that binds to an epitope identical to the epitope bound by an antibody having the amino acid sequences shown in CDR1-3 of the variable region of the heavy chain of the antibody (SEQ ID NO: 8-10), or the amino acid sequence shown in CDR3 of the variable region of the light chain of the antibody (SEQ ID NO: 5-7 or SEQ ID NO: 15-17 or SEQ ID NO: 25-27);

[0055] (4) An antibody or a binding fragment thereof that binds to the FZD10 CRD, wherein CDR1-3 of the VH of the antibody respectively comprise or consist of amino acid sequences showing 90% or more identity to the amino acid sequences shown in SEQ ID NO: 8-10 or SEQ ID NO: 18-20 or SEQ ID NO: 28-30, or CDR-L3 of the VL of the antibody respectively comprise or consist of amino acid sequences showing 90% or more identity to the amino acid sequences shown in SEQ ID NO: 5-7 or SEQ ID NO: 15-17 or SEQ ID NO: 25-27.

[0056] 2) The present invention provides an antibody or an antigen-binding fragment that binds to a class II site in a Frizzled receptor

[0057] Taking the FZD2 / 7 receptor as the research object, based on the structural characteristics of the Class II site, the inventors designed a phage display library using the publicly available hB9l9.3 antibody (SEQ ID NO: 105 - 106) as a template, such that the antibodies displayed in this library have the potential to bind to the Class II site. Meanwhile, the inventors designed a mutant protein for the Class II site in the FZD7 CRD and used this mutant protein to optimize the screening process. The phage display library was screened using the wild-type FZD7 CRD antigen in combination with the mutant antigen. In the present invention, the pF7_A5 antibody was isolated, and this antibody can bind to FZD1 / 2 / 7 simultaneously. Further, in the present invention, the crystal structure of the complex of the pF7_A5 antibody binding to the FZD2 CRD was resolved, and it was determined that pF7_A5 binds to the Class II site in the FZD2 CRD. This site includes two regions, namely, threonine at position 101 to proline at position 108 (SEQ ID NO: 97) and histidine at position 140 to glycine at position 152 (SEQ ID NO: 98). Correspondingly, in the FZD1 receptor, this epitope is respectively threonine at position 178 to proline at position 185 (SEQ ID NO: 99) and lysine at position 217 to glycine at position 229 (SEQ ID NO: 100). In the FZD7 receptor, this epitope is respectively threonine at position 111 to proline at position 118 (SEQ ID NO: 101) and asparagine at position 150 to glycine at position 162 (SEQ ID NO: 102).

[0058] Further, based on the crystal structure model, the present invention further performed directed evolution on pF7_A5 to improve its affinity and specificity without changing its binding epitope. After screening by phage display, three improved antibodies were obtained, namely, the antibody pF7_B6 that can bind to FZD1 / 2 / 7 with high affinity, the antibody sF2_C11 that specifically binds to the FZD2 receptor, and the antibody sF7_A2 that preferentially binds to the FZD7 receptor.

[0059] Specifically, the antibody or its antigen-binding fragment that binds to the Class II site in the FZD CRD has any one of the following characteristics:

[0060] (1) An antibody or an antigen-binding fragment thereof that competitively binds to an antibody having the amino acid sequences (SEQ ID NO: 38-40 or SEQ ID NO: 48-50 or SEQ ID NO: 58-60 or SEQ ID NO: 68-70) shown in CDR1-3 of the variable region of the antibody heavy chain (VH), or having the amino acid sequences (SEQ ID NO: 35-37 or SEQ ID NO: 45-47 or SEQ ID NO: 55-57 or SEQ ID NO: 65-67) shown in CDR1-3 of the variable region of the antibody light chain (VL);

[0061] (2) An antibody or an antigen-binding fragment thereof that binds to an epitope bound by an antibody having the amino acid sequences (SEQ ID NO: 38-40 or SEQ ID NO: 48-50 or SEQ ID NO: 58-60 or SEQ ID NO: 68-70) shown in CDR1-3 of the variable region of the antibody heavy chain (VH), or having the amino acid sequences (SEQ ID NO: 35-37 or SEQ ID NO: 45-47 or SEQ ID NO: 55-57 or SEQ ID NO: 65-67) shown in CDR1-3 of the variable region of the antibody light chain (VL);

[0062] (3) An antibody or an antigen-binding fragment thereof that binds to an epitope identical to the epitope bound by an antibody having the amino acid sequences (SEQ ID NO: 38-40 or SEQ ID NO: 48-50 or SEQ ID NO: 58-60 or SEQ ID NO: 68-70) shown in CDR1-3 of the variable region of the antibody heavy chain (VH), or having the amino acid sequences (SEQ ID NO: 35-37 or SEQ ID NO: 45-47 or SEQ ID NO: 55-57 or SEQ ID NO: 65-67) shown in CDR1-3 of the variable region of the antibody light chain (VL);

[0063] (4) An antibody or an antigen-binding fragment thereof that binds to FZD1 or FZD2 or FZD7, wherein CDR1-3 of the VH of the antibody respectively comprise amino acid sequences showing 90% or more identity with the amino acid sequences shown in SEQ ID NO: 38-40 or SEQ ID NO: 48-50 or SEQ ID NO: 58-60 or SEQ ID NO: 68-70, or CDR1-3 of the VL of the antibody respectively comprise amino acid sequences showing 90% or more identity with the amino acid sequences shown in SEQ ID NO: 35-37 or SEQ ID NO: 45-47 or SEQ ID NO: 55-57 or SEQ ID NO: 65-67;

[0064] (5) A high-affinity antibody or its antigen-binding fragment that binds to FZD1 or FZD2 or FZD7, wherein the CDR1-3 of the VH of the antibody respectively contain amino acid sequences showing 90% or more identity with the amino acid sequences shown in SEQ ID NO: 48-50, or the CDR1-3 of the VL of the antibody respectively contain amino acid sequences showing 90% or more identity with the amino acid sequences shown in SEQ ID NO: 45-47;

[0065] (6) An antibody or its antigen-binding fragment that specifically binds to FZD2, wherein the CDR1-3 of the VH of the antibody respectively contain amino acid sequences showing 90% or more identity with the amino acid sequences shown in SEQ ID NO: 58-60, or the CDR1-3 of the VL of the antibody respectively contain amino acid sequences showing 90% or more identity with the amino acid sequences shown in SEQ ID NO: 55-57;

[0066] (7) An antibody or its binding fragment that preferentially binds to FZD7, wherein the CDR1-3 of the VH of the antibody respectively contain amino acid sequences showing 90% or more identity with the amino acid sequences shown in SEQ ID NO: 68-70, or the CDR1-3 of the VL of the antibody respectively contain amino acid sequences showing 90% or more identity with the amino acid sequences shown in SEQ ID NO: 65-67.

[0067] Beneficial effects

[0068] On the one hand, anti-FZD10 antibodies can all bind to the FZD10 CRD antigen with high affinity. ELISA experiments showed that the EC 50 values of F10_A9, F10_E10, and F10_H7 binding to the FZD10 CRD antigen were 7.73 ± 1.07, 7.69 ± 0.39, and 16.73 ± 4.41 nM (nanomoles per liter), respectively. Surface plasmon resonance (SPR) experiments showed that the equilibrium dissociation constants (K D ) of F10_A9 and F10_H7 binding to the FZD10 CRD antigen were 6.22 and 18.88 nM, respectively.

[0069] Furthermore, the anti-FZD10 antibodies of the present invention bind directionally to the class I site in the FZD10 receptor, can directly block the binding of Wnt ligands to the FZD10 receptor, and thus effectively antagonize the Wnt signaling pathway mediated by the FZD10 receptor. Luciferase reporter gene experiments showed that F10_A9, F10_E10, and F10_H7 could all effectively inhibit the activation of the Wnt signaling pathway mediated by the FZD10 receptor, and the IC 50The values were 12.62 ± 0.90, 19.31 ± 0.92, 8.47 ± 1.37 nM respectively.

[0070] On the other hand, antibodies that bind to the FZD CRD class II sites were first identified in the present invention. The class II site antibodies and improved antibodies obtained in the present invention have high affinity and strict binding specificity. Among them, pF7_A5 and pF7_B6 can specifically bind to the FZD1 / 2 / 7 subfamily and cannot bind to FZD5 / 8. In particular, the sF2_C11 antibody specifically binds to the FZD2 receptor. Surface plasmon resonance (SPR) experiments showed that the equilibrium dissociation constants (K D ) of pF7_B6 binding to FZD1, FZD2, and FZD7 were 1.56, 0.55, and 0.18 nM respectively. The specific antibody sF2_C11 of the FZD2 receptor does not bind to the FZD1 receptor, and the affinity of this antibody for binding to the FZD2 receptor (6.51 nM) is about 100 times higher than that for binding to the FZD7 receptor (612 nM), indicating that this antibody is a single-specific antibody for the FZD2 receptor.

[0071] Furthermore, the class II site antibodies of the present invention can effectively regulate the Wnt signaling pathway. Luciferase reporter gene experiments showed that pF7_B6 can effectively inhibit the activation of the Wnt signaling pathway mediated by the FZD2 or FZD7 receptor, but does not interfere with the Wnt signaling pathway mediated by the FZD10 receptor. The IC 50 values of this antibody for inhibiting the FZD2 receptor and the FZD7 receptor were 3.33 ± 0.19 nM and 9.31 ± 0.54 nM respectively. The specific antibody sF2_C11 of the FZD2 receptor can specifically antagonize the activation of the Wnt signaling pathway mediated by the FZD2 receptor, and the IC 50 value is 13.09 ± 1.17 nM, but does not interfere with the Wnt signaling pathway mediated by the FZD7 or FZD10 receptor. Brief Description of the Drawings

[0072] Figure 1 Shows the SDS-PAGE gel electrophoresis results of the antibodies of the present invention. Lane M is the marker, and lanes 1 to 14 are the F10_A9 antibody, the F10_A9 antibody under reducing conditions, the F10_E10 antibody, the F10_E10 antibody under reducing conditions, the F10_H7 antibody, the F10_H7 antibody under reducing conditions, the pF7_A5 antibody, the pF7_A5 antibody under reducing conditions, the pF7_B6 antibody, the pF7_B6 antibody under reducing conditions, the sF2_C11 antibody, the sF2_C11 antibody under reducing conditions, the sF7_A2 antibody, and the sF7_A2 antibody under reducing conditions in sequence.

[0073] Figure 2The affinity characterization results of anti-FZD10 antibodies are shown. Figure A is the ELISA binding curves of three anti-FZD10 antibodies. Figure B is the kinetic curves of the F10_A9 antibody (left) and the F10_H7 antibody (right) binding to the FZD10 CRD antigen. The curves of different colors represent the kinetic curves of different antibody concentrations, and the black solid line is the curve fitted with the "Bivalent analyte" model.

[0074] Figure 3 The epitope characterization results of anti-FZD10 antibodies are shown. Figure A is the crystal structure of F10_A9 binding to FZD10 CRD (top), and the epitope of the F10_A9 antibody binding in FZD10 CRD (bottom). The amino acids at the epitope are marked in bold red. Figure B is the competitive ELISA results of the F10_E10 antibody and the F10_H7 antibody with the F10_A9 antibody, where the F2.I antibody is an isotype control.

[0075] Figure 4 The functional characterization results of anti-FZD10 antibodies are shown. Specifically shown are the experimental data analysis results of F10_A9, F10_E10, and F10_H7 inhibiting the activation of the FZD10-mediated Wnt signaling pathway.

[0076] Figure 5 The specificity characterization results of class II Frizzled antibodies are shown. In the present invention, the binding ability of the pF7_A5 antibody to various Frizzled receptors was detected by ELISA.

[0077] Figure 6 The epitope characterization results of class II Frizzled antibodies are shown. Figure A is the crystal structure of the class II site antibody pF7_A5 binding to FZD2 CRD. Figure B is the sequence alignment results of FZD1, FZD2, and FZD7 receptors at the class II site.

[0078] Figure 7 The affinity and specificity characterization results of the modified class II Frizzled site antibodies are shown. Figures A - C are the SPR curves of the Fab fragment of the pF7_B6 antibody binding to FZD1, FZD2, and FZD7 antigens, respectively. Figures D - F are the SPR curves of the Fab fragment of the sF2_C11 antibody binding to FZD1, FZD2, and FZD7 antigens, respectively. Figures G - I are the SPR curves of the Fab fragment of the sF7_A2 antibody binding to FZD1, FZD2, and FZD7 antigens, respectively. The curves of different colors represent the kinetic curves of different antibody concentrations, and the black solid line is the curve fitted with the "1:1 binding" model.

[0079] Figure 8Shows the functional characterization results of the improved Frizzled receptor class II site antibodies. Figures A - C are the experimental results of the pF7_B6 antibody, sF2_C11 antibody, and sF7_A2 antibody inhibiting the activation of the Wnt signaling pathway mediated by the FZD2, FZD7, and FZD10 receptors, respectively. Detailed implementation manners

[0080] To make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the following further elaborates on the present invention in detail with reference to specific embodiments and the accompanying drawings.

[0081] Example 1 Antibodies obtained by expression and purification

[0082] 1) Design primers, fuse the gene sequence of the antibody heavy chain variable region with the CH1, CH2, and CH3 regions of human IgG1 (SEQ ID NO: 72), and add a secretion signal peptide (SEQ ID NO: 71) to the N - terminus of the heavy chain variable region to construct a heavy chain expression vector. Meanwhile, fuse the gene sequence of the antibody light chain variable region with the human light chain constant region CL, where F10_A9, F10_E10, F10_H7 are fused with the κ - chain constant region (SEQ ID NO: 74), and pF7_A5, pF7_B6, sF2_C11, and sF7_A2 are fused with the λ - chain constant region (SEQ ID NO: 75), and add a secretion signal peptide (SEQ ID NO: 71) to the N - terminus of the light chain variable region.

[0083] 2) Transfect the heavy chain expression vector and the light chain expression vector into mammalian cells Expi293F TM Cells (Thermo Fisher, Cat# A14527) by transient transfection in a mass ratio of 3:2 using PEI (Polysciences, Inc. Cat# 24765). After culturing for 5 days, collect the culture medium supernatant, purify the antibody in the supernatant using a Protein A (Cytiva, Cat# GE17 - 1279 - 01) column, and perform SDS - PAGE electrophoresis. The results are as Figure 1 shown. From the supernatant, we obtained high - purity antibodies.

[0084] Example 2 Characterize the affinity and binding epitope of the anti - FZD10 antibody

[0085] 1) The binding ability of antibodies F10_A9, F10_E10, and F10_H7 to FZD10 CRD was preliminarily characterized by non-competitive ELISA: The FZD10 CRD antigen (SEQ ID NO: 84) with a human IgG Fc fusion tag was diluted to 1 μg / mL with PBS solution and added to a NUNC Maxisorb plate (Thermo Fisher, Cat# 44 - 2404) at 50 μL per well, and anchored at room temperature for 2 hours; blocked with a blocking solution (PBS solution containing 0.5% bovine serum albumin) at room temperature for two hours; washed three times with a washing solution (PBS solution containing 0.05% Tween - 20), 200 μl each time, added the serially diluted antibody solution to the microtiter plate, 50 μl per well, and incubated at room temperature for one hour; washed four times with the washing solution, then added a 1:10000 diluted horseradish peroxidase - conjugated anti - human Ig Kappa chain antibody (Hepeng Biotech), 30 μl per well, and incubated at room temperature for 30 minutes; washed six times with the washing solution, then added 120 μl of TMB solution (Beyotime) to each well, reacted for 2 to 10 minutes, and then added 50 μl of sulfuric acid solution to terminate the color reaction, and read the absorbance value of each well at 450 nm. The results are as Figure 2 shown in A. The EC 50 values of F10_A9, F10_E10, and F10_H7 binding to FZD10 CRD were 7.73 ± 1.07, 7.69 ± 0.39, and 16.73 ± 4.41 nM, respectively.

[0086] 2) The binding affinities of antibodies F10_A9 and F10_H7 to FZD10 CRD were characterized by SPR: The FZD10 CRD antigen with a human IgG Fc fusion tag was diluted to 2 μg / mL with a sodium acetate solution at pH 4.5 and coupled to one channel of a CM5 chip, coupling 30 to 100 RU of the antigen. At the same time, a control channel without protein coupling was set up and blocked with ethanolamine. The two antibodies were serially diluted with a buffer (PBS solution containing 0.05% Tween - 20), and then flowed through the above two channels at a speed of 30 μL / min respectively, while detecting the signal value (RU). After one cycle was completed, 10 mM NaOH was used to wash off the bound antibodies to regenerate the chip. All operations were completed on a Biacore T200 system. The results are as Figure 2 shown in B. Analyzed the results using the Biacore evaluation program, the equilibrium dissociation constants (K D ) of F10_A9 and F10_H7 binding to the FZD10 CRD antigen were 6.22 and 18.88 nM, respectively.

[0087] 3) Resolving the epitope of F10_A9 antibody binding to FZD10 CRD by X-ray crystallography: Co-transfect the expression vector of the antigen-binding fragment (Fab) of F10_A9 antibody (SEQ ID NO: 91-92) and the expression vector of FZD10 CRD antigen (SEQ ID NO: 90, in which an N48Q point mutation is designed to eliminate the influence of glycosylation modification on crystallization) into HEK293F cells. The heavy chain fragment of the antibody carries a histidine tag for easy purification. Five days after transfection, collect the culture medium supernatant and purify the antigen-antibody complex using a Ni-NTA affinity chromatography column (Cytiva). Perform fine purification using a Superdex 200 Increase 10 / 300 chromatography column (Cytiva). Concentrate the purified complex to a protein concentration greater than 8 mg / ml and screen using a variety of commercial crystal growth kits. Further combine and optimize according to the initial crystal formation conditions. We successfully obtained crystals with a diffraction resolution of under the conditions of 0.1 M HEPES (pH 7.0), 30% Jeffamine (pH 7.0) (Hampton Research, HR2-597) and carried out data collection. The structural model of the F10_A9-FZD10 CRD complex was resolved by molecular replacement, and it was determined that F10_A9 binds to the class I site in FZD10 CRD. This site includes two regions, namely alanine at position 72 to tyrosine at position 77 (APLVEY) (SEQ ID NO: 95) and isoleucine at position 120 to phenylalanine at position 124 (IMEQF) (SEQ ID NO: 96)( Figure 3 A).

[0088] 4) Characterization of the binding epitopes of F10_E10 and F10_H7 antibodies by competitive ELISA: Dilute the FZD10 CRD antigen with a human IgG Fc fusion tag to 1 μg / mL with PBS solution, and add it to a NUNC Maxisorb plate enzyme-linked immunosorbent assay (ELISA) plate at a volume of 50 μL per well, and anchor it at room temperature for 2 hours; use a blocking solution (PBS solution containing 0.5% bovine serum albumin) to block for two hours at room temperature; wash three times with a washing solution (PBS solution containing 0.05% Tween-20), 200 μL each time, add 50 μL of F10_A9 phage solution, and incubate at room temperature for 30 minutes; then add the antibody solution (F2.I, F10_A9, F10_E10, F10_H7) diluted to 200 nM (the amino acid sequence of the F2.I antibody is shown in SEQ ID NO: 103-104) to the ELISA plate, 50 μL per well, and incubate at room temperature for 30 minutes; then wash four times with the washing solution, and then add an anti-M13 phage antibody (Sino Biological) conjugated with horseradish peroxidase diluted 1:10,000, 30 μL per well, and incubate at room temperature for 30 minutes; wash six times with the washing solution, and then add 120 μL of TMB solution (Beyotime) to each well, react for 2 to 10 minutes, and then add 50 μL of 2M sulfuric acid solution to terminate the color reaction, and read the absorbance value of each well at 450 nm. The results show that F10_E10 and F10_H7 antibodies inhibit the binding of F10_A9 phage to FZD10, indicating that the binding epitopes of F10_E10 and F10_H7 antibodies overlap or partially overlap with the binding epitope of F10_A9( Figure 3 B).

[0089] Example 3 Characterization of the regulatory ability of anti-FZD10 antibodies on the Wnt signaling pathway

[0090] Characterize the regulatory ability of F10_A9, F10_E10, and F10_H7 antibodies on the Wnt signaling pathway by luciferase reporter gene assay. The functional experiment uses a HEK293T cell line (F127-KO) with knocked-out FZD1 / 2 / 7 receptors (refer to Voloshanenko, O., et al. (2017). "Mapping of Wnt-Frizzled interactions by multiplex CRISPR targeting of receptor gene families.") as the model cell line. Seed the F127-KO cells into a 6-well plate, inoculating 1.3×10 6 cells per well, and culture for 24 hours; use PolyJet TMTransfection reagent (Signagen, Cat# SL100688), transfection was performed according to the standard protocol. The cell confluence at the time of transfection was 70–90%, and 500 ng of the full-length expression plasmid of FZD10 (SEQ ID NO: 87), 480 ng of the TCF / LEF-Firefly Luciferase plasmid (modified from pGL3-Basic of Promega, referring to Tao, Y., et al. (2019). "Tailored tetravalent antibodies potently and specifically activate Wnt / Frizzled pathways in cells, organoids and mice.") and 20 ng of the Renilla Luciferase plasmid (Promega) were transfected simultaneously; 24 hours after transfection, the cells in the 6-well plate were transferred to a 96-well plate, and 40 μl of the mWnt3a conditioned medium (prepared referring to Willert, K., et al. (2003). "Wnt proteins are lipid-modified and can act as stem cell growth factors.") was added to stimulate the Wnt signaling pathway, and 40 μl of the serially diluted antibody solution was added simultaneously; 24 hours later, the cells were collected and detected using the Dual-Luciferase Reporter Assay Kit (Transgene). The results showed that F10_A9, F10_E10, and F10_H7 could all effectively inhibit the activation of the Wnt signaling pathway mediated by the FZD10 receptor, and the IC 50 values were 12.62 ± 0.90, 19.31 ± 0.92, and 8.47 ± 1.37 nM ( Figure 4 ).

[0091] Example 4 Characterization of the Specificity and Binding Epitope of Antibodies against Class II Sites of Frizzled Receptors

[0092] 1) The binding specificity of the antibody pF7_A5 against various Frizzled receptors was characterized by ELISA. Among them, the carboxyl terminus of the light chain of the pF7_A5 antibody carried an HA epitope tag. Different FZD CRD antigens (SEQ ID NO: 76-84) were diluted to 1 μg / mL with PBS solution, and added to a NUNC Maxisorb plate ELISA plate at a volume of 50 μL per well, and anchored at room temperature for 2 hours; blocked with a blocking solution (PBS solution containing 0.5% bovine serum albumin) at room temperature for two hours; washed three times with a washing solution (PBS solution containing 0.05% Tween-20), 200 μl each time, and the pF7_A5 antibody solution diluted to 10 nM was added to the ELISA plate, 50 μl per well, and incubated at room temperature for one hour; washed four times with the washing solution, and then added with a 1:5000 diluted horseradish peroxidase-conjugated Anti-HA tag antibody (Sino Biological), 30 μl per well, and incubated at room temperature for 30 minutes; washed six times with the washing solution, and then 120 μl of TMB solution (Beyotime) was added to each well, and the reaction was carried out for 2 to 10 minutes, and then 50 μl of sulfuric acid solution was added to terminate the color development reaction, and the absorbance value of each well at 450 nm was read. The results are as Figure 5 shown, the pF7_A5 antibody specifically binds to the FZD1 / 2 / 7 receptors.

[0093] 2) The epitope of the pF7_A5 antibody binding to FZD2 / 7 CRD was resolved by X-ray crystallography: The antigen-binding fragment (Fab) expression vector of the pF7_A5 antibody and the FZD2 CRD or FZD7 CRD antigen expression vector (SEQ ID NO: 88, 89, 93, 94) were co-transfected into HEK293F cells; 5 days after transfection, the culture medium supernatant was collected and the antigen-antibody complex was purified using a Ni-NTA affinity chromatography column (Cytiva); fine purification was carried out using a Superdex 200 Increase 10 / 300 chromatography column (Cytiva); the purified complex was concentrated to a protein concentration greater than 8 mg / ml, and various commercial crystal growth kits were used for screening; further combinatorial optimization was carried out according to the initial crystallization conditions. We successfully obtained a diffraction resolution of TM at 8% Tacsimate Crystals were obtained and data collection was performed. The structural model of the pF7_A5-FZD2CRD complex was resolved by molecular replacement, and it was determined that pF7_A5 binds to the Class II site in FZD2CRD, which consists of two regions, namely threonine at position 101 to proline at position 108 (SEQ ID NO:97) and histidine at position 140 to glycine at position 152 (SEQ ID NO:98).( Figure 6 A). Correspondingly, in the FZD1 receptor, the epitopes are threonine at position 178 to proline at position 186 (SEQ ID NO:99) and lysine at position 217 to glycine at position 229 (SEQ ID NO:100). In the FZD7 receptor, the epitopes are threonine at position 111 to proline at position 118 (SEQ ID NO:101) and asparagine at position 150 to glycine at position 162 (SEQ ID NO:102)( Figure 6 B).

[0094] Example 5 Characterization of the Affinity and Specificity of Modified Class II Site Antibodies of Frizzled Receptors

[0095] Surface Plasmon Resonance (SPR) was used to characterize the affinity of pF7_B6, sF2_C11 and sF7_A2 antibodies for FZD1 / 2 / 7CRD: The FZD1 / 2 / 7CRD antigens (SEQ ID NO:76, 77, 81) with human IgG Fc fusion tags were diluted to 2 μg / mL with sodium acetate solution at pH 4.5 and coupled to one channel of a CM5 chip, coupling 30 to 100 RU of antigen. At the same time, a control channel without protein coupling was set up and blocked with ethanolamine. The three antibodies were serially diluted with buffer (PBS solution containing 0.05% Tween-20), and then flowed through the above two channels at a speed of 30 μL / min respectively, while the signal value (RU) was detected. After one cycle was completed, the bound antibodies were washed off with 10 mM NaOH solution to regenerate the chip. All operations were completed using a BiacoreT200 system. The Biacore evaluation program was used to analyze the results, and the affinity and related kinetic parameters were as Figure 7 shown. The K on (M -1 ×s -1 ), K off (s -1 ) and K D (nM) of pF7_B6 with FZD1 were 2.00×10 6 , 3.12×10 -3 and 1.56 respectively. The K on (M -1 ×s -1), K off (s -1 ) and K D (nM) were 3.17×10 6 , 1.73×10 -3 and 0.55, K between pF7_B6 and FZD7 on (M -1 ×s -1 ), K off (s -1 ) and K D (nM) were 4.72×10 6 ,8.50×10 -4 and 0.18, K of sF2_C11 and FZD1 on (M -1 ×s -1 ), K off (s -1 ) and K D (nM) were n / a, n / a and n / a, respectively (indicating that sF2_C11 does not bind to the FZD1 receptor). The K on (M -1 ×s -1 ), K off (s -1 ) and K D (nM) were 1.87×10 6 , 1.22×10 -2 and 6.51, K of sF2_C11 and FZD7 on (M -1 ×s -1 ), K off (s -1 ) and K D (nM) were 7.63×10 5 , 0.49 and 642, K between sF7_A2 and FZD1 on (M -1 ×s -1 ), K off (s -1 ) and K D (nM) were 2.22×10 6 , 2.11×10 -2 and 9.51, K of sF7_A2 and FZD2 on (M -1 ×s -1 ), K off (s -1 ) and K D (nM) were 1.36×10 6 3.20×10-2 and 24.52, the K of sF7_A2 and FZD7 on (M -1 × s -1 ), K off (s -1 ) and K D (nM) are 6.55 × 10 6 、2.65 × 10 -2 and 4.05 respectively.

[0096] Example 6 Characterize the regulatory ability of the improved class II frizzled receptor site antibody on the Wnt signaling pathway

[0097] Use the luciferase reporter gene assay to characterize the regulatory ability of pF7_B6, sF2_C11 and sF7_A2 antibodies on the Wnt signaling pathway. Seed F127-KO cells into 6-well plates at 1.3 × 10 6 cells per well and culture for 24 hours; use PolyJet TM transfection reagent and perform transfection according to the standard protocol. The cell confluence at the time of transfection is 70–90%, and simultaneously transfect 500 ng of the full-length expression plasmid of FZD2 or FZD7 or FZD10 (SEQ ID NO: 85, 86, 87), 480 ng of the TCF / LEF-Firefly Luciferase plasmid and 20 ng of the Renilla Luciferase plasmid; 24 hours after transfection, transfer the cells in the 6-well plates to 96-well plates, add 40 μl of the mWnt3a conditioned medium to stimulate the Wnt signaling pathway, and simultaneously add 40 μl of the antibody solution with gradient dilution; 24 hours later, collect the cells and detect them using the Dual-Luciferase Reporter Assay Kit (Transgene). The results show that the IC 50 values of the pF7_B6 antibody for inhibiting the FZD2 receptor and the FZD7 receptor are 3.33 ± 0.19 nM and 9.31 ± 0.54 nM respectively. The specific antibody sF2_C11 of the FZD2 receptor can specifically antagonize the activation of the Wnt signaling pathway mediated by the FZD2 receptor, and the IC 50 value is 13.09 ± 1.17 nM, but it does not interfere with the Wnt signaling pathway mediated by the FZD7 or FZD10 receptor. The preferred antibody sF7_A2 of the FZD7 receptor has a more significant maximum inhibitory effect on the FZD7 receptor, and it can reach a 96% maximum inhibitory effect on FZD7, but only a 71% maximum inhibitory effect on the FZD2 receptor.( Figure 8 ).

[0098] Sequence

[0099] SEQ ID NO:1 Light chain of F10_A9 antibody

[0100] DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQGWPFFTFGQGTKVEIKRTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0101] SEQ ID NO:2 Variable region of the heavy chain of F10_A9 antibody

[0102] EVQLVESGGGLVQPGGSLRLSCAASGFPIRGSSIHWVRQAPGKGLEWVAATYGWPGSITYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHTYPLWALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0103] SEQ ID NO:3 Variable region of the light chain of F10_A9 antibody

[0104] DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQGWPFFTFGQGTKVEIK

[0105] SEQ ID NO:4 Variable region of the heavy chain of F10_A9 antibody

[0106] EVQLVESGGGLVQPGGSLRLSCAASGFPIRGSSIHWVRQAPGKGLEWVAATYGWPGSITYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHTYPLWALDYWGQGTLVTVSS

[0107] SEQ ID NO:5 F10_A9 antibody LCDR1

[0108] QSVSSA

[0109] SEQ ID NO:6 F10_A9 antibody LCDR2

[0110] SAS

[0111] SEQ ID NO:7 F10_A9 antibody LCDR3

[0112] QQGWPFFT

[0113] SEQ ID NO:8 F10_A9 antibody HCDR1

[0114] GFPIRGSS

[0115] SEQ ID NO:9 F10_A9 antibody HCDR2

[0116] TYGWPGSI

[0117] SEQ ID NO:10 F10_A9 antibody HCDR3

[0118] ARRHTYPLWALDY

[0119] SEQ ID NO:11 F10_E10 antibody light chain

[0120] DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQGYRPFTFGQGTKVEIKRTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0121] SEQ ID NO:12 F10_E10 antibody heavy chain

[0122] EVQLVESGGGLVQPGGSLRLSCAASGFSIPWSSIHWVRQAPGKGLEWVAATYFLTGSITYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARLHDDWRSALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0123] SEQ ID NO:13 Variable region of the light chain of F10_E10 antibody

[0124] DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQGYRPFTFGQGTKVEIK

[0125] SEQ ID NO:14 Variable region of the heavy chain of F10_E10 antibody

[0126] EVQLVESGGGLVQPGGSLRLSCAASGFSIPWSSIHWVRQAPGKGLEWVAATYFLTGSITYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARLHDDWRSALDYWGQGTLVTVSS

[0127] SEQ ID NO:15 LCDR1 of F10_E10 antibody

[0128] QSVSSA

[0129] SEQ ID NO:16 LCDR2 of F10_E10 antibody

[0130] SAS

[0131] SEQ ID NO:17 LCDR3 of F10_E10 antibody

[0132] QQGYRPFT

[0133] SEQ ID NO:18 HCDR1 of F10_E10 antibody

[0134] GFSIPWSS

[0135] SEQ ID NO:19 HCDR2 of F10_E10 antibody

[0136] TYFLTGSI

[0137] SEQ ID NO:20 HCDR3 of F10_E10 antibody

[0138] ARLHDDWRSALDY

[0139] SEQ ID NO:21 Light chain of F10_H7 antibody

[0140] DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQGGYAFTFGQGTKVEIKRTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0141] SEQ ID NO:22 Heavy chain of F10_H7 antibody

[0142] EVQLVESGGGLVQPGGSLRLSCAASGFNISTSSIHWVRQAPGKGLEWVAATYVASGSITYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARNHFNIWLALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0143] SEQ ID NO:23 Variable region of the light chain of the F10_H7 antibody

[0144] DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQGGYAFTFGQGTKVEIK

[0145] SEQ ID NO:24 Variable region of the heavy chain of the F10_H7 antibody

[0146] EVQLVESGGGLVQPGGSLRLSCAASGFNISTSSIHWVRQAPGKGLEWVAATYVASGSITYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARNHFNIWLALDYWGQGTLVTVSS

[0147] SEQ ID NO:25 F10_H7 antibody LCDR1

[0148] QSVSSA

[0149] SEQ ID NO:26 F10_H7 antibody LCDR2

[0150] SAS

[0151] SEQ ID NO:27 LCDR3 of F10_H7 antibody

[0152] QQGGYAFT

[0153] SEQ ID NO:28 HCDR1 of F10_H7 antibody

[0154] GFNISTSS

[0155] SEQ ID NO:29 HCDR2 of F10_H7 antibody

[0156] TYVASGSI

[0157] SEQ ID NO:30 HCDR3 of F10_H7 antibody

[0158] ARNHFNIWLALDY

[0159] SEQ ID NO:31 Light chain of pF7_A5 antibody

[0160] ELTQPPSVSVSPGQTARITCSGDGSIYRVFSYGWYQQKPGQAPVTLIYLNNKRPSGIPERFSGSLSGSTNTLTISGVQAEDEADYYCGSFGNAPLAFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0161] SEQ ID NO:32 Heavy chain of pF7_A5 antibody

[0162] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFNMFWVRQAPGKGLEWVAGIDDDGSYPNYGSAVKGRATISRDNSKNTLYLQMNSLRAEDTAVYYCAKSGGWALGGLITLRWIDAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0163] SEQ ID NO:33 Variable region of the light chain of pF7_A5 antibody

[0164] ELTQPPSVSVSPGQTARITCSGDGSIYRVFSYGWYQQKPGQAPVTLIYLNNKRPSGIPERFSGSLSGSTNTLTISGVQAEDEADYYCGSFGNAPLAFGGGTKLTVL

[0165] SEQ ID NO:34 Variable region of the heavy chain of pF7_A5 antibody

[0166] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFNMFWVRQAPGKGLEWVAGIDDDGSYPNYGSAVKGRATISRDNSKNTLYLQMNSLRAEDTAVYYCAKSGGWALGGLITLRWIDAWGQGTLVTVSS

[0167] SEQ ID NO:35 LCDR1 of pF7_A5 antibody

[0168] GSIYRVFS

[0169] SEQ ID NO:36 LCDR2 of pF7_A5 antibody

[0170] LNN

[0171] SEQ ID NO:37 LCDR3 of pF7_A5 antibody

[0172] GSFGNAPLA

[0173] SEQ ID NO:38 HCDR1 of pF7_A5 antibody

[0174] GFTFSSFN

[0175] SEQ ID NO:39 HCDR2 of pF7_A5 antibody

[0176] IDDDGSYP

[0177] SEQ ID NO:40 HCDR3 of pF7_A5 antibody

[0178] AKSGGWALGGLITLRWIDA

[0179] SEQ ID NO:41 Light chain of pF7_B6 antibody

[0180] ELTQPPSVSVSPGQTARITCSGDGSIYRAYLYGWYQQKPGQAPVTLIYLNNKRPSGIPERFSGSLSGSTNTLTISGVQAEDEADYYCGSFGNRPLAFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0181] SEQ ID NO:42 Heavy chain of pF7_B6 antibody

[0182] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFNMFWVRQAPGKGLEWVAGIDDDGSYPNYGSAVKGRATISRDNSKNTLYLQMNSLRAEDTAVYYCAKSGGWALGGLITLRWIDAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0183] SEQ ID NO:43 Variable region of the light chain of pF7_B6 antibody

[0184] ELTQPPSVSVSPGQTARITCSGDGSIYRAYLYGWYQQKPGQAPVTLIYLNNKRPSGIPERFSGSLSGSTNTLTISGVQAEDEADYYCGSFGNRPLAFGGGTKLTVL

[0185] SEQ ID NO:44 Variable region of the heavy chain of pF7_B6 antibody

[0186] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFNMFWVRQAPGKGLEWVAGIDDDGSYPNYGSAVKGRATISRDNSKNTLYLQMNSLRAEDTAVYYCAKSGGWALGGLITLRWIDAWGQGTLVTVSS

[0187] SEQ ID NO:45 LCDR1 of pF7_B6 antibody

[0188] GSIYRAYL

[0189] SEQ ID NO:46 LCDR2 of pF7_B6 antibody

[0190] LNN

[0191] SEQ ID NO:47 LCDR3 of pF7_B6 antibody

[0192] GSFGNRPLA

[0193] SEQ ID NO:48 HCDR1 of pF7_B6 antibody

[0194] GFTFSSFN

[0195] SEQ ID NO:49 HCDR2 of pF7_B6 antibody

[0196] IDDDGSYP

[0197] SEQ ID NO:50 HCDR3 of pF7_B6 antibody

[0198] AKSGGWALGGLITLRWIDA

[0199] SEQ ID NO:51 Light chain of sF2_C11 antibody

[0200] ELTQPPSVSVSPGQTARITCSGDGSIYEAFLYGWYQQKPGQAPVTLIYLNNKRPSGIPERFSGSLSGSTNTLTISGVQAEDEADYYCGSFGNHPRAFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0201] SEQ ID NO:52 Heavy chain of sF2_C11 antibody

[0202] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFNMFWVRQAPGKGLEWVAGIDDDGSYPNYGSAVKGRATISRDNSKNTLYLQMNSLRAEDTAVYYCAKSGGWALGGLITLRWIDAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0203] SEQ ID NO:53 Light chain variable region of sF2_C11 antibody

[0204] ELTQPPSVSVSPGQTARITCSGDGSIYEAFLYGWYQQKPGQAPVTLIYLNNKRPSGIPERFSGSLSGSTNTLTISGVQAEDEADYYCGSFGNHPRAFGGGTKLTVL

[0205] SEQ ID NO:54 Heavy chain variable region of sF2_C11 antibody

[0206] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFNMFWVRQAPGKGLEWVAGIDDDGSYPNYGSAVKGRATISRDNSKNTLYLQMNSLRAEDTAVYYCAKSGGWALGGLITLRWIDAWGQGTLVTVSS

[0207] SEQ ID NO:55 sF2_C11 antibody LCDR1

[0208] GSIYEAFL

[0209] SEQ ID NO:56 sF2_C11 antibody LCDR2

[0210] LNN

[0211] SEQ ID NO:57 LCDR3 of sF2_C11 antibody

[0212] GSFGNHPRA

[0213] SEQ ID NO:58 HCDR1 of sF2_C11 antibody

[0214] GFTFSSFN

[0215] SEQ ID NO:59 HCDR2 of sF2_C11 antibody

[0216] IDDDGSYP

[0217] SEQ ID NO:60 HCDR3 of sF2_C11 antibody

[0218] AKSGGWALGGLITLRWIDA

[0219] SEQ ID NO:61 Light chain of sF7_A2 antibody

[0220] ELTQPPSVSVSPGQTARITCSGDGSIFRAYSYGWYQQKPGQAPVTLIYLNNKRPSGIPERFSGSLSGSTNTLTISGVQAEDEADYYCGSFGNRPLAFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0221] SEQ ID NO:62 Heavy chain of sF7_A2 antibody

[0222] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFNMFWVRQAPGKGLEWVAGIDDDGSYPNYGSAVKGRATISRDNSKNTLYLQMNSLRAEDTAVYYCAKSGGWALGGLITLRWIDAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0223] SEQ ID NO:63 Light chain variable region of sF7_A2 antibody

[0224] ELTQPPSVSVSPGQTARITCSGDGSIFRAYSYGWYQQKPGQAPVTLIYLNNKRPSGIPERFSGSLSGSTNTLTISGVQAEDEADYYCGSFGNRPLAFGGGTKLTVL

[0225] SEQ ID NO:64 Heavy chain variable region of sF7_A2 antibody

[0226] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFNMFWVRQAPGKGLEWVAGIDDDGSYPNYGSAVKGRATISRDNSKNTLYLQMNSLRAEDTAVYYCAKSGGWALGGLITLRWIDAWGQGTLVTVSS

[0227] SEQ ID NO:65 LCDR1 of sF7_A2 antibody

[0228] GSIFRAYS

[0229] SEQ ID NO:66 LCDR2 of sF7_A2 antibody

[0230] LNN

[0231] SEQ ID NO:67 LCDR3 of sF7_A2 antibody

[0232] GSFGNRPLA

[0233] SEQ ID NO:68 HCDR1 of sF7_A2 antibody

[0234] GFTFSSFN

[0235] SEQ ID NO:69 HCDR2 of sF7_A2 antibody

[0236] IDDDGSYP

[0237] SEQ ID NO:70 HCDR3 of sF7_A2 antibody

[0238] AKSGGWALGGLITLRWIDA

[0239] SEQ ID NO:71 Secretory signal peptide

[0240] MNLLLILTFVAAAVA

[0241] SEQ ID NO:72 Human IgG1 CH1-CH2-CH3 sequence

[0242] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0243] SEQ ID NO:73 Human IgG1 Fc sequence

[0244] PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0245] SEQ ID NO:74 Human κ-chain constant region (κCL)

[0246] RTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0247] SEQ ID NO:75 Human λ-chain constant region (λCL)

[0248] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0249] SEQ ID NO:76 Human FZD1 CRD Fc fusion protein

[0250] MNLLLILTFVAAAVAEFQAAGQGPGQGPGPGQQPPPPPQQQQSGQQYNGERGISVPDHGYCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCTVLEQALPPCRSLCERARQGCEALMNKFGFQWPDTLKCEKFPVHGAGELCVGQNTSDKGTPTPSLLPEFWTSNPQHEDIEGRMDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH

[0251] SEQ ID NO:77 Human FZD2 CRD Fc Fusion Protein

[0252] MNLLLILTFVAAAVAAQFHGEKGISIPDHGFCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLEQAIPPCRSICERARQGCEALMNKFGFQWPERLRCEHFPRHGAEQICVGQNHSEDGAPALEDIEGRMDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH

[0253] SEQ ID NO:78 Human FZD4 CRD Fc Fusion Protein

[0254] MNLLLILTFVAAAVAEFFGDEEERRCDPIRISMCQNLGYNVTKMPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCEPVLKEFGFAWPESLNCSKFPPQNDHNHMCMEGPGLELEVLFQGPKVEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0255] SEQ ID NO:79 Human FZD5 CRD Fc Fusion Protein

[0256] MNLLLILTFVAAAVAASKAPVCQEITVPMCRGIGYNLTHMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLRFFLCSMYTPICLPDYHKPLPPCRSVCERAKAGCSPLMRQYGFAWPERMSCDRLPVLGRDAEVLCMDYNRSEATTAPPRPFPAKPEDIEGRMDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH

[0257] SEQ ID NO:80 Human FZD6 CRD Fc Fusion Protein

[0258] MNLLLILTFVAAAVAAHSLFTCEPITVPRCMKMAYNMTFFPNLMGHYDQSIAAVEMEHFLPLANLECSPNIETFLCKAFVPTCIEQIHVVPPCRKLCEKVYSDCKKLIDTFGIRWPEELECDRLQYCDETVPVTFDPHTEFLGPQKKTEQVEDIEGRMDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH

[0259] SEQ ID NO:81 Human FZD7 CRD Fc Fusion Protein

[0260] MNLLLILTFVAAAVAAQPYHGEKGISVPDHGFCQPISIPLCTDIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLRCENFPVHGAGEICVGQNTSDGSGGPGGGPTAYPTAPYLEDIEGRMDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH

[0261] SEQ ID NO:82 Human FZD8 CRD Fc Fusion Protein

[0262] MNLLLILTFVAAAVAAAAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTAAPSPPRRLPPPPPEDIEGRMDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH

[0263] SEQ ID NO:83 Human FZD9 CRD Fc Fusion Protein

[0264] MNLLLILTFVAAAVAEFLEIGRFDPERGRGAAPCQAVEIPMCRGIGYNLTRMPNLLGHTSQGEAAAELAEFAPLVQYGCHSHLRFFLCSLYAPMCTDQVSTPIPACRPMCEQARLRCAPIMEQFNFGWPDSLDCARLPTRNDPHALCMEAPENATLELEVLFQGPKVEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0265] SEQ ID NO:84 Human FZD10 CRD Fc Fusion Protein

[0266] MNLLLILTFVAAAVAAISSMDMERPGDGKCQPIEIPMCKDIGYNMTRMPNLMGHENQREAAIQLHEFAPLVEYGCHGHLRFFLCSLYAPMCTEQVSTPIPACRVMCEQARLKCSPIMEQFNFKWPDSLDCRKLPNKNDPNYLCMEAPNNGSDEPTRGEDIEGRMDPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH

[0267] SEQ ID NO:85 Full-length expression sequence of human FZD2

[0268] MRPRSALPRLLLPLLLLPAAGPAQFHGEKGISIPDHGFCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLEQAIPPCRSICERARQGCEALMNKFGFQWPERLRCEHFPRHGAEQICVGQNHSEDGAPALLTTAPPPGLQPGAGGTPGGPGGGGAPPRYATLEHPFHCPRVLKVPSYLSYKFLGERDCAAPCEPARPDGSMFFSQEETRFARLWILTWSVLCCASTFFTVTTYLVDMQRFRYPERPIIFLSGCYTMVSVAYIAGFVLQERVVCNERFSEDGYRTVVQGTKKEGCTILFMMLYFFSMASSIWWVILSLTWFLAAGMKWGHEAIEANSQYFHLAAWAVPAVKTITILAMGQIDGDLLSGVCFVGLNSLDPLRGFVLAPLFVYLFIGTSFLLAGFVSLFRIRTIMKHDGTKTEKLERLMVRIGVFSVLYTVPATIVIACYFYEQAFREHWERSWVSQHCKSLAIPCPAHYTPRMSPDFTVYMIKYLMTLIVGITSGFWIWSGKTLHSWRKFYTRLTNSRHGETTV

[0269] SEQ ID NO:86 Full-length expression sequence of human FZD7

[0270] MRDPGAAAPLSSLGLCALVLALLGALSAGAGAQPYHGEKGISVPDHGFCQPISIPLCTDIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLRCENFPVHGAGEICVGQNTSDGSGGPGGGPTAYPTAPYLPDLPFTALPPGASDGRGRPAFPFSCPRQLKVPPYLGYRFLGERDCGAPCEPGRANGLMYFKEEERRFARLWVGVWSVLCCASTLFTVLTYLVDMRRFSYPERPIIFLSGCYFMVAVAHVAGFLLEDRAVCVERFSDDGYRTVAQGTKKEGCTILFMVLYFFGMASSIWWVILSLTWFLAAGMKWGHEAIEANSQYFHLAAWAVPAVKTITILAMGQVDGDLLSGVCYVGLSSVDALRGFVLAPLFVYLFIGTSFLLAGFVSLFRIRTIMKHDGTKTEKLEKLMVRIGVFSVLYTVPATIVLACYFYEQAFREHWERTWLLQTCKSYAVPCPPGHFPPMSPDFTVFMIKYLMTMIVGITTGFWIWSGKTLQSWRRFYHRLSHSSKGETAV

[0271] SEQ ID NO:87 Full-length expression sequence of human FZD10

[0272] MQRPGPRLWLVLQVMGSCAAISSMDMERPGDGKCQPIEIPMCKDIGYNMTRMPNLMGHENQREAAIQLHEFAPLVEYGCHGHLRFFLCSLYAPMCTEQVSTPIPACRVMCEQARLKCSPIMEQFNFKWPDSLDCRKLPNKNDPNYLCMEAPNNGSDEPTRGSGLFPPLFRPQRPHSAQEHPLKDGGPGRGGCDNPGKFHHVEKSASCAPLCTPGVDVYWSREDKRFAVVWLAIWAVLCFFSSAFTVLTFLIDPARFRYPERPIIFLSMCYCVYSVGYLIRLFAGAESIACDRDSGQLYVIQEGLESTGCTLVFLVLYYFGMASSLWWVVLTLTWFLAAGKKWGHEAIEANSSYFHLAAWAIPAVKTILILVMRRVAGDELTGVCYVGSMDVNALTGFVLIPLACYLVIGTSFILSGFVALFHIRRVMKTGGENTDKLEKLMVRIGLFSVLYTVPATCVIACYFYERLNMDYWKILAAQHKCKMNNQTKTLDCLMAASIPAVEIFMVKIFMLLVVGITSGMWIWTSKTLQSWQQVCSRRLKKKSRRKPASVITSGGIYKKAQHPQKTHHGKYEIPAQSPTCV

[0273] SEQ ID NO:88 Human FZD2 CRD Expression Sequence

[0274] MNLLLILTFVAAAVAEFPDHGFCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLEQAIPPCRSICERARQGCEALMNKFGFQWPERLRCEHFPRHGAEQICVGQNHSEDGHHHHHH

[0275] SEQ ID NO:89 Human FZD7 CRD Expression Sequence

[0276] MNLLLILTFVAAAVAEFPDHGFCQPISIPLCTDIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLRCENFPVHGAGEICVGQNTSDGHHHHHH

[0277] SEQ ID NO:90 Human FZD10 CRD Expression Sequence (with N48Q Point Mutation)

[0278] MNLLLILTFVAAAVAEFMDMERPGDGKCQPIEIPMCKDIGYQMTRMPNLMGHENQREAAIQLHEFAPLVEYGCHGHLRFFLCSLYAPMCTEQVSTPIPACRVMCEQARLKCSPIMEQFNFKWPDSLDCRKLPNKNDPNYLCMEAPN

[0279] SEQ ID NO:91 Antibody F10_A9 Expression Sequence (for Preparing Complex)

[0280] MNLLLILTFVAAAVAEVQLVESGGGLVQPGGSLRLSCAASGFPIRGSSIHWVRQAPGKGLEWVAATYGWPGSITYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHTYPLWALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCHHHHHHHH

[0281] SEQ ID NO:92 Antibody F10_A9 Expression Sequence (for Preparing Complex)

[0282] MNLLLILTFVAAAVADIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQGWPFFTFGQGTKVEIKRTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0283] SEQ ID NO:93 Antibody pF7_A5 Expression Sequence (for preparing complex)

[0284] MNLLLILTFVAAAVAEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFNMFWVRQAPGKGLEWVAGIDDDGSYPNYGSAVKGRATISRDNSKNTLYLQMNSLRAEDTAVYYCAKSGGWALGGLITLRWIDAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0285] SEQ ID NO:94 Antibody pF7_A5 Expression Sequence (for preparing complex)

[0286] MNLLLILTFVAAAVAELTQPPSVSVSPGQTARITCSGDGSIYRVFSYGWYQQKPGQAPVTLIYLNNKRPSGIPERFSGSLSGSTNTLTISGVQAEDEADYYCGSFGNAPLAFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0287] SEQ ID NO:95 Epitope of Antibody F10_A9 Binding to FZD10 (Sequence at positions 72 - 77)

[0288] APLVEY

[0289] Epitope of antibody F10_A9 binding to FZD10 (sequence at positions 120 - 124), SEQ ID NO:96

[0290] IMEQF

[0291] Epitope of antibody pF7_A5 binding to FZD2 (sequence at positions 101 - 108), SEQ ID NO:97

[0292] TVLEQAIP

[0293] Epitope of antibody pF7_A5 binding to FZD2 (sequence at positions 140 - 152), SEQ ID NO:98

[0294] HFPRHGAEQICVG

[0295] Epitope of antibody pF7_A5 binding to FZD1 (sequence at positions 178 - 185), SEQ ID NO:99

[0296] TVLEQALP

[0297] Epitope of antibody pF7_A5 binding to FZD1 (sequence at positions 217 - 229), SEQ ID NO:100

[0298] KFPVHGAGELCVG

[0299] Epitope of antibody pF7_A5 binding to FZD7 (sequence at positions 111 - 118), SEQ ID NO:101

[0300] TVLDQAIP

[0301] Epitope of antibody pF7_A5 binding to FZD7 (sequence at positions 150 - 162), SEQ ID NO:102

[0302] NFPVHGAGEICVG

[0303] Light chain sequence of antibody F2.I, SEQ ID NO:103

[0304] DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQGVYLFTFGQGTKVEIKRTVAAPSVFIFPPSDSQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGS

[0305] SEQ ID NO: 104 Antibody F2.I Heavy Chain Sequence

[0306] EVQLVESGGGLVQPGGSLRLSCAASGFNIHSSSIHWVRQAPGKGLEWVAATYSSFGSITYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYHHPFGFALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0307] SEQ ID NO: 105 Antibody hB9l9.3 Light Chain Sequence

[0308] EFELTQPPSVSVSPGQTARITCSGDGSYAGSYYYGWYQQKPGQAPVTLIYYNNKRPSGIPERFSGSLSGSTNTLTISGVQAEDEADYYCGSADNSGAAFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0309] SEQ ID NO: 106 Antibody hB9l9.3 Heavy Chain Sequence

[0310] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFNMFWVRQAPGKGLEWVAGIDDDGSYPNYGSAVKGRATISRDNSKNTLYLQMNSLRAEDTAVYYCAKSGYGGSWGGYIADDIDAWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0311] The specific embodiments described above further elaborate on the objectives, technical solutions, and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. An antibody or an antigen-binding fragment thereof that binds to a frizzled receptor, wherein: the antibody comprises: HCDR1 having the sequence shown in SEQ ID NO: 58, HCDR2 having the sequence shown in SEQ ID NO: 59, HCDR3 having the sequence shown in SEQ ID NO: 60, LCDR1 having the sequence shown in SEQ ID NO: 55, LCDR2 having the sequence shown in SEQ ID NO: 56, and LCDR3 having the sequence shown in SEQ ID NO:

57.

2. The antibody or an antigen-binding fragment thereof according to claim 1, wherein, the antibody comprises: (i) a heavy chain variable region having the sequence shown in SEQ ID NO: 54, and, (ii) a light chain variable region having the sequence shown in SEQ ID NO:

53.

3. The antibody or an antigen-binding fragment thereof according to claim 1 or 2, wherein, the antibody comprises: (i) a heavy chain having the sequence shown in SEQ ID NO: 52, and, (ii) a light chain having the sequence shown in SEQ ID NO:

51.

4. The antibody or an antigen-binding fragment thereof according to claim 1 or 2, wherein, the antibody or an antigen-binding fragment thereof further comprises an Fc domain.

5. The antibody or an antigen-binding fragment thereof according to claim 4, wherein, the Fc domain is an IgG1 Fc domain.

6. The antibody or an antigen-binding fragment thereof according to claim 5, wherein, the Fc domain is a human IgG1 Fc domain.

7. The antibody or an antigen-binding fragment thereof according to claim 6, wherein, the sequence of the human IgG1 Fc domain is shown in SEQ ID NO:

73.

8. The antibody or an antigen-binding fragment thereof according to claim 1 or 2, wherein, the frizzled receptor is a human frizzled receptor.

9. A polynucleotide encoding the antibody or an antigen-binding fragment thereof according to any one of claims 1-8.

10. An expression vector comprising the polynucleotide according to claim 9 or expressing the antibody or an antigen-binding fragment thereof according to any one of claims 1-8.

11. A host cell comprising the expression vector according to claim 10, and the host cell is a host cell for expressing exogenous proteins.

12. The host cell according to claim 11, wherein, the host cell is a bacterium, yeast, insect cell, mammalian cell.

13. A method for preparing the antibody or an antigen-binding fragment thereof according to any one of claims 1-8, characterized in that, it comprises the steps of culturing the host cell according to claim 11 or 12, and recovering the antibody or an antigen-binding fragment thereof from the cell culture.

14. A pharmaceutical composition comprising the antibody or an antigen-binding fragment thereof according to any one of claims 1-8 and a pharmaceutical carrier.

15. A kit comprising the antibody or an antigen-binding fragment thereof according to any one of claims 1-8.

16. The kit according to claim 15, wherein the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof according to any one of claims 1-8.

17. The kit according to claim 16, wherein, the second antibody comprises a detectable label.

18. The kit according to claim 17, wherein, the detectable label is a radioisotope, a luminescent substance, a colored substance, an enzyme or polyethylene glycol.

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